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Sample records for human elastin mrna

  1. Molecular-level characterization of elastin-like constructs and human aortic elastin.

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    Heinz, Andrea; Schräder, Christoph U; Baud, Stéphanie; Keeley, Fred W; Mithieux, Suzanne M; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2014-09-01

    This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS(2) data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin. Copyright © 2014. Published by Elsevier B.V.

  2. Does human leukocyte elastase degrade intact skin elastin?

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

    2012-11-01

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

  3. Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism

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    TIAN Shan; HE Pei-ying; ZHANG Jian-zhong; CHEN Zhou

    2012-01-01

    Background Elastin derived peptides can regulate melanocyte precursor development.Ultraviolet irradiation,infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis.The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes.Methods A375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours.A375 human melanoma cells were randomly assigned to control,kappa elastin,and lactose pre-incubated groups.The cell viabilities were detected by the methyl thiazoleterazolium assay.Melanin content and tyrosinase activity in A375 melanoma cells were measured.The expressions of endothelin B receptor(ETBR)mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction.Results Fifty μg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control(P<0.05).Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours(P<0.05).Kappa elastin increased the expressions of ETBR and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls,respectively.When pre-incubating cells with a lactose solution(10 mmol/L),the inhibition on melanin production was 34.96% compared with the kappa elastin group(P<0.05),tyrosinase activity was inhibited by 29.93% compared with kappa elastin group(P<0.05),and the expressions of ETBR mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group,respectively.Conclusion Kappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ETBR and c-kit.The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

  4. Insights into the degradation of human elastin by matrilysin-1.

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    Heinz, Andrea; Taddese, Samuel; Sippl, Wolfgang; Neubert, Reinhard H H; Schmelzer, Christian E H

    2011-02-01

    Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describing the enzymatic turnover of matrix components helps to understand the molecular basis of disease processes. Hence, in this work, the cleavage behavior of MMP-7 with respect to its natural substrate human elastin was investigated using mass spectrometric (MS) techniques and molecular modeling. Elastin peptides in the range of 500-8000 Da released through the action of MMP-7 were analyzed and domains susceptible to proteolytic attack by MMP-7 were identified. MMP-7 was found to mainly cleave in N- and C-terminal regions of elastin's precursor, which involves linkages in domains encoded by exons 2, 3, 5-7, 26, and 30-33. In contrast, only few cleavages were found in the central part of the precursor and no cleavages in regions in elastin that are involved in cross-linking. MMP-7 shows a strong preference for Leu in P(1)' and also accepts Val, Gly, and Pro at this position, whereas Ala is not preferred at P(1)'. Analysis by molecular modeling revealed that not only the size of the amino acid residue in P(1)' but also the orientation of the neighboring P(1) residue and, thus, the orientation of the peptide bond that is cleaved influences the cleavage preference of MMP-7. Overall, this study provides an important insight into the degradation of human elastin by MMP-7 and may aid in the development of approaches to treat elastin-degrading diseases. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  5. Elastin aging and lipid oxidation products in human aorta

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    Kamelija Zarkovic

    2015-04-01

    Full Text Available Vascular aging is associated with structural and functional modifications of the arteries, and by an increase in arterial wall thickening in the intima and the media, mainly resulting from structural modifications of the extracellular matrix (ECM components. Among the factors known to accumulate with aging, advanced lipid peroxidation end products (ALEs are a hallmark of oxidative stress-associated diseases such as atherosclerosis. Aldehydes generated from the peroxidation of polyunsaturated fatty acids (PUFA, (4-hydroxynonenal, malondialdehyde, acrolein, form adducts on cellular proteins, leading to a progressive protein dysfunction with consequences in the pathophysiology of vascular aging. The contribution of these aldehydes to ECM modification is not known. This study was carried out to investigate whether aldehyde-adducts are detected in the intima and media in human aorta, whether their level is increased in vascular aging, and whether elastin fibers are a target of aldehyde-adduct formation. Immunohistological and confocal immunofluorescence studies indicate that 4-HNE-histidine-adducts accumulate in an age-related manner in the intima, media and adventitia layers of human aortas, and are mainly expressed in smooth muscle cells. In contrast, even if the structure of elastin fiber is strongly altered in the aged vessels, our results show that elastin is not or very poorly modified by 4-HNE. These data indicate a complex role for lipid peroxidation and in particular for 4-HNE in elastin homeostasis, in the vascular wall remodeling during aging and atherosclerosis development.

  6. Mapping of macrophage elastase cleavage sites in insoluble human skin elastin.

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    Taddese, Samuel; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2008-06-01

    Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.

  7. Human elastin-based recombinant biopolymers improve mesenchymal stem cell differentiation.

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    Çelebi, Betül; Cloutier, Maxime; Rabelo, Rodrigo B; Balloni, Rodrigo; Mantovani, Diego; Bandiera, Antonella

    2012-11-01

    Elastin-based polypeptides are a class of smart biopolymers representing an important model in the design of biomaterials. The combination of biomimetic materials with cells that have great plasticity provides a promising strategy for the realization of highly engineered cell-based constructs for regenerative medicine and tissue repair applications. Two recombinant biopolymers inspired by human elastin are assessed as coating agents to prepare biomimetic surfaces for cell culture. These substrates are assayed for hBM MSC culture. The coated surfaces are also characterized with AFM to evaluate the topographical features of the deposited biopolymers. The results suggest that the elastin-derived biomimetic surfaces play a stimulatory role on osteogenic differentiation of MSCs.

  8. Polymorphisms in the human tropoelastin gene modify in vitro self-assembly and mechanical properties of elastin-like polypeptides.

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    David He

    Full Text Available Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN are associated with a number of disorders including Supravalvular aortic stenosis (SVAS, Williams-Beuren syndrome (WBS and autosomal dominant cutis laxa (ADCL. Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.

