Science.gov

Sample records for daf-16 requires intact

  1. The DAF-16/FOXO transcription factor functions as a regulator of epidermal innate immunity.

    PubMed

    Zou, Cheng-Gang; Tu, Qiu; Niu, Jie; Ji, Xing-Lai; Zhang, Ke-Qin

    2013-01-01

    The Caenorhabditis elegans DAF-16 transcription factor is critical for diverse biological processes, particularly longevity and stress resistance. Disruption of the DAF-2 signaling cascade promotes DAF-16 activation, and confers resistance to killing by pathogenic bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. However, daf-16 mutants exhibit similar sensitivity to these bacteria as wild-type animals, suggesting that DAF-16 is not normally activated by these bacterial pathogens. In this report, we demonstrate that DAF-16 can be directly activated by fungal infection and wounding in wild-type animals, which is independent of the DAF-2 pathway. Fungal infection and wounding initiate the Gαq signaling cascade, leading to Ca(2+) release. Ca(2+) mediates the activation of BLI-3, a dual-oxidase, resulting in the production of reactive oxygen species (ROS). ROS then activate DAF-16 through a Ste20-like kinase-1/CST-1. Our results indicate that DAF-16 in the epidermis is required for survival after fungal infection and wounding. Thus, the EGL-30-Ca(2+)-BLI-3-CST-1-DAF-16 signaling represents a previously unknown pathway to regulate epidermal damage response. PMID:24146615

  2. Cell-nonautonomous signaling of FOXO/DAF-16 to the stem cells of Caenorhabditis elegans.

    PubMed

    Qi, Wenjing; Huang, Xu; Neumann-Haefelin, Elke; Schulze, Ekkehard; Baumeister, Ralf

    2012-01-01

    In Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells. PMID:22916022

  3. Cell-Nonautonomous Signaling of FOXO/DAF-16 to the Stem Cells of Caenorhabditis elegans

    PubMed Central

    Qi, Wenjing; Huang, Xu; Neumann-Haefelin, Elke; Schulze, Ekkehard; Baumeister, Ralf

    2012-01-01

    In Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells. PMID:22916022

  4. 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

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

    PubMed

    Kaplan, Rebecca E W; Chen, Yutao; Moore, Brad T; Jordan, James M; Maxwell, Colin S; Schindler, Adam J; Baugh, L Ryan

    2015-12-01

    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 shows

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

    PubMed

    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

  7. 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

  8. 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

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:26195740

  10. 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

    2015-08-01

    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. PMID:26195740

  11. 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

  12. 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

  13. The solution structure of the forkhead box-O DNA binding domain of Brugia malayi DAF-16a.

    PubMed

    Casper, Sarah K; Schoeller, Scott J; Zgoba, Danielle M; Phillips, Andrew J; Morien, Thomas J; Chaffee, Gary R; Sackett, Peter C; Peterson, Francis C; Crossgrove, Kirsten; Veldkamp, Christopher T

    2014-12-01

    Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF-16a, a putative ortholog of Caenorhabditis elegans DAF-16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF-16 is involved in the insulin/IGF-I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggests that B. malayi DAF-16a is a member of the FOXO family of forkhead proteins. PMID:25297652

  14. Control of cell-fate plasticity and maintenance of multipotency by DAF-16/FoxO in quiescent Caenorhabditis elegans.

    PubMed

    Karp, Xantha; Greenwald, Iva

    2013-02-01

    The Caenorhabditis elegans vulval precursor cells (VPCs) offer a paradigm for investigating how multipotency of progenitor cells is maintained during periods of quiescence. The VPCs are born in the first larval stage. When hermaphrodites are grown under favorable conditions, the EGF-mediated "inductive" signal and the LIN-12/Notch-mediated "lateral" signal confer a precise spatial pattern of distinct vulval cell fates in the third larval stage, a day after hatching. Under adverse conditions, hermaphrodites undergo a prolonged quiescent period as dauer larvae, which can endure for several months with progenitor cells such as VPCs in developmental arrest. If favorable conditions ensue, larvae recover and resume development as postdauer third stage larvae, with the same VPC spatial-patterning events as in continuously developing third stage larvae. Here, we identify several consequences of dauer life history for VPC specification. In wild-type dauers, VPCs undergo a phenomenon reminiscent of natural direct reprogramming to maintain or reestablish multipotency; they acquire an active block to signal transduction by EGF receptor and LIN-12/Notch and have a different mechanism for regulating transcription of the lateral signal. Furthermore, DAF-16/FoxO, a target of insulin/insulin-like growth factor signaling, is required to promote VPC fate plasticity during dauer and for normal vulval patterning after passage through dauer, suggesting that DAF-16/FoxO coordinates environment and life history with plasticity of cell fate. PMID:23341633

  15. 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

  16. 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

  17. 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

  18. 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. PMID:26796881

  19. Structure and developmental expression of Strongyloides stercoralis fktf-1, a proposed ortholog of daf-16 in Caenorhabditis elegans☆

    PubMed Central

    Massey, Holman C.; Nishi, Manami; Chaudhary, Kshitiz; Pakpour, Nazzy; Lok, James B.

    2013-01-01

    A forkhead transcription factor gene, fktf-1, which we propose to be orthologous to the Caenorhabditis elegans dauer-regulatory gene daf-16 has been discovered in the parasitic nematode Strongyloides stercoralis. Genomic and cDNA sequences from both species predict alternately spliced a and b message isoforms. In contrast to C. elegans, where two a isoforms, daf-16a1 and daf-16a2, are found, a single fktf-1a isoform is found in S. stercoralis. Five of the 10 introns found in the C. elegans gene are found in the proposed S. stercoralis ortholog. Functional motifs common to DAF-16 and several mammalian forkhead transcription factors are conserved in FKTF-1. These include the forkhead DNA binding domain, four Akt/protein kinase B phosphorylation sites and a C-terminal domain that may associate with factors such as the steroid receptor coactivator and other factors necessary for transcriptional regulation. An N-terminal serine-rich domain found in DAF-16A is greatly expanded in FKTF-1A. This domain is missing in DAF-16B, FKTF-1B and all mammalian orthologs. FKTF-1 shows the closest phylogenetic relationship to DAF-16 among all known mammalian and nematode forkhead transcription factors. Like its proposed Caenorhabditis ortholog, the fktf-1 message is expressed at all stages of the life cycle examined thus far. Discovery of fktf-1 indicates the presence of an insulin-like signalling pathway in S. stercoralis similar to that known to regulate dauer development in C. elegans. This pathway is a likely candidate to control infective larval arrest and reactivation as well as regulation of the switch between parasitic and free-living development in the parasite. PMID:14572516

  20. Effects of Caenorhabditis elegans sgk-1 mutations on lifespan, stress resistance, and DAF-16/FoxO regulation.

    PubMed

    Chen, Albert Tzong-Yang; Guo, Chunfang; Dumas, Kathleen J; Ashrafi, Kaveh; Hu, Patrick J

    2013-10-01

    The AGC family serine-threonine kinases Akt and Sgk are similar in primary amino acid sequence and in vitro substrate specificity, and both kinases are thought to directly phosphorylate and inhibit FoxO transcription factors. In the nematode Caenorhabditis elegans, it is well established that AKT-1 controls dauer arrest and lifespan by regulating the subcellular localization of the FoxO transcription factor DAF-16. SGK-1 is thought to act similarly to AKT-1 in lifespan control by phosphorylating and inhibiting the nuclear translocation of DAF-16/FoxO. Using sgk-1 null and gain-of-function mutants, we now provide multiple lines of evidence indicating that AKT-1 and SGK-1 influence C. elegans lifespan, stress resistance, and DAF-16/FoxO activity in fundamentally different ways. Whereas AKT-1 shortens lifespan, SGK-1 promotes longevity in a DAF-16-/FoxO-dependent manner. In contrast to AKT-1, which reduces resistance to multiple stresses, SGK-1 promotes resistance to oxidative stress and ultraviolet radiation but inhibits thermotolerance. Analysis of several DAF-16/FoxO target genes that are repressed by AKT-1 reveals that SGK-1 represses a subset of these genes while having little influence on the expression of others. Accordingly, unlike AKT-1, which promotes the cytoplasmic sequestration of DAF-16/FoxO, SGK-1 does not influence DAF-16/FoxO subcellular localization. Thus, in spite of their similar in vitro substrate specificities, Akt and Sgk influence longevity, stress resistance, and FoxO activity through distinct mechanisms in vivo. Our findings highlight the need for a re-evaluation of current paradigms of FoxO regulation by Sgk. PMID:23786484

  1. Central nervous system promotes thermotolerance via FoxO/DAF-16 activation through octopamine and acetylcholine signaling in Caenorhabditis elegans.

    PubMed

    Furuhashi, Tsubasa; Sakamoto, Kazuichi

    2016-03-25

    The autonomic nervous system (ANS) responds to many kinds of stressors to maintain homeostasis. Although the ANS is believed to regulate stress tolerance, the exact mechanism underlying this is not well understood. To understand this, we focused on longevity genes, which have functions such as lifespan extension and promotion of stress tolerance. To understand the relationship between ANS and longevity genes, we analyzed stress tolerance of Caenorhabditis elegans treated with octopamine, which has an affinity to noradrenaline in insects, and acetylcholine. Octopamine and acetylcholine did not show resistance against H2O2, but the neurotransmitters promoted thermotolerance via DAF-16. However, chronic treatment with octopamine and acetylcholine did not extend the lifespan, although DAF-16 plays an important role in longevity. In conclusion, our results show that octopamine and acetylcholine activate DAF-16 in response to stress, but chronic induction of octopamine and acetylcholine is not beneficial for increasing longevity. PMID:26903298

  2. Structural and functional characterisation of the fork head transcription factor-encoding gene, Hc-daf-16, from the parasitic nematode Haemonchus contortus (Strongylida)

    PubMed Central

    Hu, Min; Lok, James B.; Ranjit, Najju; Massey, Holman C.; Sternberg, Paul W.; Gasser, Robin B.

    2010-01-01

    Despite their phylogenetic diversity, parasitic nematodes share attributes of longevity and developmental arrest (=hypobiosis) with free-living nematodes at key points in their life cycles, particularly in larval stages responsible for establishing infection in the host. Insulin-like signalling plays crucial roles in the regulation of life span and arrest (=dauer formation) in the free-living nematode, Caenorhabditis elegans. Insulin-like signalling in C. elegans negatively regulates the fork head boxO (FoxO) transcription factor encoded by daf-16, which is linked to initiating a dauer-specific pattern of gene expression. Orthologues of daf-16 have been identified in several species of parasitic nematode. Although function has been demonstrated for an orthologue from the parasitic nematode Strongyloides stercoralis (Rhabditida), the functional capabilities of homologues/orthologues in bursate nematodes (Strongylida) are unknown. In the present study, we used a genomic approach to determine the structures of two complete daf-16 orthologues (designated Hc-daf-16.1 and Hc-daf-16.2) and their transcripts in the parasitic nematode Haemonchus contortus, and assessed their function(s) using C. elegans as a genetic surrogate. Unlike the multiple isoforms of Ce-DAF-16 and Ss-DAF-16, which are encoded by a single gene and produced by alternative splicing, mRNAs encoding the proteins Hc-DAF-16.1 and Hc-DAF-16.2 are transcribed from separate and distinct loci. Both orthologues are transcribed in all developmental stages and both sexes of H. contortus, and the inferred proteins (603 and 556 amino acids) each contain a characteristic, highly conserved fork head domain. In spite of distinct differences in genomic organisation compared with orthologues in C. elegans and S. stercoralis, genetic complementation studies demonstrated here that Hc-daf-16.2, but not Hc-daf-16.1, could restore daf-16 function to a C. elegans strain carrying a null mutation at this locus. These findings

  3. EPs7630(®) from Pelargonium sidoides increases stress resistance in Caenorhabditis elegans probably via the DAF-16/FOXO pathway.

    PubMed

    Rezaizadehnajafi, Leila; Wink, Michael

    2014-03-15

    EPs7630(®) a water alcohol extract of the roots from Pelargonium sidoides contains several secondary metabolites including highly oxygenated coumarins, various phenolics and polyphenols. Using the DPPH assay to measure antioxidant activity a free radical scavenging activity of 14.7±0.85μg/ml (IC50) was determined. As an in vivo model Caenorhabditis elegans was applied to study the effect of EPs7630(®) on stress resistance. EPs7630(®) treatment reduces intracellular hsp-16.2::GFP expression (induced by the pro-oxidant juglone) indicating that the secondary metabolites of EPs7630(®) are bioavailable and exhibit antioxidant activities in vivo. Application of EPs7630(®) (50μg/ml) to the transgenic mutant TJ356 induced the migration of the transcription factor DAF-16 from cytosol to the nucleus, suggesting a prominent role of DAF-16/FOXO in the daf-2 pathway for stress resistance. PMID:24252337

  4. 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

    2012-05-01

    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. PMID:22560223

  5. 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 PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units § 174.045 Intact stability requirements. (a)...

  6. 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

  7. 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

  8. 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...

  9. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 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...

  10. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 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...

  11. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 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...

  12. Diapause Formation and Downregulation of Insulin-Like Signaling via DAF-16/FOXO Delays Axonal Degeneration and Neuronal Loss

    PubMed Central

    Calixto, Andrea; Jara, Juan S.; Court, Felipe A.

    2012-01-01

    Axonal degeneration is a key event in the pathogenesis of neurodegenerative conditions. We show here that mec-4d triggered axonal degeneration of Caenorhabditis elegans neurons and mammalian axons share mechanistical similarities, as both are rescued by inhibition of calcium increase, mitochondrial dysfunction, and NMNAT overexpression. We then explore whether reactive oxygen species (ROS) participate in axonal degeneration and neuronal demise. C. elegans dauers have enhanced anti-ROS systems, and dauer mec-4d worms are completely protected from axonal degeneration and neuronal loss. Mechanistically, downregulation of the Insulin/IGF-1-like signaling (IIS) pathway protects neurons from degenerating in a DAF-16/FOXO–dependent manner and is related to superoxide dismutase and catalase-increased expression. Caloric restriction and systemic antioxidant treatment, which decrease oxidative damage, protect C. elegans axons from mec-4d-mediated degeneration and delay Wallerian degeneration in mice. In summary, we show that the IIS pathway is essential in maintaining neuronal homeostasis under pro-degenerative stimuli and identify ROS as a key intermediate of neuronal degeneration in vivo. Since axonal degeneration represents an early pathological event in neurodegeneration, our work identifies potential targets for therapeutic intervention in several conditions characterized by axonal loss and functional impairment. PMID:23300463

  13. 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

  14. 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

  15. 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

    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

    2014-02-01

    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

  16. Pyrroloquinoline quinone enhances the resistance to oxidative stress and extends lifespan upon DAF-16 and SKN-1 activities in C. elegans.

    PubMed

    Wu, J Z; Huang, J H; Khanabdali, R; Kalionis, B; Xia, S J; Cai, W J

    2016-07-01

    Pyrroloquinoline quinone (PQQ) is linked to fundamental biological processes such as mitochondrial biogenesis and lipid metabolism. PQQ may also function as an essential micronutrient during animal development. Recent studies have shown the therapeutic potential of PQQ for several age-related diseases due to its antioxidant capacity. However, whether PQQ can promote longevity is unknown. Here, we investigate the effects of PQQ on oxidative stress resistance as well as lifespan modulation in Caenorhabditis elegans. We find that PQQ enhances resistance to oxidative stress and extends the lifespan of C. elegans at optimal doses. The underlying molecular mechanism involves the increased activities of the primary lifespan extension transcriptional factors DAF-16/FOXO, the conserved oxidative stress-responsive transcription factor SKN-1/Nrf2, and upregulation of daf-16, skn-1 downstream targets including sod-3, hsp16.2, gst-1 and gst-10. Our findings uncover a novel role of PQQ in longevity, supporting PQQ as a possible dietary supplement for overall health improvement. PMID:27090484

  17. True navigation in migrating gulls requires intact olfactory nerves.

    PubMed

    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

  18. 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

  19. Ida-1, the Caenorhabditis elegans Orthologue of Mammalian Diabetes Autoantigen IA-2, Potentially Acts as a Common Modulator between Parkinson’s Disease and Diabetes: Role of Daf-2/Daf-16 Insulin Like Signalling Pathway

    PubMed Central

    Fatima, Soobiya; Haque, Rizwanul; Jadiya, Pooja; Shamsuzzama; Kumar, Lalit; Nazir, Aamir

    2014-01-01

    The lack of cure to age associated Parkinson’s disease (PD) has been challenging the efforts of researchers as well as health care providers. Recent evidences suggest that diabetic patients tend to show a higher future risk for PD advocating a strong correlation between PD and Diabetes, thus making it intriguing to decipher common genetic cues behind these ailments. We carried out studies on ida-1, the C. elegans orthologue of mammalian type-1 diabetes auto-antigen IA-2 towards achieving its functional workup vis-à-vis various associated endpoints of PD and Diabetes. Employing transgenic C. elegans strain expressing “human” alpha synuclein (NL5901) under normal and increased glucose concentrations, we studied aggregation of alpha synuclein, content of dopamine, expression of dopamine transporter, content of reactive oxygen species, locomotor activity, nuclear translocation of FOXO transcription factor Daf-16, and quantification of Daf2/Daf-16 mRNA. Our findings indicate that ida-1 affords protection in the studied disease conditions as absence of ida-1 resulted in higher alpha-synuclein aggregation under conditions that mimic the blood glucose levels of diabetic patients. We also observed reduced dopamine content, decreased motility, defective Daf-16 translocation and reduced expression of Daf-2 and Daf-16. Our studies establish important function of ida-1 as a modulator in Daf-2/Daf-16 insulin like signalling pathway thus possibly being a common link between PD and Diabetes. PMID:25469508

  20. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements for a sailing catamaran... Intact stability requirements for a sailing catamaran. (a) A sailing vessel that operates on protected...) A sailing vessel that operates on partially protected or exposed waters must be designed to...

  1. 46 CFR 178.325 - Intact stability requirements for a sailing vessel.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements for a sailing vessel. 178... Intact stability requirements for a sailing vessel. (a) Except as provided in paragraphs (b), (c) and (e) of this section, each sailing vessel must undergo a simplified stability proof test in...

  2. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements for a sailing catamaran... Intact stability requirements for a sailing catamaran. (a) A sailing vessel that operates on protected...) A sailing vessel that operates on partially protected or exposed waters must be designed to...

  3. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements for a sailing catamaran... Intact stability requirements for a sailing catamaran. (a) A sailing vessel that operates on protected...) A sailing vessel that operates on partially protected or exposed waters must be designed to...

  4. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements for a sailing catamaran... Intact stability requirements for a sailing catamaran. (a) A sailing vessel that operates on protected...) A sailing vessel that operates on partially protected or exposed waters must be designed to...

  5. 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. 178.320 Section 178.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER... Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part,...

  6. 46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements-monohull sailing vessels....325 Intact stability requirements—monohull sailing vessels. (a) As permitted by § 178.310(c) of this part, a monohull sailing vessel may demonstrate compliance with paragraphs (b) or (c) of this...

  7. 46 CFR 178.320 - Intact stability requirements-non-sailing vessels.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements-non-sailing vessels. 178.320 Section 178.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER... Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part,...

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

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements-monohull sailing vessels....325 Intact stability requirements—monohull sailing vessels. (a) As permitted by § 178.310(c) of this part, a monohull sailing vessel may demonstrate compliance with paragraphs (b) or (c) of this...

  9. 46 CFR 178.320 - Intact stability requirements-non-sailing vessels.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-non-sailing vessels. 178.320 Section 178.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER... Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part,...

  10. 46 CFR 178.320 - Intact stability requirements-non-sailing vessels.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements-non-sailing vessels. 178.320 Section 178.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER... Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part,...

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

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-monohull sailing vessels....325 Intact stability requirements—monohull sailing vessels. (a) As permitted by § 178.310(c) of this part, a monohull sailing vessel may demonstrate compliance with paragraphs (b) or (c) of this...

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

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements-monohull sailing vessels....325 Intact stability requirements—monohull sailing vessels. (a) As permitted by § 178.310(c) of this part, a monohull sailing vessel may demonstrate compliance with paragraphs (b) or (c) of this...

  13. 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

  14. 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...

  15. 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...

  16. 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...

  17. 46 CFR 173.063 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 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...

  18. 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...

  19. 46 CFR 171.057 - Intact stability requirements for a sailing catamaran.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements for a sailing catamaran... stability requirements for a sailing catamaran. (a) A sailing vessel that operates on protected waters must... vessel, in kilograms (pounds). X=4.88 kilograms/square meter (1.0 pounds/square foot). (b) A...

  20. ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex†

    PubMed Central

    Komarnitsky, Philip B.; Klebanow, Edward R.; Weil, P. Anthony; Denis, Clyde L.

    1998-01-01

    The yeast transcriptional activator ADR1, which is required for ADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex. PMID:9742103

  1. 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 44bp 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 44bp 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. PMID:26963674

  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. 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

    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

  4. 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

  5. B cells are required for lupus nephritis in the polygenic, Fas-intact MRL model of systemic autoimmunity.

    PubMed

    Chan, O T; Madaio, M P; Shlomchik, M J

    1999-10-01

    B cells are required for both the expression of lupus nephritis and spontaneous T cell activation/memory cell accumulation in MRL-Faslpr mice (MRL/lpr). Autoimmunity in the MRL/lpr strain is the result of Fas-deficiency and multiple background genes; however, the precise roles of background genes vs Fas-deficiency have not been fully defined. Fas-deficiency (i.e., the lpr defect) is required in B cells for optimal autoantibody expression, raising the possibility that the central role for B cells in MRL/lpr mice may not extend to MRL/+ mice and, thus, to lupus models that do not depend on Fas-deficiency ("polygenic lupus"). To address this issue, B cell-deficient, Fas-intact MRL/+ mice (JHd-MRL/) were created; and disease was evaluated in aged animals (>9 mo). The JHd-MRL/+ animals did not develop nephritis or vasculitis at a time when the B cell-intact littermates had severe disease. In addition, while activated/memory CD4+ and CD8+ T cells accumulated in B cell-intact mice, such accumulation was substantially inhibited in the absence of B cells. This effect appeared to be restricted to the MRL strain because it was not seen in B cell-deficient BALB/c mice (JHd-BALB) of similar ages. The results indicate that B cells are essential in promoting systemic autoimmunity in a Fas-independent model. Therefore, B cells have an important role in pathogenesis, generalizable to lupus models that depend on multiple genes even when Fas expression is intact. The results provide further rationale for B cell suppression as therapy for systemic lupus erythematosus. PMID:10490951

  6. IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake

    PubMed Central

    Deak, Andras T.; Blass, Sandra; Khan, Muhammad J.; Groschner, Lukas N.; Waldeck-Weiermair, Markus; Hallström, Seth; Graier, Wolfgang F.; Malli, Roland

    2014-01-01

    ABSTRACT Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER–plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains. PMID:24806964

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

    PubMed

    Lee, Jennifer K; Hu, Jerry C Y; Yamada, Soichiro; Athanasiou, Kyriacos A

    2016-02-01

    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

  9. C. elegans patched-3 is an essential gene implicated in osmoregulation and requiring an intact permease transporter domain

    PubMed Central

    Soloviev, Alexander; Gallagher, Joseph; Marnef, Aline; Kuwabara, Patricia E.

    2011-01-01

    The nematode Caenorhabditis elegans has retained a rudimentary Hedgehog (Hh) signalling pathway; Hh and Smoothened (Smo) homologs are absent, but two highly related Patched gene homologs, ptc-1 and ptc-3, and 24 ptc-related (ptr) genes are present. We previously showed that ptc-1 is essential for germ line cytokinesis. Here, we report that ptc-3 is also an essential gene; the absence of ptc-3 results in a late embryonic lethality due to an apparent defect in osmoregulation. Rescue of a ptc-3 mutant with a ptc-3::gfp translational reporter reveals that ptc-3 is dynamically expressed in multiple tissues across development. Consistent with this pattern of expression, ptc-3(RNAi) reveals an additional postembryonic requirement for ptc-3 activity. Tissue-specific promoter studies indicate that hypodermal expression of ptc-3 is required for normal development. Missense changes in key residues of the sterol sensing domain (SSD) and the permease transporter domain GxxxD/E motif reveal that the transporter domain is essential for PTC-3 activity, whereas an intact SSD is dispensable. Taken together, our studies indicate that PTC proteins have retained essential roles in C. elegans that are independent of Smoothened (Smo). These observations reveal novel, and perhaps ancestral, roles for PTC proteins. PMID:21215260

  10. Buspirone requires the intact nigrostriatal pathway to reduce the activity of the subthalamic nucleus via 5-HT1A receptors.

    PubMed

    Sagarduy, A; Llorente, J; Miguelez, C; Morera-Herreras, T; Ruiz-Ortega, J A; Ugedo, L

    2016-03-01

    The most effective treatment for Parkinson's disease (PD), l-DOPA, induces dyskinesia after prolonged use. We have previously shown that in 6-hydroxydopamine (6-OHDA) lesioned rats rendered dyskinetic by prolonged l-DOPA administration, lesion of the subthalamic nucleus (STN) reduces not only dyskinesias but also buspirone antidyskinetic effect. This study examined the effect of buspirone on STN neuron activity. Cell-attached recordings in parasagittal slices from naïve rats showed that whilst serotonin excited the majority of STN neurons, buspirone showed an inhibitory main effect but only in 27% of the studied cells which was prevented by the 5-HT1A receptor selective antagonist WAY-100635. Conversely, single-unit extracellular recordings were performed in vivo on STN neurons from four different groups, i.e., control, chronically treated with l-DOPA, 6-OHDA lesioned and lesioned treated with l-DOPA (dyskinetic) rats. In control animals, systemic-buspirone administration decreased the firing rate in a dose-dependent manner in every cell studied. This effect, prevented by WAY-100635, was absent in 6-OHDA lesioned rats and was not modified by prolonged l-DOPA administration. Altogether, buspirone in vivo reduces consistently the firing rate of the STN neurons through 5-HT1A receptors whereas ex vivo buspirone seems to affect only a small population of STN neurons. Furthermore, the lack of effect of buspirone in 6-OHDA lesioned rats, suggests the requirement of not only the activation of 5-HT1A receptors but also an intact nigrostriatal pathway for buspirone to inhibit the STN activity. PMID:26687972

  11. 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.

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

    PubMed

    Oláhová, Monika; Veal, Elizabeth A

    2015-08-01

    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

  13. 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. PMID:23986233

  14. 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.

  15. 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. PMID:24056073

  16. Monitoring Intact Viruses Using Aptamers.

    PubMed

    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

  17. (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.

  18. The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

    PubMed Central

    Seemann, J; Weber, K; Osborn, M; Parton, R G; Gerke, V

    1996-01-01

    Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes. Images PMID:8885232

  19. H(2)O(2) detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system.

    PubMed

    Staniek, K; Nohl, H

    1999-10-01

    Evaluation of the existence of superoxide radicals (O*-(2)), the site of generation and conditions required for one-e(-) transfer to oxygen from biological redox systems is a prerequisite for the understanding of the deregulation of O(2) homeostasis leading to oxidative stress. Mitochondria are increasingly considered the major O*-(2) source in a great variety of diseases and the aging process. Contradictory reports on mitochondrial O*-(2) release prompted us to critically investigate frequently used O*-(2) detection methods for their suitability. Due to the impermeability of the external mitochondrial membrane for most constituents of O*-(2) detection systems we decided to follow the stable dismutation product H(2)O(2). This metabolite was earlier shown to readily permeate into the cytosol. With the exception of tetramethylbenzidine none of the chemical reactants indicating the presence of H(2)O(2) by horseradish peroxidase-catalyzed absorbance change were suited due to solubility problems or low extinction coefficients. Tetramethylbenzidine-dependent H(2)O(2) detection was counteracted by rereduction of the dye through e(-) carriers of the respiratory chain. Although the fluorescent dyes scopoletin and homovanillic acid were found to be suited for the detection of mitochondrial H(2)O(2) release, fluorescence change was strongly affected by mitochondrial protein constituents. The present study has resolved this problem by separating the detection system from H(2)O(2)-producing mitochondria. PMID:10514548

  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. 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.

  2. 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... 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...

  3. 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...

  4. 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... 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...

  5. 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... 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...

  6. 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...

  7. 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...

  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. 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. PMID:26854551

  10. Small Particles Intact Capture Experiment (SPICE)

    NASA Astrophysics Data System (ADS)

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

    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.

  11. 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.

  12. 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.

  13. 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.

  14. 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. PMID:26873884

  15. Language and Williams syndrome: how intact is "intact"?

    PubMed

    Karmiloff-Smith, A; Grant, J; Berthoud, I; Davies, M; Howlin, P; Udwin, O

    1997-04-01

    It has been claimed that Williams syndrome (WS), a rare neurodevelopmental disorder, is characterized by serious cognitive deficits alongside intact language. The syndrome is often used as a prime example of the modularity of an innate faculty for morphosyntactic rules. We challenge this claim and hypothesize that morphosyntax, although surprisingly good given WS level of mental retardation, is by no means intact. We make an initial test of this hypothesis through an analysis of the receptive language of a group of English-speaking WS individuals on a standardized morphosyntactic test. We then present an experimental study of expressive language that examines grammatical gender assignment in French-speaking WS patients. Despite a Verbal Mental Age selected to be higher than the chronological age of the young control group, these people with WS continue even in adulthood to show clear-cut deficits in their production of an aspect of morphosyntax that normal children acquire effortlessly very early. The results of the 2 studies, one focusing on receptive language and the other on expressive language, challenge the notion that comprehension and use of morphosyntactic rules in WS individuals are intact. The Within-domain dissociations regarding the use of grammatical gender assignment across several sentence clements and their difficulties in understanding embedded sentences-two quintessentially linguistic skills-suggest that we must rethink the notion of spared, modular, language capacities in Williams syndrome. We conclude that WS language follows a different path to normal acquisition and may turn out to be more like second language learning. PMID:9180000

  16. 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. PMID:11538362

  17. Mechanics of intact bone marrow.

    PubMed

    Jansen, Lauren E; Birch, Nathan P; Schiffman, Jessica D; Crosby, Alfred J; Peyton, Shelly R

    2015-10-01

    The current knowledge of bone marrow mechanics is limited to its viscous properties, neglecting the elastic contribution of the extracellular matrix. To get a more complete view of the mechanics of marrow, we characterized intact yellow porcine bone marrow using three different, but complementary techniques: rheology, indentation, and cavitation. Our analysis shows that bone marrow is elastic, and has a large amount of intra- and inter-sample heterogeneity, with an effective Young׳s modulus ranging from 0.25 to 24.7 kPa at physiological temperature. Each testing method was consistent across matched tissue samples, and each provided unique benefits depending on user needs. We recommend bulk rheology to capture the effects of temperature on tissue elasticity and moduli, indentation for quantifying local tissue heterogeneity, and cavitation rheology for mitigating destructive sample preparation. We anticipate the knowledge of bone marrow elastic properties for building in vitro models will elucidate mechanisms involved in disease progression and regenerative medicine. PMID:26189198

  18. Teaching basic neurophysiology using intact earthworms.

    PubMed

    Kladt, Nikolay; Hanslik, Ulrike; Heinzel, Hans-Georg

    2010-01-01

    Introductory neurobiology courses face the problem that practical exercises often require expensive equipment, dissections, and a favorable student-instructor ratio. Furthermore, the duration of an experiment might exceed available time or the level of required expertise is too high to successfully complete the experiment. As a result, neurobiological experiments are commonly replaced by models and simulations, or provide only very basic experiments, such as the frog sciatic nerve preparation, which are often time consuming and tedious. Action potential recordings in giant fibers of intact earthworms (Lumbricus terrestris) circumvent many of these problems and result in a nearly 100% success rate. Originally, these experiments were introduced as classroom exercises by Charles Drewes in 1978 using awake, moving earthworms. In 1990, Hans-Georg Heinzel described further experiments using anesthetized earthworms. In this article, we focus on the application of these experiments as teaching tools for basic neurobiology courses. We describe and extend selected experiments, focusing on specific neurobiological principles with experimental protocols optimized for classroom application. Furthermore, we discuss our experience using these experiments in animal physiology and various neurobiology courses at the University of Bonn. PMID:23494516

  19. Teaching Basic Neurophysiology Using Intact Earthworms

    PubMed Central

    Kladt, Nikolay; Hanslik, Ulrike; Heinzel, Hans-Georg

    2010-01-01

    Introductory neurobiology courses face the problem that practical exercises often require expensive equipment, dissections, and a favorable student-instructor ratio. Furthermore, the duration of an experiment might exceed available time or the level of required expertise is too high to successfully complete the experiment. As a result, neurobiological experiments are commonly replaced by models and simulations, or provide only very basic experiments, such as the frog sciatic nerve preparation, which are often time consuming and tedious. Action potential recordings in giant fibers of intact earthworms (Lumbricus terrestris) circumvent many of these problems and result in a nearly 100% success rate. Originally, these experiments were introduced as classroom exercises by Charles Drewes in 1978 using awake, moving earthworms. In 1990, Hans-Georg Heinzel described further experiments using anesthetized earthworms. In this article, we focus on the application of these experiments as teaching tools for basic neurobiology courses. We describe and extend selected experiments, focusing on specific neurobiological principles with experimental protocols optimized for classroom application. Furthermore, we discuss our experience using these experiments in animal physiology and various neurobiology courses at the University of Bonn. PMID:23494516

  20. 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

  1. 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.

  2. Vitrification of intact human articular cartilage.

    PubMed

    Jomha, Nadr M; Elliott, Janet A W; Law, Garson K; Maghdoori, Babak; Forbes, J Fraser; Abazari, Alireza; Adesida, Adetola B; Laouar, Leila; Zhou, Xianpei; McGann, Locksley E

    2012-09-01

    Articular cartilage injuries do not heal and large defects result in osteoarthritis with major personal and socioeconomic costs. Osteochondral transplantation is an effective treatment for large joint defects but its use is limited by the inability to store cartilage for long periods of time. Cryopreservation/vitrification is one method to enable banking of this tissue but decades of research have been unable to successfully preserve the tissue while maintaining cartilage on its bone base - a requirement for transplantation. To address this limitation, human knee articular cartilage from total knee arthroplasty patients and deceased donors was exposed to specified concentrations of 4 different cryoprotective agents for mathematically determined periods of time at lowering temperatures. After complete exposure, the cartilage was immersed in liquid nitrogen for up to 3 months. Cell viability was 75.4 ± 12.1% determined by membrane integrity stains and confirmed with a mitochondrial assay and pellet culture documented production of sulfated glycosaminoglycans and collagen II similar to controls. This report documents successful vitrification of intact human articular cartilage on its bone base making it possible to bank this tissue indefinitely. PMID:22698720

  3. The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

    PubMed

    Moyers, J S; Bouton, A H; Parsons, S J

    1993-04-01

    Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera

  4. 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.

  5. 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.

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

    PubMed

    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. However, 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. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, 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. PMID:27626670

  7. Intact endothelial autophagy is required to maintain vascular lipid homeostasis.

    PubMed

    Torisu, Kumiko; Singh, Krishna K; Torisu, Takehiro; Lovren, Fina; Liu, Jie; Pan, Yi; Quan, Adrian; Ramadan, Azza; Al-Omran, Mohammed; Pankova, Natalie; Boyd, Shelley R; Verma, Subodh; Finkel, Toren

    2016-02-01

    The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular (125) I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE(-/-) mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age-dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease. PMID:26780888

  8. 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

  9. Lipid diffusibility in the intact erythrocyte membrane.

    PubMed Central

    Bloom, J A; Webb, W W

    1983-01-01

    The lateral diffusion of fluorescent lipid analogues in the plasma membrane of intact erythrocytes from man, mouse, rabbit, and frog has been measured by fluorescence photobleaching recovery (FPR). Intact cells from dystrophic, normoblastic, hemolytic, and spherocytotic mouse mutants; from hypercholesterolemic rabbits and humans; and from prenatal, neonatal, and juvenile mice have been compared with corresponding normals. The lateral diffusion coefficient (D) for 3,3'-dioctadecylindodicarbocyanine iodide (DiI[5]) in intact normal human erythrocytes is D = 8.2 +/- 1.2 X 10(-9) cm2/s at 25 degrees C and D = 2.1 +/- 0.4 X 10(-8) cm2/s at 37 degrees C, and varies approximately 50-fold between 1 degree and 42 degrees C. The diffusion constants of lipid analogue rhodamine-B phosphatidylethanolamine (RBPE) are about twice those of DiI[5]. The temperature dependence and magnitude of D vary by up to a factor of 3 between species and are only influenced by donor age in prenatals. DiI[5] diffusibility is not perturbed by the presence of calcium or local anesthetics or by spectrin depletion (via mutation). However, lipid-analogue diffusibility in erythrocyte ghosts may differ from intact cells. Dietary hypercholesterolemia in rabbits reduces the diffusion coefficient and eliminates the characteristic break in Arrhenius plots of D found in all other cells studied except frog. PMID:6603237

  10. 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 out of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. he headspace of an intact c...