  9. Aldosterone and mineralocorticoid receptor antagonists modulate elastin and collagen deposition in human skin.

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    Mitts, Thomas F; Bunda, Severa; Wang, Yanting; Hinek, Aleksander

    2010-10-01

    We have shown that the steroid hormone aldosterone, recognized for its action on the kidney and the cardiovascular system, also modulates deposition of extracellular matrix in human skin. We have shown that treatment of primary cultures of normal skin fibroblasts with aldosterone (10 n-1 μM), in addition to stimulation of collagen type I expression, induces elastin gene expression and elastic fiber deposition. We have further shown that the elastogenic effect of aldosterone, which can be enhanced in the presence of mineralocorticoid receptor (MR) antagonists spironolactone and eplerenone, is executed in a MR-independent manner via amplification of IGF-I receptor-mediated signaling. Because aldosterone applied alone stimulates both collagen and elastin deposition in cultures of fibroblasts and in cultures of skin explants derived from dermal stretch marks, we postulate that this steroid should be used in the treatment of damaged skin that loses its volume and elasticity. Moreover, aldosterone applied in conjunction with spironolactone or eplerenone induces matrix remodeling and exclusively enhances elastogenesis in cultures of fibroblasts and explants derived from dermal scars and keloids. We therefore propose that intra-lesional injection of these factors should be considered in therapy for disfiguring dermal lesions and especially in prevention of their recurrence after surgical excision.

  10. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

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    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-05-15

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approx. 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

  11. Prolyl hydroxylation in elastin is not random.

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    Schmelzer, Christian E H; Nagel, Marcus B M; Dziomba, Szymon; Merkher, Yulia; Sivan, Sarit S; Heinz, Andrea

    2016-10-01

    This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. The study for the first time shows that prolyl hydroxylation is highly regulated in elastin. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Evaluation of Anti-aging Compounds Using the Promoters of Elastin and Fibrillin-1 Genes Combined with a Secreted Alkaline Phosphatase Reporter in Normal Human Fibroblasts.

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    Lin, Chih-Chien; Yang, Chao-Hsun; Kuo, Wan-Ting; Chen, Cheng-Yu

    2015-01-01

    Elastic fibers are major constituents of the extracellular matrix (ECM) in dynamic tissues in the human body, and regulation of elastin and fibrillin-1 expression mediates the formation of these fibers. Traditional assays for the measurement of elastin and fibrillin-1, such as western blotting, Luna staining and immunostaining, are relatively complex and time-consuming. Thus, a relatively simple assay system that also provides rational results is urgently needed. In the study, we aimed to develop a human cell-based assay system that can be used to analyze functional compounds using the promoters of elastin (ELN) and fibrillin-1 (FBN1) genes integrated with a secreted alkaline phosphatase (SEAP) reporter in normal human fibroblast cells. We used this system to assess anti-aging compounds. We used several regulators of elastinogenesis, including retinol, coenzyme Q10, deoxyArbutin and Elestan(TM) (Manilkara multinervis leaf extract), to verify the efficacy of this assay system. Our results demonstrate that this assay system can be used as a fast and realistic method for identifying anti-aging components for future use in foods, cosmetics and drugs.

  13. Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.

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    Merrilees, Mervyn J; Falk, Ben A; Zuo, Ning; Dickinson, Michelle E; May, Barnaby C H; Wight, Thomas N

    2014-06-19

    Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Elastin in the Liver

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    Jiri Kanta

    2016-10-01

    Full Text Available A characteristic feature of liver cirrhosis is the accumulation of large amounts of connective tissue with the prevailing content of type I collagen. Elastin is a minor connective tissue component in normal liver but it is actively synthesized by hepatic stellate cells and portal fibroblasts in diseased liver. The accumulation of elastic fibers in later stages of liver fibrosis may contribute to the decreasing reversibility of the disease with advancing time. Elastin is formed by polymerization of tropoelastin monomers. It is an amorphous protein highly resistant to the action of proteases that forms the core of elastic fibers. Microfibrils surrounding the core are composed of fibrillins that bind a number of proteins involved in fiber formation. They include microfibril-associated glycoproteins (MAGPs, microfibrillar-associated proteins (MFAPs and fibulins. Lysyl oxidase (LOX and lysyl oxidase-like proteins (LOXLs are responsible for tropoelastin cross-linking and polymerization. TGF-β complexes attached to microfibrils release this cytokine and influence the behavior of the cells in the neighborhood. The role of TGF-β as the main profibrotic cytokine in the liver is well-known and the release of the cytokines of TGF-β superfamily from their storage in elastic fibers may affect the course of fibrosis. Elastic fibres are often studied in the tissues where they provide elasticity and resilience but their role is no longer viewed as purely mechanical. Tropoelastin, elastin polymer and elastin peptides resulting from partial elastin degradation influence fibroblastic and inflammatory cells as well as angiogenesis. A similar role may be performed by elastin in the liver. This article reviews the results of the research of liver elastic fibers on the backgound of the present knowledge of elastin biochemistry and physiology. The regulation of liver elastin synthesis and degradation may be important for the outcome of liver fibrosis.

  15. A custom image-based analysis tool for quantifying elastin and collagen micro-architecture in the wall of the human aorta from multi-photon microscopy.

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    Koch, Ryan G; Tsamis, Alkiviadis; D'Amore, Antonio; Wagner, William R; Watkins, Simon C; Gleason, Thomas G; Vorp, David A

    2014-03-21

    The aorta possesses a micro-architecture that imparts and supports a high degree of compliance and mechanical strength. Alteration of the quantity and/or arrangement of the main load-bearing components of this micro-architecture--the elastin and collagen fibers--leads to mechanical, and hence functional, changes associated with aortic disease and aging. Therefore, in the future, the ability to rigorously characterize the wall fiber micro-architecture could provide insight into the complicated mechanisms of aortic wall remodeling in aging and disease. Elastin and collagen fibers can be observed using state-of-the-art multi-photon microscopy. Image-analysis algorithms have been effective at characterizing fibrous constructs using various microscopy modalities. The objective of this study was to develop a custom MATLAB-language automated image-based analysis tool to describe multiple parameters of elastin and collagen micro-architecture in human soft fibrous tissue samples using multi-photon microscopy images. Human aortic tissue samples were used to develop the code. The tool smooths, cleans and equalizes fiber intensities in the image before segmenting the fibers into a binary image. The binary image is cleaned and thinned to a fiber skeleton representation of the image. The developed software analyzes the fiber skeleton to obtain intersections, fiber orientation, concentration, porosity, diameter distribution, segment length and tortuosity. In the future, the developed custom image-based analysis tool can be used to describe the micro-architecture of aortic wall samples in a variety of conditions. While this work targeted the aorta, the software has the potential to describe the architecture of other fibrous materials, tube-like networks and connective tissues.

  16. Acquired Localized Cutis Laxa due to Increased Elastin Turnover.

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    Nygaard, Rie Harboe; Maynard, Scott; Schjerling, Peter; Kjaer, Michael; Qvortrup, Klaus; Bohr, Vilhelm A; Rasmussen, Lene J; Jemec, Gregor B E; Heidenheim, Michael

    2016-01-01

    Cutis laxa is a rare disease characterized by abnormal skin wrinkling and laxity, due to decreased elastin synthesis or structural extracellular matrix defects. We have explored elastin metabolism in a case of adult onset cutis laxa localized to the upper body of a woman. For this purpose, we obtained skin biopsies from affected and unaffected skin areas of the patient and analyzed these with microscopy, polymerase chain reaction, western blotting and cell culture experiments. Skin from the affected area lacked elastin fibers in electron microscopy but had higher mRNA expression of elastin and total RNA. Levels of an apparent tropoelastin degradation product were higher in the affected area. Fibroblast cultures from the affected area were able to produce elastin and showed higher proliferation and survival after oxidative and UVB stress compared to fibroblasts from the unaffected area. In conclusion, we report a case of acquired localized cutis laxa with a lack of elastic fibers in the skin of the patient's upper body. The lack of elastic fibers in the affected skin was combined with increased mRNA expression and protein levels of elastin. These findings indicate that elastin synthesis was increased but did not lead to deposited elastic fibers in the tissue.

  17. Acquired Localized Cutis Laxa due to Increased Elastin Turnover

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    Rie Harboe Nygaard

    2016-02-01

    Full Text Available Cutis laxa is a rare disease characterized by abnormal skin wrinkling and laxity, due to decreased elastin synthesis or structural extracellular matrix defects. We have explored elastin metabolism in a case of adult onset cutis laxa localized to the upper body of a woman. For this purpose, we obtained skin biopsies from affected and unaffected skin areas of the patient and analyzed these with microscopy, polymerase chain reaction, western blotting and cell culture experiments. Skin from the affected area lacked elastin fibers in electron microscopy but had higher mRNA expression of elastin and total RNA. Levels of an apparent tropoelastin degradation product were higher in the affected area. Fibroblast cultures from the affected area were able to produce elastin and showed higher proliferation and survival after oxidative and UVB stress compared to fibroblasts from the unaffected area. In conclusion, we report a case of acquired localized cutis laxa with a lack of elastic fibers in the skin of the patient's upper body. The lack of elastic fibers in the affected skin was combined with increased mRNA expression and protein levels of elastin. These findings indicate that elastin synthesis was increased but did not lead to deposited elastic fibers in the tissue.

  18. Biophysical characterization of a de novo elastin

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    Greenland, Kelly Nicole

    Natural human elastin is found in tissue such as the lungs, arteries, and skin. This protein is formed at birth with no mechanism present to repair or supplement the initial quantity formed. As a result, the functionality and durability of elastin's elasticity is critically important. To date, the mechanics of this ability to stretch and recoil is not fully understood. This study utilizes de novo protein design to create a small library of simplistic versions of elastin-like proteins, demonstrate the elastin-like proteins, maintain elastin's functionality, and inquire into its structure using solution nuclear magnetic resonance (NMR). Elastin is formed from cross-linked tropoelastin. Therefore, the first generation of designed proteins consisted of one protein that utilized homogony of interspecies tropoelastin by using three common domains, two hydrophobic and one cross-linking domains. Basic modifications were made to open the hydrophobic region and also to make the protein easier to purify and characterize. The designed protein maintained its functionality, self-aggregating as the temperature increased. Uniquely, the protein remained self-aggregated as the temperature returned below the critical transition temperature. Self-aggregation was additionally induced by increasing salt concentrations and by modifying the pH. The protein appeared to have little secondary structure when studied with solution NMR. These results fueled a second generation of designed elastin-like proteins. This generation contained variations designed to study the cross-linking domain, one specific hydrophobic domain, and the effect of the length of the elastin-like protein. The cross-linking domain in one variation has been significantly modified while the flanking hydrophobic domains have remained unchanged. This characterization of this protein will answer questions regarding the specificity of the homologous nature of the cross-linking domain of tropoelastin across species. A second

  19. Elastin distribution in the normal uterus, uterine leiomyomas, adenomyosis and adenomyomas: a comparison.

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    Zheng, Wei-Qiang; Ma, Rong; Zheng, Jian-Ming; Gong, Zhi-Jing

    2006-04-01

    To describe the histologic distribution of elastin in the nonpregnant human uterus, uterine leiomyomas, adenomyosis and adenomyomas. Uteri were obtained from women undergoing hysterectomy for benign conditions, including 26 cases of uterine leiomyomas, 24 cases of adenomyosis, 18 adenomyomas and 6 cases of autopsy specimens. Specific histochemical staining techniques were employed in order to demonstrate the distribution of elastin. The distribution of elastin components in the uterus was markedly uneven and showed a decreasing gradient from outer to inner myometrium. No elastin was present within leiomyomas, adenomyomas or adenomyosis. The distribution of elastin may help explain the normal function of the myometrium in labor. It implies that the uneven distribution of elastin components and absence of elastin within leiomyomas, adenomyomas and adenomyosis could be of some clinical significance. The altered elastin distribution in disease states may help explain such symptoms as dysmenorrhea in uterine endometriosis.