  11. 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 ...

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

    PubMed

    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

  13. 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

  14. 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. PMID:24630119

  15. 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.

  16. Measuring mitochondrial function in intact cardiac myocytes

    PubMed Central

    Dedkova, Elena N.; Blatter, Lothar A.

    2011-01-01

    Mitochondria are involved in cellular functions that go beyond the traditional role of these organelles as the power plants of the cell. Mitochondria have been implicated in several human diseases, including cardiac dysfunction, and play a role in the aging process. Many aspects of our knowledge of mitochondria stem from studies performed on the isolated organelle. Their relative inaccessibility imposes experimental difficulties to study mitochondria in their natural environment – the cytosol of intact cells – and has hampered a comprehensive understanding of the plethora of mitochondrial functions. Here we review currently available methods to study mitochondrial function in intact cardiomyocytes. These methods primarily use different flavors of fluorescent dyes and genetically encoded fluorescent proteins in conjunction with high-resolution imaging techniques. We review methods to study mitochondrial morphology, mitochondrial membrane potential, Ca2+ and Na+ signaling, mitochondrial pH regulation, redox state and ROS production, NO signaling, oxygen consumption, ATP generation and the activity of the mitochondrial permeability transition pore. Where appropriate we complement this review on intact myocytes with seminal studies that were performed on isolated mitochondria, permeabilized cells, and in whole hearts. PMID:21964191

  17. Multi-View Intact Space Learning.

    PubMed

    Xu, Chang; Tao, Dacheng; Xu, Chao

    2015-12-01

    It is practical to assume that an individual view is unlikely to be sufficient for effective multi-view learning. Therefore, integration of multi-view information is both valuable and necessary. In this paper, we propose the Multi-view Intact Space Learning (MISL) algorithm, which integrates the encoded complementary information in multiple views to discover a latent intact representation of the data. Even though each view on its own is insufficient, we show theoretically that by combing multiple views we can obtain abundant information for latent intact space learning. Employing the Cauchy loss (a technique used in statistical learning) as the error measurement strengthens robustness to outliers. We propose a new definition of multi-view stability and then derive the generalization error bound based on multi-view stability and Rademacher complexity, and show that the complementarity between multiple views is beneficial for the stability and generalization. MISL is efficiently optimized using a novel Iteratively Reweight Residuals (IRR) technique, whose convergence is theoretically analyzed. Experiments on synthetic data and real-world datasets demonstrate that MISL is an effective and promising algorithm for practical applications. PMID:26539856

  18. Radioimmunoassay for intact Gross mouse leukemia virus.

    PubMed Central

    Yalow, R S; Gross, L

    1976-01-01

    A radioimmunoassay for intact Gross leukemia virus has been developed using 125I-labeled Gross virus grown in tissue culture and guinea pig antisera to Gross virus grown either in tissue culture or harvested from leukemic C3H(f) mice. Separation of bound from free labeled virus was effected using the double antibody method. The assay can detect fewer than 10(8) virus particles and has been used to measure the viral content of individual organs from inoculated leukemic C3H(f) mice and from Ak mice with spontaneous leukemia. Organs from noninoculated healthy C3H(f) mice crossreacted poorly in the system, virus generally being detectable only in the thymus and spleen and at low concentration. In some of the inoculated C3H(f) leukemic mice the viral content of as little as 0.5 mul of plasma is measurable. That this assay is for intact virus and not for soluble antigens of the viral envelope was proven by the observation that the immunoreactive material of plasma and extracts from thymus and liver of leukemic mice has a buoyant denisty in sucrose of 1.17-1.18 g/ml, corresponding to that of intact virus grown in tissue culture. With this sensitivity it may now be possible to quantitate viral concentrations in tissue and body fluids from the time of inoculation through the development of obvious pathology. PMID:1066697

  19. 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

  20. 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

  1. High frequency oscillations in the intact brain

    PubMed Central

    Buzsáki, György; da Silva, Fernando Lopes

    2016-01-01

    High frequency oscillations (HFOs) constitute a novel trend in neurophysiology that is fascinating neuroscientists in general, and epileptologists in particular. But what are HFOs? What is the frequency range of HFOs? Are there different types of HFOs, physiological and pathological? How are HFOs generated? Can HFOs represent temporal codes for cognitive processes? These questions are pressing and this symposium volume attempts to give constructive answers. As a prelude to this exciting discussion, we summarize the physiological high frequency patterns in the intact brain, concentrating mainly on hippocampal patterns, where the mechanisms of high frequency oscillations are perhaps best understood. PMID:22449727

  2. 7 CFR 160.29 - Containers to remain intact.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Containers to remain intact. 160.29 Section 160.29... STANDARDS FOR NAVAL STORES Analysis, Inspection, and Grading on Request § 160.29 Containers to remain intact... the containers holding such naval stores remain intact as sampled until the analysis,...

  3. 50 CFR 622.38 - Landing fish intact.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 10 2011-10-01 2011-10-01 false Landing fish intact. 622.38 Section 622... § 622.38 Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish on that vessel in the EEZ are maintained intact and, if taken from the EEZ,...

  4. 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

  5. Intact Protein Quantitation Using Pseudoisobaric Dimethyl Labeling.

    PubMed

    Fang, Houqin; Xiao, Kaijie; Li, Yunhui; Yu, Fan; Liu, Yan; Xue, Bingbing; Tian, Zhixin

    2016-07-19

    Protein structural and functional studies rely on complete qualitative and quantitative information on protein species (proteoforms); thus, it is important to quantify differentially expressed proteins at their molecular level. Here we report our development of universal pseudoisobaric dimethyl labeling (pIDL) of amino groups at both the N-terminal and lysine residues for relative quantitation of intact proteins. Initial proof-of-principle study was conducted on standard protein myoglobin and hepatocellular proteomes (HepG2 vs LO2). The amino groups from both the N-terminal and lysine were dimethylated with HXHO (X = (13)C or C) and NaBY3CN (Y = H or D). At the standard protein level, labeling efficiency, effect of product ion size, and mass resolution on quantitation accuracy were explored; and a good linear quantitation dynamic range up to 50-fold was obtained. For the hepatocellular proteome samples, 33 proteins were quantified with RSD ≤ 10% from one-dimensional reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis of the 1:1 mixed samples. The method in this study can be extended to quantitation of other intact proteome systems. The universal "one-pot" dimethyl labeling of all the amino groups in a protein without the need of preblocking of those on the lysine residues is made possible by protein identification and quantitation analysis using ProteinGoggle 2.0 with customized databases of both precursor and product ions containing heavy isotopes. PMID:27359340

  6. 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.…

  7. 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

  8. 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.

  9. 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. PMID:16627698

  10. 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

  11. Effects of intact versus non-intact families on adolescent head injury rehabilitation.

    PubMed

    Barry, P; Clark, D

    1992-01-01

    Forty-one children, aged 8-18 years, were admitted to a comprehensive in-patient head injury rehabilitation facility over a 5-year period. Over half (59%) came from families with only one biological parent in the home. Data from intact versus non-intact families suggest that the children from the latter were significantly younger and remained as in-patients significantly longer than their counterparts. The severity of injuries and incidence of premorbid psychosocial documentation were equivalent for the two groups. The implications for treatment and future research are discussed. A previous version of this paper was presented at the 97th convention of the American Psychological Association in New Orleans on August 13, 1989. The authors are grateful to Dr Mark Ylvisaker for his comments and suggestions. PMID:1581746

  12. 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

  13. 33. TURBINE HALL, ORIGINAL TURBO GENERATOR, INTACT CONDENSER Philadelphia ...

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

    33. TURBINE HALL, ORIGINAL TURBO GENERATOR, INTACT CONDENSER - Philadelphia Electric Company, Richmond Power Station, Southeast end of Lewis Street along Delaware River, Philadelphia, Philadelphia County, PA

  14. Development of a novel immunoassay specific for mouse intact proinsulin.

    PubMed

    Imai, Sunao; Takahashi, Tatsuya; Naito, Shoichi; Yamauchi, Akira; Okada, Chihiro; Notsu, Yoshihide; Sakikawa, Ikue; Hatanaka, Michiyoshi; Iwasaki, Takanori; Morita, Atsushi; Fujii, Ikuo; Yamane, Shoji

    2015-09-01

    The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM. PMID:26026387

  15. Nitrite Uptake into Intact Pea Chloroplasts 1

    PubMed Central

    Brunswick, Pamela; Cresswell, Christopher F.

    1988-01-01

    The relationship between net nitrite uptake and its reduction in intact pea chloroplasts was investigated employing electron transport regulators, uncouplers, and photophosphorylation inhibitors. Observations confirmed the dependence of nitrite uptake on stromal pH and nitrite reduction but also suggested a partial dependance upon PSI phosphorylation. It was also suggested that ammonia stimulates nitrogen assimilation in the dark by association with stromal protons. Inhibition of nitrite uptake by N-ethylmaleimide and dinitrofluorobenzene could not be completely attributed to their inhibition of carbon dioxide fixation. Other protein binding reagents which inhibited photosynthesis showed no effect on nitrite uptake, except for p-chlormercuribenzoate which stimulated nitrite uptake. The results with N-ethylmaleimide and dinitrofluorobenzene tended to support the proposed presence of a protein permeation channel for nitrite uptake in addition to HNO2 penetration. On the basis of a lack of effect by known anion uptake inhibitors, it was concluded that the nitrite uptake mechanism was distinct from that of phosphate and chloride/sulfate transport. PMID:16665917

  16. 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

  17. 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...

  18. 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...

  19. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  20. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  1. 50 CFR 622.38 - Landing fish intact.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Landing fish intact. 622.38 Section 622.38... Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish... specified in paragraphs (c) and (d) of this section. Such fish may be eviscerated, gilled, and scaled,...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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)...

  7. 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)...

  8. 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

  9. 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

  10. 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

    2016-03-16

    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. PMID:26786854

  11. 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...

  12. 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...

  13. IntAct: an open source molecular interaction database

    PubMed Central

    Hermjakob, Henning; Montecchi-Palazzi, Luisa; Lewington, Chris; Mudali, Sugath; Kerrien, Samuel; Orchard, Sandra; Vingron, Martin; Roechert, Bernd; Roepstorff, Peter; Valencia, Alfonso; Margalit, Hanah; Armstrong, John; Bairoch, Amos; Cesareni, Gianni; Sherman, David; Apweiler, Rolf

    2004-01-01

    IntAct provides an open source database and toolkit for the storage, presentation and analysis of protein interactions. The web interface provides both textual and graphical representations of protein interactions, and allows exploring interaction networks in the context of the GO annotations of the interacting proteins. A web service allows direct computational access to retrieve interaction networks in XML format. IntAct currently contains ∼2200 binary and complex interactions imported from the literature and curated in collaboration with the Swiss-Prot team, making intensive use of controlled vocabularies to ensure data consistency. All IntAct software, data and controlled vocabularies are available at http://www.ebi.ac.uk/intact. PMID:14681455

  14. IntAct: an open source molecular interaction database.

    PubMed

    Hermjakob, Henning; Montecchi-Palazzi, Luisa; Lewington, Chris; Mudali, Sugath; Kerrien, Samuel; Orchard, Sandra; Vingron, Martin; Roechert, Bernd; Roepstorff, Peter; Valencia, Alfonso; Margalit, Hanah; Armstrong, John; Bairoch, Amos; Cesareni, Gianni; Sherman, David; Apweiler, Rolf

    2004-01-01

    IntAct provides an open source database and toolkit for the storage, presentation and analysis of protein interactions. The web interface provides both textual and graphical representations of protein interactions, and allows exploring interaction networks in the context of the GO annotations of the interacting proteins. A web service allows direct computational access to retrieve interaction networks in XML format. IntAct currently contains approximately 2200 binary and complex interactions imported from the literature and curated in collaboration with the Swiss-Prot team, making intensive use of controlled vocabularies to ensure data consistency. All IntAct software, data and controlled vocabularies are available at http://www.ebi.ac.uk/intact. PMID:14681455

  15. 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

  16. Vesta is not an intact protoplanet

    NASA Astrophysics Data System (ADS)

    Consolmagno, G.; Turrini, D.; Golabek, G.; Svetsov, V.; Sirono, S.; Tsiganis, K.

    2014-07-01

    The Dawn mission was designed to explore ''remnant intact protoplanets from the earliest epoch of solar system formation'' [1]. However, models of Vesta composed of an iron core, olivine mantle, and HED crust in chondritic proportions cannot match the joint constraints from Dawn [1] of Vesta's density, core size, and the extremely limited presence of exposed olivine on its surface. Vesta has a mean density of 3456 kg/m3 and its surface composition is well matched by howardites. The Dawn gravity data suggest a nickel-iron core of radius 110 km and density 7500--7800 kg/m3. The Rheasilvia impact basin, formed within a pre-existing large basin, Veneneia, should have excavated material from a depth of 50 km to 80 km or more below Vesta's surface [2]. If the howardite crust were thinner than 50--80 km, a significant amount of olivine-rich material, derived from depth, would have been exposed within this basin; models suggest that olivine would also be distributed both on Vesta's surface and in space as meteorite-source Vestoids. Such olivine is rare on Vesta, among the Vestoids, or in our meteorite collection. Vesta's density is similar to an L chondrite, but the Na and K abundances in Vesta are strongly depleted compared to chondrites and the average metal content of an L chondrite, 8.4% by mass, would give a core radius less than 90 km. A 110 km radius metallic core, via the Dawn data, represents 15% of Vesta's mass. The Mg/Al ratio in cosmic abundances is about 10:1, but roughly 1:1 within the eucrites; thus if Vesta started with cosmic abundances, the eucrites can only represent 10% of the parent body total mass. Likewise the 10 x chondritic rare earth trace elements (REE) abundance seen in most eucrites demands that, regardless of formation mechanism, these basalts were crystallized from a melt representing 10% of the mass of the source region [3]. Thus the howardite crust of a chondritic HED parent body, mixing all the available eucritic and diogenitic material

  17. 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. PMID:25059822

  18. Structural and molecular interrogation of intact biological systems.

    PubMed

    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

    2013-05-16

    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

  19. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium

    PubMed Central

    Nickell, William T; Kleene, Nancy K; Kleene, Steven J

    2007-01-01

    When olfactory receptor neurons respond to odours, a depolarizing Cl− efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl− against an electrochemical gradient. In isolated olfactory receptor neurons, the Na+–K+–2Cl− cotransporter NKCC1 is essential for Cl− accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl− efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl− accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl− transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl− transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl− transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl−/HCO3− exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na+–Cl− cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl−/HCO3− exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl− component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl− accumulation in olfactory receptor neurons. PMID:17656441

  20. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium.

    PubMed

    Nickell, William T; Kleene, Nancy K; Kleene, Steven J

    2007-09-15

    When olfactory receptor neurons respond to odours, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl(-) efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl(-) accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl(-) transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl(-) transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl(-) transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl(-)/HCO(3)(-) exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na(+)-Cl(-) cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl(-)/HCO(3)(-) exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl(-) component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl(-) accumulation in olfactory receptor neurons. PMID:17656441

  1. Physical adsorption analysis of intact supported MFI zeolite membranes.

    PubMed

    Hammond, Karl D; Tompsett, Geoffrey A; Auerbach, Scott M; Conner, W Curtis

    2007-07-31

    We compare the adsorption properties of intact supported silicalite membranes with those of silicalite powder and of alumina supports using nitrogen and argon as adsorbates at 77 K. We disentangle contributions from the membrane and support and find that the support contributes significantly to the total quantity adsorbed due to its relative thickness. The micropore-filling regions of the adsorption isotherms of the powder and the supported membrane are nearly identical for the membranes studied, but the isotherms differ at higher pressures--the supported membranes exhibit a much higher quantity adsorbed than the powders. Despite this difference, no hysteresis is observed in the membrane isotherms, indicating a lack of mesoporosity (pores in the 2-50 nm range) in either membrane or support for this preparation. We estimate argon transport fluxes at steady state by assuming surface diffusion with both a constant and concentration-dependent Maxwell-Stefan diffusion coefficient in the zeolite and the support. Further, we use the respective adsorption isotherms to determine the thermodynamic correction factors--that is, the ratios of the Fick and Maxwell-Stefan diffusion coefficients--required to solve the diffusion equation. The estimated argon flux is virtually the same using adsorption data from powders and membranes. For the relatively thick supports used in our study (approximately 2 mm), we find that the support exerts a much greater influence on the predicted fluxes for a wide range of values of the ratio of the support to zeolite diffusion coefficients. We emphasize that the results are specific to the architecture of the supported membranes studied, and thus, the results should be interpreted accordingly. PMID:17602679

  2. 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

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

    NASA Astrophysics Data System (ADS)

    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.

    2016-03-01

    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 3-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.

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

    PubMed

    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 LLO(1-491) 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 LLO(90-91)/CD8(+) and LLO(189-201)/CD4(+) specific immune responses. However, only endosomes containing the Ctsd-processed LLO(1-491) 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 LLO(1-491) 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 LLO(1-491) form that might be accessible for MHC class I and class II antigen presentation. PMID:22003433

  5. Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale

    PubMed Central

    Durbin, Kenneth R.; Tran, John C.; Zamdborg, Leonid; Sweet, Steve M. M.; Catherman, Adam D.; Lee, Ji Eun; Li, Mingxi; Kellie, John F.; Kelleher, Neil L.

    2011-01-01

    Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale. PMID:20848673

  6. Single-Cell Phenotyping within Transparent Intact Tissue Through Whole-Body Clearing

    PubMed Central

    Yang, Bin; Treweek, Jennifer B.; Kulkarni, Rajan P.; Deverman, Benjamin E.; Chen, Chun-Kan; Lubeck, Eric; Shah, Sheel; Cai, Long; Gradinaru, Viviana

    2014-01-01

    SUMMARY Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically-transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT, a protocol for passive tissue clearing and immunostaining of intact organs; RIMS, a refractive index matching media for imaging thick tissue; and PARS, a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies. PMID:25088144

  7. 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,…

  8. ROOTBOX FOR QUANTITATIVE OBSERVATIONS ON INTACT ENTIRE ROOT SYSTEMS

    EPA Science Inventory

    A rootbox is described which allows observation of an intact, entire root system. oots are sandwiched against a plexiglass surface by a nylon mesh that is impermeable to roots, but permeable to water and nutrients. o quantify root growth non-destructively, roots of different size...

  9. 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.

  10. 50 CFR 622.381 - 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 Coastal Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia, king mackerel, and Spanish mackerel in or from the Gulf, Mid-Atlantic, or South Atlantic EEZ, except...

  11. 50 CFR 622.381 - 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 Coastal Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia, king mackerel, and Spanish mackerel in or from the Gulf, Mid-Atlantic, or South Atlantic EEZ, except...

  12. Pharmacokinetics of erythropoietin in intact and anephric dogs

    SciTech Connect

    Fu, J.S.; Lertora, J.J.; Brookins, J.; Rice, J.C.; Fisher, J.W.

    1988-06-01

    The present studies were performed to determine the pharmacokinetic parameters of erythropoietin in intact and anephric dogs by use of unlabeled crude native erythropoietin (nEp) and iodine 125-labeled purified recombinant erythropoietin (rEp) given by intravenous infusion for 15 minutes. Sephadex G-75 gel filtration was used to confirm that the 125I-rEp molecule remained iodinated in dog plasma during the 24-hour period of these studies. The plasma disappearance of erythropoietin conformed to a biexponential equation for both nEp and 125I-rEp, with the central compartment being larger than the peripheral compartment. The mean distribution half-life of 75.3 +/- 21.2 minutes for nEp was significantly (p less than 0.05) longer than that of 125I-rEp (23.7 +/- 5.0 minutes) in intact dogs. The intercompartmental clearance (CIic) for nEp (0.018 +/- 0.006 L/kg/hr) was significantly smaller than that of 125I-rEp (0.068 +/- 0.018 L/kg/hr) in intact dogs (p less than 0.05). There were no significant differences in apparent volume of distribution, elimination half-life, and elimination clearance (CIe) for nEp and rEp in intact dogs. The mean elimination half-life for 125I-rEp in intact dogs (9.0 +/- 0.6 hours) and anephric dogs (13.8 +/- 1.4 hours) was significantly different (p less than 0.05). The CIe for 125I-rEp in anephric dogs (0.008 +/- 0.001 L/kg/hr) was significantly (p less than 0.05) smaller than that of 125I-rEp in intact dogs (0.011 +/- 0.001 L/kg/hr). There were no significant differences in apparent volume of distribution, distribution half-life, and CIic for 125I-rEp in intact and anephric dogs.

  13. Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry

    PubMed Central

    Mazur, Matthew T.; Cardasis, Helene L.; Spellman, Daniel S.; Liaw, Andy; Yates, Nathan A.; Hendrickson, Ronald C.

    2010-01-01

    Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of “unknown” proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL3. The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 → 3)-Gal-β(1 → 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level. PMID:20388904

  14. 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…

  15. High-resolution magic-angle-spinning NMR spectroscopy of intact tissue.

    PubMed

    Giskeødegård, Guro F; Cao, Maria D; Bathen, Tone F

    2015-01-01

    High-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy is a nondestructive technique that is used to obtain the metabolite profile of a tissue sample. This method requires minimal sample preparation. However, it is important to handle the sample with care and keep it frozen during preparation to minimize degradation. Here, we describe a typical protocol for HR-MAS analysis of intact tissue. We also include examples of typical pulse sequence programs and quantification methods that are used today. PMID:25677145

  16. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  17. 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.

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

    SciTech Connect

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

    2009-05-08

    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.

  19. 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.

  20. 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

  1. Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

    PubMed Central

    Ng, Kiaw Kiaw; Motoda, Yoko; Watanabe, Satoru; Sofiman Othman, Ahmad; Kigawa, Takanori; Kodama, Yutaka; Numata, Keiji

    2016-01-01

    In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology. PMID:27100681

  2. Physical and Biological Interactions of Intact Watersheds: Are they relevant?

    NASA Astrophysics Data System (ADS)

    Bryant, M.

    2005-05-01

    Intact watersheds are rare or absent throughout most of the northern hemisphere. Southeast Alaska is an exception with many forested watersheds that either have not been or are lightly disturbed by human activity. The Kadashan River, a short (less than 20 km) river in an old growth watershed on Chichagof Island is one example of an intact watershed. Natural processes from the headwaters to the ocean interact to create a salmon rich environment. Steep headwater streams are sources for sediment recruitment, nutrients (organic material and invertebrates), and large wood. Large riparian trees entering the stream provide long term storage of sediment, create pools, and link the main stream to off-channel habitats. A natural discharge regime that floods a complex riparian zone with abundant beaver ponds is a key process that maintains this complex system. Examples of these processes and salmonid populations in the Kadashan River illustrate how they interact to support one of the more productive watersheds in southeast Alaska. Understanding and quantifying processes in intact watersheds provide an opportunity to test major river paradigms and provide reference points for watershed restoration projects elsewhere.

  3. Carboxylated and intact osteocalcin predict adiponectin concentration in hemodialyzed patients.

    PubMed

    Kuźniewski, Marek; Fedak, Danuta; Dumnicka, Paulina; Kapusta, Maria; Stępień, Ewa; Chowaniec, Eve; Krzanowska, Katarzyna; Krzanowski, Marcin; Chmiel, Grzegorz; Solnica, Bogdan; Sułowicz, Władysław

    2016-01-01

    Purpose Disrupted bone metabolism in patients with chronic kidney disease (CKD) is associated with elevated concentrations of biochemical bone markers. Recently, animal studies show the role of osteocalcin in energy metabolism, which is partially confirmed in humans. The aim of our study was to evaluate the relationships between serum concentrations of bone markers and indices of energy metabolism in CKD patients on maintenance hemodialysis; in particular, the relationship between various forms of osteocalcin and adiponectin. Patients and methods The cross-sectional study included 155 hemodialyzed stage 5 CKD patients. Serum concentrations of glucose, insulin, adiponectin, bone alkaline phosphatase (bALP), tartrate resistant acid phosphatase (TRAP), carboxylated (cOC), undercarboxylated (ucOC), and intact osteocalcin (OC) were determined. Results In total cohort, bALP, TRAP, cOC, and ucOC negatively correlated with BMI. All analyzed bone markers positively correlated with adiponectin in total cohort and in men. In multiple linear regression analysis including all patients, log(cOC) and log(intact OC) were the only bone markers that predicted log(adiponectin) (beta = 0.22; p = 0.016 and beta = 0.26; p = 0.010) independently of sex, dialysis vintage, CRP, insulin, iPTH concentrations, BMI, and age. Conclusions Our data confirm the positive association between cOC, intact OC, and adiponectin concentrations in CKD patients on maintenance hemodialysis. PMID:26822199

  4. 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

  5. A chamber for applying pressure to roots of intact plants.

    PubMed

    Gee, G W

    1973-11-01

    A chamber was designed to apply up to 20 bars pressure to roots of intact plants. The unique features of this chamber are a split top arrangement to permit enclosing roots of intact plants within the chamber, a circulation coil to control temperature of rooting media, and a valve arrangement to permit changing solution without disturbing the plant. Changes in transpiration in response to changes in the pressure applied to roots of intact pepper plants illustrate one use of the equipment. Well watered plants at low light (0.05 langley/min) were observed to exude water from the leaf margins when 5 bars pressure was applied to the roots. When roots were cut off, a 1 bar pressure caused exudation. Plants with cooled roots or plants in dry soil did not exude water when as much as 6 bars pressure was applied. Transient response of transpiration rates to sudden application and release of pressure was observed in pepper and bean plants but not in rhododendron. The magnitude of this transient response was highly dependent upon light intensity and CO(2) concentration in the aerial environment. PMID:16658586

  6. Modified madigan prostatectomy: a procedure preserved prostatic urethra intact.

    PubMed

    Lu, Jun; Ye, Zhangqun; Hu, Weilie

    2005-01-01

    A total of 92 patients with benign prostatic hyperplasia (BPH) were subjected to modified Madigan prostatectomy (MPC) for a much satisfactory effect in open prostatectomy surgery. Exposing anterior prostatic urethra near the bladder neck and conjunct cystotomy modified the MPC procedure. This modified procedure preserved prostatic urethra intact and could also deal with intracystic lesions at the same time. The intact of prostatic urethra was kept completely or largely in 86 cases. The amount of blood loss during modified procedure was less. The mean operative time was 105 min. Seventy patients had been followed up for 3-24 months. The postoperative average Qmax was 19. 2 ml/s. The cystourethrography revealed that the urethra and bladder neck were intact in 10 patients postoperatively. Furthermore, the prostatic urethra was obviously wider after modified MPC. The modified MPC can reduce the occurrence of urethra injury and enlarge the MPC indications. The modified technique is easy to perform with less complications and much satisfactory clinical result. PMID:16201285

  7. Laser valvotomy with balloon valvoplasty for pulmonary atresia with intact ventricular septum: five years' experience.

    PubMed Central

    Gibbs, J. L.; Blackburn, M. E.; Uzun, O.; Dickinson, D. F.; Parsons, J. M.; Chatrath, R. R.

    1997-01-01

    OBJECTIVE: To assess immediate and medium term results of transcatheter laser valvotomy with balloon valvoplasty in selected infants with pulmonary atresia and intact ventricular septum. DESIGN: Prospective study. SETTING: Tertiary cardiac unit. PATIENTS: All infants with pulmonary atresia and intact septum with no more than minor tricuspid valve hypoplasia referred between November 1990 and June 1995. Laser valvotomy was attempted in nine infants of median age 4-5 days and median weight 3.6 kg. INTERVENTION: The pulmonary valve was perforated using a 0.018 inch fibreoptic guidewire attached to a NdYag laser and introduced through a catheter positioned beneath the valve. After perforation the valve was dilated with progressively larger balloons. MAIN OUTCOME MEASURES: Successful laser valvotomy and balloon dilatation, complications, pulse oximetry, right ventricular outflow velocities, and need for surgical treatment. RESULTS: Valvotomy was successful in all but one case, the failure being due to laser breakdown. After perforation the valve was dilated to 6-8 mm diameter. Prostaglandin E was withdrawn immediately in six of the eight duct dependent infants, and 28 and 49 days later in two. No patient required an aortopulmonary shunt. Two patients had repeat valvoplasty at 20 days and three months of age, respectively; one required infundibular resection and closure of the atrial septum at age four and one is awaiting similar treatment. CONCLUSIONS: Laser valvotomy with balloon valvoplasty is safe and effective treatment for selected patients with pulmonary atresia and intact ventricular septum and should be considered as first line treatment in place of surgical valvotomy. Images PMID:9093038

  8. Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

    PubMed Central

    Amin, Nirav M.; Greco, Todd M.; Kuchenbrod, Lauren M.; Rigney, Maggie M.; Chung, Mei-I; Wallingford, John B.; Cristea, Ileana M.; Conlon, Frank L.

    2014-01-01

    The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development. PMID:24496632

  9. On Plant Detection of Intact Tomato Fruits Using Image Analysis and Machine Learning Methods

    PubMed Central

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

    2014-01-01

    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. PMID:25010694

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

    PubMed

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

    2014-01-01

    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. PMID:25010694

  11. 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

  12. Effects of reactive Mn(III)-oxalate complexes on structurally intact plant cell walls

    NASA Astrophysics Data System (ADS)

    Summering, J. A.; Keiluweit, M.; Goni, M. A.; Nico, P. S.; Kleber, M.

    2011-12-01

    Lignin components in the in plant litter are commonly assumed to have longer residence times in soil than many other compounds, which are supposedly, more easily degradable. The supposed resistance of lignin compounds to decomposition is generally attributed to the complex chain of biochemical steps required to create footholds in the non-porous structure of ligno-cellulose in cell walls. Interestingly, Mn(III) complexes have shown the ability to degrade ligno-cellulose. Mn(III) chelated by ligands such as oxalate are soluble oxidizers with a high affinity for lignin structures. Here we determined (i) the formation and decay kinetics of the Mn(III)-oxalate complexes in aqueous solution and (ii) the effects that these complexes have on intact ligno-cellulose. UV/vis spectroscopy and iodometric titrations confirmed the transient nature of Mn(III)-oxalate complexes with decay rates being in the order of hours. Zinnia elegans tracheary elements - a model ligno-cellulose substrate - were treated with Mn(III)-oxalate complexes in a newly developed flow-through reactor. Soluble decomposition products released during the treatment were analyzed by GC/MS and the degree of cell integrity was measured by cell counts, pre- and post-treatment counts indicate a decrease in intact Zinnia elegans as a result of Mn(III)-treatment. GC/MS results showed the release of a multitude of solubilized lignin breakdown products from plant cell walls. We conclude that Mn(III)-oxalate complexes have the ability to lyse intact plant cells and solubilize lignin. Lignin decomposition may thus be seen as resource dependent, with Mn(III) a powerful resource that should be abundant in terrestrial characterized by frequent redox fluctuations.

  13. Approximating the isometric force-calcium relation of intact frog muscle using skinned fibers.

    PubMed Central

    Maughan, D W; Molloy, J E; Brotto, M A; Godt, R E

    1995-01-01

    In previous papers we used estimates of the composition of frog muscle and calculations involving the likely fixed charge density in myofibrils to propose bathing solutions for skinned fibers, which best mimic the normal intracellular milieu of intact muscle fibers. We tested predictions of this calculation using measurements of the potential across the boundary of skinned frog muscle fibers bathed in this solution. The average potential was -3.1 mV, close to that predicted from a simple Donnan equilibrium. The contribution of ATP hydrolysis to a diffusion potential was probably small because addition of 1 mM vanadate to the solution decreased the fiber actomyosin ATPase rate (measured by high-performance liquid chromatography) by at least 73% but had little effect on the measured potential. Using these solutions, we obtained force-pCa curves from mechanically skinned fibers at three different temperatures, allowing the solution pH to change with temperature in the same fashion as the intracellular pH of intact fibers varies with temperature. The bath concentration of Ca2+ required for half-maximal activation of isometric force was 1.45 microM (22 degrees C, pH 7.18), 2.58 microM (16 degrees C, pH 7.25), and 3.36 microM (5 degrees C, pH 7.59). The [Ca2+] at the threshold of activation at 16 degrees C was approximately 1 microM, in good agreement with estimates of threshold [Ca2+] in intact frog muscle fibers. PMID:8534819

  14. 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. PMID:25430370

  15. 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.

  16. 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.

  17. 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. PMID:6300565

  18. 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.

  19. 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. PMID:26788309

  20. 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

  1. Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings

    PubMed Central

    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 only18 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. PMID:26158488

  2. 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

  3. 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. PMID:26871636

  4. A method for immunolabeling neurons in intact cuticularized insect appendages.

    PubMed

    Ehrhardt, Erica; Kleele, Tatjana; Boyan, George

    2015-06-01

    The antennae of the grasshopper Schistocerca gregaria possess a pair of nerve pathways which are established by so-called pioneer neurons early in embryonic development. Subsequently, sensory cell clusters mediating olfaction, flight, optomotor responses, and phase changes differentiate from the antennal epithelium at stereotypic locations and direct their axons onto those of the pioneers to then project to the brain. Early in embryonic development, before the antennae become cuticularized, immunolabeling can be used to follow axogenesis in these pioneers and sensory cells. At later stages, immunolabeling becomes problematical as the cuticle is laid down and masks internal antigen sites. In order to immunolabel the nervous system of cuticularized late embryonic and first instar grasshopper antennae, we modified a procedure known as sonication in which the appendage is exposed to ultrasound thereby rendering it porous to antibodies. Comparisons of the immunolabeled nervous system of sectioned and sonicated antennae show that the cellular organization of sensory clusters and their axon projections is intact. The expression patterns of neuron-specific, microtubule-specific, and proliferative cell-specific labels in treated antennae are consistent with those reported for earlier developmental stages where sonication is not necessary, suggesting that these molecular epitopes are also conserved. The method ensures reliable immunolabeling in intact, cuticularized appendages so that the peripheral nervous system can be reconstructed directly via confocal microscopy throughout development. PMID:25868908

  5. Nondestructive time-of-capture, location and velocity sensing in intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter

    1994-01-01

    Velocity sensing of hypervelocity particles was a significant field of interest from the 1960s to 1970s. Generically, velocity sensing has been approached by either of two methods: first, detecting direct plasma or light emissions onto a solid surface, or second, performing time-of-flight by film penetration, or by detecting charged particles passing a static electric field. Impact plasma techniques required the destruction of the particles. Multiple-film penetrations were also destructive, especially for small particles. There is a need for an integrated, reliable, and simple location and velocity sensor compatible to our intact capture underdense medium without contributing additional damage to the captured particle. Since cometary dust, like cosmic dust, is essentially randomly distributed, knowing the time of the dust capture and the location of the capture on the collector permits the identification of the specific dust particle captured. Velocity information helps to determine the trajectory of the dust particle. Our discovery of a very suitable acoustic sensor and fruitful experimental results have led to the realization of a flight velocity sensor with intact capture underdense media.

  6. 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. PMID:25466049

  7. Mechanism of dioxin action: receptor-enhancer interactions in intact cells.

    PubMed Central

    Wu, L; Whitlock, J P

    1993-01-01

    We have used a ligation-mediated polymerase chain reaction technique to analyze protein-DNA interactions at a dioxin-responsive enhancer upstream of the CYP1A1 gene in intact mouse hepatoma cells. In its inactive state, the enhancer binds few, if any, proteins within the major DNA groove in vivo. Thus, the inactive enhancer is relatively inaccessible to DNA-binding proteins. Exposure of cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to the binding of the liganded Ah receptor at six sites within the major DNA groove of the enhancer. The receptor-enhancer interactions occur rapidly and do not require ongoing transcription, consistent with their role in regulating CYP1A1 gene expression. The liganded receptor, which is a heteromer composed of at least two basic helix-loop-helix proteins, is probably the only DNA-binding transcription factor necessary to activate the enhancer in vivo. The small size and irregular distribution of receptor binding sites suggest that chromatin structure imposes substantial steric constraints upon the function of the receptor-enhancer system in intact cells. Images PMID:8382788

  8. An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

    PubMed Central

    Dunston, David; Ogura, Tatsuya; Lin, Weihong

    2013-01-01

    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. PMID:23963491

  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. PMID:2098347

  10. Quantitative assessment of the degree of lipid unsaturation in intact Mortierella by Raman microspectroscopy.

    PubMed

    Münchberg, Ute; Wagner, Lysett; Rohrer, Carsten; Voigt, Kerstin; Rösch, Petra; Jahreis, Gerhard; Popp, Jürgen

    2015-05-01

    Fungi of the genus Mortierella can accumulate large amounts of unusual lipids depending on species, strain, and growth conditions. Fast and easy determination of key parameters of lipid quality for these samples is required. In this contribution, we apply Raman microspectroscopy to determine the degree of unsaturation for fungal lipids directly inside intact hyphae without elaborate sample handling. Six Mortierella species were grown under varying conditions, and Raman spectra of single lipid vesicles were acquired. From the spectra, we calculate a peak intensity ratio I(1270 cm(-1))/I(1445 cm(-1)) from the signals of =CH and -CH2/-CH3 groups, respectively. This ratio is linked to the iodine value (IV) using spectra of reference compounds with known IV. IVs of fungal samples are compared to gas chromatography results. Values from both methods are in good accordance. Lipid composition is found to vary between the investigated species, with Mortierella alpina having the most unsaturated lipid (IV up to 280) and Mortierella exigua the least unsaturated (IV as low as 70). We find Raman microspectroscopy a suitable tool to determine the IV reliably, fast, and easily inside intact hyphae without extensive sample handling or treatment. The method can also be transferred to other microscopic samples. PMID:25757824

  11. Complexing of amino acids to DNA by chromate in intact cells.

    PubMed Central

    Voitkun, V; Zhitkovich, A; Costa, M

    1994-01-01

    Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7843108

  12. Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO21

    PubMed Central

    Criswell, Jerome G.; Havelka, Ulysses D.; Quebedeaux, Bruno; Hardy, Ralph W. F.