  20. Cues for cellular assembly of vascular elastin networks

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    Kothapalli, Chandrasekhar R.

    Elastin, a structural protein distributed in the extracellular matrix of vascular tissues is critical to the maintenance of vascular mechanics, besides regulation of cell-signaling pathways involved in injury response and morphogenesis. Thus, congenital absence or disease-mediated degradation of vascular elastin and its malformation within native vessels due to innately poor elastin synthesis by adult vascular cells compromise vascular homeostasis. Current elastin regenerative strategies using tissue engineering principles are limited by the progressive destabilization of tropoelastin mRNA expression in adult vascular cells and the unavailability of scaffolds that can provide cellular cues necessary to up-regulate elastin synthesis and regenerate faithful mimics of native elastin. Since our earlier studies demonstrated the elastogenic utility of hyaluronan (HA)-based cues, we have currently sought to identify a unique set of culture conditions based on HA fragments (0.756-2000 kDa), growth factors (TGF-beta1, IGF-1) and other biomolecules (Cu2+ ions, LOX), which will together enhance synthesis, crosslinking, maturation and fibrous elastin matrix formation by adult SMCs, under both healthy and inflammatory conditions. It was observed that TGF-beta1 (1 ng/mL) together with HA oligomers (0.2 microg/mL) synergistically suppressed SMC proliferation, enhanced tropoelastin (8-fold) and matrix elastin synthesis (5.5-fold), besides improving matrix yield (4.5-fold), possibly by increasing production and activity of lysyl oxidase (LOX). Though addition of IGF-1 alone did not offer any advantage, HA fragments (20-200 kDa) in the presence of IGF-1 stimulated tropoelastin and soluble elastin synthesis more than 2.2-fold, with HMW HA contributing for ˜5-fold increase in crosslinked matrix elastin synthesis. Similarly, 0.1 M of Cu2+ ions, alone or together with HA fragments stimulated synthesis of tropoelastin (4-fold) and crosslinked matrix elastin (4.5-fold), via increases in

  1. Elastin as a nonthrombogenic biomaterial.

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    Waterhouse, Anna; Wise, Steven G; Ng, Martin K C; Weiss, Anthony S

    2011-04-01

    Surface-induced thrombosis is a significant issue for artificial blood-contacting materials used in the treatment of cardiovascular diseases. The development of biomaterials and tissue-engineered constructs that mimic the vasculature represents a way to overcome this problem. Elastin is an extracellular matrix macromolecule that imparts arterial elasticity where it comprises up to 50% of the nonhydrated mass of the vessel. In addition to its critical role in maintaining vessel integrity and elastic properties under pulsatile flow, elastin plays an important role in signaling and regulating luminal endothelial cells and smooth muscle cells in the arterial wall. Despite its well-established significance in the vasculature and its growing use as a biomaterial in tissue engineering, the hemocompatibility of elastin is often overlooked. Past studies pointing to the potential of arterial elastin and decellularized elastin as nonthrombogenic materials have begun to be realized, with elastin scaffolds and coatings displaying increased hemocomptibility. This review explores the mechanisms of elastin's nonthrombogenicity and highlights the current problems limiting its wider application as a biomaterial. We discuss the benefits of constructing biomaterials encompassing the relevant mechanical and biological features of elastin to provide enhanced hemocompatibility to biomaterials.

  2. The protein and lipid composition of arterial elastin and its relationship to lipid accumulation in the atherosclerotic plaque.

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    Kramsch, D M; Franzblau, C; Hollander, W

    1971-08-01

    Elastin preparations from intimal layers and the media of normal and atherosclerotic human aortae were analyzed for protein and lipid content. In atherosclerotic aortae, elastin from plaques was compared with elastin from adjacent normal appearing areas of the same aorta. Arterial elastin purified by alkaline extraction appeared to be a protein-lipid complex containing free and ester cholesterol, phospholipids, and triglycerides. The lipid component of normal arterial elastin was small (1-2%). With increasing severity of atherosclerosis, there was a progressive accumulation of lipid in intimal elastin from plaques, reaching a mean lipid content of 37% in severe plaques. The increase in the lipid content of plaque elastic preparations was mainly due to large increases in cholesterol, over 80% of which was cholesteryl ester. This deposition of cholesterol in plaque elastin accounted for 20-34% of the total cholesterol content of the plaque. The increased lipid deposition in plaque elastin was associated with alterations in the amino acid composition of plaque elastin. In elastin from plaque intima, the following polar amino acids were increased significantly: aspartic acid, threonine, serine, glutamic acid, lysine, histidine, and arginine; whereas, cross-linking amino acids: desmosine, isodesmosine, and lysinonorleucine were decreased significantly. The amino acid and lipid composition of elastin from normal appearing aortic areas was comparable to that of normal arterial elastin except for intimal elastin directly adjacent to and medial elastin directly below the most severe plaques.The data indicate that the focal lipid deposition in early atherosclerotic plaques is due to a large extent to lipid accumulations in altered elastin protein of localized intimal areas. Continued lipid deposition in altered elastin appears to contribute substantially to the progressive lipid accumulation in the plaque. The study suggests that elastin of intimal elastic membranes may play

  3. Use of a collagen-elastin matrix as transport carrier system to transfer proliferating epidermal cells to human dermis in vitro.

    Science.gov (United States)

    Waaijman, Taco; Breetveld, Melanie; Ulrich, Magda; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2010-01-01

    This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10-14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed

  4. Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Leurgans, Thomas M; Spronck, Bart

    2017-01-01

    The impact of disease related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared to frequen......The impact of disease related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared...