    1976-01-01

    The N2-fixing legume nodule requires O2 for ATP production; however, the O2 sensitivity of nitrogenase dictates a requirement for a low pO2 inside the nodule. The effects of long term exposures to various pO2s on N2[C2H2] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO2 of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O2 initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO2 of 0.32 atmospheres had little effect on N2[C2H2] fixation; a pO2 of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O2 controls by 95 hours. Re-exposure to ambient pO2 of plants adapted to nonambient pO2s reduced N2[C2H2] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO2 conditions, and a time period was required to readapt to ambient O2. It is concluded that the N2[C2H2]-fixing system of intact soybean plants is able to adapt to a wide range of external pO2s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO2 optimal for nitrogenase activity. PMID:16659731

  13. Head direction cell activity in the anterodorsal thalamus requires intact supragenual nuclei

    PubMed Central

    Clark, Benjamin J.; Brown, Joel E.

    2012-01-01

    Neural activity in several limbic areas varies as a function of the animal's head direction (HD) in the horizontal plane. Lesions of the vestibular periphery abolish this HD cell signal, suggesting an essential role for vestibular afference in HD signal generation. The organization of brain stem pathways conveying vestibular information to the HD circuit is poorly understood; however, recent anatomical work has identified the supragenual nucleus (SGN) as a putative relay. To test this hypothesis, we made lesions of the SGN in rats and screened for HD cells in the anterodorsal thalamus. In animals with complete bilateral lesions, the overall number of HD cells was significantly reduced relative to control animals. In animals with unilateral lesions of the SGN, directional activity was present, but the preferred firing directions of these cells were unstable and less influenced by the rotation of an environmental landmark. In addition, we found that preferred directions displayed large directional shifts when animals foraged for food in a darkened environment and when they were navigating from a familiar environment to a novel one, suggesting that the SGN plays a critical role in projecting essential self-motion (idiothetic) information to the HD cell circuit. PMID:22875899

  14. 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.

  15. An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin.

    PubMed Central

    Schiavo, G; Papini, E; Genna, G; Montecucco, C

    1990-01-01

    Tetanus toxin is composed of a heavy chain (100 kDa) and a light chain (50 kDa) held together by a single interchain disulfide bridge. An additional intrachain disulfide is present in the carboxy-terminal part of the heavy chain. Reduction of the two disulfide bonds in tetanus toxin with both chemical and proteinaceous reducing agents was studied. Dithiothreitol and 2-mercaptoethanol cleaved both the inter- and intrachain disulfide bridges of the toxin, while glutathione and cysteine were ineffective. Specific reduction of the single interchain disulfide link was achieved with the thioredoxin-thioredoxin reductase system, thus indicating that this bond is exposed at the protein surface. Also, dead or permeabilized cells were able to reduce the toxin. Such reduced toxin bound to neuronal membranes as well as the native toxin but was not neurotoxic. These findings open the possibility that reduction by cytoplasmic agents released by dead cells contributes to detoxification of tetanus toxin. Moreover, together with the notion that the light chain is the active form of the toxin in the cytoplasm, these results suggest that the interchain disulfide bond of tetanus toxin plays a role in nerve cell penetration. Images PMID:2254033

  16. Head direction cell activity in the anterodorsal thalamus requires intact supragenual nuclei.

    PubMed

    Clark, Benjamin J; Brown, Joel E; Taube, Jeffrey S

    2012-11-01

    Neural activity in several limbic areas varies as a function of the animal's head direction (HD) in the horizontal plane. Lesions of the vestibular periphery abolish this HD cell signal, suggesting an essential role for vestibular afference in HD signal generation. The organization of brain stem pathways conveying vestibular information to the HD circuit is poorly understood; however, recent anatomical work has identified the supragenual nucleus (SGN) as a putative relay. To test this hypothesis, we made lesions of the SGN in rats and screened for HD cells in the anterodorsal thalamus. In animals with complete bilateral lesions, the overall number of HD cells was significantly reduced relative to control animals. In animals with unilateral lesions of the SGN, directional activity was present, but the preferred firing directions of these cells were unstable and less influenced by the rotation of an environmental landmark. In addition, we found that preferred directions displayed large directional shifts when animals foraged for food in a darkened environment and when they were navigating from a familiar environment to a novel one, suggesting that the SGN plays a critical role in projecting essential self-motion (idiothetic) information to the HD cell circuit. PMID:22875899

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Intact and long-lasting repetition priming in amnesia.

    PubMed

    Cave, C B; Squire, L R

    1992-05-01

    In 2 experiments, we evaluated the ability of amnesic patients to exhibit long-lasting perceptual priming after a single exposure to pictures. Ss named pictures as quickly as possible on a single occasion, and later named the same pictures mixed with new pictures. In Experiment 1, amnesic patients exhibited fully intact priming effects lasting at least 7 days. In Experiment 2, the priming effect for both groups was shown to depend on both highly specific visual information and on less visual, more conceptual information. In contrast, recognition memory was severely impaired in the patients, as assessed by both accuracy and response time. The results provide the first report of a long-lasting priming effect in amnesic patients, based on a single encounter, which occurs as strongly in the patients as in normal Ss. Together with other recent findings, the results suggest that long-lasting priming and recognition memory depend on separate brain systems. PMID:1534352

  19. 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

  20. Semipermeability of the nuclear membrane in the intact cell.

    PubMed

    HARDING, C V; FELDHERR, C

    1959-07-20

    The osmotic properties of nuclei in intact cells were studied by injecting solutions into the cytoplasm of amphibian oocytes. Subsequent changes in nuclear volume were recorded photographically. The injection of solutions containing polyvinylpyrrolidone or bovine serum albumin caused changes in nuclear volume which were related to the colloid osmotic pressure of the solution injected. The concentration in which no significant nuclear volume change occurred (the isotonic range) was 1.0 to 1.5 per cent polyvinylpyrrolidone (2.0 to 3.75 x 10(-4)M). 2 per cent bovine serum albumin had no significant effect on nuclear volume, whereas 4 per cent caused a significant decrease. The significance of these findings is discussed in terms of the permeability characteristics of the nuclear membrane. PMID:13664918

  1. Simple Genome Editing of Rodent Intact Embryos by Electroporation

    PubMed Central

    Kaneko, Takehito; Mashimo, Tomoji

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing. PMID:26556280

  2. Intact quasiparticles at an unconventional quantum critical point

    NASA Astrophysics Data System (ADS)

    Sutherland, M. L.; O'Farrell, E. C. T.; Toews, W. H.; Dunn, J.; Kuga, K.; Nakatsuji, S.; Machida, Y.; Izawa, K.; Hill, R. W.

    2015-07-01

    We report measurements of in-plane electrical and thermal transport properties in the limit T →0 near the unconventional quantum critical point in the heavy-fermion metal β -YbAlB4 . The high Kondo temperature TK≃200 K in this material allows us to probe transport extremely close to the critical point, at unusually small values of T /TK<5 ×10-4 . Here we find that the Wiedemann-Franz law is obeyed at the lowest temperatures, implying that the Landau quasiparticles remain intact in the critical region. At finite temperatures we observe a non-Fermi-liquid T -linear dependence of inelastic-scattering processes to energies lower than those previously accessed. These processes have a weaker temperature dependence than in comparable heavy fermion quantum critical systems, revealing a temperature scale of T ˜0.3 K which signals a sudden change in the character of the inelastic scattering.

  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. Ischaemic stroke with intact atrial septum--exclude arteriovenous malformations.

    PubMed

    Doering, Friederike; Eicken, Andreas; Hess, John

    2014-02-01

    A 44-year-old woman was referred to our centre for interventional cardiac catheterisation. The diagnostic work-up after a preceding ischaemic stroke led to the assumption of a patent foramen ovale due to a positive bubble study. Before the planned percutaneous closure of the patent foramen ovale, we performed a second bubble study, which showed an intact atrial septum. However, after two to three heart cycles bubbles could be detected in the left atrium, assuming a right-to-left shunt of an extracardiac origin most likely in the lung. We therefore performed cardiac catheterisation, yielding a pulmonary arteriovenous malformation in the lower lobe of the right lung. This was successfully closed interventionally by placing a Cook coil, as well as several plugs into the malformation and feeding vessels. PMID:23347820

  5. In vivo measurement of muscle output in intact Drosophila.

    PubMed

    Elliott, Christopher J H; Sparrow, John C

    2012-01-01

    We describe our methods for analysing muscle function in a whole intact small insect, taking advantage of a simple flexible optical beam to produce an inexpensive transducer with wide application. We review our previous data measuring the response to a single action potential driven muscle twitch to explore jumping behaviour in Drosophila melanogaster. In the fruitfly, where the sophisticated and powerful genetic toolbox is being widely employed to investigate neuromuscular function, we further demonstrate the use of the apparatus to analyse in detail, within whole flies, neuronal and muscle mutations affecting activation of muscle contraction in the jump muscle. We have now extended the use of the apparatus to record the muscle forces during larval and other aspects of adult locomotion. The robustness, simplicity and versatility of the apparatus are key to these measurements. PMID:22037247

  6. 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.

  7. 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

  8. Nanoplasmonic Quantitative Detection of Intact Viruses from Unprocessed Whole Blood

    PubMed Central

    Inci, Fatih; Tokel, Onur; Wang, ShuQi; Gurkan, Umut Atakan; Tasoglu, Savas; Kuritzkes, Daniel R.; Demirci, Utkan

    2013-01-01

    Infectious diseases such as HIV and Hepatitis B infection pose an omnipresent threat to global health. Reliable, fast, accurate and sensitive platforms that can be deployed at the point-of-care (POC) in multiple settings, such as airports and offices for detection of infectious pathogens are essential for the management of epidemics and possible biological attacks. To the best of our knowledge, no viral load technology adaptable to the POC settings exists today due to critical technical and biological challenges. Here, we present for the first time a broadly applicable technology for quantitative, nanoplasmonic-based intact virus detection at clinically relevant concentrations. The sensing platform is based on unique nanoplasmonic properties of nanoparticles utilizing immobilized antibodies to selectively capture rapidly evolving viral subtypes. We demonstrate the capture, detection and quantification of multiple HIV subtypes (A, B, C, D, E, G, and subtype panel) with high repeatability, sensitivity and specificity down to 98 ± 39 copies/mL (i.e., subtype D) using spiked whole blood samples and clinical discarded HIV-infected patient whole blood samples validated by the gold standard, i.e., RT-qPCR. This platform technology offers an assay time of 1 hour and 10 minutes (1 hour for capture, 10 minutes for detection and data analysis). The presented platform is also able to capture intact viruses at high efficiency using immuno-surface chemistry approaches directly from whole blood samples without any sample preprocessing steps such as spin-down or sorting. Evidence is presented showing the system to be accurate, repeatable and reliable. Additionally, the presented platform technology can be broadly adapted to detect other pathogens having reasonably well-described biomarkers by adapting the surface chemistry. Thus, this broadly applicable detection platform holds great promise to be implemented potentially at POC settings, hospital and primary care settings. PMID

  9. Fluorescence-based screening of cytochrome P450 activities in intact cells.

    PubMed

    Donato, M Teresa; Gómez-Lechón, M José

    2013-01-01

    Fluorimetric methods to assess cytochrome P450 (P450) activities that do not require metabolite separation have been developed. These methods make use of non- or low-fluorescent P450 substrates that produce highly fluorescent metabolites in aqueous solutions. The assays are based on the direct incubation of intact cells in culture with appropriate fluorogenic probe substrates, followed by fluorimetric quantification of the product formed and released into incubation medium. We describe a battery of fluorescence assays for rapid measurement of the activity of nine P450s involved in drug metabolism. For each individual P450 activity the probe showing the best properties (highest metabolic rates, lowest background fluorescence) has been selected. Fluorescence-based assays are highly sensitive and allow the simultaneous activity assessments of cells cultured in 96-well plates, using plate readers, with notable reductions in costs, time, and cells, thus enhancing sample throughput. PMID:23475674

  10. Masseter flap for reconstruction of defects after excision of buccal mucosa cancers with intact mandible.

    PubMed

    Chaturvedi, Pankaj; Lerra, Sandeep; Ustad, Farheen; Pai, Prathamesh S; Chaukar, Devendra A; D'Cruz, Anil K

    2015-01-01

    Among the reconstructive options available for buccal mucosa defects with an intact mandible, free flap with microvascular anastomosis is the best option. However, in the developing world, with poor resources, limited infrastructure, and high patient load, this cannot be offered to all patients. We report on the success of the masseter flap for reconstruction of such defects in carefully selected patients. Despite some known limitations, this flap is easy to learn and carries acceptable complications. The results of this flap may not be comparable to those of microvascular reconstructions, but they are better than those from other options such as skin graft, nasolabial flap, submental flap, etc., in terms of surgical time required, no donor site morbidity, and minimal aesthetic deformity. PMID:26535825

  11. 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

  12. 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). PMID:21468941

  13. Three-dimensional imaging of crystalline inclusions embedded in intact maize stalks.

    PubMed

    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

  14. 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.

  15. 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

  16. Surgical management of special cases of intractable Meniere's disease: unilateral cases with intact canals and bilateral cases.

    PubMed

    Kitahara, Tadashi; Kondoh, Kazumasa; Morihana, Tetsuo; Okumura, Shin-ichi; Mishiro, Yasuo; Kubo, Takeshi

    2004-05-01

    If a clinician seeks to allow patients with vertigo to return to work as soon as possible, it is very important to determine the appearance of vestibular symptoms during convalescence just after treatment, as well as the long-term results. Apprehensive patients with vertigo may undergo severe psychological torment if treatment requires long-term rest in bed before they can return to daily life. In this paper, we observed postoperative vestibular symptoms (subjective sensation and objective nystagmus) in 50 patients with intractable Meniere's disease, including cases from our previous preliminary report, during the period of convalescence just after endolymphatic sac drainage and steroid instillation surgery (EDSS). All symptoms were eliminated within 8 days after EDSS. There was no significant difference in the duration of any vestibular symptoms between bilateral (n = 8) and unilateral cases (n = 42). This result indicates that EDSS could be as safe a treatment for bilateral Meniere's disease as for unilateral disease. In unilateral cases with intact semicircular canal function (n = 17), postoperative evoked vestibular sensation, positional, and positioning (Dix-Hallpike) nystagmus disappeared significantly earlier than in those with canal paresis (n = 25). This result indicates that EDSS could keep the vestibular peripheral function of patients with unilateral Meniere's disease with intact canals quite stable after surgery. Therefore, EDSS could be recommended as an initial, less-invasive surgical treatment for intractable Meniere's disease, especially in unilateral cases with intact canals and in bilateral cases. PMID:15174769

  17. 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

    2016-06-01

    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. PMID:27111496

  18. A mechanistic code for intact and defective nuclear fuel element performance

    NASA Astrophysics Data System (ADS)

    Shaheen, Khaled

    During reactor operation, nuclear fuel elements experience an environment featuring high radiation, temperature, and pressure. Predicting in-reactor performance of nuclear fuel elements constitutes a complex multi-physics problem, one that requires numerical codes to be solved. Fuel element performance codes have been developed for different reactor and fuel designs. Most of these codes simulate fuel elements using one-or quasi-two-dimensional geometries, and some codes are only applicable to steady state but not transient behaviour and vice versa. Moreover, while many conceptual and empirical separate-effects models exist for defective fuel behaviour, wherein the sheath is breached allowing coolant ingress and fission gas escape, there have been few attempts to predict defective fuel behaviour in the context of a mechanistic fuel performance code. Therefore, a mechanistic fuel performance code, called FORCE (Fuel Operational peRformance Computations in an Element) is proposed for the time-dependent behaviour of intact and defective CANDU nuclear fuel elements. The code, which is implemented in the COMSOL Multiphysics commercial software package, simulates the fuel, sheath, and fuel-to-sheath gap in a radial-axial geometry. For intact fuel performance, the code couples models for heat transport, fission gas production and diffusion, and structural deformation of the fuel and sheath. The code is extended to defective fuel performance by integrating an adapted version of a previously developed fuel oxidation model, and a model for the release of radioactive fission product gases from the fuel to the coolant. The FORCE code has been verified against the ELESTRES-IST and ELESIM industrial code for its predictions of intact fuel performance. For defective fuel behaviour, the code has been validated against coulometric titration data for oxygen-to-metal ratio in defective fuel elements from commercial reactors, while also being compared to a conceptual oxidation model

  19. 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.

  20. Intact alternation performance in high lethality suicide attempters.

    PubMed

    Keilp, John G; Wyatt, Gwinne; Gorlyn, Marianne; Oquendo, Maria A; Burke, Ainsley K; John Mann, J

    2014-09-30

    Suicide attempters often perform poorly on tasks linked to ventral prefrontal cortical (VPFC) function. Object Alternation (OA) - a VPFC probe - has not been used in these studies. In this study, currently depressed medication-free past suicide attempters whose most severe attempt was of high (n=31) vs. low (n=64) lethality, 114 medication-free depressed non-attempters, and 86 non-patients completed a computerized OA task. Participants also completed comparison tasks assessing the discriminant validity of OA (Wisconsin Card Sort), its concurrent validity relative to tasks associated with past attempt status (computerized Stroop task, Buschke Selective Reminding Test), and its construct validity as a VPFC measure (Go-No Go and Iowa Gambling Task). Against expectations, high lethality suicide attempters - the majority of whom used non-violent methods in their attempts with some planning - outperformed other depressed groups on OA, with no group differences observed on Wisconsin Card Sort. Despite intact performance on OA, past attempters exhibited deficits on the Stroop and Buschke. OA performance was associated with performance on Go-No Go and Iowa Gambling, confirming that OA measures a similar construct. VPFC dysfunction may not be a characteristic of all suicide attempters, especially those who make more carefully planned, non-violent - though potentially lethal - attempts. PMID:24878299

  1. Action perception is intact in autism spectrum disorder.

    PubMed

    Cusack, James P; Williams, Justin H G; Neri, Peter

    2015-02-01

    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

  2. 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

  3. Spontaneous postpartum rupture of an intact uterus: a case report.

    PubMed

    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

  4. Lipidation of intact proteins produces highly immunogenic vaccine candidates.

    PubMed

    Zeng, Weiguang; Eriksson, Emily M; Lew, Andrew; Jackson, David C

    2011-01-01

    In this study we investigate the feasibility of generating self-adjuvanting vaccines capable of inducing high titre antibody responses following the covalent attachment of the TLR2 agonist Pam(2)Cys to intact proteins. Three Pam(2)Cys-based lipid moieties were prepared which contain a solubilising spacer composed of either lysine residues or polyethyleneglycol. A model protein, hen egg white lysozyme (HEL), was lipidated individually with each of these lipid modules and the immunogenicity of the lipidated species studied in mice by measuring antibody responses. We found that lipidated HEL elicited antibodies which is much stronger than the responses obtained when the HEL was administered in Freund's adjuvant or in Alum. Little or no antibody was elicited by the lipidated HEL in CD4 T cell-deficient mice indicating that the antibody response is T cell dependent. Furthermore, the lipidated protein elicited similar antibody responses in two different strains of mice indicating that sufficient helper T cell epitopes are available to enable antibody production across the histocompatability barrier. In a similar way, lipidated bovine insulin was found to be highly immunogenic in mice despite the largely conserved sequences of bovine and murine insulin. The results provide evidence that lipidation of proteins provides a simple and safe method for the manufacture of soluble self-adjuvanting protein-based vaccines. PMID:21056473

  5. Some Factors Regulating Auxin Translocation in Intact Bean Seedlings 1

    PubMed Central

    Long, John; Basler, Eddie

    1973-01-01

    Several factors which influence the translocation patterns of stem-injected indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid in bean seedlings (Phaseolus vulgaris L. cv. Stringless Greenpod) were characterized. The acropetal translocation of auxin from the site of injection is markedly sensitive to concentration in the range of 1.0 to 5.0 micrograms per plant. The antiauxin p-chlorophenoxyisobutyric acid enhanced translocation of 2,4,5-trichlorophenoxyacetic acid to the growing shoots and primary leaves. Translocation to the roots was unaffected by p-chlorophenoxyisobutyric acid while leaching of 2,4,5-trichlorophenoxyacetic acid into the nutrient solution was enhanced slightly. Steam girdling experiments revealed that translocation to the primary leaves was in the xylem. The protein synthesis inhibitor, cycloheximide, inhibited accumulation of 2,4,5-trichlorophenoxyacetic acid in young shoots, epicotyls, and roots and enhanced accumulation in the primary leaves. The relative exchangeability of auxin between xylem and phloem is discussed in terms of regulation of auxin movements in intact bean seedlings. Images PMID:16658273

  6. Hypoplastic left heart syndrome with intact atrial septum: case report

    PubMed Central

    Cester, Maddalena; Nanhorngue, Kimta; Pascoli, Irene; Garofano, Greta; Surico, Nicola; Paternoster, Delia Maria

    2007-01-01

    Summary Objectives Hypoplastic left heart syndrome (HLHS) with an intact atrial septum (IAS) is a rare finding, reported in only 1% of pathologic specimens with hypoplasia of the aortic tract complex. In newborns with left heart obstruction, the existence of an interatrial communication is very important for oxygenated blood to be distributed to the body and to prevent pulmonary congestion. The ability to predict prenatally restriction of the atrial defect may allow earlier surgery to be planned. Methods We report a case of prenatal diagnosis of HLHS with a complete premature closure of the foramen ovale that was not detected by prenatal echocardiography. Results and conclusion The management of neonates with HLHS in the first days of life is crucial to the results of the first stage of the Norwood procedure. We suggest that delivery of the mother close to surgical centre and avoiding neonatal transfer improve the results, but stabilisation with prostaglandins and balancing of the systemic and pulmonary resistances are also important. A restrictive or closed atrial septal defect may contribute to haemodynamic instability in the first days of life. The ability to predict this complication prenatally may help in the immediate postnatal management of the affected infant. PMID:22470823

  7. 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

  8. Genetic disruptions of Drosophila Pavlovian learning leave extinction learning intact.

    PubMed

    Qin, H; Dubnau, J

    2010-03-01

    Individuals who experience traumatic events may develop persistent posttraumatic stress disorder. Patients with this disorder are commonly treated with exposure therapy, which has had limited long-term success. In experimental neurobiology, fear extinction is a model for exposure therapy. In this behavioral paradigm, animals are repeatedly exposed in a safe environment to the fearful stimulus, which leads to greatly reduced fear. Studying animal models of extinction already has lead to better therapeutic strategies and development of new candidate drugs. Lack of a powerful genetic model of extinction, however, has limited progress in identifying underlying molecular and genetic factors. In this study, we established a robust behavioral paradigm to study the short-term effect (acquisition) of extinction in Drosophila melanogaster. We focused on the extinction of olfactory aversive 1-day memory with a task that has been the main workhorse for genetics of memory in flies. Using this paradigm, we show that extinction can inhibit each of two genetically distinct forms of consolidated memory. We then used a series of single-gene mutants with known impact on associative learning to examine the effects on extinction. We find that extinction is intact in each of these mutants, suggesting that extinction learning relies on different molecular mechanisms than does Pavlovian learning. PMID:20015341

  9. 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.

  10. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  11. 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.

  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

    2016-01-01

    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

  13. 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

  14. 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. PMID:22784911

  15. 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. PMID:24667273

  16. Flux profiling of photosynthetic carbon metabolism in intact plants.

    PubMed

    Heise, Robert; Arrivault, Stéphanie; Szecowka, Marek; Tohge, Takayuki; Nunes-Nesi, Adriano; Stitt, Mark; Nikoloski, Zoran; Fernie, Alisdair R

    2014-08-01

    Flux analysis has been carried out in plants for decades, but technical innovations are now enabling it to be carried out in photosynthetic tissues in a more precise fashion with respect to the number of metabolites measured. Here we describe a protocol, using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS), to resolve intracellular fluxes of the central carbon metabolism in illuminated intact Arabidopsis thaliana rosettes using the time course of the unlabeled fractions in 40 major constituents of the metabolome after switching to (13)CO2. We additionally simplify modeling assumptions, specifically to cope with the presence of multiple cellular compartments. We summarize all steps in this 8-10-week-long process, including setting up the chamber; harvesting; liquid extraction and subsequent handling of sample plant material to chemical derivatization procedures such as silylation and methoxymation (necessary for gas chromatography only); choosing instrumentation settings and evaluating the resultant chromatogram in terms of both unlabeled and labeled peaks. Furthermore, we describe how quantitative insights can be gained by estimating both benchmark and previously unknown fluxes from collected data sets. PMID:24992096

  17. 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.

  18. 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.

  19. 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. PMID:26673971

  20. Stimulation of the cerebral cortex in the intact human subject

    NASA Astrophysics Data System (ADS)

    Merton, P. A.; Morton, H. B.

    1980-05-01

    One of the most fertile methods of investigating the brain is to stimulate a part of it electrically and observe the results. So far, however, use of the method in man has been restricted by the necessity of opening the skull surgically to apply the electrodes. Much could be done, both with healthy subjects and with neurological patients, if it were feasible to stimulate through electrodes on the scalp, although the localization of the stimulus on the cortex will always be much less sharp than with electrodes on the brain surface. In an intact man, however, the brain is protected from electricity by the skull and by the scalp, both of which normally offer considerable resistance. Furthermore, the cerebral cortex does not have a particularly low electrical threshold. It is probably for these reasons (despite an occasional contrary claim1) that attempts to stimulate the brain by applying stimuli from conventional stimulators to the scalp have been stopped by pain or have otherwise failed. These obstacles have now begun to yield. Recently, it was found that, on stimulating muscles in the human hand2 without any special preparation of the skin, the effective resistance fell to low values if brief but very high voltage shocks were used. Applying the same technique to the head, it has now proved possible at the first attempt to stimulate two areas of the human cortex, without undue discomfort.

  1. Therapeutic Ultrasound to Non-Invasively Create Intra-Cardiac Communications in an Intact Animal Model

    PubMed Central

    Owens, Gabe E.; Miller, Ryan M.; Ensing, Greg; Ives, Kimberly; Gordon, David; Ludomirsky, Achi; Xu, Zhen

    2010-01-01

    Objective To determine if pulsed cavitational ultrasound therapy (histotripsy) can accurately and safely generate ventricular septal defects (VSDs) through the intact chest of a neonatal animal, with the eventual goal of developing a non-invasive technique of creating intra-cardiac communications in patients with congenital heart disease. Background Histotripsy is an innovative ultrasonic technique that generates demarcated, mechanical tissue fractionation utilizing high intensity ultrasound pulses. Previous work has shown that histotripsy can create atrial septal defects in a beating heart in an open-chest canine model. Methods Nine neonatal pigs were treated with transcutaneous histotripsy targeting the ventricular septum. Ultrasound pulses of 5μs duration at a peak negative pressure of 13 MPa and a pulse repetition frequency of 1 kHz were generated by a 1 MHz focused transducer. The procedure was guided by real-time ultrasound imaging. Results VSDs were created in all pigs with diameters ranging from 2–6.5mm. Six pigs were euthanized within 2 hrs of treatment, while 3 were recovered and maintained for 2–3 days to evaluate lesion maturation and clinical side effects. There were only transient clinical effects and pathology revealed mild collateral damage around the VSD with no significant damage to other cardiac or extra-cardiac structures. Conclusions Histotripsy can accurately and safely generate VSDs through the intact chest in a neonatal animal model. These results suggest that with further advances, histotripsy can be a useful, non-invasive technique to create intra-cardiac communications, which currently require invasive catheter-based or surgical procedures, to clinically stabilize newborn infants with complex congenital heart disease. PMID:20853366

  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. Long-term intact rock strength and critical in situ stresses in near-surface bedrock

    NASA Astrophysics Data System (ADS)

    Leith, K.; Moore, J.; Amann, F.; Loew, S.

    2012-04-01

    Near-surface in situ stresses in excess of gravitational or tectonic loading are frequently encountered in natural landscapes, although the question of their origin is rarely discussed in detail. These high near-surface stresses are common to both cratonic shield and alpine regions, and their relationship to landscape or landform development has been widely commented on, as they are often associated with the development of exfoliation or sheeting fractures. We suggest these high near-surface stresses originate from the elastic and thermoelastic response of bedrock to exhumation, and reflect a regional stress regime no longer in agreement with either Andersonian stress states or Byerlee's law, but instead limited by the long-term intact rock strength. Although sufficiently rapid extensional strain, or well-oriented Reverse faults are generally assumed to limit the development of such stresses, we suggest that in the absence of these factors, an upper limit to long-term in situ stresses may be defined by the onset of inelastic strain facilitated through micro-cracking, as the rock itself is not able to maintain further increases in differential stress. Recent studies have made significant progress in understanding critical aspects of this system, in particular, the long-term behaviour of bedrock maintaining high differential stresses at shallow depths (100's of m) and the potential for micro-crack development in response to exhumation and thermoelastic relaxation. Our analysis of global in-situ stress measurements indicates good agreement between maximum differential stress magnitudes in compressional regimes and laboratory-derived threshold stresses required to initiate micro-cracking in intact rock. This correlation appears to hold for confining stresses up to 30 MPa (approximately 1 km depth) and indicates that the long-term cohesive component of rock strength may dominate the behaviour of high quality rock masses, and therefore define an upper-bound for in situ

  4. Mechanism and base specificity of DNA breakage in intact cells by neocarzinostatin

    SciTech Connect

    Kappen, L.S.; Ellenberger, T.E.; Goldberg, I.H.

    1987-01-27

    When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by (1) reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of /sup 32/P from (..gamma..-/sup 32/P)ATP by polynucleotide kinase and (2) treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO/sub 4/ termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the /sup 32/P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP >> CMP > GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells. Furthermore, in cells depleted of glutathione, DNA strand breakage was greatly reduced, confirming thiol involvement in the activation of the drug in cells, as in the vitro reaction.

  5. Using the intact hand for objective assessment of phantom hand-perception.

    PubMed

    Sumitani, Masahiko; Yozu, Arito; Tomioka, Toshiya; Yamada, Yoshitsugu; Miyauchi, Satoru

    2010-03-01

    After amputation, most patients experience a phenomenon known as a phantom limb (PL). A variety of PL experiences appear to be associated with neural plasticity within the CNS. However, due to the subjective nature of PL experiences, there was no definitive way to reliably assess PL experiences other than using patients' direct reports. Here, we were able to obtain patients' indirect responses to PL experiences, for a more objective evaluation. First, we conducted a study with normals and 17 non-PL patients experiencing pathological pain in one hand. We took digital photographs of their affected and unaffected hands, altered the sizes of the images digitally, and then asked each subject to choose the image that most closely matched the actual size of their own hands (from a series of images presented on a video screen). Subjective size perceptions of the hands were homologous, regardless of the pathological condition of one hand (p<0.0001, Spearman R(2)=0.82). Next, we used the same method for total 19 patients with a phantom hand. The intact hand-size perception was linearly correlated with phantom hand-size perception (weighted linear regression analysis: p<0.0001, R(2)=0.75, adjusted R(2)=0.73, F-value=50.1, degree of freedom=18). Thus, without requiring a subjective description about PL, the patients' evaluation of the intact hand-size precisely but indirectly indicated whether the PL was perceived to be telescoped (shrunken), normal or enlarged. This more objective evaluation of PL phenomena could become a key tool for disentangling the neural mechanisms involved. PMID:19477667

  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. Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

    NASA Astrophysics Data System (ADS)

    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.

  8. The effects of silver nanoparticles on intact wastewater biofilms

    PubMed Central

    Sheng, Zhiya; Van Nostrand, Joy D.; Zhou, Jizhong; Liu, Yang

    2015-01-01

    Silver nanoparticles (Ag-NPs) have strong antibacterial properties, which may adversely affect biological wastewater treatment processes. To determine the overall effect, intact biofilm samples were collected from the rotating biological contactor at the local wastewater treatment plant and treated with 200 mg Ag/L Ag-NPs for 24 h. The biofilm uptake of Ag-NPs was monitored with transmission electron microscopy. Forty-five minutes after Ag-NP application, Ag-NPs were seen in the biofilm extracellular polymeric substances (EPS). After 24 h, Ag-NPs had entered certain microbial cells, while other cells contained no observable Ag-NPs. Some cells were dying after the uptake of Ag-NPs. However, there was no significant reduction in cultivable bacteria in the biofilms, based on heterotrophic plate counts (HPC). While this may indicate that wastewater biofilms are highly resistant to Ag-NPs, the HPC represents only a small portion of the total microbial population. To further investigate the effects of Ag-NPs, a GeoChip microarray was used to directly detect changes in the functional gene structure of the microbial community in the biofilm. A clear decrease (34.6% decreases in gene number) in gene diversity was evident in the GeoChip analysis. However, the complete loss of any specific gene was rare. Most gene families present in both treated and untreated biofilms. However, this doesn’t necessarily mean that there was no change in these families. Signal intensity decreased in certain variants in each family while other variants increased to compensate the effects of Ag-NPs. The results indicate that Ag-NP treatment decreased microbial community diversity but did not significantly affect the microbial community function. This provides direct evidence for the functional redundancy of microbial community in engineered ecosystems such as wastewater biofilms. PMID:26217316

  9. Spatial variations in membrane properties in the intact rat lens.

    PubMed Central

    Baldo, G J; Mathias, R T

    1992-01-01

    We have used linear frequency domain techniques to measure impedance at various locations and depths in the intact rat lens. The data are used to obtain best-fit solutions to a new electrical model based on lens structure, allowing us to estimate localized conductances of surface cell membranes (Gs), fiber cell membranes (gm), and gap junctions (Gj) as functions of position. We find that gm is small and fairly uniform throughout the lens (2.02 +/- 0.58 microS/cm2); for the anterior surface-epithelial cells Gs = 1.26 +/- 0.19 mS/cm2; for the posterior surface differentiating fiber cells Gs = 0.46 +/- 0.04 mS/cm2. Thus, Gs varies about the equator in a stepwise fashion. Gj between fiber cells at locations interior to 80% of the radius is fairly uniform (0.75 S/cm2); but in the outer 20% Gj varies smoothly and symmetrically from both poles (0.66 S/cm2) to equator (5.95 S/cm2). This pattern of variation in Gj is similar to the pattern of inward and outward currents reported by Robinson and Patterson (1983. Curr. Eye Res. 2:843-847). We therefore suggest that the nonuniform distribution of functional gap junctions, not the surface cell conductance or Na/K pumps, may be responsible for directing these current flows. Gap junctional uncoupling during exposure to elevated calcium and acidification was also examined. High calcium (20 mM, with the calcium ionophore A23187) produced modest (twofold) irreversible uncoupling along with large, irreversible decreases in membrane potential. We did not pursue this further. Acidification with 20 and 100% CO2-bubbled Tyrode's produced 5- and 15-fold reversible uncoupling, respectively, only in the outer 20% of the lens radius. The remaining inner 80% of the lens gap junctions seemed resistant to the acidification and did not uncouple. PMID:1420894

  10. 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.

  11. Partial characterization of cyclic electron transport in intact chloroplasts

    SciTech Connect

    Crowther, D.; Hind, G.