  5. Selenoprotein P mRNA expression in human hepatic tissues

    Institute of Scientific and Technical Information of China (English)

    Chun-Li Li; Ke-Jun Nan; Tao Tian; Chen-Guang Sui; Yan-Fang Liu

    2007-01-01

    AIM: To investigate the expression of Selenoprotein P mRNA (SePmRNA) in tissues of normal liver, liver cirrhosis and hepatocellular carcinoma (HCC), and its relationship with HCC occurrence and development.METHODS: The expression of SePmRNA in tissues of normal liver, liver cirrhosis and HCC were detected by in situ hybridization using a cDNA probe.RESULTS: The enzyme digesting products of pBluescript-Human Selenoprotein P were evaluated by electrophoresis.The positive expression of SePmRNA was found in the tissues of normal liver,liver cirrhosis and HCC.The expression of SeP mRNA was found in hepatic interstitial substance,especially in endothelial cells and lymphocytes of vasculature.The positive rate of SePmRNA in normal liver tissue was 84.6% (11/13) and the positive signals appeared in the nucleus and cytoplasm,mostly in the nucleolus,and the staining granules were larger in the nucleolus and around the nucleus.The positive rate of SePmRNA in liver cirrhosis tissue was 45.O% (9/20) and the positive signals were mainly in the nucleolus and cytoplasm,being less around the nucleus and inner nucleus than that in normal liver tissue. The positive rate of SePmRNA in HCC tissue was 30.0% (9/30) and the positive signals were in the cytoplasm, but less in the nucleus.CONCLUSION: SePmRNA expression in the tissues of normal liver and HCC is significantly different (84.6% vs 30.0%, P = 0.003), suggesting that SeP might play a role in the occurrence and development of HCC.

  6. mRNA related to insulin family in human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  7. Human cytomegalovirus induces alteration of (-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  8. Evaluation of Elastin/Collagen Content in Human Dermis in-Vivo by Multiphoton Tomography—Variation with Depth and Correlation with Aging

    Directory of Open Access Journals (Sweden)

    Jean-Christophe Pittet

    2014-08-01

    Full Text Available The aim of this study was to evaluate the influence of the depth of the dermis on the measured collagen and elastin levels and to establish the correlation between the amount of these two extracellular matrix (ECM components and age. Multiphoton Microscopy (MPM that measures the autofluorescence (AF and second harmonic generation (SHG was used to quantify the levels of elastin and collagen and to determine the SAAID (SHG-to-AF Aging Index of Dermis at two different skin depths. A 50 MHz ultrasound scanner was used for the calculation of the Sub Epidermal Non Echogenic Band (SENEB. The measurements of the skin mechanical properties were done with a cutometer. All measurements were performed on two groups of 30 healthy female volunteers. The MPM showed a decrease of the quantity of collagen and elastin as a function of depth of the dermis as well as age. The SAAID was lower for the older skin in the deeper dermis. Ultrasound imaging revealed a significant decrease of SENEB as a function of aging. The mechanical properties confirmed a loss of cutaneous elasticity and firmness. Although multiphoton microscopy is a powerful technique to study the characteristics of the dermis and its age-related damage, the location of the measurements (depth remains very important for the validation of these variations. These variations do not seem to be homogeneous according to the part of the dermis that is studied.

  9. [Application of elastin in biomedical materials].

    Science.gov (United States)

    Chang, Decai; Wang, Xiaoli; Hou, Xin; Yao, Kangde

    2008-12-01

    Elastin is a natural biomedical material of great potential. Being endowed with the special crosslinking and hydrophobic structure, elastin retains many good properties such as good elasticity, ductibility, biocompatibility, biodegradability and so on. Nowadays, elastin as a material, which is gradually attracting people' s attention in the biomedical materials field, has been used as tissue engineering scaffolds, derma substitutes and other biomedical materials. In this context, a systematic review on the characteristics of elastin as a biomedical material and on the actuality of its application is presented. Future developments of elastin in the field of biomedical applications are also discussed.

  10. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  11. Functionally Distinct Tendons From Elastin Haploinsufficient Mice Exhibit Mild Stiffening and Tendon-Specific Structural Alteration.

    Science.gov (United States)

    Eekhoff, Jeremy D; Fang, Fei; Kahan, Lindsey G; Espinosa, Gabriela; Cocciolone, Austin J; Wagenseil, Jessica E; Mecham, Robert P; Lake, Spencer P

    2017-11-01

    Elastic fibers are present in low quantities in tendon, where they are located both within fascicles near tenocytes and more broadly in the interfascicular matrix (IFM). While elastic fibers have long been known to be significant in the mechanics of elastin-rich tissue (i.e., vasculature, skin, lungs), recent studies have suggested a mechanical role for elastic fibers in tendons that is dependent on specific tendon function. However, the exact contribution of elastin to properties of different types of tendons (e.g., positional, energy-storing) remains unknown. Therefore, this study purposed to evaluate the role of elastin in the mechanical properties and collagen alignment of functionally distinct supraspinatus tendons (SSTs) and Achilles tendons (ATs) from elastin haploinsufficient (HET) and wild type (WT) mice. Despite the significant decrease in elastin in HET tendons, a slight increase in linear stiffness of both tendons was the only significant mechanical effect of elastin haploinsufficiency. Additionally, there were significant changes in collagen nanostructure and subtle alteration to collagen alignment in the AT but not the SST. Hence, elastin may play only a minor role in tendon mechanical properties. Alternatively, larger changes to tendon mechanics may have been mitigated by developmental compensation of HET tendons and/or the role of elastic fibers may be less prominent in smaller mouse tendons compared to the larger bovine and human tendons evaluated in previous studies. Further research will be necessary to fully elucidate the influence of various elastic fiber components on structure-function relationships in functionally distinct tendons.