    1980-10-15

    Turnover of the cyclic electron transfer chain around photosystem I in intact chloroplasts was induced by addition of sodium dithionite after poisoning with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A substantial permeability barrier to dithionite allowed redox poising to a level sufficiently negative to active, but not overreduce, the cycle. Spectral changes could thus be studied without interference from photosystem II reactions. Illumination by repetitive single-turnover flashes showed the participation in the cycle of cytochromes f and b/sub 563/ with an apparent 1:1 stoichiometry. The rise of the flash-induced electrochromic bandshift showed a fast phase with rise time < 10 ..mu..s and a slow phase with rise time variable in the millisecond range. The slow phase had an amplitude equal to that of the fast phase and occurred only when electron transfer between cytochromes b/sub 563/ and f was uninhibited. A kinetic correlation was observed between the rise of the slow phase and the rereduction of cytochrome f, whereas cytochrome b/sub 563/ reoxidation was slower than both. Redox titrations of the appearance of the slow rise in the P518 response showed that it was only observed on repetitive flashes when a component of midpoint potential approx. = -55mV(pH 8.1), n = 2, was reduced before the flash. A comparison is drawn between this protonmotive electron transfer cycle and that of the purple nonsulfur bacterium Rhodopseudomonas capsulata; possible arrangements of electron carriers in the photosystem I cycle are discussed, and a modified Q cycle is proposed to account for the properties observed.

  12. The effects of silver nanoparticles on intact wastewater biofilms.

    PubMed

    Sheng, Zhiya; Van Nostrand, Joy D; Zhou, Jizhong; Liu, Yang

    2015-01-01

    Silver nanoparticles (Ag-NPs) have strong antibacterial properties, which may adversely affect biological wastewater treatment processes. To determine the overall effect, intact biofilm samples were collected from the rotating biological contactor at the local wastewater treatment plant and treated with 200 mg Ag/L Ag-NPs for 24 h. The biofilm uptake of Ag-NPs was monitored with transmission electron microscopy. Forty-five minutes after Ag-NP application, Ag-NPs were seen in the biofilm extracellular polymeric substances (EPS). After 24 h, Ag-NPs had entered certain microbial cells, while other cells contained no observable Ag-NPs. Some cells were dying after the uptake of Ag-NPs. However, there was no significant reduction in cultivable bacteria in the biofilms, based on heterotrophic plate counts (HPC). While this may indicate that wastewater biofilms are highly resistant to Ag-NPs, the HPC represents only a small portion of the total microbial population. To further investigate the effects of Ag-NPs, a GeoChip microarray was used to directly detect changes in the functional gene structure of the microbial community in the biofilm. A clear decrease (34.6% decreases in gene number) in gene diversity was evident in the GeoChip analysis. However, the complete loss of any specific gene was rare. Most gene families present in both treated and untreated biofilms. However, this doesn't necessarily mean that there was no change in these families. Signal intensity decreased in certain variants in each family while other variants increased to compensate the effects of Ag-NPs. The results indicate that Ag-NP treatment decreased microbial community diversity but did not significantly affect the microbial community function. This provides direct evidence for the functional redundancy of microbial community in engineered ecosystems such as wastewater biofilms. PMID:26217316

  13. 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

  14. Voltage clamping single cells in intact malpighian tubules of mosquitoes.

    PubMed

    Masia, R; Aneshansley, D; Nagel, W; Nachman, R J; Beyenbach, K W

    2000-10-01

    Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways. PMID:10997925

  15. 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.

  16. Assessing Fatigability in Mobility-Intact Older Adults

    PubMed Central

    Simonsick, Eleanor M.; Schrack, Jennifer A.; Glynn, Nancy W.; Ferrucci, Luigi

    2014-01-01

    OBJECTIVES Evaluate criterion validity of two measures of fatigability defined as performance deterioration or perceived effort to perform a standardized task. DESIGN Cross-sectional analysis of data from the Baltimore Longitudinal Study of Aging (BLSA). SETTING National Institute on Aging, Intramural Research Program, Clinical Research Unit, Baltimore, Maryland. PARTICIPANTS Six hundred five men (53.7%) and women (aged 65–97) participating in the BLSA eligible for endurance walk testing without a walking aid. MEASUREMENTS Fatigability was assessed using (1) completion status and lap times from a 400m-walk performed “as quickly as possible” and (2) perceived exertion rating using the Borg scale (range 6–20) following 5 minutes of treadmill walking at 1.5mph (0.67m/s). Criterion measures include self-report of tiredness, level of weakness and energy in past month and walking ability and objective measures of usual and fast gait speed, time to complete 10 chair stands and grip strength. Co-variates include age, race, sex, obesity, smoking status and walking activity. RESULTS Among mobility intact older persons, 23% exhibited performance deterioration (i.e., slowed or stopped) during the 400m-walk and1/3 reported more than very light exertion following a 5-minute slow walk. Slowing was strongly associated with self-reported fatigue and walking ability, but weakly associated with performance-based mobility measures. High perceived exertion was associated with tiredness and weakness and reported and observed mobility deficits. CONCLUSION Slowing down may have low sensitivity for identifying fatigability in older persons, but ascertaining perceived exertion during a defined workload shows promise. In seemingly healthy, motivated individuals, fatigue and fatigability were common and may impact socially meaningful mobility behaviors. Assessment of fatigability in well-elderly examinations may help identify threats to independent functioning earlier in the decline

  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. 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. PMID:24626162

  19. 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. PMID:23609185

  20. 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... 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...

  1. 46 CFR 28.545 - Intact stability when using lifting gear.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 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...

  2. 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...

  3. 46 CFR 28.545 - Intact stability when using lifting gear.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 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. 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...

  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. 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. PMID:27259943

  7. Following rotator cuff tears, the remaining (intact) tendons are mechanically altered

    PubMed Central

    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 utilized an animal model of multiple rotator cuff tendon tears to investigate alterations in the remaining (intact) tendon mechanical properties at 4 and 8 weeks post-injury. Twenty-four animals served as uninjured controls, while seventy-two were divided among the tendon detachment groups (supraspinatus tendon detachment, supraspinatus+infraspinatus tendon detachment, supraspinatus+subscapularis tendon detachment). We found the remaining (intact) rotator cuff tendons have decreased mechanical properties in the presence of rotator cuff tears. Remaining (intact) subscapularis and infraspinatus tendon cross-sectional area increased, while tendon modulus decreased after both one and two tendon tears. Additionally, the remaining (intact) tendon cross-sectional areas continued to increase with time post-injury. These alterations could potentially lead to further tendon damage and tear progression. PMID:19095175

  8. 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." PMID:26112060

  9. 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

  10. 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

  11. Intact fetal ovarian cord formation promotes mouse oocyte survival and development

    PubMed Central

    2010-01-01

    Background Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined. Results Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles. Conclusions Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the human fetal ovary, the

  12. Induced lactation with a dopamine antagonist in mares: different responses between ovariectomized and intact mares.

    PubMed

    Guillaume, D; Chavatte-Palmer, P; Combarnous, Y; Duchamp, G; Martinat, N; Nagy, P; Daels, P F

    2003-10-01

    The aim of this study was to compare the effects of treatment with repeated injections of sulpiride (a dopamine D2 antagonist) on prolactin secretion and induced lactation in ovariectomized and intact adult mares and to verify if this induction was possible at the beginning and at the end of the birth season. Two experiments were carried out in September [experiment (expt) 1], and in March (expt 2), in France (48 degrees N). In expt 1, three groups of five mares were tested: intact-control, intact-treated and ovariectomized-treated mares. In expt 2, mares previously subjected to artificial photoperiod were assigned in two groups: four intact-control and five intact-treated mares. The cyclicity of intact mares was previously synchronized with PGF2alpha injections, then all the mares were in the follicular phase at the beginning of treatment. Sulpiride was intramuscularly injected (0.5 mg/kg of BW), twice a day. Mares were milked at 7:30, 11:45, 16:00 and 20:15 hours. Blood samples were collected every day during the treatment for progesterone, total oestrogen and prolactin assays. In the two experiments, only treated intact mares produced milk, with a large inter-animal variability. Prolactin increase after sulpiride treatment was not so great in the ovariectomized-treated mares as in the intact-treated mares. The total correlations between prolactin, progesterone, oestrogen plasma concentrations and daily milk production were significant (0.57, 0.25, 0.17 respectively). This induction of lactation can be performed during the entire birth season in intact mares, but not in ovariectomized mares, indicating that steroids are necessary for this induction in mares treated by dopamine D2 antagonist. PMID:12950692

  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. 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

    2016-01-01

    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

  15. Evidence of a Low Stromal Mg2+ Concentration in Intact Chloroplasts in the Dark

    PubMed Central

    Portis, Archie R.

    1981-01-01

    The loss of Mg2+ upon the addition of the ionophore A23187 in the dark was prevented by less than 0.1 millimolar MgCl2 with intact chloroplasts suspended in a sorbitol medium, but required 1 to 3 millimolar MgCl2 if the chloroplasts were in a K+ -gluconate medium. Measurements of stromal pH in the dark indicated that, in the K+ -gluconate medium, the stromal pH is nearly the same as that of the medium, whereas in the sorbitol medium it is much more acidic as reported previously. These observations suggest that the free Mg2+ concentration in the stroma in the dark is between 1 and 3 millimolar. Other experiments on the inihibition by A23187 of CO2 fixation in the light and in a system capable of catalyzing CO2 fixation in the dark, and on the Mg2+ binding properties of thylakoid membranes, are consistent with this conclusion. The results provide further support for the hypothesis that light-induced Mg2+ concentration changes occur in the stroma that are important in the light-dark regulation of CO2 fixation. PMID:16661806

  16. 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

  17. Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement.

    PubMed

    Thurlow, Sophie E; Kilgour, David P; Campopiano, Dominic J; Mackay, C Logan; Langridge-Smith, Pat R R; Clarke, David J; Campbell, Colin J

    2016-03-01

    Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential (E(⊕)) for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods, but top-down mass spectrometry recently has been shown to enable the quantification of E(⊕) for thiol oxidations. In this paper, we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E(⊕) for the thiols in both E. coli Thioredoxin 1 and human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using liquid chromatography-mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E(⊕) measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists. PMID:26881737

  18. Fast immuno-labeling by electrophoretically driven infiltration for intact tissue imaging

    PubMed Central

    Li, Jun; Czajkowsky, Daniel M.; Li, Xiaowei; Shao, Zhifeng

    2015-01-01

    Recently developed tissue clearing techniques, where the tissue is embedded within a hydrogel, have revolutionized our ability to resolve fine cellular structures in nearly intact tissues. However, the slow rate of penetration of antibodies within this hydrogel-tissue matrix has become a significantly limiting factor in many experiments, as thick tissues often require weeks to months to be adequately labeled. Increasing the pore size of this matrix has been investigated as a possible solution, but with only modest success. Here, we have systematically examined the diffusional behavior of antibodies and other typically used immuno-labels within this hydrogel-tissue matrix and, surprisingly, found that infiltration occurs at rates similar to those of diffusion in free solution. Therefore, changing the pore size of the matrix would be expected to afford only limited improvement and, instead, some means of active transport is necessary. We show that an electrophoretically-driven approach decreases the delivery time of antibodies by more than 800-fold over simple diffusion, without incurring structural damage. These results, together with the high quality of the images obtained with this method, demonstrate the advantage of this approach, thus significantly broadening the practical range of samples that can now be investigated by whole-mount tissue clearing methods. PMID:26013317

  19. Consummatory feeding behavior to amino acids in intact and anosmic channel catfish Ictalurus punctatus.

    PubMed

    Valentincic, T; Caprio, J

    1994-05-01

    The entire sequence of feeding behavior patterns exhibited by intact and anosmic channel catfish to food extracts was also released by single amino acids. L-arginine (> 10(-6) M), L-alanine (> 10(-6) M), and L-proline (> 10(-4) M) were each highly effective at releasing consummatory behavior patterns, such as turning, increasing pumping of water across the gill arches, and biting-snapping. Swallowing required solid objects, whereas rhythmic movement of the hyoid was released by > 10(-2) M L-arginine alone. For the biting-snapping behavior, the number of bites depended upon both the number of eddies containing the amino acid above the behavioral threshold concentration and the amino acid applied. Multiple eddies of > 10(-3) M L-proline and L-alanine provoked up to 25 bites per test; however, the most effective stimulus for releasing biting-snapping behavior at low concentrations was L-arginine (behavioral threshold 3 x 10(-7) M). In comparison to 10(-4) M L-alanine and L-arginine, other amino acids were less effective stimuli. PMID:8022905

  20. 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.

  1. 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. PMID:7478927

  2. Estimation of Elastic Modulus of Intact Rocks by Artificial Neural Network

    NASA Astrophysics Data System (ADS)

    Ocak, Ibrahim; Seker, Sadi Evren

    2012-11-01

    The modulus of elasticity of intact rock ( E i) is an important rock property that is used as an input parameter in the design stage of engineering projects such as dams, slopes, foundations, tunnel constructions and mining excavations. However, it is sometimes difficult to determine the modulus of elasticity in laboratory tests because high-quality cores are required. For this reason, various methods for predicting E i have been popular research topics in recently published literature. In this study, the relationships between the uniaxial compressive strength, unit weight ( γ) and E i for different types of rocks were analyzed, employing an artificial neural network and 195 data obtained from laboratory tests carried out on cores obtained from drilling holes within the area of three metro lines in Istanbul, Turkey. Software was developed in Java language using Weka class libraries for the study. To determine the prediction capacity of the proposed technique, the root-mean-square error and the root relative squared error indices were calculated as 0.191 and 92.587, respectively. Both coefficients indicate that the prediction capacity of the study is high for practical use.

  3. 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

  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. Direct observation of local xylem embolisms induced by soil drying in intact Zea mays leaves.

    PubMed

    Ryu, Jeongeun; Hwang, Bae Geun; Kim, Yangmin X; Lee, Sang Joon

    2016-04-01

    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

  6. 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

  7. Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs1,2,3

    PubMed Central

    Epp, Jonathan R.; Niibori, Yosuke; (Liz) Hsiang, Hwa-Lin; Mercaldo, Valentina; Deisseroth, Karl

    2015-01-01

    Abstract The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology—anatomical and functional—connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy. PMID:26464982

  8. Mechanism of adenovirus-mediated endosome lysis: role of the intact adenovirus capsid structure.

    PubMed

    Seth, P

    1994-12-15

    Adenoviruses have been previously shown to enhance the delivery of many ligands including proteins and plasmid DNAs to the cells. The key biochemical step during this process is the ability of adenovirus to disrupt (lyse) the endosome membrane releasing the co-internalized virus and the other ligands into the cytosol (Seth et al, 1986, In: Adenovirus attachment and entry into cells, pp 191-195, American Society for Microbiology, Washington, D.C.). To understand the role of the adenovirus proteins involved in the endosome lysis, it is further shown here that empty capsids of adenovirus also possess this membrane vesicle lytic activity; though the activity is about 5-times lower than the adenovirus. Incubation of adenovirus with low concentration of ionic detergent or brief exposure to 45 degrees C destroyed this lytic activity without affecting the adenovirus binding to cell surface receptor, suggesting the lytic activity of adenovirus to be of enzymatic nature. However, exposing adenovirus to conditions that can disrupt adenovirus capsid structure such as heating at 65 degrees C, treating with 0.5% SDS, treating with different proteases, dialyzing against no glycerol buffer, treating with 6 M urea or with 10% pyridine, and sonication destroyed the adenovirus-associated lytic activity. Results suggest the requirement of an intact capsid structure for adenovirus-mediated lysis of the endosome. PMID:7802664

  9. 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.…

  10. High Resolution CZE-MS Quantitative Characterization of Intact Biopharmaceutical Proteins: Proteoforms of Interferon-β1.

    PubMed

    Bush, David R; Zang, Li; Belov, Arseniy M; Ivanov, Alexander R; Karger, Barry L

    2016-01-19

    New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-β1 (Avonex, rhIFN-β1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms. PMID:26641950

  11. An intact small animal model of myocardial ischemia-reperfusion: Characterization of metabolic changes by hyperpolarized 13C MR spectroscopy.

    PubMed

    Yoshihara, Hikari A I; Bastiaansen, Jessica A M; Berthonneche, Corinne; Comment, Arnaud; Schwitter, Juerg

    2015-12-15

    Hyperpolarized carbon-13 magnetic resonance spectroscopy ((13)C MRS) enables the sensitive and noninvasive assessment of the metabolic changes occurring during myocardial ischemia-reperfusion. Ischemia-reperfusion models using hyperpolarized (13)C MRS are established in heart preparations ex vivo and in large animals in vivo, but an in vivo model in small animals would be advantageous to allow the study of reperfusion metabolism with neuroendocrine and inflammatory responses intact with the option to perform a greater number of experiments. A novel intact rat model of ischemia-reperfusion is presented that incorporates hyperpolarized (13)C MRS to characterize reperfusion metabolism. Typically, in an in vivo model, a tissue input function (TIF) is required to account for apparent changes in the metabolism of injected hyperpolarized [1-(13)C]pyruvate resulting from changes in perfusion. Whereas the measurement of a TIF by metabolic imaging is particularly challenging in small animals, the ratios of downstream metabolites can be used as an alternative. The ratio of [(13)C]bicarbonate:[1-(13)C]lactate (RatioBic/Lac) measured within 1-2 min after coronary release decreased vs. baseline in ischemic rats (n = 10, 15-min occlusion, controls: n = 10; P = 0.017 for interaction, 2-way ANOVA). The decrease in oxidative pyruvate metabolism [RatioBic/Lac(Ischemia)/RatioBic/Lac(Baseline)] modestly correlated with area at risk (r = 0.66; P = 0.002). Hyperpolarized (13)C MRS was also used to examine alanine production during ischemia, which is observed in ex vivo models, but no significant change was noted; metrics incorporating [1-(13)C]alanine did not substantially improve the discrimination of ischemic-reperfused myocardium from nonischemic myocardium. This intact rat model, which mimics the human situation of reperfused myocardial infarction, could be highly valuable for the testing of new drugs to treat reperfusion injury, thereby facilitating translational research. PMID

  12. 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).

  13. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

    PubMed

    Hartig, P R; Raftery, M A

    1979-04-01

    Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes. PMID:427105

  14. 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.

  15. 40 CFR 261.41 - Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. 261.41 Section 261.41 Protection of... HAZARDOUS WASTE Exclusions/Exemptions § 261.41 Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. (a) Persons who export used, intact CRTs for reuse must send a...

  16. 40 CFR 261.41 - Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. 261.41 Section 261.41 Protection of... HAZARDOUS WASTE Exclusions/Exemptions § 261.41 Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. (a) Persons who export used, intact CRTs for reuse must send a...

  17. 40 CFR 261.41 - Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. 261.41 Section 261.41 Protection of... HAZARDOUS WASTE Exclusions/Exemptions § 261.41 Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. (a) Persons who export used, intact CRTs for reuse must send a...

  18. 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... HAZARDOUS WASTE Exclusions/Exemptions § 261.41 Notification and Recordkeeping for Used, Intact Cathode Ray Tubes (CRTs) Exported for Reuse. (a) Persons who export used, intact CRTs for reuse must send a...

  19. 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…

  20. Ultrasensitive assay for measuring the intact (1-39) ACTH molecule: an asset in depression research.

    PubMed

    Maes, M; Meltzer, H; Blockx, P; Cosyns, P; Calabrese, J

    1994-01-01

    This study investigates the utility for depression research of an assay for intact (1-39) adrenocorticotropic hormone (ACTH) versus that of a previously employed (i.e., 1-17, 1-24 sequences) ACTH assay. ACTH plasma levels were measured using two different ACTH assays (labeled as intact versus nonintact) in 10 minor and 27 major depressed subjects undergoing the combined dexamethasone suppression (DST) and corticotropin-releasing hormone (CRH) test. Intact--but not nonintact--ACTH values were significantly correlated with the severity of illness. Major depressed subjects exhibited significantly higher post-DST+CRH intact ACTH values than minor depressives, whereas nonintact ACTH values were not significantly different between these groups. Post-DST+CRH intact ACTH values were significantly more closely related to post-DST+CRH cortisol than nonintact ACTH values. It is concluded that the assay of the intact ACTH molecule is an asset in depression research and should replace the previous less specific and sensitive ACTH assays. PMID:8047242

  1. Lactobacillus reuteri 6475 Increases Bone Density in Intact Females Only under an Inflammatory Setting

    PubMed Central

    Collins, Fraser L.; Irwin, Regina; Bierhalter, Hayley; Schepper, Jonathan; Britton, Robert A.

    2016-01-01

    Background & Aims We previously demonstrated that short-term oral administration of the probiotic Lactobacillus reuteri 6475 enhanced bone density in male but not female mice. We also established that L. reuteri 6475 enhanced bone health and prevented bone loss in estrogen-deficient female mice. In this study, we tested whether a mild inflammatory state and/or a long-term treatment with the probiotic was required to promote a positive bone effect in estrogen-sufficient female mice. Methods A mild inflammatory state was induced in female mice by dorsal surgical incision (DSI). Following DSI animals were orally supplemented with L. reuteri or vehicle control for a period of 8 weeks. Gene expression was measured in the intestine and bone marrow by qPCR. Distal femoral bone density and architecture was analyzed by micro-CT. Results We report that 8 weeks after DSI there is a significant increase in the weight of spleen, thymus and visceral (retroperitoneal) fat pads. Expression of intestinal cytokines and tight junction proteins are also altered 8 weeks post-DSI. Interestingly, L. reuteri treatment was found to display both intestinal region- and inflammation-dependent effects. Unexpectedly we identified that 1) L. reuteri treatment increased bone density in females but only in those that underwent DSI and 2) DSI benefited cortical bone parameters. In the bone marrow, dorsal surgery induced CD4+ T cell numbers, a response that was unaffected by L. reuteri treatment, whereas expression of RANKL, OPG and IL-10 were significantly affected by L. reuteri treatment. Conclusion Our data reveals a previously unappreciated effect of a mild surgical procedure causing a long-lasting effect on inflammatory gene expression in the gut and the bone. Additionally, we demonstrate that in intact female mice, the beneficial effect of L. reuteri on bone requires an elevated inflammatory status. PMID:27058036

  2. Electrophysiological recording in the brain of intact adult zebrafish.

    PubMed

    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

  3. 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.

  4. CRITICALITY CALCULATION FOR THE MOST REACTIVE DEGRADED CONFIGURATIONS OF THE FFTF SNF CODISPOSAL WP CONTAINING AN INTACT IDENT-69 CONTAINER

    SciTech Connect

    D.R. Moscalu

    2002-08-28

    The objective of this calculation is to perform additional degraded mode criticality evaluations of the Department of Energy's (DOE) Fast Flux Test Facility (FFTF) Spent Nuclear Fuel (SNF) codisposed in a 5-Defense High-Level Waste (5-DHLW) Waste Package (WP). The scope of this calculation is limited to the most reactive degraded configurations of the codisposal WP with an almost intact Ident-69 container (breached and flooded but otherwise non-degraded) containing intact FFTF SNF pins. The configurations have been identified in a previous analysis (CRWMS M&O 1999a) and the present evaluations include additional relevant information that was left out of the original calculations. The additional information describes the exact distribution of fissile material in each container (DOE 2002a). The effects of the changes that have been included in the baseline design of the codisposal WP (CRWMS M&O 2000) are also investigated. The calculation determines the effective neutron multiplication factor (k{sub eff}) for selected degraded mode internal configurations of the codisposal waste package. These calculations will support the demonstration of the technical viability of the design solution adopted for disposing of MOX (FFTF) spent nuclear fuel in the potential repository. This calculation is subject to the Quality Assurance Requirements and Description (QARD) (DOE 2002b) per the activity evaluation under work package number P6212310M2 in the technical work plan TWP-MGR-MD-000010 REV 01 (BSC 2002).

  5. [Analysis and stability of suxamethonium chloride. 1. Detection and quantitative determination of the intact active agent with its degradation products].

    PubMed

    Döge, G; Pohloudek-Fabini, R; Kottke, D

    1982-10-01

    The authors developed methods permitting to follow the course of degradation in injectable solutions of suxamethonium chloride. The qualitative detection of the active agent in the presence of degradation products is achieved by thin-layer chromatography and spraying with Dragendorff's reagent and bromocresol green indicator solution. The same separation technique may be used for determining with sufficient accuracy the intact agent in the eluate, using the method of Okken and Haas. Furthermore, a modified Draganic procedure (which is based on the spectrophotometric determination of the hydrolysis products succinic acid monocholine chloride and succinic acid as a Cu-benzidine complex) is suited for estimating the degree of degradation. No previous separation is required because the molar extinction efficients of the monoester and of the acid are in agreement, so that the degree of degradation can be calculated from the sum of the molar concentrations. The value of these methods is demonstrated by analyzing experimental mixtures of the intact agent and degradation products. PMID:7178216

  6. 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. PMID:27254279

  7. Gibberellin metabolism in isolated pea fruit tissue and intact fruits

    SciTech Connect

    Maki, S.; Brenner, M.L. )

    1989-04-01

    Gibberellins (GAs) have been shown by others to be required for normal development of pea fruit. Whether the pericarp of the developing pea fruit produces GAs in situ is not known. To determine if the pericarp has the capacity to produce GAs during fruit growth, the metabolism of the first two committed GAs in the biosynthetic pathway, ({sup 14}C)GA{sub 12}-aldehyde and ({sup 14}C)GA{sub 12} was examined in tissue obtained from pollinated, parthenocarpic, and control fruit over 4 days from treatment. ({sup 14}C)GA{sub 12}-aldehyde was converted primarily to conjugates, including ({sup 14}C)GA{sub 12}-aldehyde conjugate. ({sup 14}C)GA{sub 12} was converted to ({sup 14}C)GA{sub 53} in all tissue, but by day 4 only tissue from pollinated or parthenocarpic fruits showed sustained formation of ({sup 14}C)GA{sub 53}. When ({sup 14}C)GA{sub 12} is applied to 4-day-old fruits attached to the plants, the major product obtained after 24 hours is ({sup 14}C)GA{sub 20} (as identified by GC-MS). No transport to the developing seed was observed. These results indicate that the elongating fruit tissue has the capacity to produce GAs.

  8. Choline oxidation by intact chloroplasts isolated directly from spinach leaves

    SciTech Connect

    Weigel, P.; Hanson, A.D.

    1986-04-01

    Illuminated chloroplasts derived from spinach leaf protoplasts synthesize betaine from choline via the intermediate betaine aldehyde (BAL) (PNAS 82:3678). Photosynthetically active chloroplasts isolated directly from spinach leaves oxidized (/sup 14/C)choline in the light at rates 10 times higher (25-80 nmol/mg chl b) than protoplast-derived chloroplasts. Up to 20% of the (/sup 14/C)choline supplied during a 30 min incubation was oxidized in the light; the main product was (/sup 14/C)BAL. Rates of (/sup 14/C)choline oxidation in darkness were only 5-30% of rates in light. Light-dependent (/sup 14/C)choline oxidation was abolished by DCMU and 5 mM DTT. Pre-illumination of the chloroplasts did not promote (/sup 14/C)choline oxidation in darkness. The uncouplers nigericin and CCCP at concentrations which eliminated CO/sub 2/-dependent O/sub 2/ evolution did not affect (/sup 14/C)choline oxidation in the light. They hypothesize that (/sup 14/C)choline oxidation is not dependent upon light activation of an enzymatic system or upon the electrochemical proton gradient but requires an oxidant generated in the light.

  9. Nuclear import of an intact preassembled proteasome particle

    PubMed Central

    Savulescu, Anca F.; Shorer, Hagai; Kleifeld, Oded; Cohen, Ilana; Gruber, Rita; Glickman, Michael H.; Harel, Amnon

    2011-01-01

    The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated “+” factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development. PMID:21289101

  10. Neural Circuit Recording from an Intact Cockroach Nervous System

    PubMed Central

    Titlow, Josh S.; Majeed, Zana R.; Hartman, H. Bernard; Burns, Ellen; Cooper, Robin L.

    2013-01-01

    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. PMID:24300738

  11. Experimental validation of finite element models of intact and implanted composite hemipelvises using digital image correlation.

    PubMed

    Ghosh, Rajesh; Gupta, Sanjay; Dickinson, Alexander; Browne, Martin

    2012-08-01

    A detailed understanding of the changes in load transfer due to implantation is necessary to identify potential failure mechanisms of orthopedic implants. Computational finite element (FE) models provide full field data on intact and implanted bone structures, but their validity must be assessed for clinical relevance. The aim of this study was to test the validity of FE predicted strain distributions for the intact and implanted pelvis using the digital image correlation (DIC) strain measurement technique. FE models of an in vitro hemipelvis test setup were produced, both intact and implanted with an acetabular cup. Strain predictions were compared to DIC and strain rosette measurements. Regression analysis indicated a strong linear relationship between the measured and predicted strains, with a high correlation coefficient (R = 0.956 intact, 0.938 implanted) and a low standard error of the estimate (SE = 69.53 με, 75.09 με). Moreover, close agreement between the strain rosette and DIC measurements improved confidence in the validity of the DIC technique. The FE model therefore was supported as a valid predictor of the measured strain distribution in the intact and implanted composite pelvis models, confirming its suitability for further computational investigations. PMID:22938356

  12. Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry

    SciTech Connect

    Sharma, Seema; Simpson, David C.; Tolic, Nikola; Jaitly, Navdeep; Mayampurath, Anoop M.; Smith, Richard D.; Pasa-Tolic, Liljiana

    2007-02-01

    We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.

  13. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    PubMed

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. PMID:26171817

  14. Parasite load in intact and ulcerative skin of dogs with leishmaniais.

    PubMed

    Silva, Francine Maria de França; Santos, Edna Michely de Sá; Torres, Sandra Maria; Yamasak, Elise Miyuki; Ramos, Rafael Antonio Nascimento; Alves, Leucio Câmara

    2016-03-01

    The skin is the site of inoculation of Leishmania spp. in susceptible hosts, and consequently dermatopathies, especially ulcerative dermatitis, are the main clinical signs observed. The aim of this study was to assess parasitism of the skin (intact and ulcerated) among dogs that were naturally infected by Leishmania spp., through immunohistochemical analysis. Skin fragments (intact and ulcerated) were collected from 13 dogs with positive parasitological (bone marrow aspiration and exfoliative skin) and serological examinations (ELISA S7® Biogene) forLeishmania spp. These samples were processed using the immunohistochemical technique, involving the streptavidin-peroxidase complex. Ulcerative lesions were mainly observed on the elbows (53.84%; 7/13), nostrils (15.38%; 2/13), ears (23.07%; 3/13) and wings of the ilium (7.69%; 1/13). A severe parasite load was detected in 46.15% and 76.92% of the intact and ulcerated skin samples tested, respectively. The parasite load on ulcerated skin was statistically higher than on intact skin (p = 0.0221). These results indicate that the intact and ulcerated skin may host a high parasite load of amastigote forms of Leishmania spp., which can favor the transmission of the parasite. PMID:27007246

  15. Imaging cardiomyocytes in intact tissue with a remote focusing microscope

    NASA Astrophysics Data System (ADS)

    Corbett, A. D.; Burton, R. A. B.; Bub, G.; Wilson, T.

    2015-03-01

    In cardiac imaging, the spacing between sub-cellular sarcomere structures is of great importance to physiologists in understanding muscle design and performance. Making accurate measurements of the sarcomere length (SL) presents a significant imaging challenge owing to the size of the SL (~2μm) and its naturally low variability (<6%), requiring a high level of precision to determine subtle changes between heart disease models. Moreover, measurements of SL from traditional two-photon imaging have so far been ambiguous to within a factor of cos(α), where α is the inclination of the tissue with respect to the focal plane. By remotely focussing a customised two-photon microscope, it is possible to image heart cells at two oblique angles within 200ms. The oblique images uniquely resolve the tissue inclination ambiguity and reduce the variance of SL measures by as much as 23%. This improved precision is crucial in discerning between pathological models of chronic hypertension. As well as improving measurement precision, the distribution of α across the field of view provides additional structural information which can be related to disease morphology. To validate this new imaging protocol, the value ofα calculated from the oblique planes provided the input to a rigid model cell which was used to predict the appearance of the cell in the conventional focal plane. The comparison of the model to the image data provided a confidence metric for our measurements. Finally, by considering the optical transfer function, the range of cell orientations for which the method is valid could be calculated.

  16. Estradiol enhances retention but not organization of hippocampus-dependent memory in intact male mice.

    PubMed

    Al Abed, Alice Shaam; Sellami, Azza; Brayda-Bruno, Laurent; Lamothe, Valérie; Noguès, Xavier; Potier, Mylène; Bennetau-Pelissero, Catherine; Marighetto, Aline

    2016-07-01

    Because estrogens have mostly been studied in gonadectomized females, effects of chronic exposure to environmental estrogens in the general population are underestimated. Estrogens can enhance hippocampus-dependent memory through the modulation of information storage. However, declarative memory, the hippocampus-dependent memory of facts and events, demands more than abilities to retain information. Specifically, memory of repetitive events of everyday life such as "where I parked" requires abilities to organize/update memories to prevent proactive interference from similar memories of previous "parking events". Whether such organizational processes are estrogen-sensitive is unknown. We here studied, in intact young and aged adult mice, drinking-water (1μM) estradiol effects on both retention and organizational components of hippocampus-dependent memory, using a radial-maze task of everyday-like memory. Demand on retention vs organization was manipulated by varying the time-interval separating repetitions of similar events. Estradiol increased performance in young and aged mice under minimized organizational demand, but failed to improve the age-associated memory impairment and diminished performance in young mice under high organizational demand. In fact, estradiol prolonged mnemonic retention of successive events without improving organization abilities, hence resulted in more proactive interference from irrelevant memories. c-Fos imaging of testing-induced brain activations showed that the deterioration of young memory was associated with dentate gyrus dysconnectivity, reminiscent of that seen in aged mice. Our findings support the view that estradiol is promnesic but also reveal that such property can paradoxically impair memory. These findings have important outcomes regarding health issues relative to the impact of environmental estrogens in the general population. PMID:27038677

  17. A remote acceptance probe and illumination configuration for spectral assessment of internal attributes of intact fruit

    NASA Astrophysics Data System (ADS)

    Greensill, Colin V.; Walsh, Kerry B.

    2000-12-01

    Near infrared spectroscopy can be employed in the non-invasive assessment of intact fruit for eating quality attributes such as soluble solid content (SSC). Rapid sorting is dependent on a suitable non-contact geometry of fruit, light source and detector assembly, optimized for a given fruit commodity. An optical system was designed with reference to distribution of SSC and light penetration into rockmelon fruit. SSC of mesocarp tissue was not significantly different over the greater part of the proximal-distal axis of the fruit, particularly in the vicinity of the fruit equator. There was also no consistent variation in SSC of mesocarp tissue with respect to radial position of sampling. Mesocarp SSC was higher (~3% w/v) closer to the seed cavity. The optical sampling system was therefore designed to assess an equatorial position on the fruit. Light penetrating a rockmelon fruit was empirically assessed to be diffuse at a depth of <15 mm from the fruit surface. Signal decreased in an exponential proportionality with depth into the fruit, but was still detectable at depths in excess of the seed cavity of rockmelons. A partial transmittance optical design was employed, with a collimated light source interrupted by a central light stop, and a detector viewing the shadowed region of the sample. This system did not physically contact the sample. It was compared to a system with a light excluding `contacting' shroud between the detector and the fruit surface. The performance of calibrations generated using the non-contact configuration was not significantly different than for the configuration requiring contact.

  18. Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

    PubMed

    Wang, Liyuan; Cheng, Guyue; Ren, Yuxia; Dai, Zheng; Zhao, Zhong-Shu; Liu, Feng; Li, Shiyong; Wei, Yahan; Xiong, Jing; Tang, Xiao-Feng; Tang, Bing

    2015-05-01

    Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and β-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes. PMID:25412577

  19. Non-invasive integrative analysis of contraction energetics in intact beating heart.

    PubMed

    Deschodt-Arsac, Véronique; Calmettes, Guillaume; Gouspillou, Gilles; Chapolard, Mathilde; Raffard, Gérard; Rouland, Richard; Jais, Pierre; Haissaguerre, Michel; Dos Santos, Pierre; Diolez, Philippe

    2013-01-01

    The comprehensive study of human pathologies has revealed the complexity of the interactions involved in cardiovascular physiology. The recent validation of system's biology approaches - like our Modular Control and Regulation Analysis (MoCA) - motivates the current interest for new integrative and non-invasive analyses that could be used for medical study of human heart contraction energetics. By considering heart energetics as a supply-demand system, MoCA gives access to integrated organ function and brings out a new type of information, the "elasticities", which describe in situ the regulation of both energy demand and supply by cellular energetic status. These regulations determine the internal control of contraction energetics and may therefore be a key to the understanding of the links between molecular events in pathologies and whole organ function/dysfunction. A wider application to the effects of cardiac drugs in conjunction with the direct study of heart pathologies may be considered in the near future. MoCA can potentially be used not only to detect the origin of the defects associated with the pathology (elasticity analyses), but also to provide a quantitative description of how these defects influence global heart function (regulation analysis) and therefore open new therapeutic perspectives. Several key examples of current applications to intact isolated beating heart are presented in this paper. The future application to human pathologies will require the use of non-invasive NMR techniques for the simultaneous measurement of energy status ((31)P NMR) and heart contractile activity (3D MRI). This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. PMID:22789933

  20. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  1. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice.

    PubMed

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  2. Retinal ganglion cell axons regenerate in the presence of intact sensory fibres.

    PubMed

    King, Carolyn; Bartlett, Carole; Sauvé, Yves; Lund, Ray; Dunlop, Sarah; Beazley, Lyn

    2006-02-01

    A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053+/-223) and conventional grafts (1323+/-881; P>0.05). The results indicate that an intact sensory environment supports central axon regeneration. PMID:16407770

  3. Advanced CLARITY for rapid and high-resolution imaging of intact tissues

    PubMed Central

    Tomer, Raju; Ye, Li; Hsueh, Brian; Deisseroth, Karl

    2014-01-01

    CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe next-generation protocols spanning multiple dimensions of the CLARITY workflow, ranging from a novel approach to simple, reliable, and efficient lipid removal without electrophoretic instrumentation (passive CLARITY), to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy or COLM) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. These methods may find application in the structural and molecular analysis of large assembled biological systems such as the intact mammalian brain. PMID:24945384

  4. Incidence of specific absolute neurocognitive impairment in globally intact children with histories of early severe deprivation

    PubMed Central

    Behen, Michael E.; Helder, Emily; Rothermel, Robert; Solomon, Katherine; Chugani, Harry T.

    2008-01-01

    Postnatal deprivation is associated with neurocognitive delay/dysfunction. Although “catch up” in global cognition following adoption has been reported, this study examined the incidence of specific absolute impairment in adopted children with intact global cognitive functioning. Eighty-five children (38 males, mean age=112.8, SD=30.3 months; range 61−209 months) raised from birth in orphanages underwent comprehensive neuropsychological evaluation. Fifty-four were deemed globally intact (IQ>85). Of those deemed globally intact, 46 percent evidenced absolute impairment in at least one domain of functioning. Duration of stay in the orphanage was directly associated with incidence of impairment and number of domains affected. A substantial proportion of participants evidenced persistent, absolute impairment in one or more domains of neurocognitive function despite integrity of basic intellectual functions. PMID:18686074

  5. A search for intact lava tubes on the Moon: Possible lunar base habitats

    NASA Technical Reports Server (NTRS)

    Coombs, Cassandra R.; Hawke, B. Ray

    1992-01-01

    We have surveyed lunar sinuous rilles and other volcanic features in an effort to locate intact lava tubes that could be used to house an advanced lunar base. Criteria were established for identifying intact tube segments. Sixty-seven tube candidates within 20 rilles were identified on the lunar nearside. The rilles, located in four mare regions, varied in size and sinuosity. We identified four rilles that exhibited particularly strong evidence for the existence of intact lava tube segments. These are located in the following areas: (1) south of Gruithuisen K, (2) in the Marius Hills region, (3) in the southeastern Mare Serenitatis, and (4) in the eastern Mare Serenitatis. We rated each of the 67 probable tube segments for lunar base suitability based on its dimensions, stability, location, and access to lunar resources. Nine tube segments associated with three separate rilles are considered prime candidates for use as part of an advanced lunar base.

  6. Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells.

    PubMed

    Chen, Chun-Rong; Salazar, Larry M; McLachlan, Sandra M; Rapoport, Basil

    2015-07-01

    The TSH receptor (TSHR) extracellular domain (ECD) comprises a N-terminal leucine-rich repeat domain and an hinge region (HR), the latter contributing to ligand binding and critical for receptor activation. The crystal structure of the leucine-rich repeat domain component has been solved, but previous attempts to generate conformationally intact complete ECD or the isolated HR component for structural analysis have failed. The TSHR HR contains a C-peptide segment that is removed during spontaneous TSHR intramolecular cleavage into disulfide linked A- and B-subunits. We hypothesized that deletion of the redundant C-peptide would overcome the obstacle to generating conformationally intact TSHR ECD protein. Indeed, lacking the C-peptide region, the TSHR ECD (termed ECD-D1) and the isolated HR (termed HR-D1) were secreted into medium of insect cells infected with baculoviruses coding for these modified proteins. The identities of TSHR ECD-D1 and HR-D1 were confirmed by ELISA and immunoblotting using TSHR-specific monoclonal antibodies. The TSHR-ECD-D1 in conditioned medium was folded correctly, as demonstrated by its ability to inhibit radiolabeled TSH binding to the TSH holoreceptor. The TSHR ECD-D1 purification was accomplished in a single step using a TSHR monoclonal antibody affinity column, whereas the HR-D1 required a multistep protocol with a low yield. In conclusion, we report a novel approach to generate the TSHR ECD, as well as the isolated HR in insect cells, the former in sufficient amounts for structural studies. However, such studies will require previous complexing of the ECD with a ligand such as TSH or a thyroid-stimulating antibody. PMID:25860033

  7. Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

    SciTech Connect

    Wu, Si; Brown, Joseph N.; Tolic, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J.; Pevzner, Pavel A.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2014-05-31

    There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

  8. Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear magnetic resonance.

    PubMed

    Greiner, J V; Kopp, S J; Gillette, T E; Glonek, T

    1983-05-01

    The principal low molecular weight phosphatic metabolites of the intact cornea were identified and quantitated nondestructively by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy. As part of this analytical procedure, the intracorneal pH was approximated from the resonance shift position of inorganic orthophosphate. In addition the metabolic and pH stability of incubated corneas at 37 C in MK medium was evaluated during an 8-hr time course and compared to similar dynamic analyses performed on corneas with denuded endothelium. Perchloric acid extracts prepared from these same corneas were analyzed by P-31 NMR to verify the metabolite peak assignments and to quantitate the concentrations of minor corneal metabolites. The concentrations of phosphatic metabolites of the cornea, including three previously unidentified phosphorus-containing substances, were determined for freshly excised corneas. The initial corneal spectroscopic profile was not altered by removal of the endothelium. At 37 C the MK media-incubated intact whole corneas experienced a time-dependent decline in ATP levels with a concomitant rise in inorganic orthophosphate; however, the tissue levels of the other principal phosphatic metabolites were not altered by prolonged incubation. In contrast, removal of the endothelial layer of the cornea-induced progressive metabolic deterioration of intact corneas characterized, most prominantly, by time-dependent declines in ATP and glycerol 3-phosphorylcholine levels and concomitant increases in ADP and inorganic orthophosphate levels relative to intact whole corneas. This study has established the feasibility of monitoring the metabolic status of intact rabbit corneas nondestructively and noninvasively. As such, P-31 NMR spectroscopy offers a promising method that may enable analysis of the metabolic viability of intact human donor corneas to provide a basis for selecting donor corneas for transplantation. PMID:6840999

  9. Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

    PubMed Central

    2014-01-01

    Background The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. Methods We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. Results The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. Conclusions The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a

  10. Intact mariner-like element in tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae).

    PubMed

    Ren, X; Park, Y; Miller, T A

    2006-12-01

    An intact mariner-like element was isolated from the genome of tobacco budworm, Heliothis virescens. This is the first report of an intact mariner element after the initial identification of Mos1 from Drosophila mauritiana. The full-length Hvmar1 has 30 bp inverted terminal repeats and a complete 1065 bp open reading frame (ORF) encoding the transposase with a 'D,D(34)D' motif in the catalytic domain. Polymerase chain reaction results show that at least one insertion of the Hvmar1 element is conserved in this Heliothis strain. Phylogenetic analysis demonstrated that Hvmar1 belongs to the irritans subfamily. PMID:17201767

  11. Fluorescence imaging of endothelial cellular responses in the intact lung microvasculature

    NASA Astrophysics Data System (ADS)

    Kuebler, Wolfgang M.; Brueckl, Corinna; Kuehnl, Andreas; Ying, Xiaoyou; Bhattacharya, Jahar

    2001-05-01

    Endothelial cells outline the blood vessel wall and contribute pivotally to the regulation of numerous microvascular characteristics such as permeability, vascular tone, vasomotion, or cell-cell interactions. These functions are mediated by second messenger responses and intracellular signal transduction cascades within the endothelial cells. Imaging of endothelial cellular and subcellular responses has yet been limited to studies in isolated or cultured cells, which cannot reflect dynamic physiologic or pathologic cellular interactions in the intact microcirculation. Combining micropuncture and microcatheter approaches with in vitro fluorescence imaging techniques, we have developed methods for imaging endothelial cellular responses of intact pulmonary microvessels in situ.

  12. 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

    ... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) IDENTIFICATION AND LISTING OF HAZARDOUS WASTE Exclusions/Exemptions § 261.41 Notification and Recordkeeping for Used, Intact Cathode...

  13. Differentiation between ligand trapping into intact cells and binding on muscarinic receptors.

    PubMed

    Gossuin, A; Maloteaux, J M; Trouet, A; Laduron, P

    1984-05-22

    Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells. PMID:6722181

  14. Predictors of Attachment Security in Preschool Children from Intact and Divorced Families

    ERIC Educational Resources Information Center

    Nair, Hira; Murray, Ann D.

    2005-01-01

    The authors selected 58 mother-child dyads from divorced and intact families to participate in a study on the impact of divorce on preschoolers' attachment security. The authors explored pathways that lead to security of attachment. They found that mothers from divorced families were younger, had lower income levels, and had lower levels of…

  15. Adolescents from One Parent, Stepparent and Intact Families: Emotional Problems and Suicide Attempts.

    ERIC Educational Resources Information Center

    Garnefski, Nadia; Diekstra, Rene F. W.

    1997-01-01

    Investigated differences among adolescents (N=13,953) living in intact families, one-parent families, and stepparent families. Results indicate that adolescents from one parent and stepparent families reported lower self-esteem, more symptoms of anxiety and loneliness, more depressed mood, more suicidal thoughts, and more suicide attempts than…

  16. The Father-Child Relationship, Parenting Styles, and Adolescent Risk Behaviors in Intact Families

    ERIC Educational Resources Information Center

    Bronte-Tinkew, Jacinta; Moore, Kristin A.; Carrano, Jennifer

    2006-01-01

    The father-child relationship and father's parenting style are examined as predictors of first delinquency and substance use, using data from the National Longitudinal Study of Youth 1997, Rounds 1 to 3 (N = 5,345), among adolescents in intact families. Discrete time logistic regressions indicate that a more positive father-child relationship…

  17. How-to-Do-It: Cytokinin Induced Cell Division & Differentiation Using Intact Plants.

    ERIC Educational Resources Information Center

    Bohnsack, Charles W.

    1989-01-01

    Presents a procedure by which cytokinins are used to induce a population of dividing and differentiating cells on the cut surface of the roots of an intact plant. Includes the method used, results, and suggestions for a variety of variables that may be tested. (RT)

  18. Phosphorus leaching through intact soil cores as influenced by type and duration of manure application

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaching of phosphorus (P) in manure-amended soils has received increased attention as a significant source of non-point source P pollution. Intact soil cores were collected from fields on a farm in Southern New York to test the effects of long-term dairy or poultry manure application on P leaching....

  19. Intact and fragmented intracellular immunoglobulin in a case of non-secretory myeloma.

    PubMed Central

    Whicher, J T; Davies, J D; Grayburn, J A

    1975-01-01

    A patient is described with myeloma without paraproteinaemia or Bence Jones proteinuria in whom the tumour cells have been shown to contain monoclonal immunoglobulin. The use of immunoelectrophoresis and polyacrylamide gel electrophoresis has established, apparently for the first time, that the immunoglobulin components are present in the form of intact molecules and free light chains. Images PMID:1123437

  20. High incidence of intact or fragmented immunoglobulin in urine of patients with multiple myeloma.

    PubMed

    Kraj, Maria; Kruk, Barbara; Lech-Marańda, Ewa; Warzocha, Krzysztof; Prochorec-Sobieszek, Monika

    2015-01-01

    In this prospective study we determined the incidence of intact/fragmented immunoglobulin and Bence Jones protein in urine immunofixation using Sebia reagents and HydrasysTM 2 apparatus and compared the results to concentrations of serum free light chains (FLC) assessed using Siemens BNTM II nephelometer and the immunoassay Freelite (Binding Site) in 289 patients with multiple myeloma at diagnosis. It was found that in one third of IgG, IgA and IgD myeloma patients, intact/fragmented immunoglobulin can be detected in urine and is connected with impaired renal function and reduced survival. Urine immunofixation detects monoclonal protein (FLC and intact/fragmented immunoglobulin) in 66-79% of IgG and IgA myeloma patients while serum FLC immunoassay detect it in 82-94% of IgG and IgA myeloma patients. However, the latter method is inadequate for detection of intact/fragmented immunoglobulin in urine. Serum FLC immunoassay and urine immunofixation are complementary methods in diagnosing and monitoring monoclonal protein in patients with myeloma. PMID:25860239

  1. Raman spectroscopy of normal oral buccal mucosa tissues: study on intact and incised biopsies

    NASA Astrophysics Data System (ADS)

    Deshmukh, Atul; Singh, S. P.; Chaturvedi, Pankaj; Krishna, C. Murali

    2011-12-01

    Oral squamous cell carcinoma is one of among the top 10 malignancies. Optical spectroscopy, including Raman, is being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex vivo tissues. Spectral features showed predominance of lipids and proteins in normal and cancer conditions, respectively, which were attributed to membrane lipids and surface proteins. In view of recent developments in deep tissue Raman spectroscopy, we have recorded Raman spectra from superior and inferior surfaces of 10 normal oral tissues on intact, as well as incised, biopsies after separation of epithelium from connective tissue. Spectral variations and similarities among different groups were explored by unsupervised (principal component analysis) and supervised (linear discriminant analysis, factorial discriminant analysis) methodologies. Clusters of spectra from superior and inferior surfaces of intact tissues show a high overlap; whereas spectra from separated epithelium and connective tissue sections yielded clear clusters, though they also overlap on clusters of intact tissues. Spectra of all four groups of normal tissues gave exclusive clusters when tested against malignant spectra. Thus, this study demonstrates that spectra recorded from the superior surface of an intact tissue may have contributions from deeper layers but has no bearing from the classification of a malignant tissues point of view.

  2. Recent advances in the use of non-destructive near infrared spectroscopy on intact olive fruits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this review is to illustrate the state of the art in the use of non-destructive near infrared (NIR) spectroscopy for quality evaluation of intact fruit in the olive industry. First, the most recent studies regarding the application of non-destructive NIR spectroscopy methods to asse...

  3. Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina.

    PubMed

    Kombrink, E; Wöber, G

    1980-07-01

    A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,L-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm(-3). Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm(-3). Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each. PMID:24306242

  4. Adolescents' Perception of the Ideal Mate: Its Relationship to Parental Characteristics in Intact and Nonintact Families

    ERIC Educational Resources Information Center

    Lazar, Amnon; Guttmann, Joseph

    2004-01-01

    The relation of adolescents' experiencing parental divorce, their perception of parents' characteristics, and their perception of the ideal mate's characteristics was investigated. One hundred adolescents from intact families and 79 from nonintact families were asked to rank both the degree to which each of 40 personality traits characterized…

  5. THE ENDOCRINE PROFILE OF INTACT FEMALE RATS ON THE DAY OF PROESTRUS FOLLOWING EXPOSURE TO ATRAZINE

    EPA Science Inventory

    The Endocrine Profile of Intact Female Rats on the Day of Proestrus Following Exposure to Atrazine.
    RL Cooper, A Buckalew, SC Laws and TE Stoker
    Endocrinology Branch, RTD, NHEERL, ORD, U.S. EPA, RTP, NC, 27711.

    The chlorotriazine herbicide, atrazine, has been sho...

  6. [Biodegradation of Pyrene by Intact Cells and Spores of Brevibacillus brevis].

    PubMed

    Liu, Zhi-chen; Ye, Jin-shao; Peng, Hui; Liu, Ze-hua; Deng, Ting-jin; Yin, Hua; Liao, Li-ping

    2015-05-01

    Biodegradation characteristics of pyrene by intact cells and spores of Brevibacillus brevis were investigated. The results revealed that the degradation efficiency of 1 mg · L(-1) pyrene by B. brevis intact cells reached 53% within 5 d. Four hydroxy metabolites were detected by LC-MS/MS during the degradation of pyrene by intact cells and intracellular enzymes, including 1-hydroxy-pyrene, 9- phenanthrol, α-naphthol and β-naphthol. The amount of products first increased and then declined in the degradation system with intact cells, while the macromolecular metabolites showed a continuous accumulation tendency in the degradation system with intracellular enzymes, proving that some degrading enzymes of B. brevis could be induced by the products generated during the degradation, and then the enzymes induced could further degrade these degradation products. The amount of cells germinated from spores in MSM containing pyrene was 1.5 x 10(9) cells · L(-1), and the degradation rate of pyrene (1 mg · L(-1)) within 5 d reached 15%. PMID:26314128

  7. Feasibility study of utilizing simplified near infrared imaging for detecting fruit fly larvae in intact fruit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Following the previous research to classify intact mangoes infested with oriental fruit fly from the control ones using near infrared (NIR) spectra acquired by a spot-type handheld NIR instrument, an attempt to improve the sensitivity of the system by employing NIR imaging technology was conducted. ...

  8. Micron surface-enhanced Raman spectroscopy of intact biological organisms and model systems

    SciTech Connect

    Todd, E.A.; Morris, M.D.

    1994-05-01

    Surface-enhanced Raman spectra have been obtained within intact zebrafish embryos and inside the 500-fL pores of a Nucleopore filter membrane with the use of coated microelectrodes with 1-3 {mu}m active silver tip diameters. The spectra obtained demonstrate the microelectrode`s ability to penetrate biological membranes as well as restricted volumes. 8 refs., 5 figs.

  9. Operative findings of conductive hearing loss with intact tympanic membrane and normal temporal bone computed tomography.

    PubMed

    Kim, Se-Hyung; Cho, Yang-Sun; Kim, Hye Jeong; Kim, Hyung-Jin

    2014-06-01

    Despite recent technological advances in diagnostic methods including imaging technology, it is often difficult to establish a preoperative diagnosis of conductive hearing loss (CHL) in patients with an intact tympanic membrane (TM). Especially, in patients with a normal temporal bone computed tomography (TBCT), preoperative diagnosis is more difficult. We investigated middle ear disorders encountered in patients with CHL involving an intact TM and normal TBCT. We also analyzed the surgical results with special reference to the pathology. We reviewed the medical records of 365 patients with intact TM, who underwent exploratory tympanotomy for CHL. Fifty nine patients (67 ears, eight bilateral surgeries) had a normal preoperative TBCT findings reported by neuro-radiologists. Demographic data, otologic history, TM findings, preoperative imaging findings, intraoperative findings, and pre- and postoperative audiologic data were obtained and analyzed. Exploration was performed most frequently in the second and fifth decades. The most common postoperative diagnosis was stapedial fixation with non-progressive hearing loss. The most commonly performed hearing-restoring procedure was stapedotomy with piston wire prosthesis insertion. Various types of hearing-restoring procedures during exploration resulted in effective hearing improvement, especially with better outcome in the ossicular chain fixation group. In patients with CHL who have intact TM and normal TBCT, we should consider an exploratory tympanotomy for exact diagnosis and hearing improvement. Information of the common operative findings from this study may help in preoperative counseling. PMID:23744181

  10. Family Relationships and the Psychosocial Adjustment of School-Aged Children in Intact Families

    ERIC Educational Resources Information Center

    Hakvoort, Esther M.; Bos, Henny M. W.; Van Balen, Frank; Hermanns, Jo M. A.

    2010-01-01

    The authors investigated whether the quality of three family relationships (i.e., marital, parent-child, sibling) in intact families are associated with each other and with children's psychosocial adjustment. Data were collected by means of maternal and child reports (N = 88) using standardized instruments (i.e., Marital Satisfaction Scale,…

  11. Redox Activities and ROS, NO and Phenylpropanoids Production by Axenically Cultured Intact Olive Seedling Roots after Interaction with a Mycorrhizal or a Pathogenic Fungus

    PubMed Central

    Espinosa, Francisco; Garrido, Inmaculada; Ortega, Alfonso; Casimiro, Ilda; Álvarez-Tinaut, Mª Carmen

    2014-01-01

    Roots of intact olive seedlings, axenically cultured, were alternatively placed in contact with Rhizophagus irregularis (mycorrhizal) or Verticillim dahliae (pathogenic) fungi. MeJA treatments were also included. In vivo redox activities in the apoplast of the intact roots (anion superoxide generation, superoxide dismutase and peroxidase activities) were measured. All our results showed that apoplastic redox activities of intact seedling roots in contact with the compatible mycorrhizal fungus were clearly attenuated in comparison with the pathogenic fungus or treated with MeJA, even at the early stages of treatment used. Total phenolics, flavonoids and phenylpropanoid glycosides were also quantified. Roots in contact with the mycorrhizal fungus did not enhance the biosynthesis of phenolic compounds with respect to controls, while those in contact with the pathogenic one significantly enhanced the biosynthesis of all phenolic fractions measured. Reactive oxygen species and nitric oxid accumulation in roots were examined by fluorescence microscopy. All of them presented much higher accumulation in roots in contact with the pathogenic than with the mycorrhizal fungus. Altogether these results indicate that intact olive seedling roots clearly differentiated between mycorrhizal and pathogenic fungi, attenuating defense reactions against the first to facilitate its establishment, while inducing a strong and sustained defense reaction against the second. Both reactive oxygen and nitrogen species seemed to be involved in these responses from the first moments of contact. However, further investigations are required to clarify the proposed crosstalk between them and their respective roles in these responses since fluorescence images of roots revealed that reactive oxygen species were mainly accumulated in the apoplast (congruently with the measured redox activities in this compartment) while nitric oxid was mainly stored in the cytosol. PMID:24967716

  12. Quantification of Intact and Truncated Stromal Cell-Derived Factor-1α in Circulation by Immunoaffinity Enrichment and Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Wang, Weixun; Choi, Bernard K.; Li, Wenyu; Lao, Zhege; Lee, Anita Y. H.; Souza, Sandra C.; Yates, Nathan A.; Kowalski, Timothy; Pocai, Alessandro; Cohen, Lucinda H.

    2014-04-01

    Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form. Using top-down strategy in quantification, we demonstrated the unique advantage of keeping SDF-1α's two disulfide bridges intact in the analysis. To achieve the optimal sensitivity required for quantification of intact and truncated SDF-1α at endogenous levels in blood, we coupled nano-flow tandem mass spectrometry with antibody-based affinity enrichment. The assay has a quantitative range of 20 pmol/L to 20 nmol/L in human plasma as well as in rhesus monkey plasma. With only slight modification, the same assay can be used to quantify SDF-1α in mice. Using two in vivo animal studies as examples, we demonstrated that it was critical to differentiate intact SDF-1α from its truncated form in the analysis of biomarkers for pharmacologic inhibition of DPP-IV activity. These novel methods enable translational research on suppression of SDF-1 inactivation with DPP-IV inhibition and can be applied to relevant clinical samples in the future to yield new insights on change of SDF-1α levels in disease settings and in response to therapeutic interventions.

  13. Quantification of intact and truncated stromal cell-derived factor-1α in circulation by immunoaffinity enrichment and tandem mass spectrometry.

    PubMed

    Wang, Weixun; Choi, Bernard K; Li, Wenyu; Lao, Zhege; Lee, Anita Y H; Souza, Sandra C; Yates, Nathan A; Kowalski, Timothy; Pocai, Alessandro; Cohen, Lucinda H

    2014-04-01

    Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form. Using top-down strategy in quantification, we demonstrated the unique advantage of keeping SDF-1α's two disulfide bridges intact in the analysis. To achieve the optimal sensitivity required for quantification of intact and truncated SDF-1α at endogenous levels in blood, we coupled nano-flow tandem mass spectrometry with antibody-based affinity enrichment. The assay has a quantitative range of 20 pmol/L to 20 nmol/L in human plasma as well as in rhesus monkey plasma. With only slight modification, the same assay can be used to quantify SDF-1α in mice. Using two in vivo animal studies as examples, we demonstrated that it was critical to differentiate intact SDF-1α from its truncated form in the analysis of biomarkers for pharmacologic inhibition of DPP-IV activity. These novel methods enable translational research on suppression of SDF-1 inactivation with DPP-IV inhibition and can be applied to relevant clinical samples in the future to yield new insights on change of SDF-1α levels in disease settings and in response to therapeutic interventions. PMID:24500701

  14. Simultaneous bicompartmental bucket-handle meniscal tears with intact anterior cruciate ligament: a case report

    PubMed Central

    2010-01-01

    Introduction Bucket handle tear of the menisci is a common type of lesion resulting from injury to the knee joint. Bucket handle injury of both menisci in almost all cases is associated with a lesion to either the anterior or the posterior cruciate ligament of the knee joint. We describe a case of acute bucket-handle tear of the medial and lateral menisci with intact anterior and posterior cruciate ligaments in a dancer. To the best of our knowledge, there are no previous reports of this type of injury in the literature. Case presentation A 28-year-old Caucasian Greek woman presented to the emergency department after sustaining an injury to her right knee during dancing. An MRI evaluation demonstrated tears in both menisci of the right knee, while the anterior and posterior cruciate ligaments were found to be intact. A partial medial and lateral meniscectomy was then performed. At a follow-up examination six months after her injury, clinical tests demonstrated that our patient's right knee was stable, had a full range of motion and had no tenderness. She was satisfied with the outcome of the operation and returned to her pre-injury activities. Conclusion We present the first case in the literature that describes a combined bucket-handle injury of both the medial and lateral menisci with an intact anterior cruciate ligament. The clinical examination of the anterior cruciate ligament was unremarkable, with no signs of deficiency or rupture. The posterior cruciate ligament was also intact. On magnetic resonance imaging, the ligaments were visualised as intact in all their length. These findings were confirmed by arthroscopic evaluation. PMID:20181059

  15. Degradation of fluorescent and radiolabelled sphingomyelins in intact cells by a non-lysosomal pathway.

    PubMed

    Levade, T; Vidal, F; Vermeersch, S; Andrieu, N; Gatt, S; Salvayre, R

    1995-10-01

    The aim of the present study was to investigate the role of the entitled neutral, sphingomyelinase in the non-lysosomal pathway of sphingomyelin degradation by intact cells (Spence et al. (1983) J. Biol. Chem. 258, 8595-8600; Levade et al. (1991) J. Biol. Chem. 266, 13519-13529). The uptake and degradation of sphingomyelin by intact living cells was studied using cell lines exhibiting a wide range of activity levels of acid, lysosomal and neutral sphingomyelinases as determined in vitro on cell homogenates by their respective standard assays. For this purpose, neuroblastoma, skin fibroblasts, lymphoid and leukemic cell lines, some of them derived from patients with Niemann-Pick disease (deficient in the acid, lysosomal sphingomyelinase) were incubated with radioactive, [oleoyl-3H]sphingomyelin or fluorescent, pyrene-sulfonylaminoundecanoyl-sphingomyelin. Either compound was taken up by a pathway which was not receptor-mediated and hydrolyzed by all intact cells, including those derived from Niemann-Pick disease patients. Moreover, their degradation by the intact cells was not inhibited by treatment with chloroquine, indicating hydrolysis by a non-lysosomal sphingomyelinase. The intracellular sphingomyelin degradation rates showed no correlation with the activity of the 'classical' neutral sphingomyelinase as determined in vitro. In particular, fibroblasts derived from Niemann-Pick patients lacking the lysosomal sphingomyelinase, and having no detectable in vitro activity of the 'classical' neutral sphingomyelinase, were able to degrade the exogenously supplied sphingomyelins. Indeed, in vitro these cells were shown to exhibit neutral, magnesium- and dithiothreitol-dependent sphingomyelinase activities, that might contribute to the non-lysosomal pathway for sphingomyelin degradation to ceramide in intact cells. PMID:7548198

  16. Body condition and habitat use by Hermann's tortoises in burnt and intact habitats

    PubMed Central

    Lecq, S.; Ballouard, J.-M.; Caron, S.; Livoreil, B.; Seynaeve, V.; Matthieu, L.-A.; Bonnet, X.

    2014-01-01

    In Mediterranean regions, fires threaten terrestrial tortoises. Nevertheless, varying proportions of adults survive fire; these surviving individuals can play a central role for population recovery. The regions devastated by fire often include important habitat of Hermann's tortoises (Testudo hermanni hermanni), so assessing the ability of survivors to persist is essential for conserving the species. Body-condition indices provide an integrative estimate of how well individuals cope with environmental variations and impacts, including fires. Between 2002 and 2009, we monitored Hermann's tortoises in intact and burnt habitats in southeastern France. In summer 2003, a strong fire ravaged half of the surveyed zone, providing an opportunity to compare body condition of tortoises between intact and burnt areas over time. Six years later, the impact of fire on vegetation was still marked; large trees were abundant in the intact area, whereas open shrub vegetation prevailed in the burnt area. In both areas, the mean body condition of tortoises fluctuated over time; however, there were no differences between the two areas. A radio-tracking experiment demonstrated that individuals from each area were residents, and not vagrants commuting between areas. We also assessed changes in body condition and microhabitat use in radio-tracked individuals. We found no significant differences between the tortoises living in the burnt and intact areas, despite subtle differences in habitat use. In conclusion: (i) surviving tortoises in an area ravaged by fire can maintain their body condition like individuals living in an intact area, and thus, individuals from burnt areas should not be translocated to supposedly better areas; and (ii) depopulated burnt areas are likely to be appropriate for population-augmentation programmes.

  17. Reorganization of Intact Descending Motor Circuits to Replace Lost Connections After Injury.

    PubMed

    Fink, Kathren L; Cafferty, William B J

    2016-04-01

    Neurons have a limited capacity to regenerate in the adult central nervous system (CNS). The inability of damaged axons to re-establish original circuits results in permanent functional impairment after spinal cord injury (SCI). Despite abortive regeneration of axotomized CNS neurons, limited spontaneous recovery of motor function emerges after partial SCI in humans and experimental rodent models of SCI. It is hypothesized that this spontaneous functional recovery is the result of the reorganization of descending motor pathways spared by the injury, suggesting that plasticity of intact circuits is a potent alternative conduit to enhance functional recovery after SCI. In support of this hypothesis, several studies have shown that after unilateral corticospinal tract (CST) lesion (unilateral pyramidotomy), the intact CST functionally sprouts into the denervated side of the spinal cord. Furthermore, pharmacologic and genetic methods that enhance the intrinsic growth capacity of adult neurons or block extracellular growth inhibitors are effective at significantly enhancing intact CST reorganization and recovery of motor function. Owing to its importance in controlling fine motor behavior in primates, the CST is the most widely studied descending motor pathway; however, additional studies in rodents have shown that plasticity within other spared descending motor pathways, including the rubrospinal tract, raphespinal tract, and reticulospinal tract, can also result in restoration of function after incomplete SCI. Identifying the molecular mechanisms that drive plasticity within intact circuits is crucial in developing novel, potent, and specific therapeutics to restore function after SCI. In this review we discuss the evidence supporting a focus on exploring the capacity of intact motor circuits to functionally repair the damaged CNS after SCI. PMID:26846379

  18. Glucagon and insulin responses after ingestion of different amounts of intact and hydrolysed proteins.

    PubMed

    Claessens, Mandy; Saris, Wim H M; van Baak, Marleen A

    2008-07-01

    Ingestion of dietary protein is known to induce both insulin and glucagon secretion. These responses may be affected by the dose and the form (intact or hydrolysed) in which protein is ingested. The aim of the study was to investigate the effect of different amounts of intact protein and protein hydrolysate of a vegetable (soya) and animal (whey) protein on insulin and glucagon responses and to study the effect of increasing protein loads for both intact protein and protein hydrolysate in man. The study employed a repeated-measures design with Latin-square randomisation and single-blind trials. Twelve healthy non-obese males ingested three doses (0.3, 0.4 and 0.6 g/kg body weight) of intact soya protein (SPI) and soya protein hydrolysate (SPH). Another group of twelve healthy male subjects ingested three doses (0.3, 0.4 and 0.6 g/kg body weight) of intact whey protein (WPI) and whey protein hydrolysate (WPH). Blood was sampled before (t = 0) and 15, 30, 60, 90 and 120 min after protein ingestion for insulin, glucagon and glucose determination. SPI induced a higher total area under the curve for insulin and glucagon than SPH while no difference between WPI and WPH was found. Insulin and glucagon responses increased with increasing protein load for SPI, SPH, WPI and WPH, but the effect was more pronounced for glucagon. A higher dose of protein or its hydrolysate will result in a lower insulin:glucagon ratio, an important parameter for the control of postprandial substrate metabolism. In conclusion, insulin and glucagon responses were protein and hydrolysate specific. PMID:18167171

  19. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

    PubMed

    Kellie, John F; Higgs, Richard E; Ryder, John W; Major, Anthony; Beach, Thomas G; Adler, Charles H; Merchant, Kalpana; Knierman, Michael D

    2014-01-01

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03). PMID:25052239

  20. Quantitative Measurement of Intact Alpha-Synuclein Proteoforms from Post-Mortem Control and Parkinson's Disease Brain Tissue by Intact Protein Mass Spectrometry

    PubMed Central

    Kellie, John F.; Higgs, Richard E.; Ryder, John W.; Major, Anthony; Beach, Thomas G.; Adler, Charles H.; Merchant, Kalpana; Knierman, Michael D.

    2014-01-01

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03). PMID:25052239

  1. Quantitative Measurement of Intact Alpha-Synuclein Proteoforms from Post-Mortem Control and Parkinson's Disease Brain Tissue by Intact Protein Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Kellie, John F.; Higgs, Richard E.; Ryder, John W.; Major, Anthony; Beach, Thomas G.; Adler, Charles H.; Merchant, Kalpana; Knierman, Michael D.

    2014-07-01

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

  2. Multi-Axis Prosthetic Knee Resembles Alpine Skiing Movements of an Intact Leg

    PubMed Central

    Demšar, Ivan; Duhovnik, Jože; Lešnik, Blaž; Supej, Matej

    2015-01-01

    The purpose of the study was to analyse the flexion angles of the ski boot, ankle and knee joints of an above-knee prosthesis and to compare them with an intact leg and a control group of skiers. One subject with an above-knee amputation of the right leg and eight healthy subjects simulated the movement of a skiing turn by performing two-leg squats in laboratory conditions. By adding additional loads in proportion to body weight (BW; +1/3 BW, +2/3 BW, +3/3 BW), various skiing regimes were simulated. Change of Flexion Angle (CoFA) and Range of Motion (RoM) in the ski boot, ankle and knee joints were calculated and compared. An average RoM in the skiing boot on the side of prosthesis (4.4 ± 1.1°) was significantly lower compared to an intact leg (5.9 ± 1.8°) and the control group (6.5 ± 2.3°). In the ankle joint, the average RoM was determined to be 13.2±2.9° in the prosthesis, 12.7 ± 2.8° in an intact leg and 14.8±3.6 in the control group. However, the RoM of the knee joint in the prosthesis (42.2 ± 4.2°) was significantly larger than that of the intact leg (34.7 ± 4.4°). The average RoM of the knee joint in the control group was 47.8 ± 5.4°. The influences of additional loads on the kinematics of the lower extremities were different on the side of the prosthesis and on the intact leg. In contrast, additional loads did not produce any significant differences in the control group. Although different CoFAs in the ski boot, ankle and knee joints were used, an above-knee prosthesis with a built-in multi-axis prosthetic knee enables comparable leg kinematics in simulated alpine skiing. Key points The RoM in the ski boot on the side of the prosthetic leg was smaller than the RoM of the intact leg and the control group of healthy subjects. The RoM in the ankle joint of prosthetic leg was comparable to that of the intact leg and the control group of healthy subjects. The RoM in the prosthetic knee joint was greater than the RoM in the knee joint of the

  3. Intact immune defenses are required for mice to resist the ts-4 vaccine strain of Toxoplasma gondii.

    PubMed Central

    Sayles, P C; Johnson, L L

    1996-01-01

    The ts-4 strain of Toxoplasma gondii is a temperature-sensitive mutant that fails to grow at 40 degrees C in vitro. Unlike mildly virulent cyst-forming strains, which can cause fatal chronic infections in certain mouse strains, ts-4 has been widely used to vaccinate mice against virulent T. gondii and is a valuable tool with which to investigate mechanisms of acquired resistance to this parasite. In this report, the basis for the avirulence of ts-4 is analyzed. It is shown that ts-4 is able to persist long-term in vivo in mildly immunocompromised mice, which rules out an intrinsic growth defect as a reason for avirulence. ts-4 does not induce body temperatures in mice as high as that needed to kill it in vitro. Moreover, the mild fevers elicited in resistant B6 mice are also seen in susceptible C57BL/6 scid/scid mice. However, ts-4 elicits strong preimmune defenses, dependent on gamma interferon, which are needed by mice to survive acute infection. Furthermore, CD4+ and CD8+ T-cell-dependent acquired immunity is essential for long-term survival of ts-4-infected mice. PMID:8757838

  4. HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

    PubMed

    Migita, Keisuke; Ozaki, Taku; Shimoyama, Shuji; Yamada, Junko; Nikaido, Yoshikazu; Furukawa, Tomonori; Shiba, Yuko; Egan, Terrance M; Ueno, Shinya

    2016-08-01

    P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus. PMID:27301716

  5. 46 CFR 171.055 - Intact stability requirements for a monohull sailing vessel or a monohull auxiliary sailing vessel.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... condition of loading and operation: (1) Plot the righting arm curve on Graphs 171.055 (b), (c), and (d) or... arm curve must be truncated as shown on Graph 171.055(a). (3) Plot an assumed heeling arm curve on Graph 171.055(b) that satisfies the following conditions: (i) The assumed heeling arm curve must...