  12. Glucose Suppresses Biological Ferroelectricity in Aortic Elastin

    Science.gov (United States)

    Liu, Yuanming; Wang, Yunjie; Chow, Ming-Jay; Chen, Nataly Q.; Ma, Feiyue; Zhang, Yanhang; Li, Jiangyu

    2013-01-01

    Elastin is an intriguing extracellular matrix protein present in all connective tissues of vertebrates, rendering essential elasticity to connective tissues subjected to repeated physiological stresses. Using piezoresponse force microscopy, we show that the polarity of aortic elastin is switchable by an electrical field, which may be associated with the recently discovered biological ferroelectricity in the aorta. More interestingly, it is discovered that the switching in aortic elastin is largely suppressed by glucose treatment, which appears to freeze the internal asymmetric polar structures of elastin, making it much harder to switch, or suppressing the switching completely. Such loss of ferroelectricity could have important physiological and pathological implications from aging to arteriosclerosis that are closely related to glycation of elastin. PMID:23679639

  13. Glucose Suppresses Biological Ferroelectricity in Aortic Elastin

    Science.gov (United States)

    Liu, Yuanming; Wang, Yunjie; Chow, Ming-Jay; Chen, Nataly Q.; Ma, Feiyue; Zhang, Yanhang; Li, Jiangyu

    2013-04-01

    Elastin is an intriguing extracellular matrix protein present in all connective tissues of vertebrates, rendering essential elasticity to connective tissues subjected to repeated physiological stresses. Using piezoresponse force microscopy, we show that the polarity of aortic elastin is switchable by an electrical field, which may be associated with the recently discovered biological ferroelectricity in the aorta. More interestingly, it is discovered that the switching in aortic elastin is largely suppressed by glucose treatment, which appears to freeze the internal asymmetric polar structures of elastin, making it much harder to switch, or suppressing the switching completely. Such loss of ferroelectricity could have important physiological and pathological implications from aging to arteriosclerosis that are closely related to glycation of elastin.

  14. In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials.

    Science.gov (United States)

    Heinz, Andrea; Ruttkies, Christoph K H; Jahreis, Günther; Schräder, Christoph U; Wichapong, Kanin; Sippl, Wolfgang; Keeley, Fred W; Neubert, Reinhard H H; Schmelzer, Christian E H

    2013-04-01

    Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials. The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

    Institute of Scientific and Technical Information of China (English)

    朱长虹; 田红

    2001-01-01

    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  16. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  17. Stable RNA hairpins in 88 coding regions of human mRNA

    Institute of Scientific and Technical Information of China (English)

    PAN Min; WANG Chuanming; LIU Ciquan

    2004-01-01

    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  18. Endothelin converting-enzyme-1 mRNA expression in human cardiovascular disease

    NARCIS (Netherlands)

    Bohnemeier, H; Pinto, YM; Horkay, F; Toth, M; Juhasz-Nagy, A; Orzechowski, HD; Bohm, M; Paul, M

    1998-01-01

    Endothelin-1 converting-enzyme (ECE-1) cleaves the precursor, big-endothelin-1, to the active peptide endothelin-1. The aim of this study was to investigate whether ECE-1 mRNA expression is modified in human cardiovascular disease. Tissue samples from the left human atrium were analyzed for ECE-1 ex

  19. mRNA transfection of mouse and human neural stem cell cultures.

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  20. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR......-based poly(A) test and in vitro poly(A) assay. Taken together, our results suggest that hCPEB1 and hGLD-2 are antagonizing factors regulating p53 mRNA stability....

  1. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry

    2011-01-01

    concentrations of AMH, inhibin-B, progesterone and estradiol. Androgen Receptor mRNA expression in granulosa cells, and the FF content of androgens, both showed a highly significant positive association with to the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  2. Detection of histidine decarboxylase mRNA in human vascular smooth muscle and endothelial cells.

    Science.gov (United States)

    Tippens, A S; Gruetter, C A

    2004-06-01

    The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA. HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively. Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons. These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

  3. Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

    Science.gov (United States)

    Muiznieks, Lisa D; Reichheld, Sean E; Sitarz, Eva E; Miao, Ming; Keeley, Fred W

    2015-10-01

    Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties.

  4. Fingerprinting desmosine-containing elastin peptides.

    Science.gov (United States)

    Schräder, Christoph U; Heinz, Andrea; Majovsky, Petra; Schmelzer, Christian E H

    2015-05-01

    Elastin is a vital protein of the extracellular matrix of jawed vertebrates and provides elasticity to numerous tissues. It is secreted in the form of its soluble precursor tropoelastin, which is subsequently cross-linked in the course of the elastic fiber assembly. The process involves the formation of the two tetrafunctional amino acids desmosine (DES) and isodesmosine (IDES), which are unique to elastin. The resulting high degree of cross-linking confers remarkable properties, including mechanical integrity, insolubility, and long-term stability to the protein. These characteristics hinder the structural elucidation of mature elastin. However, MS(2) data of linear and cross-linked peptides released by proteolysis can provide indirect insights into the structure of elastin. In this study, we performed energy-resolved collision-induced dissociation experiments of DES, IDES, their derivatives, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type CxHyN, suggesting that the pyridinium core of DES/IDES remains intact even at relatively high collision energies. The finding of these specific product ions enabled the development of a similarity-based scoring algorithm that was successfully applied on LC-MS/MS data of bovine elastin digests for the identification of DES-/IDES-cross-linked peptides. This approach facilitates the straightforward investigation of native cross-links in elastin.