  6. 46 CFR 171.055 - Intact stability requirements for a monohull sailing vessel or a monohull auxiliary sailing vessel.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... condition of loading and operation: (1) Plot the righting arm curve on Graphs 171.055 (b), (c), and (d) or... arm curve must be truncated as shown on Graph 171.055(a). (3) Plot an assumed heeling arm curve on Graph 171.055(b) that satisfies the following conditions: (i) The assumed heeling arm curve must...

  7. 46 CFR 171.055 - Intact stability requirements for a monohull sailing vessel or a monohull auxiliary sailing vessel.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... condition of loading and operation: (1) Plot the righting arm curve on Graphs 171.055 (b), (c), and (d) or... arm curve must be truncated as shown on Graph 171.055(a). (3) Plot an assumed heeling arm curve on... corresponding to the angle at which deck edge immersion first occurs. (4) Plot an assumed heeling arm curve...

  8. 46 CFR 171.055 - Intact stability requirements for a monohull sailing vessel or a monohull auxiliary sailing vessel.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... condition of loading and operation: (1) Plot the righting arm curve on Graphs 171.055 (b), (c), and (d) or... arm curve must be truncated as shown on Graph 171.055(a). (3) Plot an assumed heeling arm curve on... corresponding to the angle at which deck edge immersion first occurs. (4) Plot an assumed heeling arm curve...

  9. 46 CFR 171.055 - Intact stability requirements for a monohull sailing vessel or a monohull auxiliary sailing vessel.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... condition of loading and operation: (1) Plot the righting arm curve on Graphs 171.055 (b), (c), and (d) or... arm curve must be truncated as shown on Graph 171.055(a). (3) Plot an assumed heeling arm curve on... corresponding to the angle at which deck edge immersion first occurs. (4) Plot an assumed heeling arm curve...

  10. Welfare of entire male pigs is improved by socialising piglets and keeping intact groups until slaughter.

    PubMed

    Rydhmer, L; Hansson, M; Lundström, K; Brunius, C; Andersson, K

    2013-09-01

    In today's production systems, pigs raised for slaughter are mixed many times, resulting in stress and fighting. The negative consequences of mixing are probably more severe with entire males than with castrates, as they fight more. In this project, we studied a system without castration where entire male pigs met unfamiliar pigs only once. Piglets from two litters were allowed to visit each other from circa 2 weeks of age through an opening between the farrowing pens. Entire males from these litters were kept in intact groups from weaning and onwards, and they were slaughtered pen-wise in intact groups. Control pigs were raised and weaned in their litters and mixed with unknown pigs when moved to the growing-finishing unit. They were slaughtered by split marketing based on individual weight. In total, 96 entire males from 24 litters were studied. Activity and social interactions of pigs were studied by direct observations on three observation occasions per pen for pigs kept in intact groups and four occasions for control pigs. All pigs were inspected for skin lesions during raising and at slaughter. Results showed that fewer pigs in intact groups were resting (17.1% v. 28.5%; P = 0.044) and they showed less aggressive behaviour (16.1 v. 27.7 number of interactions per hour; P = 0.001) than control pigs when moved to the growing-finishing unit. They also got fewer skin lesions compared with control pigs (15 v. 35; P < 0.001). Consequently, control pigs tended to grow slower during the 1st week after mixing; however, growth rate during the whole growing-finishing phase did not differ between treatments (P = 0.205). Control pigs directed more aggressive behaviour towards non-litter mates than towards litter mates during the whole growing-finishing phase, whereas pigs from the other treatment made no difference between litter mates and other familiar pigs. At 67 kg, there was more sexual behaviour (mounting) among control pigs (7.6 v. 3.4; P = 0.033), but after

  11. Evidence does not support absorption of intact solid lipid nanoparticles via oral delivery

    NASA Astrophysics Data System (ADS)

    Hu, Xiongwei; Fan, Wufa; Yu, Zhou; Lu, Yi; Qi, Jianping; Zhang, Jian; Dong, Xiaochun; Zhao, Weili; Wu, Wei

    2016-03-01

    Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and simulated mixed micelles (SMMs) to intestinal mucus, but no evidence of penetration of integral nanocarriers. Both SLNs and SMMs exhibit significant cellular uptake, but fail to penetrate cell monolayers. Confocal laser scanning microscopy reveals that nanocarriers mainly concentrate on the surface of the monolayers, and no evidence of penetration of intact vehicles can be obtained. The mucous layer acts as a barrier to the penetration of both SLNs and SMMs. Both bile salt-decoration and SMM formulation help to strengthen the interaction with biomembranes. It is concluded that evidence does not support absorption of intact SLNs via oral delivery.Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and

  12. FATE OF TN5 MUTANTS OF ROOT GROWTH-INHIBITING PSEUDOMONAS SP. IN INTACT SOIL-CORE MICROCOSMS

    EPA Science Inventory

    Transposon Tn5 mutants of a wheat root growth-inhibiting nonfluorescent Pseudomonas sp. were inoculated into intact soil-core microcosms to determine the utility of intact soil cores for evaluating the fate and transport of microorganisms in agricultural ecosystems. ransposon Tn5...

  13. Ethanol vapor and saprophytic yeast treatments reduce decay and maintain quality of intact and fresh-cut cherries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate the use of an ethanol vapor release pad and a saprophytic yeast (Cryptococcus infirmo-miniatum) to reduce decay and maintain postharvest quality of intact or fresh-cut sweet cherries (Prunus avium L.) cv. Lapins and Bing. Intact or fresh-cut fruit were pac...

  14. 40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 26 2011-07-01 2011-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet...

  15. 40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet...

  16. 40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 27 2013-07-01 2013-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet...

  17. 40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 27 2012-07-01 2012-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet...

  18. 40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 26 2014-07-01 2014-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet...

  19. Successful Repair of Hypoplastic Left Heart Syndrome With Intact Atrial Septum, Congenital Diaphragm Hernia, and Anomalous Origin of Coronary Artery: Defying the Odds.

    PubMed

    Sathanandam, Shyam; Kumar, T K Susheel; Feliz, Alexander; Knott-Craig, Christopher J

    2016-07-01

    We report a case of an infant who was postnatally diagnosed with hypoplastic left heart syndrome and an intact atrial septum who underwent emergent atrial decompression followed by the Norwood operation. She was also found to have a congenital diaphragmatic hernia on the left side and a congenital eventration of the right diaphragm, both requiring surgical repair. She was later found to have an anomalous origin of the left circumflex coronary artery from the right pulmonary artery that was ligated at the time of the bilateral bidirectional Glenn operation. She is currently thriving at home, defying all odds. PMID:27343534

  20. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.

    PubMed

    Barbosa, Joana S; Sanchez-Gonzalez, Rosario; Di Giaimo, Rossella; Baumgart, Emily Violette; Theis, Fabian J; Götz, Magdalena; Ninkovic, Jovica

    2015-05-15

    Adult neural stem cells are the source for restoring injured brain tissue. We used repetitive imaging to follow single stem cells in the intact and injured adult zebrafish telencephalon in vivo and found that neurons are generated by both direct conversions of stem cells into postmitotic neurons and via intermediate progenitors amplifying the neuronal output. We observed an imbalance of direct conversion consuming the stem cells and asymmetric and symmetric self-renewing divisions, leading to depletion of stem cells over time. After brain injury, neuronal progenitors are recruited to the injury site. These progenitors are generated by symmetric divisions that deplete the pool of stem cells, a mode of neurogenesis absent in the intact telencephalon. Our analysis revealed changes in the behavior of stem cells underlying generation of additional neurons during regeneration. PMID:25977550

  1. Occurrence of iridoid glycosides in in vitro cultures and intact plants of Scrophularia nodosa L.

    PubMed

    Sesterhenn, Katja; Distl, Melanie; Wink, Michael

    2007-03-01

    Shoot, root, and callus cultures of Scrophularia nodosa L. (Scrophulariaceae) were established and cultivated in vitro. Iridoid glycosides, such as harpagoside, aucubin, and catalpol were identified by LC-ESI-MS and their contents determined by HPLC. For comparison intact plants of S. nodosa were analysed. In shoot cultures slightly lower amounts of detectable iridoid glycosides (4.36% dry weight) were determined than in the field grown plants (4.88%). Concentration of harpagoside was highest in leaves of field plants (1.05%) and in flowers of in vitro plantlets (1.10%). For aucubin the highest amount was found in the leaves of in vitro plantlets (1.67%) whereas the levels of aucubin in the leaves of field plants were remarkably lower. Catalpol was produced as a trace compound in intact plants and shoot cultures. Callus and root cultures were apparently not able to synthesise iridoid glycosides. PMID:16972093

  2. Cutaneous mast cell maturation does not depend on an intact bone marrow microenvironment

    SciTech Connect

    Charley, M.R.; Mikhael, A.; Sontheimer, R.D.; Gilliam, J.N.; Bennett, M.

    1984-01-01

    A study was made to determine whether the maturation of murine cutaneous mast cells from stem cells depends on an intact bone marrow microenvironment. Normal bone marrow cells (+/+) were infused into 2 groups of mast cell-deficient mice: WBB6F1-W/Wv mice and /sup 89/Sr-pretreated W/Wv mice. /sup 89/Sr is a long-lived bone-seeking radioisotope which provides continuous irradiation of the marrow and thereby ablates the marrow microenvironment. Skin biopsies revealed that the /sup 89/Sr-pretreated mice and the controls had repopulated their skin with mast cells equally well. Natural killer cell function was significantly depressed in the /sup 89/Sr-treated mice, confirming that the marrow microenvironment had been functionally altered. It appears that, although the precursors for cutaneous mast cells are marrow derived, they do not need an intact marrow microenvironment for maturation.

  3. Intact and Degraded Component Criticality Calculations of N Reactors Spent Nuclear Fuel

    SciTech Connect

    L. Angers

    2001-01-31

    The objective of this calculation is to perform intact and degraded mode criticality evaluations of the Department of Energy's (DOE) N Reactor Spent Nuclear Fuel codisposed in a 2-Defense High-Level Waste (2-DHLW)/2-Multi-Canister Overpack (MCO) Waste Package (WP) and emplaced in a monitored geologic repository (MGR) (see Attachment I). The scope of this calculation is limited to the determination of the effective neutron multiplication factor (k{sub eff}) for both intact and degraded mode internal configurations of the codisposal waste package. This calculation will support the analysis that will be performed to demonstrate the technical viability for disposing of U-metal (N Reactor) spent nuclear fuel in the potential MGR.

  4. A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

    SciTech Connect

    Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang; Ito, Etsuro; Bertino, J.R. )

    1988-01-01

    A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with ({sup 14}C)chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of {sup 14}C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated {sup 14}C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.

  5. Space research with intact organisms: The role of Space Station Freedom

    NASA Technical Reports Server (NTRS)

    Phillips, Robert W.; Haddy, Francis J.

    1993-01-01

    The study of intact organisms has provided biologists with a good working knowledge of most of the common organisms that have evolved in the 1 g environment of Earth. Reasonably accurate predictions can be made about organismal responses to most stimuli on Earth. To extend this knowledge to life without gravity, we must have access to the space environment for prolonged periods. Space Station Freedom will provide a facility with which to begin this type of research. Spaceflight research to date has been limited to relatively short-term exposures that have been informative but incomplete. This paper provides a brief background of known changes that have occurred in intact organisms in the space environment and proposes the kinds of experiments that are needed to expand our knowledge of life on Earth and in space.

  6. Characterization of elemental sulfur in isolated intact spinach chloroplasts. [Spinacia oleracea L

    SciTech Connect

    Joyard, J.; Douce, R. ); Forest, E. ); Blee, E. )

    1988-12-01

    Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of {sup 35}SO{sub 4}{sup 2{minus}} resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. The authors have identified this compound as the most stable form (S{sub 8}) of elemental sulfur (S{sup 0}, valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S{sup 0}) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S{sub 8} under the experimental conditions used for analysis.

  7. Anabolic-androgenic steroid effects on the sexual behavior of intact male rats.

    PubMed

    Clark, A S; Harrold, E V; Fast, A S

    1997-02-01

    Six separate experiments were conducted which examined the effects of long-term administration of anabolic-androgenic steroid (AAS) compounds on the sexual behavior of gonadally intact male rats. The six AAS compounds analyzed in this study were 17alpha-methyltestosterone, methandrostenolone, nandrolone decanoate, stanozolol, oxymetholone, and testosterone cypionate. In each experiment, subjects received daily injections of a high, medium, or low dose of the AAS compound, or the oil vehicle, for 12 weeks. Sexual behavior was quantified weekly. Twelve weeks of administration of the high dose of three AAS compounds, 17alpha-methyltestosterone, stanozolol, and oxymetholone, eliminated male sexual behavior. These treatments also suppressed serum testosterone levels. The remaining compounds had minimal effects on sexual behavior at any dose. Thus, in intact male rats the six AAS compounds examined in these studies evoked a range of behavioral and endocrine responses that varied as a function of the specific compound and dose administered. PMID:9109597

  8. Resonant Column Apparatus Tests on Intact and Jointed Rock Specimens with Numerical Modelling Validation

    NASA Astrophysics Data System (ADS)

    Perino, A.; Barla, G.

    2015-01-01

    The determination of rock mass dynamic parameters including the influence of joints/discontinuities on wave propagation is of interest for solving problems in geophysics, rock protective engineering, rock dynamics and earthquake engineering. This topic is covered in this paper by means of laboratory tests on intact and jointed rock specimens performed with the resonant column apparatus (RCA). Attention is dedicated to the determination, with this equipment, generally used for testing soils, of the shear modulus at small strain and the damping ratio of intact and jointed rock specimens. A correction procedure based on the RCA tests performed on aluminium specimens is described. The results of the tests performed are analysed in detail. Three-dimensional distinct element method modelling is used to evaluate the applicability of the RCA and the correctness of the laboratory tests performed. A comparison with the results obtained using the scattering matrix method is also presented.

  9. Blood epididymal barrier to (/sup 3/H)-inulin in intact and vasectomized hamsters

    SciTech Connect

    Turner, T.T.; D'Addario, D.A.; Howards, S.S.

    1981-09-01

    The net transport of (/sup 3/H)-inulin into the fluids of the hamster seminiferous and caput, corpus, and cauda epididymal tubules was examined in both intact animals and those vasectomized 10 months previously. Mean isotope concentrations in reproductive tract tubule fluids did not exceeded 10 per cent of blood plasma isotope concentrations during the experiment. There were no significant differences in net transport of (/sup 3/H)-inulin into any of the tubule fluids sampled. Ten months after vasectomy, the seminiferous tubule, and all regions of the epididymal tubule retain the capacity to exclude (/sup 3/H)-insulin. Thus in the hamster 10 months after vasectomy, the blood testis and blood epididymal barriers to inulin are intact.

  10. Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits.

    PubMed

    Lerner, Talia N; Shilyansky, Carrie; Davidson, Thomas J; Evans, Kathryn E; Beier, Kevin T; Zalocusky, Kelly A; Crow, Ailey K; Malenka, Robert C; Luo, Liqun; Tomer, Raju; Deisseroth, Karl

    2015-07-30

    Recent progress in understanding the diversity of midbrain dopamine neurons has highlighted the importance--and the challenges--of defining mammalian neuronal cell types. Although neurons may be best categorized using inclusive criteria spanning biophysical properties, wiring of inputs, wiring of outputs, and activity during behavior, linking all of these measurements to cell types within the intact brains of living mammals has been difficult. Here, using an array of intact-brain circuit interrogation tools, including CLARITY, COLM, optogenetics, viral tracing, and fiber photometry, we explore the diversity of dopamine neurons within the substantia nigra pars compacta (SNc). We identify two parallel nigrostriatal dopamine neuron subpopulations differing in biophysical properties, input wiring, output wiring to dorsomedial striatum (DMS) versus dorsolateral striatum (DLS), and natural activity patterns during free behavior. Our results reveal independently operating nigrostriatal information streams, with implications for understanding the logic of dopaminergic feedback circuits and the diversity of mammalian neuronal cell types. PMID:26232229

  11. Co-existence of intact stemness and priming of neural differentiation programs in mES cells lacking Trim71

    PubMed Central

    Mitschka, Sibylle; Ulas, Thomas; Goller, Tobias; Schneider, Karin; Egert, Angela; Mertens, Jérôme; Brüstle, Oliver; Schorle, Hubert; Beyer, Marc; Klee, Kathrin; Xue, Jia; Günther, Patrick; Bassler, Kevin; Schultze, Joachim L.; Kolanus, Waldemar

    2015-01-01

    Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long been suggested to be mutually exclusive. However, with the identification of many new components of these networks ranging from epigenetic, transcriptional, and translational to even post-translational mechanisms, the cellular states of pluripotency and early differentiation might not be strictly bi-modal, but differentiating stem cells appear to go through phases of simultaneous expression of stemness and differentiation genes. Translational regulators such as RNA binding proteins (RBPs) and micro RNAs (miRNAs) might be prime candidates for guiding a cell from pluripotency to differentiation. Using Trim71, one of two members of the Tripartite motif (Trim) protein family with RNA binding activity expressed in murine ES cells, we demonstrate that Trim71 is not involved in regulatory networks of pluripotency but regulates neural differentiation. Loss of Trim71 in mES cells leaves stemness and self-maintenance of these cells intact, but many genes required for neural development are up-regulated at the same time. Concordantly, Trim71−/− mES show increased neural marker expression following treatment with retinoic acid. Our findings strongly suggest that Trim71 keeps priming steps of differentiation in check, which do not pre-require a loss of the pluripotency network in ES cells. PMID:26057209

  12. Forest Loss in Protected Areas and Intact Forest Landscapes: A Global Analysis

    PubMed Central

    Heino, Matias; Kummu, Matti; Makkonen, Marika; Mulligan, Mark; Verburg, Peter H.; Jalava, Mika; Räsänen, Timo A.

    2015-01-01

    In spite of the high importance of forests, global forest loss has remained alarmingly high during the last decades. Forest loss at a global scale has been unveiled with increasingly finer spatial resolution, but the forest extent and loss in protected areas (PAs) and in large intact forest landscapes (IFLs) have not so far been systematically assessed. Moreover, the impact of protection on preserving the IFLs is not well understood. In this study we conducted a consistent assessment of the global forest loss in PAs and IFLs over the period 2000–2012. We used recently published global remote sensing based spatial forest cover change data, being a uniform and consistent dataset over space and time, together with global datasets on PAs’ and IFLs’ locations. Our analyses revealed that on a global scale 3% of the protected forest, 2.5% of the intact forest, and 1.5% of the protected intact forest were lost during the study period. These forest loss rates are relatively high compared to global total forest loss of 5% for the same time period. The variation in forest losses and in protection effect was large among geographical regions and countries. In some regions the loss in protected forests exceeded 5% (e.g. in Australia and Oceania, and North America) and the relative forest loss was higher inside protected areas than outside those areas (e.g. in Mongolia and parts of Africa, Central Asia, and Europe). At the same time, protection was found to prevent forest loss in several countries (e.g. in South America and Southeast Asia). Globally, high area-weighted forest loss rates of protected and intact forests were associated with high gross domestic product and in the case of protected forests also with high proportions of agricultural land. Our findings reinforce the need for improved understanding of the reasons for the high forest losses in PAs and IFLs and strategies to prevent further losses. PMID:26466348

  13. Temperature dependence of 1H NMR relaxation time, T2, for intact and neoplastic plant tissues

    NASA Astrophysics Data System (ADS)

    Lewa, Czesław J.; Lewa, Maria

    Temperature dependences of the spin-spin proton relaxation time, T2, have been shown for normal and tumorous tissues collected from kalus culture Nicotiana tabacum and from the plant Kalanchoe daigremontiana. For neoplastic plant tissues, time T2 was increased compared to that for intact plants, a finding similar to that for animal and human tissues. The temperature dependences obtained were compared to analogous relations observed with animal tissues.

  14. Fourier transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium Azospirillum brasilense

    NASA Astrophysics Data System (ADS)

    Kamnev, A. A.; Ristić, M.; Antonyuk, L. P.; Chernyshev, A. V.; Ignatov, V. V.

    1997-06-01

    The data of Fourier transform infrared (FTIR) spectroscopic measurements performed on intact cells of the soil nitrogen-fixing bacterium Azospirillum brasilense grown in a standard medium and under the conditions of an increased metal uptake are compared and discussed. The structural FTIR information obtained is considered together with atomic absorption spectrometry (AAS) data on the content of metal cations in the bacterial cells. Some methodological aspects concerning preparation of bacterial cell samples for FTIR measurements are also discussed.

  15. Analysis of Aerobic Respiration in Intact Skeletal Muscle Tissue by Microplate-Based Respirometry.

    PubMed

    Shintaku, Jonathan; Guttridge, Denis C

    2016-01-01

    Mitochondrial function is a key component of skeletal muscle health, and its dysfunction has been associated with a wide variety of diseases. Microplate-based respirometry measures aerobic respiration of live cells through extracellular changes in oxygen concentration. Here, we describe a methodology to measure aerobic respiration of intact murine skeletal muscle tissue. The tissues are not cultured, permeabilized, or enzymatically dissociated to single fibers, so there is minimal experimental manipulation affecting the samples prior to acquiring measurements. PMID:27492183

  16. Electrophysiological model of intact and processed plant tissues: cell disintegration criteria.

    PubMed

    Angersbach, A; Heinz, V; Knorr, D

    1999-01-01

    Frequency versus conductivity relationships of food cell system, based on impedance measurements as characterized by polarization effects of the Maxwell-Wagner type at intact membrane interfaces, are presented. The electrical properties of a biological membrane (represented as a resistor and capacitor) are responsible for the dependence of the total conductivity of the cell system on the alternating current frequency. Based on an equivalent circuit model of a single plant cell, the electrical conductivity spectrum of the cell system in intact plant tissue (potato, carrot, banana, and apple) was determined in a frequency range between 3 kHz and 50 MHz. The electrical properties of a cell system with different ratios of intact/ruptured cells could also be predicted on the basis of a description of a cell system consisting of elementary layers with regularly distributed intact and ruptured cells as well as of extracellular compartments. This simple determination of the degree of cell permeabilization (cell disintegration index, p(o)) is based upon electric conductivity changes in the cell sample. For accurate calculations of p(o), the sample conductivities before and after treatment, obtained at low- (f(l)) and high-frequency (f(h)) ranges of the so-called beta-dispersion, were used. In this study with plant cell systems, characteristic conductivities used were measured at frequencies f(l) = 3 kHz and f(h) = 12.5 MHz. The disintegration index was used to analyze the degree of cell disruption after different treatments (such as mechanical disruption, heating, freeze-thaw cycles, application of electric field pulses, and enzymatic treatment) of the plant tissues. PMID:10441367

  17. Nitrovasodilator-induced relaxation and tolerance development in porcine vena cordis magna: dependence on intact endothelium.

    PubMed Central

    Kojda, G.; Beck, J. K.; Meyer, W.; Noack, E.

    1994-01-01

    1. Isolated segments of porcine vena cordis magna exhibited a reproducible contractile activity upon application of prostaglandin F2 alpha (PGF2 alpha) or KCl, that was independent of the presence of intact endothelium. Substance P (3 nM) elicited strictly endothelium-dependent relaxations amounting to 46.1 +/- 1.4% (n = 206) of contractions induced by 10 microM PGF2 alpha. 2. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a compound that spontaneously liberates nitric oxide, concentration-dependently relaxed PGF2 alpha-precontracted (50 microM) venous segments. Tolerance induction (incubation with 100 microM SNAP for 30 min) within the same segments resulted in a 3 fold attenuation of this effect, which was not further reduced after additional preincubation with glyceryl trinitrate (GTN). Removal of endothelium or the presence of N omega-nitro-L-arginine methylester (L-NAME) significantly improved the potency of SNAP before and after tolerance induction. 3. Concentration-dependent relaxations induced by GTN in non-tolerant veins were similar in the presence and absence of endothelium but much more reduced in tolerant endothelium-denuded (75 fold) compared to intact (20 fold) segments. In contrast, the presence of L-NAME significantly improved GTN-activity solely in non-tolerant veins, which, therefore, also resulted in a more pronounced attenuation of activity due to tolerance induction (100 fold). Preincubation of intact veins with SNAP also reduced GTN-activity but to a lesser extent (10 fold). 4. The more delayed but much longer, and compared to GTN somewhat weaker, acting new nitrovasodilator N-(3-nitrato-pivaloyl)-1-cysteineethylester (SPM 3672) was more potent in denuded than intact non-tolerant venous segments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7521258

  18. Musculoskeletal Response to Whole-Body Vibration During Fracture Healing in Intact and Ovariectomized Rats

    PubMed Central

    Stuermer, Ewa K.; Werner, Carsten; Wicke, Michael; Kolios, Leila; Sehmisch, Stephan; Tezval, Mohammad; Utesch, Clara; Mangal, Orzala; Zimmer, Sebastian; Dullin, Christian; Stuermer, Klaus M.

    2010-01-01

    This study investigated the effect of vibration on bone healing and muscle in intact and ovariectomized rats. Thirty ovariectomized (at 3 months of age) and 30 intact 5-month old female Sprague-Dawley rats underwent bilateral metaphyseal osteotomy of tibia. Five days later, half of the ovariectomized and of the intact rats were exposed to whole-body vertical vibration (90 Hz, 0.5 mm, 4 × g acceleration) for 15 min twice a day during 30 days. The other animals did not undergo vibration. After decapitation of rats, one tibia was used for computed tomographic, biomechanical, and histological analyses; the other was used for gene expression analyses of alkaline phosphatase (Alp), osteocalcin (Oc), tartrate-resistant acid phosphatase 1, and insulinlike growth factor 1. Serum Alp and Oc were measured. Mitochondrial activity, fiber area and distribution, and capillary densities were analyzed in M. gastrocnemius and M. longissimus. We found that vibration had no effect on body weight and food intake, but it improved cortical and callus densities (97 vs. 99%, 72 vs. 81%), trabecular structure (9 vs. 14 trabecular nodes), blood supply (1.7 vs. 2.1 capillaries/fiber), and oxidative metabolism (17 vs. 23 pmol O2/s/mg) in ovariectomized rats. Vibration generally increased muscle fiber size. Tibia biomechanical properties were diminished after vibration. Oc gene expression was higher in vibrated rats. Serum Alp was increased in ovariectomized rats. In ovariectomized rats, vibration resulted in an earlier bridging; in intact rats, callus bridging occurred later after vibration. The chosen vibration regimen (90 Hz, 0.5 mm, 4 × g acceleration, 15 min twice a day) was effective in improving musculoskeletal tissues in ovariectomized rats but was not optimal for fracture healing. PMID:20532877

  19. In vitro bioactive properties of intact and enzymatically hydrolysed whey protein: targeting the enteroinsular axis.

    PubMed

    Power-Grant, O; Bruen, C; Brennan, L; Giblin, L; Jakeman, P; FitzGerald, R J

    2015-03-01

    Enzymatically hydrolysed milk proteins have a variety of biofunctional effects some of which may be beneficial in the management of type 2 diabetes mellitus. The purpose of this study was to evaluate the effect of commercially available intact and hydrolysed whey protein ingredients (DH 32, DH 45) on markers of the enteroinsular axis (glucagon like peptide-1 secretion, dipeptidyl peptidase IV inhibition, insulin secretion and antioxidant activity) before and after simulated gastrointestinal digestion (SGID). A whey protein hydrolysate, DH32, significantly enhanced (P < 0.05) insulin secretion from BRIN BD11 β-cells compared to the positive control (16.7 mM glucose and 10 mM Ala). The whey protein hydrolysates inhibited dipeptidyl peptidase IV activity, yielding half maximal inhibitory concentration values (IC50) of 1.5 ± 0.1 and 1.1 ± 0.1 mg mL(-1) for the DH 32 and DH 45, samples respectively, and were significantly more potent than the intact whey (P < 0.05). Enzymatic hydrolysis of whey protein significantly enhanced (P < 0.05) its antioxidant activity compared to intact whey, as measured by the oxygen radical absorbance capacity assay (ORAC). This antioxidant activity was maintained (DH 32, P > 0.05) or enhanced (DH 45, P < 0.05) following SGID. Intact whey stimulated GLP-1 secretion from enteroendocrine cells compared to vehicle control (P < 0.05). This data confirm that whey proteins and peptides can act through multiple targets within the enteroinsular axis and as such may have glucoregulatory potential. PMID:25666373

  20. Ingested soluble CD14 from milk is transferred intact into the blood of newborn rats

    PubMed Central

    Ward, Tonya L.; Spencer, William J.; Davis, Laura D. R.; Harrold, JoAnn; Mack, David R.; Altosaar, Illimar

    2016-01-01

    Background Milk contains immunological constituents that comprise an edible immune system conveyed from mother to newborn. Soluble Cluster of Differentiation 14 (sCD14) is a protein found in significant quantities in human milk (~8–29 μg/ml). At a tenfold lower concentration in the blood (~3 μg/ml), the most notable role of sCD14 is to sequester lipopolysaccharide of Gram-negative bacteria from immune cells. Methods To explore the pharmacodynamics of this milk protein and its biological fate, the biodistribution of radiolabeled sCD14 (14C, 125I) was monitored in 10 d old rat pups. Results Up to 3.4 ± 2.2% of the radiolabeled-sCD14 administered was observed, intact, in the pup blood for up to 8 h post-ingestion. Additionally, 30.3 ± 13.0% of the radiolabeled-sCD14 administered was observed degraded in the stomach at 8 h post-ingestion. A reservoir of intact, administered sCD14 (3.2 ± 0.3%), however, remained in the stomach at 8 h post-ingestion. Intact sCD14 was observed in the small intestine at 5.5 ± 1.6% of the dose fed at 8h post-ingestion. Conclusions The presence of intact sCD14 in the blood and gastrointestinal tract of newborns post-ingestion has implications in the development of allergies, obesity and other inflammation-related pathogeneses later in life. PMID:24232637

  1. Accumulation and diffusion of crystallin inside single fiber cells in intact chicken embryo lenses.

    PubMed Central

    Peetermans, J A; Foy, B D; Tanaka, T

    1987-01-01

    The use of microscope laser light-scattering spectroscopy allows for the measurement of dynamic properties of intracellular particles inside single fiber cells at different locations in the intact chicken embryo lens. Profiles of the diffusive properties of the delta-crystallin proteins across the lens are reported for developing chickens from day 5 to day 37. A clear decrease of the diffusion is observed in the lens nucleus relative to the cortex beginning with day 10. Images PMID:3470754

  2. Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer.

    PubMed

    Wang, Evelyn H; Combe, Peter C; Schug, Kevin A

    2016-05-01

    Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.Graphical Abstract. PMID:26956437

  3. Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

    2016-03-01

    Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

  4. Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

    2016-05-01

    Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

  5. The Structure Of Intact Side Tissue Loss Based On FTIR Spectroscopic Measurements

    NASA Astrophysics Data System (ADS)

    Hussain, N.; Al-Hadithi, K. O.; Jaafar, M. S.

    2009-09-01

    Laser applications in dentistry were strongly evolved during the last three decades. Among those applications are laser ablation of dental hard tissue, caries inhibition treatments by localized surface heating, and surface conditioning for bonding. In addition, infra-red lasers are ideally suited for the selective and precise removal of carious dental hard tissue while minimizing the healthy tissue loss. In the present study we applied laser spectroscopy technique FTIR for the study of the structure of intact side tissue of teeth. The aim of the recent work is to study the effect of race and sex (genealogy) on the structure of intact side tissue loss. Our sample consists of twenty Malay females' teeth where the FTIR has been applied. The data show a decrease in the amounts of main substances (like Hydroxyapatite crystals ([Ca5(PO4)3(OH)4], CaF2) than those in healthy teeth. The measured spectra represent the enamel with the characteristic peaks due to the phosphate group in carbonated, hydroxyapatite at 1000 cm-1 and two small peaks near 1500 cm-1 due to the carbonate group. The data explains the effect of the several factors on the intact side tissue loss.

  6. Evidence does not support absorption of intact solid lipid nanoparticles via oral delivery.

    PubMed

    Hu, Xiongwei; Fan, Wufa; Yu, Zhou; Lu, Yi; Qi, Jianping; Zhang, Jian; Dong, Xiaochun; Zhao, Weili; Wu, Wei

    2016-04-01

    Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and simulated mixed micelles (SMMs) to intestinal mucus, but no evidence of penetration of integral nanocarriers. Both SLNs and SMMs exhibit significant cellular uptake, but fail to penetrate cell monolayers. Confocal laser scanning microscopy reveals that nanocarriers mainly concentrate on the surface of the monolayers, and no evidence of penetration of intact vehicles can be obtained. The mucous layer acts as a barrier to the penetration of both SLNs and SMMs. Both bile salt-decoration and SMM formulation help to strengthen the interaction with biomembranes. It is concluded that evidence does not support absorption of intact SLNs via oral delivery. PMID:26725649

  7. Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

    PubMed

    Reinhold, A K; Batti, L; Bilbao, D; Buness, A; Rittner, H L; Heppenstall, P A

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. PMID:25880204

  8. Ribosomal ribonucleic acid isolated from Salmonella typhimurium: absence of the intact 23S species.

    PubMed Central

    Winkler, M E

    1979-01-01

    Ribonucleic acid (RNA) isolated by four distinct methods and from a variety of Salmonella typhimurium strains lacked intact 23S ribosomal RNA (rRNA). On sucrose gradients which minimize aggregation, the vast majority of S. typhimurium rRNA sedimented as a 16S peak with a 14S shoulder. RNA from this region of the gradient was resolved into three discrete bands by electrophoresis in formamide. Two very minor S. typhimurium RNA peaks were resolved at 21S and 10S on sucrose gradients, and each peak formed discrete bands in electrophoresis. It is concluded that if S. typhimurium does possess an intact 23S rRNA species, this species is extremely "labile." The absence of isolatable S. typhimurium 23S rRNA possibly reflected in vivo processing of the rRNA before isolation. Under certain conditions, S. typhimurium rRNA formed discrete aggregates which sedimented similarly to intact Escherichia coli 23S rRNA. Images PMID:383696

  9. Prolactin is associated with the development of photorefractoriness in intact, castrated, and testosterone-implanted starlings

    SciTech Connect

    Goldsmith, A.R.; Nicholls, T.J.

    1984-05-01

    Using radioimmunoassays, prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured in the plasma of intact, castrated, and testosterone-implanted male starlings. Two groups of birds (intact and castrated males) were transferred when photosensitive from short (8-hr) to long (16-hr) day lengths; in both cases plasma prolactin levels increased steadily reaching a peak after 5 weeks of photostimulation, at the time of the onset of photorefractoriness. Three groups (intacts, castrates, and castrates implanted with Silastic capsules containing testosterone) were exposed to day lengths increasing by 30 min per week from 8 to 16 hr. Again, prolactin levels increased in a similar fashion in all three groups at the time of refractoriness, which occurred when the day length reached 15-16 hr. Thus the timing of photorefractoriness and the associated rise in prolactin secretion which occur in response to photostimulation do not depend upon the presence of the gonads and are not affected when testosterone is maintained at a constant high level. Nor is the increase in prolactin altered when the cycle of gonadotrophin secretion which normally precedes it is completely suppressed by the implantation of a testosterone capsule. It would seem that prolactin secretion in the starling is stimulated by transfer from short to long day lengths, but not as a consequence of high gonadotrophin or androgen secretion rates.