  5. Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Palumbo, Fabio Salvatore [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); Pitarresi, Giovanna, E-mail: giovanna.pitarresi@unipa.it [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); Institute of Biophysics at Palermo, Italian National Research Council, Via Ugo La Malfa 153, 90146 Palermo (Italy); Fiorica, Calogero [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); Rigogliuso, Salvatrice; Ghersi, Giulio [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Biologia Cellulare, Università degli Studi di Palermo, Viale delle Scienze ed. 16, 90128, Palermo (Italy); Giammona, Gaetano [Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123, Palermo (Italy); IBIM-CNR, Via Ugo La Malfa 153, 90146 Palermo (Italy)

    2013-07-01

    In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control. - Highlights: ► Hyaluronic acid (HA) has been functionalized with ethylenediamine (EDA). ► Amino groups of HA-EDA allow the reaction with α-elastin and ethylene glycol diglycidyl ether (EGDGE). ► Chemical scaffolds of HA-EDA-graft-α-elastin/EGDGE have been characterized. ► The presence of α-elastin affects porosity, swelling and enzymatic degradation of scaffolds. ► The presence of α-elastin improves attachment, viability and proliferation of fibroblasts and smooth muscle cells.

  6. Tissue-specific mRNA expression profiles of human solute carrier 35 transporters.

    Science.gov (United States)

    Nishimura, Masuhiro; Suzuki, Satoshi; Satoh, Tetsuo; Naito, Shinsaku

    2009-01-01

    Pairs of forward and reverse primers and TaqMan probes specific to each of 23 human solute carrier 35 (SLC35) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The mRNA expression of SLC35As, SLC35Bs, SLC35Cs, SLC35D1, SLC35D2, SLC35Es, and SLC35F5 was found to be ubiquitous in both adult and fetal tissues. SLC35D3 mRNA was expressed at the highest levels in the adult retina. SLC35F1 mRNA was expressed at high levels in the adult and fetal brain. SLC35F2 mRNA was expressed at the highest levels in the adult salivary gland. Both SLC35F3 and SLC35F4 mRNAs were expressed at the highest levels in the adult cerebellum. Further, individual differences in the mRNA expression levels of human SLC35 transporters in the liver were also evaluated. Our newly determined expression profiles were used to study the gene expression in 31 adult human tissues, 6 fetal human tissues, and 3 cell lines, and tissues with high transcriptional activity for human SLC35 transporters were identified. These results are expected to be valuable for research concerning the clinical diagnosis of disease.

  7. The rules and impact of nonsense-mediated mRNA decay in human cancers.

    Science.gov (United States)

    Lindeboom, Rik G H; Supek, Fran; Lehner, Ben

    2016-10-01

    Premature termination codons (PTCs) cause a large proportion of inherited human genetic diseases. PTC-containing transcripts can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). However, the efficiency of NMD varies; it is inefficient when a PTC is located downstream of the last exon junction complex (EJC). We used matched exome and transcriptome data from 9,769 human tumors to systematically elucidate the rules of NMD targeting in human cells. An integrated model incorporating multiple rules beyond the canonical EJC model explains approximately three-fourths of the non-random variance in NMD efficiency across thousands of PTCs. We also show that dosage compensation may sometimes mask the effects of NMD. Applying the NMD model identifies signatures of both positive and negative selection on NMD-triggering mutations in human tumors and provides a classification for tumor-suppressor genes.

  8. Angiotensin II receptor mRNA expression and vasoconstriction in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Pantev, Emil; Emilson, Malin;

    2004-01-01

    Angiotensin II is a potent vasoconstrictor that is implicated in the pathogenesis of hypertension, heart failure and atherosclerosis. In the present study, angiotensin II receptor mRNA expression levels were quantified by real-time polymerase chain reaction and the vasocontractile responses...... to angiotensin II were characterised by in vitro pharmacology in endothelium-denuded human coronary arteries. Angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptor mRNA expression levels were significantly down-regulated in arteries from patients with heart failure as compared to controls. The angiotensin II...

  9. Ischemic heart disease induces upregulation of endothelin receptor mRNA in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;

    2004-01-01

    and controls using real-time polymerase chain reaction (real-time PCR). In addition, the suitability of organ culture as a model mimicking endothelin receptor changes in cardiovascular disease was evaluated by in vitro pharmacology and real-time PCR. Endothelin ETA and ETB receptor mRNA levels were......Endothelin has been implicated in the pathogenesis of ischemic heart disease and congestive heart failure. The aims were to quantify endothelin type A (ETA) and type B (ETB) receptor mRNA levels in human coronary arteries from patients with ischemic heart disease, congestive heart failure...

  10. Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    林茂芳; 魏国庆; 黄河; 蔡真

    2004-01-01

    Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

  11. The influence of elastin-like recombinant polymer on the self-renewing potential of a 3D tissue equivalent derived from human lamina propria fibroblasts and oral epithelial cells.

    Science.gov (United States)

    Kinikoglu, Beste; Rodríguez-Cabello, José Carlos; Damour, Odile; Hasirci, Vasif

    2011-09-01

    Three-dimensional epithelial tissue equivalents tend to lose their self-renewing potential progressively during culture as their epithelial cells lose their proliferative capacity with time. Even though the tissue engineered construct can mimic the native tissue well, it rapidly degrades after implantation due to the insufficient number of proliferating cells in the equivalent. In the present study we demonstrate for the first time that the use of an elastin-like recombinant polymer (ELR) engineered to contain the cell adhesion peptide RGD can result in a 3D tissue equivalent with high self-renewing potential, containing as many proliferative cells as the native tissue itself. The 3D tissue equivalent was reconstructed by the coculture of human lamina propria fibroblasts and oral epithelial cells in the nanofibrous ELR-collagen scaffold. Histological, immunohistological and transmission electron microscopic analyses of this oral mucosa equivalent demonstrated the expression of markers characteristic of epithelial proliferation (Ki67) and differentiation (keratin 13), and also the presence of a pluristratified epithelium and an ultrastructurally well-organized basement membrane expressing laminin 332. The synthesis of new extracellular matrix by the fibroblasts was also demonstrated. The scaffold proposed here presents great potential for tissue engineering applications, and also for studies of epithelial proliferation, and epithelial disorders including carcinogenesis.