  10. Reorganization of the Intact Somatosensory Cortex Immediately after Spinal Cord Injury

    PubMed Central

    Humanes-Valera, Desire; Aguilar, Juan; Foffani, Guglielmo

    2013-01-01

    Sensory deafferentation produces extensive reorganization of the corresponding deafferented cortex. Little is known, however, about the role of the adjacent intact cortex in this reorganization. Here we show that a complete thoracic transection of the spinal cord immediately increases the responses of the intact forepaw cortex to forepaw stimuli (above the level of the lesion) in anesthetized rats. These increased forepaw responses were independent of the global changes in cortical state induced by the spinal cord transection described in our previous work (Aguilar et al., J Neurosci 2010), as the responses increased both when the cortex was in a silent state (down-state) or in an active state (up-state). The increased responses in the intact forepaw cortex correlated with increased responses in the deafferented hindpaw cortex, suggesting that they could represent different points of view of the same immediate state-independent functional reorganization of the primary somatosensory cortex after spinal cord injury. Collectively, the results of the present study and of our previous study suggest that both state-dependent and state-independent mechanisms can jointly contribute to cortical reorganization immediately after spinal cord injury. PMID:23922771

  11. Visualization of the intact interface between neural tissue and implanted microelectrode arrays.

    PubMed

    Holecko, Matthew M; Williams, Justin C; Massia, Stephen P

    2005-12-01

    This research presents immunohistochemical strategies for assessing the interactions at the immediate interface between micro-scale implanted devices and the surrounding brain tissue during inflammatory astrogliotic reactions. This includes preparation, microscopy and analysis techniques for obtaining images of the intimate contact between neural cells and the surface of implantable micro-electromechanical systems (MEMS) devices. The ability to visualize the intact interface between an implant and the surrounding tissue allows researchers to examine tissue that is unchanged from its native implanted state. Conversely, current popular techniques involve removing the implant. This tends to cause damage to the tissue immediately surrounding the implant and can hinder one's ability to differentiate inflammatory responses to the implant versus physical damage occurring from removal of the implant from the tissue. Due to advances in microscopy and staining techniques, it is now possible to visualize the intact tissue-implant interface. This paper presents the development of imaging techniques for visualizing the intact interface between neural tissue and implanted devices. This is particularly important for understanding both the acute and chronic neuroinflammatory responses to devices intended for long-term use in a prosthetic system. Non-functional, unbonded devices were imaged in vitro and in vivo at different times post-implantation via a range of techniques. Using these techniques, detailed interactions could be seen between delicate cellular processes and the electrode surface, which would have been destroyed using conventional histology processes. PMID:16317233

  12. Antecedents of Intact Cognition and Dementia at Age 90: A Prospective Study

    PubMed Central

    Vaillant, George E.; Okereke, Olivia I; Mukamal, Kenneth; Waldinger, Robert J.

    2014-01-01

    Objectives To examine the possible antecedents of both dementia and sustained intact cognition at age 90 among men who underwent a prospective, multidisciplinary assessment from age 19 to 90, with little attrition. Methods A prospective 20-year reassessment of the 196 (out of 268) former Harvard College sophomores who survived until age 70. Begun in 1939 the Study gathered measurements of childhood environment, dominant personality traits, and objective mental and physical health over time, smoking in pack years, alcohol abuse and depression. Questionnaires were obtained every two years and physical exams every five years. Cognitive status was assessed at ages 80, 85 and 90. Results Despite addressing a wide variety health, behavioral and social factors over the lifespan, we observed few predictors with strong association with either intact cognition at age 90 (n = 40) or dementia (n = 44). Univariate analysis revealed seven suggestive predictors of intact cognition at age 90 or of dementia: warm childhood relationship with mother, exercise at age 60, high maternal education, young age of mother at subject’s birth, low BMI, good physical health at 60, and late retirement. Only the first 3 variables: warm childhood relationship with mother, exercise at age 60, and high maternal education remained significant with logistic regression. Conclusions In this prospective study of long-lived, highly educated men several well-known putative predictors of AD did not distinguish those who over the next 20 years developed dementia from those with unimpaired cognition until age 90. PMID:24733646

  13. Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction.

    PubMed Central

    Neubauer, S; Horn, M; Naumann, A; Tian, R; Hu, K; Laser, M; Friedrich, J; Gaudron, P; Schnackerz, K; Ingwall, J S

    1995-01-01

    The purpose of this study was to test the hypothesis that energy metabolism is impaired in residual intact myocardium of chronically infarcted rat heart, contributing to contractile dysfunction. Myocardial infarction (MI) was induced in rats by coronary artery ligation. Hearts were isolated 8 wk later and buffer-perfused isovolumically. MI hearts showed reduced left ventricular developed pressure, but oxygen consumption was unchanged. High-energy phosphate contents were measured chemically and by 31P-NMR spectroscopy. In residual intact left ventricular tissue, ATP was unchanged after MI, while creatine phosphate was reduced by 31%. Total creatine kinase (CK) activity was reduced by 17%, the fetal CK isoenzymes BB and MB increased, while the "adult" mitochondrial CK isoenzyme activity decreased by 44%. Total creatine content decreased by 35%. Phosphoryl exchange between ATP and creatine phosphate, measured by 31P-NMR magnetization transfer, fell by 50% in MI hearts. Thus, energy reserve is substantially impaired in residual intact myocardium of chronically infarcted rats. Because phosphoryl exchange was still five times higher than ATP synthesis rates calculated from oxygen consumption, phosphoryl transfer via CK may not limit baseline contractile performance 2 mo after MI. In contrast, when MI hearts were subjected to acute stress (hypoxia), mechanical recovery during reoxygenation was impaired, suggesting that reduced energy reserve contributes to increased susceptibility of MI hearts to acute metabolic stress. PMID:7883957

  14. Differential Transcriptional Profiling of Damaged and Intact Adjacent Dorsal Root Ganglia Neurons in Neuropathic Pain

    PubMed Central

    Reinhold, A. K.; Batti, L.; Bilbao, D.; Buness, A.; Rittner, H. L.; Heppenstall, P. A.

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and “bystanders,” thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. PMID:25880204

  15. The Effect of Self-Transcendence on Depression in Cognitively Intact Nursing Home Patients

    PubMed Central

    Haugan, Gørill; Innstrand, Siw Tone

    2012-01-01

    Aims. This study's aim was to test the effects of self-transcendence on depression among cognitively intact nursing home patients. Background. Depression is considered the most frequent mental disorder among the elderly population. Specifically, the depression rate among nursing home patients is three to four times higher than that among community-dwelling elderly. Therefore, finding new and alternative ways to prevent and decrease depression is of great importance for nursing home patients' well-being. Self-transcendence is related to spiritual as well as nonspiritual factors, and it is described as a correlate and resource for well-being among vulnerable populations and at the end of life. Methods. A two-factor construct of the self-transcendence scale (interpersonal and intrapersonal) and the hospital anxiety and depression scale (HADS) was applied. A sample of 202 cognitively intact nursing home patients in central Norway was selected to respond to the questionnaires in 2008/2009. Results. A hypothesized SEM model demonstrated significant direct relationships and total effects of self-transcendence on depression. Conclusion and Implication for Practice. Facilitating patients' self-transcendence, both interpersonally and intrapersonally, might decrease depression among cognitively intact nursing home patients. PMID:23738199

  16. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation: a randomised, controlled, cross-over study.

    PubMed

    Bendtsen, Line Q; Lorenzen, Janne K; Gomes, Sisse; Liaset, Bjørn; Holst, Jens J; Ritz, Christian; Reitelseder, Søren; Sjödin, Anders; Astrup, Arne

    2014-10-28

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic dietary treatments that varied in protein source. The study was conducted in a respiration chamber, where EE, substrate oxidation and subjective appetite were measured over 24 h at three independent visits. Moreover, blood and urine samples were collected from the participants. The results showed no differences in 24 h and postprandial EE or appetite regulation. However, lipid oxidation, estimated from the respiratory quotient (RQ), was found to be higher after consumption of IW than after consumption of HC during daytime (P= 0·014) as well as during the time after the breakfast meal (P= 0·008) when the food was provided. Likewise, NEFA concentrations were found to be higher after consumption of IW than after consumption of HC and IC (P< 0·01). However, there was no overall difference in the concentration of insulin or glucagon-like peptide 1. In conclusion, dietary treatments when served as high-protein mixed meals induced similar effects on EE and appetite regulation, except for lipid oxidation, where RQ values suggest that it is higher after consumption of IW than after consumption of HC. PMID:25191896

  17. Airway compliance and dynamics explain the apparent discrepancy in length adaptation between intact airways and smooth muscle strips.

    PubMed

    Dowie, Jackson; Ansell, Thomas K; Noble, Peter B; Donovan, Graham M

    2016-01-01

    Length adaptation is a phenomenon observed in airway smooth muscle (ASM) wherein over time there is a shift in the length-tension curve. There is potential for length adaptation to play an important role in airway constriction and airway hyper-responsiveness in asthma. Recent results by Ansell et al., 2015 (JAP 2014 10.1152/japplphysiol.00724.2014) have cast doubt on this role by testing for length adaptation using an intact airway preparation, rather than strips of ASM. Using this technique they found no evidence for length adaptation in intact airways. Here we attempt to resolve this apparent discrepancy by constructing a minimal mathematical model of the intact airway, including ASM which follows the classic length-tension curve and undergoes length adaptation. This allows us to show that (1) no evidence of length adaptation should be expected in large, cartilaginous, intact airways; (2) even in highly compliant peripheral airways, or at more compliant regions of the pressure-volume curve of large airways, the effect of length adaptation would be modest and at best marginally detectable in intact airways; (3) the key parameters which control the appearance of length adaptation in intact airways are airway compliance and the relaxation timescale. The results of this mathematical simulation suggest that length adaptation observed at the level of the isolated ASM may not clearly manifest in the normal intact airway. PMID:26376002

  18. New genes that extend Caenorhabditis elegans' lifespan in response to reproductive signals.

    PubMed

    McCormick, Mark; Chen, Kan; Ramaswamy, Priya; Kenyon, Cynthia

    2012-04-01

    In Caenorhabditis elegans and Drosophila, removing germline stem cells increases lifespan. In C. elegans, this lifespan extension requires DAF-16, a FOXO transcription factor, and DAF-12, a nuclear hormone receptor. To better understand the regulatory relationships between DAF-16 and DAF-12, we used microarray analysis to identify downstream genes. We found that these two transcription factors influence the expression of distinct but overlapping sets of genes in response to loss of the germline. In addition, we identified several new genes that are required for loss of the germline to increase lifespan. One, phi-62, encodes a conserved, predicted RNA-binding protein. PHI-62 influences DAF-16-dependent transcription, possibly by collaborating with TCER-1, a putative transcription elongation factor, and FTT-2, a 14-3-3 protein known to bind DAF-16. Three other genes encode proteins involved in lipid metabolism; one is a triacylglycerol lipase, and another is an acyl-CoA reductase. These genes do not noticeably affect bulk fat storage levels; therefore, we propose a model in which they may influence production of a lifespan-extending signal or metabolite. PMID:22081913

  19. Photothermal Multispectral Image Cytometry for Quantitative Histology of Nanoparticles and Micrometastasis in Intact, Stained and Laser Burned Tissues

    PubMed Central

    Nedosekin, Dmitry A.; Shashkov, Evgeny V.; Galanzha, Ekaterina I.; Hennings, Leah; Zharov, Vladimir P.

    2012-01-01

    There is a rapidly growing interest in the advanced analysis of histological data and the development of appropriate detection technologies, including mapping of nanoparticle distributions in tissue in nanomedicine applications. We evaluated photothermal (PT) scanning cytometry for color-coded imaging, spectral identification, and quantitative detection of individual nanoparticles and abnormal cells in histological samples with and without staining. Using this tool, individual carbon nanotubes, gold nanorods, and melanoma cells with intrinsic melanin markers were identified in unstained (e.g. sentinel lymph nodes) and conventionally-stained tissues. In addition, we introduced a spectral burning technique for histology through selective laser bleaching areas with nondesired absorption background and nanobubble-based PT signal amplification. The obtained data demonstrated the promise of PT cytometry in the analysis of low-absorption samples and mapping of various individual nanoparticles' distribution that would be impossible with existing assays. Comparison of PT cytometry and photoacoustic (PA) cytometry previously, developed by us, revealed that these methods supplement each other with a sensitivity advantage (up to 10-fold) of contactless PT technique in assessment of thin (≤100 μm) histological samples, while PA imaging provides characterization of thicker samples which, however, requires an acoustic contact with transducers. A potential of high-speed integrated PT–PA cytometry for rapid examination of both intact and stained heterogeneous tissues with high sensitivity at the zepromolar concentration level is further highlighted. PMID:20949577

  20. Parameter-based assessment of disturbed and intact components of visual attention in children with developmental dyslexia.

    PubMed

    Bogon, Johanna; Finke, Kathrin; Schulte-Körne, Gerd; Müller, Hermann J; Schneider, Werner X; Stenneken, Prisca

    2014-09-01

    People with developmental dyslexia (DD) have been shown to be impaired in tasks that require the processing of multiple visual elements in parallel. It has been suggested that this deficit originates from disturbed visual attentional functions. The parameter-based assessment of visual attention based on Bundesen's () theory of visual attention allows one to identify and quantify the underlying deficits. The present study provides the first groupwise comparison between children with DD (n = 12; mean age 9.84 years) and typically developing children (n = 12; mean age 9.87 years) with regard to intact and disturbed components of visual attention. From the performance on whole- and partial-report tasks, we derived individual parameter estimates of four different parameters: visual processing speed, storage capacity of visual short-term memory (VSTM), laterality of attentional weighting and efficiency of top-down control. Groupwise comparisons revealed that general attentional resources, processing speed and VSTM storage capacity, are impaired in children with DD compared to typically developing children. In contrast, selectivity parameters, laterality of attentional selection and attentional top-down control did not differ between these groups. Relating the current findings to previous results, obtained in highly comparable methodological settings, from single cases of children with DD, and from a group of adults with DD, we conclude that slowed perceptual processing speed is a primary visual attentional deficit in DD. Furthermore, reduced VSTM storage capacity seems to modulate the difficulties in written language processing imposed by the disorder. PMID:24576161

  1. [Anesthesia Management of a Patient with Pulmonary Atresia, Intact Ventricular Septum, Major Aortopulmonary Collateral Artery and Tetralogy of Fallot].

    PubMed

    Hitosugi, Takashi; Tsukamoto, Masanori; Ishii, Kentaro; Kadowaki, Masanori; Fujiwara, Shigeki; Yokoyama, Takeshi

    2016-03-01

    The patient was a 6-year-old girl with pulmonary atresia, intact ventricular septum and major aortopulmonary collateral artery with tetralogy of Fallot Her Sp(O2) was around 60% under room air, and she could not walk long. She underwent dental treatment under general anesthesia. Invasive monitoring using pulmonary artery catheter should have been avoided, since the risk of monitoring greatly exceeds that of the treatment. The patient entered the operating room with her mother, and anesthesia was induced with intravenous midazolam, propofol and vecuronium. She was intubated orally first and impedance cardiography monitoring was started. FI(O2) was maintained at 0.5-1.0. Increases in airway pressure and Pa(CO2) were appropriately avoided. Dental treatment is important for infants with cardiac disease not only to reduce their pain, but also to reduce the risk of infection. It often requires general anesthesia. We have to conduct it with less invasiveness and less stress. PMID:27097511

  2. Trends in the Quality of Treatment for Patients With Intact Cervical Cancer in the United States, 1999 Through 2011

    SciTech Connect

    Smith, Grace L.; Jiang, Jing; Giordano, Sharon H.; Meyer, Larissa A.; Eifel, Patricia J.

    2015-06-01

    Purpose: High-quality treatment for intact cervical cancer requires external radiation therapy, brachytherapy, and chemotherapy, carefully sequenced and completed without delays. We sought to determine how frequently current treatment meets quality benchmarks and whether new technologies have influenced patterns of care. Methods and Materials: By searching diagnosis and procedure claims in MarketScan, an employment-based health care claims database, we identified 1508 patients with nonmetastatic, intact cervical cancer treated from 1999 to 2011, who were <65 years of age and received >10 fractions of radiation. Treatments received were identified using procedure codes and compared with 3 quality benchmarks: receipt of brachytherapy, receipt of chemotherapy, and radiation treatment duration not exceeding 63 days. The Cochran-Armitage test was used to evaluate temporal trends. Results: Seventy-eight percent of patients (n=1182) received brachytherapy, with brachytherapy receipt stable over time (Cochran-Armitage P{sub trend}=.15). Among patients who received brachytherapy, 66% had high–dose rate and 34% had low–dose rate treatment, although use of high–dose rate brachytherapy steadily increased to 75% by 2011 (P{sub trend}<.001). Eighteen percent of patients (n=278) received intensity modulated radiation therapy (IMRT), and IMRT receipt increased to 37% by 2011 (P{sub trend}<.001). Only 2.5% of patients (n=38) received IMRT in the setting of brachytherapy omission. Overall, 79% of patients (n=1185) received chemotherapy, and chemotherapy receipt increased to 84% by 2011 (P{sub trend}<.001). Median radiation treatment duration was 56 days (interquartile range, 47-65 days); however, duration exceeded 63 days in 36% of patients (n=543). Although 98% of patients received at least 1 benchmark treatment, only 44% received treatment that met all 3 benchmarks. With more stringent indicators (brachytherapy, ≥4 chemotherapy cycles, and duration not exceeding 56

  3. Genetic risk for Alzheimer's disease alters the five-year trajectory of semantic memory activation in cognitively intact elders.

    PubMed

    Rao, Stephen M; Bonner-Jackson, Aaron; Nielson, Kristy A; Seidenberg, Michael; Smith, J Carson; Woodard, John L; Durgerian, Sally

    2015-05-01

    Healthy aging is associated with cognitive declines typically accompanied by increased task-related brain activity in comparison to younger counterparts. The Scaffolding Theory of Aging and Cognition (STAC) (Park and Reuter-Lorenz, 2009; Reuter-Lorenz and Park, 2014) posits that compensatory brain processes are responsible for maintaining normal cognitive performance in older adults, despite accumulation of aging-related neural damage. Cross-sectional studies indicate that cognitively intact elders at genetic risk for Alzheimer's disease (AD) demonstrate patterns of increased brain activity compared to low risk elders, suggesting that compensation represents an early response to AD-associated pathology. Whether this compensatory response persists or declines with the onset of cognitive impairment can only be addressed using a longitudinal design. The current prospective, 5-year longitudinal study examined brain activation in APOE ε4 carriers (N=24) and non-carriers (N=21). All participants, ages 65-85 and cognitively intact at study entry, underwent task-activated fMRI, structural MRI, and neuropsychological assessments at baseline, 18, and 57 months. fMRI activation was measured in response to a semantic memory task requiring participants to discriminate famous from non-famous names. Results indicated that the trajectory of change in brain activation while performing this semantic memory task differed between APOE ε4 carriers and non-carriers. The APOE ε4 group exhibited greater activation than the Low Risk group at baseline, but they subsequently showed a progressive decline in activation during the follow-up periods with corresponding emergence of episodic memory loss and hippocampal atrophy. In contrast, the non-carriers demonstrated a gradual increase in activation over the 5-year period. Our results are consistent with the STAC model by demonstrating that compensation varies with the severity of underlying neural damage and can be exhausted with the onset

  4. Human skin penetration of gold nanoparticles through intact and damaged skin.

    PubMed

    Filon, Francesca Larese; Crosera, Matteo; Adami, Gianpiero; Bovenzi, Massimo; Rossi, Federica; Maina, Giovanni

    2011-12-01

    Gold nanoparticles (AuNPs) are produced for many applications but there is a lack of available data on their skin absorption. Experiments were performed using the Franz diffusion cell method with intact and damaged human skin. A physiological solution was used as receiving phase and 0.5 mL (1st exp) and 1.5 mL (2nd exp) of a solution containing 100 mg L⁻¹ of AuNPs (15 and 45 μg cm⁻², respectively) was applied as donor phase to the outer surface of the skin for 24 h. Skin absorption was dose dependent. Mean gold content of 214.0 ± 43.7 ng cm⁻² and 187.7 ± 50.2 ng cm⁻² were found in the receiving solutions of cells where the AuNPs solution was applied in higher concentration on intact skin (8 Franz cells) and on damaged skin (8 Franz cells), respectively. Twenty-four hours gold flux permeation was 7.8 ± 2.0 ng cm⁻² h⁻¹ and 7.1 ± 2.5 ng cm⁻² h⁻¹ in intact and damaged skin, respectively, with a lag time less than 1 hour. Transmission Electron Microscope analysis on skin samples and chemical analysis using Inductively Coupled Plasma-Mass Spectrometry demonstrated the presence of AuNPs into epidermis and dermis. This study showed that AuNPs are able to penetrate the human skin in an in vitro diffusion cell system. PMID:21319954

  5. Spatially rearranged object parts can facilitate perception of intact whole objects

    PubMed Central

    Cacciamani, Laura; Ayars, Alisabeth A.; Peterson, Mary A.

    2014-01-01

    The familiarity of an object depends on the spatial arrangement of its parts; when the parts are spatially rearranged, they form a novel, unrecognizable configuration. Yet the same collection of parts comprises both the familiar and novel configuration. Is it possible that the collection of familiar parts activates a representation of the intact familiar configuration even when they are spatially rearranged? We presented novel configurations as primes before test displays that assayed effects on figure-ground perception from memories of intact familiar objects. In our test displays, two equal-area regions shared a central border; one region depicted a portion of a familiar object. Previous research with such displays has shown that participants are more likely to perceive the region depicting a familiar object as the figure and the abutting region as its ground when the familiar object is depicted in its upright orientation rather than upside down. The novel primes comprised either the same or a different collection of parts as the familiar object in the test display (part-rearranged and control primes, respectively). We found that participants were more likely to perceive the familiar region as figure in upright vs. inverted displays following part-rearranged primes but not control primes. Thus, priming with a novel configuration comprising the same familiar parts as the upcoming figure-ground display facilitated orientation-dependent effects of object memories on figure assignment. Similar results were obtained when the spatially rearranged collection of parts was suggested on the groundside of the prime's border, suggesting that familiar parts in novel configurations access the representation of their corresponding intact whole object before figure assignment. These data demonstrate that familiar parts access memories of familiar objects even when they are arranged in a novel configuration. PMID:24904495

  6. Dosimetric comparison of intensity-modulated solutions for intact prostate cancer

    SciTech Connect

    Neill, Cory J.

    2014-01-01

    The purpose of this study is intended to investigate the implementation of a modified class solution for intact prostate intensity-modulated radiation therapy (IMRT). The class solution uses 2 additional optimization structures intended to increase target conformity and decrease unnecessary dose to healthy tissue. A total of 10 randomly selected intact prostate IMRT patients were chosen for this retrospective study. Each of the original IMRT plans was compared with a modified class solution. The class solution implemented 2 additional optimization structures. The 95{sub O}PT was intended to increase target conformity, and the Avoidance{sub 3}780 was intended to reduce normal tissue. Each plan was evaluated for minimum, maximum, and mean doses to the target. Additionally, mean normal tissue dose, total monitor units (MUs), and segments were investigated. Conformity index and normal healthy index were also compared. All comparisons were evaluated using a paired t-test using GraphPad software. Evaluations of MUs; segments; minimum, maximum, mean target doses; mean normal tissue dose; and conformity index did not demonstrate a significant difference between the modified class solution and the original plans. However, evaluation of healthy tissue conformity index indicated a significant difference. Overall, 70% of the original plans failed to demonstrate a satisfactory score (< 0.6) of properly sparing normal healthy tissue, whereas 70% of the modified plans exhibited a satisfactory score (> 0.6). Most (90%) of the modified plans demonstrated a greater number of segments than the compared original plan. A modified class solution provides a good starting point for planning intact prostate cancer. The addition of the Avoidance{sub 3}780 structure increases the healthy tissue conformity index score.

  7. Correlations Between Physical and Hydraulic Properties and Uranium Desorption in Contaminated, Intact Sediment Cores

    NASA Astrophysics Data System (ADS)

    Rockhold, M. L.; Oostrom, M.; Wietsma, T. W.; Zachara, J. M.

    2010-12-01

    An unlined disposal pond in the 300 Area of the Hanford Site received uranium-bearing liquid effluents associated with nuclear reactor fuel rod processing from 1943 to 1975. Contaminated sediments from the base and sides of the former pond were excavated and removed from the site in the early 1990s, but a uranium plume has persisted in the groundwater at concentrations exceeding the drinking water standard. The former process pond is located adjacent to the Columbia River and seasonal fluctuations in the river stage and water table provide a mechanism for resupplying residual uranium from the vadose zone to the groundwater when the lower vadose zone is periodically rewetted. Intact cores were collected from the site for measurements of physical, hydraulic, and geochemical properties. Multistep outflow experiments were also performed on the intact cores to determine permeability-saturation-capillary pressure relations. Pore water displaced during these experiments for two of the vadose zone cores was also analyzed for uranium. For a core containing finer-textured sediment classified as muddy sandy gravel, and a core containing coarser-textured sediment classified as gravel, the relative aqueous uranium concentrations increased by factors of 8.3 and 1.5, respectively, as the cores were desaturated and progressively smaller pore-size classes were drained. Aqueous concentrations of uranium in the extracted pore waters were up to 115 times higher than the current drinking water standard of 30 ppb. These results confirm that there is a continuing source of uranium in the vadose zone at the site, and are consistent with a hypothesis that the persistence of the groundwater uranium plume is also associated, in part, with rate-limited mass transfer from finer-textured sediments. The data from these and several other intact cores from the site are evaluated to explore relationships between physical and hydraulic properties and uranium desorption characteristics.

  8. Prototypic and Arkypallidal Neurons in the Dopamine-Intact External Globus Pallidus

    PubMed Central

    Abdi, Azzedine; Mallet, Nicolas; Mohamed, Foad Y.; Sharott, Andrew; Dodson, Paul D.; Nakamura, Kouichi C.; Suri, Sana; Avery, Sophie V.; Larvin, Joseph T.; Garas, Farid N.; Garas, Shady N.; Vinciati, Federica; Morin, Stéphanie; Bezard, Erwan

    2015-01-01

    Studies in dopamine-depleted rats indicate that the external globus pallidus (GPe) contains two main types of GABAergic projection cell; so-called “prototypic” and “arkypallidal” neurons. Here, we used correlative anatomical and electrophysiological approaches in rats to determine whether and how this dichotomous organization applies to the dopamine-intact GPe. Prototypic neurons coexpressed the transcription factors Nkx2-1 and Lhx6, comprised approximately two-thirds of all GPe neurons, and were the major GPe cell type innervating the subthalamic nucleus (STN). In contrast, arkypallidal neurons expressed the transcription factor FoxP2, constituted just over one-fourth of GPe neurons, and innervated the striatum but not STN. In anesthetized dopamine-intact rats, molecularly identified prototypic neurons fired at relatively high rates and with high regularity, regardless of brain state (slow-wave activity or spontaneous activation). On average, arkypallidal neurons fired at lower rates and regularities than prototypic neurons, and the two cell types could be further distinguished by the temporal coupling of their firing to ongoing cortical oscillations. Complementing the activity differences observed in vivo, the autonomous firing of identified arkypallidal neurons in vitro was slower and more variable than that of prototypic neurons, which tallied with arkypallidal neurons displaying lower amplitudes of a “persistent” sodium current important for such pacemaking. Arkypallidal neurons also exhibited weaker driven and rebound firing compared with prototypic neurons. In conclusion, our data support the concept that a dichotomous functional organization, as actioned by arkypallidal and prototypic neurons with specialized molecular, structural, and physiological properties, is fundamental to the operations of the dopamine-intact GPe. PMID:25926446

  9. Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation

    SciTech Connect

    Cao, Li; Tolic, Nikola; Qu, Yi; Meng, Da; Zhao, Rui; Zhang, Qibin; Moore, Ronald J.; Zink, Erika M.; Lipton, Mary S.; Pasa-Tolic, Ljiljana; Wu, Si

    2014-01-15

    Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30% to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE valuespreferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thus enabling the putative glycan structure characterization. Also, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. On the contrary, HCD with higher NCE values preferentially fragmented the peptide backbone and thus provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N-linked and O-linked glycopeptides in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (including six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both, the peptide and the glycan, thus enabling comprehensive structural characterization of glycoproteins in a single LC-MS/MS analysis.

  10. Small Angle Neutron-Scattering Studies of the Core Structure of Intact Neurosecretory Vesicles.

    NASA Astrophysics Data System (ADS)

    Krueger, Susan Takacs

    Small angle neutron scattering (SANS) was used to study the state of the dense cores within intact neurosecretory vesicles. These vesicles transport the neurophysin proteins, along with their associated hormones, oxytocin or vasopressin, from the posterior pituitary gland to the bloodstream, where the entire vesicle contents are released. Knowledge of the vesicle core structure is important in developing an understanding of this release mechanism. Since the core constituents exist in a dense state at concentrations which cannot be reproduced (in solution) in the laboratory, a new method was developed to determine the core structure from SANS experiments performed on intact neurosecretory vesicles. These studies were complemented by biochemical assays performed to determine the role, if any, played by phospholipids in the interactions between the core constituents. H_2O/D_2 O ratio in the solvent can be adjusted, using the method of contrast variation, such that the scattering due to the vesicle membranes is minimized, thus emphasizing the scattering originating from the cores. The applicability of this method for examining the interior of biological vesicles was tested by performing an initial study on human red blood cells, which are similar in structure to other biological vesicles. Changes in intermolecular hemoglobin interactions, occurring when the ionic strength of the solvent was varied or when the cells were deoxygenated, were examined. The results agreed with those expected for dense protein solutions, indicating that the method developed was suitable for the study of hemoglobin within the cells. Similar SANS studies were then performed on intact neurosecretory vesicles. The experimental results were inconsistent with model calculations which assumed that the cores consisted of small, densely-packed particles or large, globular aggregates. Although a unique model could not be determined, the data suggest that the core constituents form long aggregates of

  11. An intact dorsomedial posterior arcuate nucleus is not necessary for photoperiodic responses in Siberian hamsters.

    PubMed

    Teubner, Brett J W; Leitner, Claudia; Thomas, Michael A; Ryu, Vitaly; Bartness, Timothy J

    2015-04-01

    Seasonal responses of many animal species are triggered by changes in daylength and its transduction into a neuroendocrine signal by the pineal gland through the nocturnal duration of melatonin (MEL) release. The precise central sites necessary to receive, transduce, and relay the short day (SD) fall-winter MEL signals into seasonal responses and changes in physiology and behavior are unclear. In Siberian hamsters, SDs trigger decreases in body and lipid mass, testicular regression and pelage color changes. Several candidate genes and their central sites of expression have been proposed as components of the MEL transduction system with considerable recent focus on the arcuate nucleus (ARC) and its component, the dorsomedial posterior arcuate nucleus (dmpARC). This site has been postulated as a critical relay of SD information through the modulation of a variety of neurochemicals/receptors important for the control of energy balance. Here the necessity of an intact dmpARC for SD responses was tested by making electrolytic lesions of the Siberian hamster dmpARC and then exposing them to either long days (LD) or SDs for 12wks. The SD typical decreases in body and fat mass, food intake, testicular volume, serum testosterone concentrations, pelage color change and increased UCP-1 protein expression (a proxy for brown adipose tissue thermogenesis) all occurred despite the lack of an intact dmpARC. Although the Siberian hamster dmpARC contains photoperiod-modulated constituents, these data demonstrate that an intact dmpARC is not necessary for SD responses and not integral to the seasonal energy- and reproductive-related responses measured here. PMID:25647158

  12. Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

    PubMed

    Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth

    2016-06-01

    Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted. PMID:27044871

  13. Sediment properties and CO2 efflux from intact and cleared temperate mangrove forests

    NASA Astrophysics Data System (ADS)

    Bulmer, R. H.; Lundquist, C. J.; Schwendenmann, L.

    2015-10-01

    Temperate mangrove forests in New Zealand have increased in area over recent decades. Expansion of temperate mangroves in New Zealand is associated with perceived loss of other estuarine habitats, and decreased recreational and amenity values, resulting in clearing of mangrove forests. In the tropics, changes in sediment characteristics and carbon efflux have been reported following mangrove clearance. This is the first study in temperate mangrove (Avicennia marina) forests investigating the impact of clearing on sediment CO2 efflux and associated biotic and abiotic factors. Sediment CO2 efflux rates from intact (168.5 ± 45.8 mmol m-2 d-1) and cleared (133.9 ± 37.2 mmol m-2 d-1) mangrove forests in New Zealand are comparable to rates measured in tropical mangrove forests. We did not find a significant difference in sediment CO2 efflux rates between intact and cleared temperate mangrove forests. Pre-shading the sediment for more than 30 min prior to dark chamber measurements was found to have no significant effect on sediment CO2 efflux. This suggests that the continuation of photosynthetic CO2 uptake by biofilm communities was not occurring after placement of dark chambers. Rather, above-ground mangrove biomass, sediment temperature and chlorophyll a concentration were the main factors explaining the variability in sediment CO2 efflux in intact mangrove forests. The main factors influencing sediment CO2 efflux in cleared mangrove forest sites were sediment organic carbon concentration, nitrogen concentration and sediment grain size. Our results show that greater consideration should be given regarding the rate of carbon released from mangrove forest following clearance and the relative contribution to global carbon emissions.

  14. Hindered transport of macromolecules in isolated glomeruli. I. Diffusion across intact and cell-free capillaries.

    PubMed Central

    Edwards, A; Deen, W M; Daniels, B S

    1997-01-01

    The filtrate formed by renal glomerular capillaries must pass through a layer of endothelial cells, the glomerular basement membrane (GBM), and a layer of epithelial cells, arranged in series. To elucidate the relative resistances of the GBM and cell layers to movement of uncharged macromolecules, we measured the diffusional permeabilities of intact and cell-free capillaries to narrow fractions of Ficoll with Stokes-Einstein radii ranging from 3.0 to 6.2 nm. Glomeruli were isolated from rat kidneys, and diffusion of fluorescein-labeled Ficoll across the walls of single capillary loops was monitored with a confocal microscopy technique. In half of the experiments the glomeruli were treated first to remove the cells, leaving skeletons that retained the general shape of the glomerulus and consisted almost entirely of GBM. The diffusional permeability of cell-free capillaries to Ficoll was approximately 10 to 20 times that of intact capillaries, depending on molecular size. Taking into account the blockage of much of the GBM surface by cells, the contribution of the GBM to the diffusional resistance of the intact barrier was calculated to be 13% to 26% of the total, increasing with molecular size. Thus, the GBM contribution, although smaller than that of the cells, was not negligible. The structure that is most likely to be responsible for the cellular part of the diffusional resistance is the slit diaphragm, which spans the filtration slit between epithelial foot processes. A novel hydrodynamic model was developed to relate the diffusional resistance of the slit diaphragm to its structure, which was idealized as a single layer of cylindrical fibers in a ladder-like arrangement. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 5 PMID:8994605

  15. Palladium nanoparticles exposure: Evaluation of permeation through damaged and intact human skin.

    PubMed

    Larese Filon, Francesca; Crosera, Matteo; Mauro, Marcella; Baracchini, Elena; Bovenzi, Massimo; Montini, Tiziano; Fornasiero, Paolo; Adami, Gianpiero

    2016-07-01

    The intensified use of palladium nanoparticles (PdNPs) in many chemical reactions, jewellery, electronic devices, in car catalytic converters and in biomedical applications lead to a significant increase in palladium exposure. Pd can cause allergic contact dermatitis when in contact with the skin. However, there is still a lack of toxicological data related to nano-structured palladium and information on human cutaneous absorption. In fact, PdNPs, can be absorbed through the skin in higher amounts than bulk Pd because NPs can release more ions. In our study, we evaluated the absorption of PdNPs, with a size of 10.7 ± 2.8 nm, using intact and damaged human skin in Franz cells. 0.60 mg cm(-2) of PdNPs were applied on skin surface for 24 h. Pd concentrations in the receiving solutions at the end of experiments were 0.098 ± 0.067 μg cm(-2) and 1.06 ± 0.44 μg cm(-2) in intact skin and damaged skin, respectively. Pd flux permeation after 24 h was 0.005 ± 0.003 μg cm(-2) h(-1) and 0.057 ± 0.030 μg cm(-2) h(-1) and lag time 4.8 ± 1.7 and 4.2 ± 3.6 h, for intact and damaged skin respectively. This study indicates that Pd can penetrate human skin. PMID:27131807

  16. Mechanical analysis of the human cadaveric thoracic spine with intact rib cage.

    PubMed

    Mannen, Erin M; Anderson, John T; Arnold, Paul M; Friis, Elizabeth A

    2015-07-16

    The goal of this study was to characterize the overall in-plane and basic coupled motion of a cadaveric human thoracic spine with intact true ribs. Researchers are becoming increasingly interested in the thoracic spine due to both the high prevalence of injury and pain in the region and also innovative surgical techniques that utilize the rib cage. Computational models can be useful tools to predict loading patterns and understand effects of surgical procedures or medical devices, but they are often limited by insufficient cadaveric input data. In this study, pure moments to ±5 Nm were applied in flexion-extension, lateral bending, and axial rotation to seven human cadaveric thoracic spine specimens (T1-T12) with intact true ribs to determine symmetry of in-plane motion, differences in neutral and elastic zone motion and stiffness, and significance of out-of-plane rotations and translations. Results showed that lateral bending and axial rotation exhibited symmetric motion, neutral and elastic zone motion and stiffness values were significantly different for all modes of bending (p<0.05), and out-of-plane rotations and translations were greater than zero for most rotations and translations. Overall in-plane rotations were 7.7±3.4° in flexion, 9.6±3.7° in extension, 23.3±8.4° in lateral bending, and 26.3±12.2° in axial rotation. Results of this study could provide inputs or validation comparisons for computational models. Future studies should characterize coupled motion patterns and local and regional level biomechanics of cadaveric human thoracic spines with intact true ribs. PMID:25912664

  17. Analysis of responses to glyceryl trinitrate and sodium nitrite in the intact chest rat.

    PubMed

    Nossaman, Bobby D; Pankey, Edward A; Badejo, Adeleke R; Casey, David B; Uppu, Satvika; Murthy, Subramanyam N; Kadowitz, Philip J

    2012-05-15

    Responses to glyceryl trinitrate/nitroglycerin (GTN), S-nitrosoglutathione (GSNO), and sodium nitrite were compared in the intact chest rat. The iv injections of GTN, sodium nitrite, and GSNO produced dose-dependent decreases in pulmonary and systemic arterial pressures. In as much as cardiac output was not reduced, the decreases in pulmonary and systemic arterial pressures indicate that GTN, sodium nitrite, and GSNO have significant vasodilator activity in the pulmonary and systemic vascular beds in the rat. Responses to GTN were attenuated by cyanamide, but not allopurinol, whereas responses to nitrite formed by the metabolism of GTN were attenuated by allopurinol and cyanamide. The results with allopurinol and cyanamide suggest that only mitochondrial aldehyde dehydrogenase is involved in the bioactivation of GTN, sodium nitrite, and GSNO, whereas both pathways are involved in the bioactivation of nitrite anion in the intact rat. The comparison of vasodilator activity indicates that GSNO and GTN are more than 1000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures in the rat. Following administration of 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ), responses to GTN were significantly attenuated, indicating that responses are mediated by the activation of soluble guanylyl cyclase. These data suggest that the reduction of nitrite to nitric oxide formed from the metabolism of GTN, cannot account for the vasodilator activity of GTN in the intact rat and that another mechanism; perhaps the formation of an S-NO, may mediate the vasodilator response to GTN in this species. PMID:22465477

  18. Analysis of Responses to Glyceryl Trinitrate and Sodium Nitrite in the Intact Chest Rat

    PubMed Central

    Nossaman, Bobby D.; Pankey, Edward A.; Badejo, Adeleke R.; Casey, David B.; Uppu, Satvika; Murthy, Subramanyam N.; Kadowitz, Philip J.

    2012-01-01

    Responses to glyceryl trinitrate/nitroglycerin (GTN), S-nitrosoglutathione (GSNO), and sodium nitrite were compared in the intact chest rat. The iv injections of GTN, sodium nitrite, and GSNO produced dose-dependent decreases in pulmonary and systemic arterial pressures. In as much as cardiac output was not reduced, the decreases in pulmonary and systemic arterial pressures indicate that GTN, sodium nitrite, and GSNO have significant vasodilator activity in the pulmonary and systemic vascular beds in the rat. Responses to GTN were attenuated by cyanamide, but not allopurinol, whereas responses to nitrite formed by the metabolism of GTN were attenuated by allopurinol and cyanamide. The results with allopurinol and cyanamide suggest that only mitochondrial aldehyde dehydrogenase is involved in the bioactivation of GTN, sodium nitrite, and GSNO, whereas both pathways are involved in the bioactivation of nitrite anion in the intact rat. The comparison of vasodilator activity indicates that GSNO and GTN are more than 1000 fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures in the rat. Following administration of 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ), responses to GTN were significantly attenuated, indicating that responses are mediated by the activation of soluble guanylyl cyclase. These data suggest that the reduction of nitrite to nitric oxide formed from the metabolism of GTN, cannot account for the vasodilator activity of GTN in the intact rat and that another mechanism; perhaps the formation of an S-NO, may mediate the vasodilator response to GTN in this species. PMID:22465477

  19. Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat

    EPA Science Inventory

    Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and te...

  20. INTACT SOIL-CORE MICROCOSMS FOR EVALUATING THE FATE AND ECOLOGICAL IMPACT OF THE RELEASE OF GENETICALLY ENGINEERED MICROORGANISMS

    EPA Science Inventory

    Intact soil-core microcosms were studied to determine their applicability for evaluating the transport, survival and potential ecosystem effects of genetically engineered microorganisms before they are released into the environment. oi1-core microcosms were planted with wheat and...

  1. Evaluation of codisposal viability of MOX (FFTF) DOE-owned fuel: Phase 1 -- Intact mode calculations

    SciTech Connect

    Goluoglu, S.; Davis, J.W.; Montierth, L.M.

    1999-07-01

    The authors provide the intact criticality information that supports the disposal of spent nuclear fuel (SNF) from the US Department of Energy's (DOE's) Fast Flux Test Facility (FFTF) in the potential Monitored Geologic Repository at Yucca Mountain. FFTF is one of more than 250 forms of DOE-owned SNF. Because of the variety of the DOE SNF, the National Spent Nuclear Fuel Program has designated nine representative fuel groups for disposal criticality analyses based on fuel matrix, primary fissile isotope, and enrichment. The FFTF fuel is representative of the mixed-oxide fuel (MOX) group.

  2. Microsporidial spores can cross the intact Descemet membrane in deep stromal infection.

    PubMed

    Murthy, Somasheila I; Sangit, Vishram A; Rathi, Varsha M; Vemuganti, Geeta K

    2013-01-01

    We report a rare case of a deep stromal keratitis with a chronic indolent course, diagnosed as microsporidial keratitis from corneal scrapings. The patient's condition worsened despite medical therapy and penetrating keratoplasty was performed. The histopathology of the corneal tissue revealed multiple microsporidial spores in the posterior stroma and the endothelial exudates, whereas there was no clinical or histopathological breach in Descemet's membrane. This is the second report in the literature to report that micropsoridial spores can cross the intact Descemet's membrane. PMID:23580858

  3. Borane-Catalyzed Reductive α-Silylation of Conjugated Esters and Amides Leaving Carbonyl Groups Intact.

    PubMed

    Kim, Youngchan; Chang, Sukbok

    2016-01-01

    Described herein is the development of the B(C6F5)3-catalyzed hydrosilylation of α,β-unsaturated esters and amides to afford synthetically valuable α-silyl carbonyl products. The α-silylation occurs chemoselectively, thus leaving the labile carbonyl groups intact. The reaction features a broad scope of both acyclic and cyclic substrates, and the synthetic utility of the obtained α-silyl carbonyl products is also demonstrated. Mechanistic studies revealed two operative steps: fast 1,4-hydrosilylation of conjugated carbonyls and then slow silyl group migration of a silyl ether intermediate. PMID:26549843

  4. Identification of periplasmic nitrate reductase Mo(V) EPR signals in intact cells of Paracoccus denitrificans.

    PubMed

    Sears, H J; Bennett, B; Spiro, S; Thomson, A J; Richardson, D J

    1995-08-15

    EPR spectroscopy has been successfully used to detect signals due to molybdenum (V) and ferric iron in intact cells of aerobically grown Paracoccus denitrificans. The signals are ascribed to the catalytic molybdenum centre and to the haem iron of the periplasmic nitrate reductase. These signals are absent from a mutant strain deficient in this enzyme. The Mo(V) signal is due to the High-g Split species which has been well characterized in the purified enzyme. This confirms that the High-g Split is the physiologically relevant signal of a number observed in the previous work on the purified enzyme. PMID:7646461

  5. Incorporation of phosphatidylglycerol into murein lipoprotein in intact cells of Salmonella typhimurium by phospholipid vesicle fusion.

    PubMed Central

    Chattopadhyay, P K; Lai, J S; Wu, H C

    1979-01-01

    The biosynthesis of the diglyceride moiety of murein lipoprotein was studied by fusion of labeled phospholipid vesicles with intact cells of Salmonella typhimurium. Phosphatidylglycerol was found to be an excellent donor for the glyceryl moiety in lipoprotein, whereas phosphatidylethanolamine and cardiolipin were not. The incorporation of radioactivity from monoacyl-phosphatidylglycerol into lipoprotein can be attributed to its conversion to phosphatidylglycerol. The results strongly support our hypothesis that the glyceryl residue covalently linked to murein lipoprotein is derived from the nonacylated glycerol moiety of phosphatidylglycerol. PMID:368018

  6. Enhanced intensity discrimination in the intact ear of adults with unilateral deafness.

    PubMed

    Maslin, Michael R D; Taylor, Melissa; Plack, Christopher J; Munro, Kevin J

    2015-06-01

    Physiological measures of neural activity in the auditory cortex have revealed plasticity following unilateral deafness. Central projections from the remaining ear reorganize to produce a stronger cortical response than normal. However, little is known about the perceptual consequences of this increase. One possibility is improved sound intensity discrimination. Intensity difference limens were measured in 11 individuals with unilateral deafness that were previously shown to exhibit increased cortical activity to sounds heard by the intact ear. Significantly smaller mean difference limens were observed compared with controls. These results provide evidence of the perceptual consequences of plasticity in humans following unilateral deafness. PMID:26093448

  7. Advances and techniques to measure cGMP in intact cardiomyocytes.

    PubMed

    Götz, Konrad R; Nikolaev, Viacheslav O

    2013-01-01

    Förster resonance energy transfer (FRET)-based biosensors are powerful tools for real-time monitoring of signaling events in intact cells using fluorescence microscopy. Here, we describe a highly sensitive method which allows FRET-based measurements of the second messenger cGMP in adult mouse ventricular myocytes. Such measurements have been challenging before, primarily due to relatively low cGMP concentrations in cardiomyocytes and limited sensitivity of the available biosensors. With our new technique, one can reliably measure dynamic changes in cGMP upon stimulation of myocytes with natriuretic peptides and other physiological and pharmacological ligands. PMID:23709029

  8. Factors Affecting the Extraction of Intact Ribonucleic Acid from Plant Tissues Containing Interfering Phenolic Compounds

    PubMed Central

    Newbury, H. John; Possingham, John V.

    1977-01-01

    Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions. PMID:16660134

  9. Multiphoton imaging to distinguish grana and starch inside an intact leaf

    NASA Astrophysics Data System (ADS)

    Chen, Mei-Yu; Zhuo, Guan-Yu; Chen, Po-Fu; Wu, Pei-Chun; Liu, Tzu-Ming; Chu, Shi-Wei

    2013-02-01

    We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

  10. Scanning spectrophotometry for the dynamic study of tissue respiration in intact organs.

    PubMed

    Jarry, G; Marzouki, L; Debray, S; Ghesquiere, S; Besson, B; Hung, B M; Laurent, D

    1986-04-01

    For use with intact perfused organs a spectrophotometer system has been developed, both for dual-wavelength absorption measurements and for spectral scanning. A monochromator is used for illumination and scanning in the visible and near infrared. Optic fibres conduct light to the specimen under examination and from the specimen to a detecting photomultiplier. System control is exercised by a microcomputer, which also processes the collected data. The performance of the system on isolated perfused rat heart is demonstrated, in the spectral scanning mode and in the dual-wavelength mode, by studying simultaneously the kinetics of cytochrome aa3, and myoglobin oxidation-reduction. PMID:3012201

  11. Pulmonary atresia with intact ventricular septum and hypoplastic right ventricle in an Arabian foal.

    PubMed

    Krüger, M U; Wünschmann, A; Ward, C; Stauthammer, C D

    2016-09-01

    Pulmonary atresia with intact ventricular septum, rudimentary tricuspid valve, hypoplastic right ventricle, and right-to-left atrial shunting were identified in a four-day-old, male Arabian foal with clinical signs of cyanotic heart disease. Pulmonary blood flow was apparently derived from a ductus arteriosus. Echocardiographic evaluation revealed the majority of cardiac abnormalities and also findings compatible with right-sided congestive heart failure. Congenital cardiac defects have a high incidence in this breed, and this is the first description of this combination of congenital cardiac defects. PMID:27283083

  12. Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells.

    PubMed

    King, Nicholas M P; Methawasin, Methajit; Nedrud, Joshua; Harrell, Nicholas; Chung, Charles S; Helmes, Michiel; Granzier, Henk

    2011-01-01

    A carbon fiber-based cell attachment and force measurement system was used to measure the diastolic stress-sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm(2) at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm(2) (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca(2+)] from below to above 100 nM. Mean active stress was significantly increased at [Ca(2+)] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm(2) at 100 nM [Ca(2+)] (pCa 7.0) and ∼1.3 mN/mm(2) at 175 nM Ca(2+) (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell's mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm(2)/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure. PMID:21187335

  13. In Vitro Study of Cytophysiological Characteristics of Multinuclear Macrophages from Intact and BCG-Infected Mice.

    PubMed

    Il'in, D A; Arkhipov, S A; Shkurupy, V A

    2016-03-01

    Peritoneal macrophages were isolated from intact and BCG-infected BALB/c mice and explanted in vitro. Multinuclear macrophages formed in these cultures differed by the number of nuclei, expression of apoptosis inductors and regulators (TNF-α, p53 protein, caspase 3, and Bcl-2 protein), and cytophysiological characteristics (phagocytic activity, ROS generation, and antimycobacterial properties). Our results indicate that the formation of multinuclear macrophages is accompanied by induction of apoptosis (p53 signaling pathway) and appearance of multinuclear macrophage-derived cells characterized by high phagocytic and antimycobacterial activity. PMID:27021088

  14. GPQuest: A Spectral Library Matching Algorithm for Site-Specific Assignment of Tandem Mass Spectra to Intact N-glycopeptides.

    PubMed

    Toghi Eshghi, Shadi; Shah, Punit; Yang, Weiming; Li, Xingde; Zhang, Hui

    2015-01-01

    Glycoprotein changes occur in not only protein abundance but also the occupancy of each glycosylation site by different glycoforms during biological or pathological processes. Recent advances in mass spectrometry instrumentation and techniques have facilitated analysis of intact glycopeptides in complex biological samples by allowing the users to generate spectra of intact glycopeptides with glycans attached to each specific glycosylation site. However, assigning these spectra, leading to identification of the glycopeptides, is challenging. Here, we report an algorithm, named GPQuest, for site-specific identification of intact glycopeptides using higher-energy collisional dissociation (HCD) fragmentation of complex samples. In this algorithm, a spectral library of glycosite-containing peptides in the sample was built by analyzing the isolated glycosite-containing peptides using HCD LC-MS/MS. Spectra of intact glycopeptides were selected by using glycan oxonium ions as signature ions for glycopeptide spectra. These oxonium-ion-containing spectra were then compared with the spectral library generated from glycosite-containing peptides, resulting in assignment of each intact glycopeptide MS/MS spectrum to a specific glycosite-containing peptide. The glycan occupying each glycosite was determined by matching the mass difference between the precursor ion of intact glycopeptide and the glycosite-containing peptide to a glycan database. Using GPQuest, we analyzed LC-MS/MS spectra of protein extracts from prostate tumor LNCaP cells. Without enrichment of glycopeptides from global tryptic peptides and at a false discovery rate of 1%, 1008 glycan-containing MS/MS spectra were assigned to 769 unique intact N-linked glycopeptides, representing 344 N-linked glycosites with 57 different N-glycans. Spectral library matching using GPQuest assigns the HCD LC-MS/MS generated spectra of intact glycopeptides in an automated and high-throughput manner. Additionally, spectral library

  15. Delayed fluorescence spectra of intact leaves photoexcited by sunlight measured with a multichannel Fourier-transform chemiluminescence spectrometer

    NASA Astrophysics Data System (ADS)

    Akita, Saeka; Yano, Ayako; Ishii, Hiroshi; Satoh, Chikahiro; Akai, Nobuyuki; Nakata, Munetaka

    2013-06-01

    Delayed fluorescence spectra of intact leaves of Green pak choi (Brassica rapa var. chinensis) were measured with a multichannel Fourier-transform chemiluminescence spectrometer, which we developed recently. The intact samples, photoexcited by sunlight without artificial light sources, showed delayed fluorescence around 740 nm with a lifetime of ˜6 s. The observed spectra were deconvoluted into two Gaussian bands: the delayed fluorescence from photosystem II and photosystem I complexes. Their relative intensities depended on the chlorophyll concentration, but their wavelengths were unchanged.

  16. Measurement of intact-core length of atomizing liquid jets by image deconvolution

    NASA Astrophysics Data System (ADS)

    Woodward, Roger; Burch, Robert; Kuo, Kenneth; Cheung, Fan-Bill

    1993-11-01

    The investigation of liquid jet breakup and spray development is critical to the understanding of combustion phenomena in liquid propellant rocket engines. Much work has been done to characterize low-speed liquid jet breakup and dilute sprays, but atomizing jets and dense sprays have yielded few quantitative measurements due to their high liquid load fractions and hence their optical opacity. Focus was on a characteristic of the primary breakup process of round liquid jets, namely the length of the intact-liquid core. The specific application considered is that of shear-coaxial-type rocket engine injectors in which liquid oxygen is injected through the center post while high velocity gaseous hydrogen is injected through a concentric annulus, providing a shear force to the liquid jet surface. Real-time x ray radiography, capable of imaging through the dense two-phase region surrounding the liquid core, is used to make the measurements. The intact-liquid-core length data were obtained and interpreted using two conceptually different methods to illustrate the effects of chamber pressure, gas-to-liquid momentum ratio, and cavitation.

  17. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier

    PubMed Central

    Dreier, Jes; Sørensen, Jens A.; Brewer, Jonathan R.

    2016-01-01

    In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers. PMID:26751684

  18. Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures.

    PubMed

    Tian, Yuwei; Han, Linjie; Buckner, Adam C; Ruotolo, Brandon T

    2015-11-17

    Monoclonal antibodies (mAbs) are among the fastest growing class of therapeutics due to their high specificity and low incidence of side effects. Unlike most drugs, mAbs are complex macromolecules (∼150 kDa), leading to a host of quality control and characterization challenges inherent in their development. Recently, we introduced a new approach for the analysis of the intact proteins based on ion mobility-mass spectrometry (IM-MS). Our protocol involves the collision induced unfolding (CIU) of intact antibodies, where collisional heating in the gas-phase is used to generate unfolded antibody forms, which are subsequently separated by IM and then analyzed by MS. Collisional energy is added to the antibody ions in a stepwise fashion, and "fingerprint plots" are created that track the amount of unfolding undergone as a function of the energy imparted to the ions prior to IM separation. In this report, we have used these fingerprints to rapidly distinguish between antibody isoforms, possessing different numbers and/or patterns of disulfide bonding and general levels of glycosylation. In addition, we validate our CIU protocols through control experiments and systematic statistical evaluations of CIU reproducibility. We conclude by projecting the impact of our approach for antibody-related drug discovery and development applications. PMID:26471104

  19. Human blindsight is mediated by an intact geniculo-extrastriate pathway.

    PubMed

    Ajina, Sara; Pestilli, Franco; Rokem, Ariel; Kennard, Christopher; Bridge, Holly

    2015-01-01

    Although damage to the primary visual cortex (V1) causes hemianopia, many patients retain some residual vision; known as blindsight. We show that blindsight may be facilitated by an intact white-matter pathway between the lateral geniculate nucleus and motion area hMT+. Visual psychophysics, diffusion-weighted magnetic resonance imaging and fibre tractography were applied in 17 patients with V1 damage acquired during adulthood and 9 age-matched controls. Individuals with V1 damage were subdivided into blindsight positive (preserved residual vision) and negative (no residual vision) according to psychophysical performance. All blindsight positive individuals showed intact geniculo-hMT+ pathways, while this pathway was significantly impaired or not measurable in blindsight negative individuals. Two white matter pathways previously implicated in blindsight: (i) superior colliculus to hMT+ and (ii) between hMT+ in each hemisphere were not consistently present in blindsight positive cases. Understanding the visual pathways crucial for residual vision may direct future rehabilitation strategies for hemianopia patients. PMID:26485034

  20. Imaging the Intact Mouse Cornea Using Coherent Anti-Stokes Raman scattering (CARS)

    PubMed Central

    Ammar, David A.; Lei, Tim C.; Kahook, Malik Y.; Masihzadeh, Omid

    2013-01-01

    Purpose. The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon–hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents. Methods. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed. Results. Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers. Conclusions. The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques. PMID:23821187

  1. High-resolution in vivo imaging of mouse brain through the intact skull

    PubMed Central

    Park, Jung-Hoon; Sun, Wei; Cui, Meng

    2015-01-01

    Multiphoton microscopy is the current method of choice for in vivo deep-tissue imaging. The long laser wavelength suffers less scattering, and the 3D-confined excitation permits the use of scattered signal light. However, the imaging depth is still limited because of the complex refractive index distribution of biological tissue, which scrambles the incident light and destroys the optical focus needed for high resolution imaging. Here, we demonstrate a wavefront-shaping scheme that allows clear imaging through extremely turbid biological tissue, such as the skull, over an extended corrected field of view (FOV). The complex wavefront correction is obtained and directly conjugated to the turbid layer in a noninvasive manner. Using this technique, we demonstrate in vivo submicron-resolution imaging of neural dendrites and microglia dynamics through the intact skulls of adult mice. This is the first observation, to our knowledge, of dynamic morphological changes of microglia through the intact skull, allowing truly noninvasive studies of microglial immune activities free from external perturbations. PMID:26170286

  2. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium

    PubMed Central

    Wilson, Calum; Saunter, Christopher D.; Girkin, John M.; McCarron, John G.

    2016-01-01

    Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca2+ concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca2+ signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca2+ signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.—Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium. PMID:26873937

  3. Displaced Medial and Lateral Bucket Handle Meniscal Tears With Intact ACL and PCL.

    PubMed

    Boody, Barrett S; Omar, Imran M; Hill, James A

    2015-08-01

    Bucket handle lesions are vertical longitudinal tears in the meniscus that may displace centrally into the respective medial or lateral compartment, frequently causing mechanical symptoms, including pain, perceived instability, and mechanical locking. Bucket handle meniscal tears are most commonly from a traumatic etiology and are frequently found with concomitant anterior cruciate ligament (ACL) injuries. Multiple imaging signs and associations have been described for the diagnosis of bucket handle meniscus tears, including coronal truncation, absent bow tie sign, double posterior cruciate ligament (PCL), double ACL, displacement of the bucket handle fragment, and disproportionate posterior horn signs. Among meniscal pathology encountered on magnetic resonance imaging or during arthroscopy, bucket handle meniscal tears are infrequent occurrences. Furthermore, the occurrence of displaced medial and lateral bucket handle tears found on imaging and during arthroscopy is very uncommon and is only sparsely reported in the literature. When displaced medial and lateral bucket handle meniscal segments are visualized within the intercondylar notch along with the ACL and PCL, the radiologic findings are referred to as the "quadruple cruciate" sign or the "Jack and Jill lesion." Of the few case reports described in the literature, only one noted displaced medial and lateral bucket handle meniscus tears with an intact ACL and PCL. The current case report outlines a similar rare case of the quadruple cruciate sign: displaced medial and lateral bucket handle meniscal tears located within the intercondylar notch and an intact ACL and PCL. PMID:26270763

  4. Near infrared spectroscopy (NIRS) for on-line determination of quality parameters in intact olives.

    PubMed

    Salguero-Chaparro, Lourdes; Baeten, Vincent; Fernández-Pierna, Juan A; Peña-Rodríguez, Francisco

    2013-08-15

    The acidity, moisture and fat content in intact olive fruits were determined on-line using a NIR diode array instrument, operating on a conveyor belt. Four sets of calibrations models were obtained by means of different combinations from samples collected during 2009-2010 and 2010-2011, using full-cross and external validation. Several preprocessing treatments such as derivatives and scatter correction were investigated by using the root mean square error of cross-validation (RMSECV) and prediction (RMSEP), as control parameters. The results obtained showed RMSECV values of 2.54-3.26 for moisture, 2.35-2.71 for fat content and 2.50-3.26 for acidity parameters, depending on the calibration model developed. Calibrations for moisture, fat content and acidity gave residual predictive deviation (RPD) values of 2.76, 2.37 and 1.60, respectively. Although, it is concluded that the on-line NIRS prediction results were acceptable for the three parameters measured in intact olive samples in movement, the models developed must be improved in order to increase their accuracy before final NIRS implementation at mills. PMID:23561217

  5. Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

    PubMed Central

    Nyberg, Lena K.; Quaderi, Saair; Emilsson, Gustav; Karami, Nahid; Lagerstedt, Erik; Müller, Vilhelm; Noble, Charleston; Hammarberg, Susanna; Nilsson, Adam N.; Sjöberg, Fei; Fritzsche, Joachim; Kristiansson, Erik; Sandegren, Linus; Ambjörnsson, Tobias; Westerlund, Fredrik

    2016-01-01

    The rapid spread of antibiotic resistance – currently one of the greatest threats to human health according to WHO – is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics. PMID:27460437

  6. Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

    SciTech Connect

    McIntyre, D.A.; Harlander, S.K. )

    1989-03-01

    The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit. Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 {mu}s to 1 s). Transformation efficiencies of 10{sup 3} transformants per {mu}g of DNA were obtained when dense suspensions (final concentration, 5 {times} 10{sup 10} CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30{degree}C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.

  7. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

    PubMed

    Dreier, Jes; Sørensen, Jens A; Brewer, Jonathan R

    2016-01-01

    In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers. PMID:26751684

  8. Oestrogen and progesterone receptor expression in subtypes of canine mammary tumours in intact and ovariectomised dogs.

    PubMed

    Mainenti, M; Rasotto, R; Carnier, P; Zappulli, V

    2014-10-01

    The objective of this study was to investigate as a potential prognostic indicator the relationship between histological subtype of canine mammary tumours (CMTs) and oestrogen-α (ORα) and progesterone (PR) receptor expression. Using immunohistochemistry, receptor expression in neoplastic epithelial cells was assessed in 12 different subtypes in 113 CMTs (34 benign, 79 malignant) and 101 surrounding normal tissues. Sixty-eight and 45 CMTs were from intact and ovariectomised bitches, respectively. Histological subtype strongly influenced ORα/PR expression: simple and complex adenomas as well as simple tubular carcinomas exhibited the greatest expression, whereas immunohistochemical labelling for these receptors was weakest in carcinoma and malignant myoepitheliomas, as well as in solid/anaplastic carcinomas and comedocarcinomas. Receptor expression was generally higher in benign relative to malignant neoplasms, and in the latter it was significantly lower in ovariectomised vs. intact bitches. Lymphatic invasion, mitotic index, nodule diameter, and tumour grade were significantly associated with ORα/PR expression. Although not found to be an independent prognostic indicator, tumours from dogs with <10% cells with ORα/PR expression had a poorer prognosis. Lymphatic invasion, the state of the margins of excision, and mitotic index were found to be independent prognostic indicators. Overall, the results suggest that differences in histological subtype and whether or not a bitch has been ovariectomised should be considered when evaluating the significance of ORα and PR expression in CMTs. PMID:24980810

  9. Web and database software for identification of intact proteins using "top down" mass spectrometry.

    PubMed

    Taylor, Gregory K; Kim, Yong-Bin; Forbes, Andrew J; Meng, Fanyu; McCarthy, Ryan; Kelleher, Neil L

    2003-08-15

    For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/). PMID:14632120

  10. Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules.

    PubMed

    Nyberg, Lena K; Quaderi, Saair; Emilsson, Gustav; Karami, Nahid; Lagerstedt, Erik; Müller, Vilhelm; Noble, Charleston; Hammarberg, Susanna; Nilsson, Adam N; Sjöberg, Fei; Fritzsche, Joachim; Kristiansson, Erik; Sandegren, Linus; Ambjörnsson, Tobias; Westerlund, Fredrik

    2016-01-01

    The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics. PMID:27460437

  11. Three-Dimensional Optical Mapping of Nanoparticle Distribution in Intact Tissues.

    PubMed

    Sindhwani, Shrey; Syed, Abdullah Muhammad; Wilhelm, Stefan; Glancy, Dylan R; Chen, Yih Yang; Dobosz, Michael; Chan, Warren C W

    2016-05-24

    The role of tissue architecture in mediating nanoparticle transport, targeting, and biological effects is unknown due to the lack of tools for imaging nanomaterials in whole organs. Here, we developed a rapid optical mapping technique to image nanomaterials in intact organs ex vivo and in three-dimensions (3D). We engineered a high-throughput electrophoretic flow device to simultaneously transform up to 48 tissues into optically transparent structures, allowing subcellular imaging of nanomaterials more than 1 mm deep into tissues which is 25-fold greater than current techniques. A key finding is that nanomaterials can be retained in the processed tissue by chemical cross-linking of surface adsorbed serum proteins to the tissue matrix, which enables nanomaterials to be imaged with respect to cells, blood vessels, and other structures. We developed a computational algorithm to analyze and quantitatively map nanomaterial distribution. This method can be universally applied to visualize the distribution and interactions of materials in whole tissues and animals including such applications as the imaging of nanomaterials, tissue engineered constructs, and biosensors within their intact biological environment. PMID:27101355

  12. Measurement of intact-core length of atomizing liquid jets by image deconvolution

    NASA Technical Reports Server (NTRS)

    Woodward, Roger; Burch, Robert; Kuo, Kenneth; Cheung, Fan-Bill

    1993-01-01

    The investigation of liquid jet breakup and spray development is critical to the understanding of combustion phenomena in liquid propellant rocket engines. Much work has been done to characterize low-speed liquid jet breakup and dilute sprays, but atomizing jets and dense sprays have yielded few quantitative measurements due to their high liquid load fractions and hence their optical opacity. Focus was on a characteristic of the primary breakup process of round liquid jets, namely the length of the intact-liquid core. The specific application considered is that of shear-coaxial-type rocket engine injectors in which liquid oxygen is injected through the center post while high velocity gaseous hydrogen is injected through a concentric annulus, providing a shear force to the liquid jet surface. Real-time x ray radiography, capable of imaging through the dense two-phase region surrounding the liquid core, is used to make the measurements. The intact-liquid-core length data were obtained and interpreted using two conceptually different methods to illustrate the effects of chamber pressure, gas-to-liquid momentum ratio, and cavitation.

  13. Exogenous Testosterone, Aging, and Changes in Behavioral Response of Gonadally Intact Male Mice

    PubMed Central

    Onaolapo, Olakunle J.; Onaolapo, Adejoke Y.; Omololu, Tope A.; Oludimu, Adedunke T.; Segun-Busari, Toluwalase; Omoleke, Taofeeq

    2016-01-01

    This study tested the hypothesis that aging significantly affects the influence of exogenous testosterone on neurobehavior in gonadally intact male mice. Groups of prepubertal and aged male mice received daily vehicle or testosterone propionate (TP; 2.5 or 5.0 mg/kg intraperitoneal [i.p.]) for 21 days. Behaviors were assessed on days 1 and 21. Weight gain was significant in prepubertal mice. Locomotion and rearing increased in prepubertal mice after first dose and decreased after last dose of TP. Rearing was suppressed in aged mice throughout. Suppression of grooming occurred in both age groups at day 21. Significant increase in working memory in both age groups was seen in the radial-arm maze (at specific doses) and in prepubertal mice in the Y-maze. Elevated plus maze test showed mixed anxiolytic/anxiogenic effects. Aged mice had higher serum testosterone. In conclusion, age is an important determinant for the influence of exogenous testosterone on behavior in gonadally intact male mice. PMID:27158222

  14. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

    PubMed

    Wilson, Calum; Saunter, Christopher D; Girkin, John M; McCarron, John G

    2016-05-01

    Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium. PMID:26873937

  15. A laboratory experiment of intact polar lipid degradation in sandy sediments

    NASA Astrophysics Data System (ADS)

    Logemann, J.; Graue, J.; Köster, J.; Engelen, B.; Rullkötter, J.; Cypionka, H.

    2011-03-01

    Intact polar lipids (IPLs) are considered biomarkers for living biomass. Their degradation in marine sediments, however, is poorly understood and complicates interpretation of their occurrence in geological samples. To investigate the turnover of IPLs, a degradation experiment with anoxic sandy sediments from the North Sea was conducted. Intact cells of two organisms that do not naturally occur in North Sea sediments were chosen as IPL sources: (i) Saccharomyces cerevisiae, representative for ester-bound acyl lipids that also occur in Bacteria, and (ii) the archaeon Haloferax volcanii, representative for ether-bound isoprenoid lipids. Surprisingly, IPLs with phosphoester-bound head groups showed approximately the same degradation rate as IPLs with glycosidic head groups. Furthermore, the results indicate a relatively fast degradation of S. cerevisiae IPLs with ester-bound moieties (analogs of bacterial membrane lipids) and no significant degradation of archaeal IPLs with ether-bound moieties. Pore water and 16S rRNA-based DGGE analysis showed only a minor influence of the IPL source on microbial metabolism and community profiles. Due to our results, the IPL-based quantification of Archaea and Bacteria should be interpreted with caution.

  16. A laboratory experiment of intact polar lipid degradation in sandy sediments

    NASA Astrophysics Data System (ADS)

    Logemann, J.; Graue, J.; Köster, J.; Engelen, B.; Rullkötter, J.; Cypionka, H.

    2011-09-01

    Intact polar lipids (IPLs) are considered biomarkers for living biomass. Their degradation in marine sediments, however, is poorly understood and complicates interpretation of their occurrence in geological samples. To investigate the turnover of IPLs, a degradation experiment with anoxic sandy sediments from the North Sea was conducted. Intact cells of two organisms that do not naturally occur in North Sea sediments were chosen as IPL sources: (i) Saccharomyces cerevisiae, representative for ester-bound acyl lipids that also occur in Bacteria, and (ii) the archaeon Haloferax volcanii, representative for ether-bound isoprenoid lipids. Surprisingly, IPLs with phosphoester-bound head groups showed approximately the same degradation rate as IPLs with glycosidic head groups. Furthermore, the results indicate a relatively fast degradation of S. cerevisiae IPLs with ester-bound moieties (analogs of bacterial membrane lipids) and no significant degradation of archaeal IPLs with ether-bound moieties. Pore water and 16S rRNA-based DGGE analysis showed only a minor influence of the IPL source on microbial metabolism and community profiles. Due to our results, the IPL-based quantification of Archaea and Bacteria should be interpreted with caution.

  17. Intact mass analysis of monoclonal antibodies by capillary electrophoresis-Mass spectrometry.

    PubMed

    Han, Mei; Rock, Brooke M; Pearson, Josh T; Rock, Dan A

    2016-02-01

    Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC-MS for separation and intact mass determination of mAbs and antibody-based therapeutics. PMID:26751590

  18. Multivariate calibration of the degree of crystallinity in intact pellets by X-ray powder diffraction.

    PubMed

    Nikowitz, Krisztina; Domján, Attila; Pintye-Hódi, Klára; Regdon, Géza

    2016-04-11

    XRPD is the method of choice to determine crystalline content in an amorphous environment. While several studies describe its use on powders, little information is available on its performance on finished products. The method's use may be limited not only by the need of sample pretreatment and its validation but also by the propensity of some materials to recrystallize when exposed to heat or mechanical stress. In this work the authors describe an attempt at constructing a model based on the XRPD measurement of intact layered pellets using univariate methods based on peak heights and PLS regression. Results indicate that neither the goodness-of-fit (below 0.9 for all tested variables), nor the RMSEC values (above 5 for all tested variables) of any model based on peak height were good enough to consider them for everyday use. PLS regression however provided a model with improved characteristics (R(2)=0.9581, RMSEC=3.04) despite the low API content and individual loading characteristics also reflected the validity of the model. PLS analysis also indicated that a specific sample may be different in some formulation characteristic that did not register on other examinations. This further indicates the method's usefulness in the analysis of intact dosage forms. PMID:26899205

  19. A method of purifying intact complement factor H from human plasma.

    PubMed

    Wang, Feng-Mei; Yu, Feng; Zhao, Ming-Hui

    2013-10-01

    The aim of this study was to establish a method of purifying intact complement factor H (CFH) from human plasma. CFH was isolated from human plasma by polyethylene glycol (PEG) precipitation, following three sequential chromatographic columns, which consisted of l-lysine Sepharose column, Resource Q column and Sephacryl S-300 High Resolution HiPrep 16/60 column. All the above steps were performed at 4°C by Fast Protein Liquid Chromatography (FPLC) AKTA Purifier 10 with Frac-900. Identification of the purified CFH was confirmed by SDS-PAGE and Western blot. The following functions of the purified CFH were further analyzed compared with the commercial CFH in vitro: (1) binding ability with C3b; (2) binding ability with mCRP; (3) the protecting function of the hemolysis of sheep red blood cells; (4) the cofactor role for complement factor I-mediated proteolytic inactivation of C3b. Homogeneous CFH was purified from the plasma fraction through the above four steps. The purity and the functions of the purified CFH were comparable to the commercial CFH. The yield of CFH was 26±3% in our study. Compared with previous methods, our method was high yield with high purity. We established a stable and feasible system for purifying intact CFH, which could be used in the lab and clinical investigations. PMID:23906520

  20. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.

    PubMed

    Schwarz, Martin K; Scherbarth, Annemarie; Sprengel, Rolf; Engelhardt, Johann; Theer, Patrick; Giese, Guenter

    2015-01-01

    In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain. PMID:25993380