  12. Reduced fructosamine-3-kinase activity and its mRNA in human distal colorectal carcinoma.

    Science.gov (United States)

    Notarnicola, M; Caruso, Maria G; Tutino, V; Guerra, V; Frisullo, S; Altomare, D F; Misciagna, G

    2010-09-01

    Fructosamine-3-Kinase (FN3K) is an enzyme phosphorilating fructoselysine (FL) residues on glycated proteins, resulting in the production of protein-bound FL-3-phosphate. The pathological role of the non-enzymatic modification of proteins by reducing sugars has become increasingly evident in various types of disorders, including the cancer. In this study, our aim was to study FN3K enzyme activity, as well as its mRNA in human colorectal cancer (CRC). Thirty consecutive CRC patients undergoing surgery of the colon were enrolled in the study. FN3K enzymatic activity and gene expression were analyzed using a radiometric assay and quantitative RT-PCR, respectively. FN3K is a functionally active enzyme in human colon tissue, without significant differences between normal mucosa and cancer. The mean level of FN3K mRNA was significantly lower in cancer than in the corresponding normal colorectal mucosa The colorectal tumors located on the left side showed lower levels of both enzymatic activity and mRNA FN3K than tumors located in the right side of colon. This paper is the first studying FN3K enzyme activity in human CRC, showing a significant relationship between enzymatic activity, its mRNA and tumor side.

  13. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    Science.gov (United States)

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  14. The human mRNA encoding the Goodpasture antigen is alternatively spliced.

    Science.gov (United States)

    Bernal, D; Quinones, S; Saus, J

    1993-06-05

    The noncollagenous (NC1) domain of the human collagen alpha 3(IV)-chain is the primary target of autoantibodies produced in Goodpasture syndrome and, therefore, has been designated as the Goodpasture antigen. In this report, we show that Goodpasture antigen mRNA undergoes processing to at least two alternatively spliced forms in a variety of human tissues, resulting in the exclusion of sequence encoded by either one or two exons. Interestingly, no alternatively spliced forms were observed in bovine or rat tissues. The derived amino acid sequences of the two variant mRNA forms are identical and significantly shorter than that arising from the complete Goodpasture antigen mRNA. They lack the carboxyl-terminal region contributing to the formation of the Goodpasture epitope and all but one of the cysteines found in the complete form. These sequence characteristics suggest that, if translated, the variant Goodpasture antigen is likely to be defective in triple helix formation and no longer reactive with Goodpasture autoantibodies. Although each tissue expressing Goodpasture antigen displayed a specific mRNA pattern, the complete form was always the most abundant and was present at levels apparently unrelated to whether or not the organ of origin is a potential target in Goodpasture syndrome. Furthermore, the antigen sequence was identical in the kidneys of normal and Goodpasture-affected individuals, and no major differences in the expression of the complete and spliced forms were observed.

  15. Fourier transform infrared spectroscopy to quantify collagen and elastin in an in vitro model of extracellular matrix degradation in aorta.

    Science.gov (United States)

    Cheheltani, Rabee; McGoverin, Cushla M; Rao, Jayashree; Vorp, David A; Kiani, Mohammad F; Pleshko, Nancy

    2014-06-21

    Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues.

  16. Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hu; Feng-Hua Chen; Yi-Rong Li; Lin Wang

    2004-01-01

    AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay,to detect human telomerase reverse transcriptase (hTERT)messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and dinicopathological parameters in patients with gastric cancer.METHODS: A real-time quantitative RT-PCR (RQ-PCR)based on TaqMan fluorescence methodoloogy and the LightCyder system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100× (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, χ2 test and Fisher's exact test.RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27±0.89 and 0.93±0.18,respectively (t= 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation.CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

  17. The action of neutrophil serine proteases on elastin and its precursor.

    Science.gov (United States)

    Heinz, Andrea; Jung, Michael C; Jahreis, Günther; Rusciani, Anthony; Duca, Laurent; Debelle, Laurent; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2012-01-01

    This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  18. Interaction between fatty acid and the elastin network

    NARCIS (Netherlands)

    Vreeswijk, van J.

    1995-01-01

    The aim of the present study was to investigate the interaction between salts of fatty acids (FAS) and elastin. Absorption of fatty acids in elastin may affect the elasticity of elastin-containing tissue. Such phenomena could, for instance, be of relevance for the understanding of the

  19. Interaction between fatty acid salts and the elastin network.

    NARCIS (Netherlands)

    Vreeswijk, van J.

    1995-01-01

    The aim of the present study was to investigate the interaction between salts of fatty acids (FAS) and elastin. Absorption of fatty acids in elastin may affect the elasticity of elastin-containing tissue. Such phenomena could, for instance, be of relevance for the understanding of the formation of a

  20. Stimulus responsive elastin biopolymers: applications in medicine and biotechnology

    Science.gov (United States)

    Chilkoti, Ashutosh; Christensen, Trine; MacKay, J Andrew

    2013-01-01

    Elastin-like polypeptides (ELPs) are artificial polypeptides, derived from Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide repeats found in human tropoelastin, that reversibly coacervate above a critical temperature. Genetically encodable ELPs are monodisperse, stimuli responsive, and biocompatible, properties that make them attractive for drug delivery and tissue engineering. The potential of ELPs to self-assemble into nanostructures in response to environmental triggers is another interesting feature of these polypeptides that promises to lead to a host of new applications. PMID:17055770

  1. Resistance artery creatine kinase mRNA and blood pressure in humans.

    Science.gov (United States)

    Karamat, Fares A; Oudman, Inge; Ris-Stalpers, Carrie; Afink, Gijs B; Keijser, Remco; Clark, Joseph F; van Montfrans, Gert A; Brewster, Lizzy M

    2014-01-01

    Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

  2. Transcription pattern of UL131A-128 mRNA in clinical strains of human cytomegalovirus

    Indian Academy of Sciences (India)

    Zhengrong Sun; Gaowei Ren; Yanping Ma; Ning Wang; Yaohua Ji; Ying Qi; Mali Li; Rong He; Qiang Ruan

    2010-09-01

    Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by sequencing. Mean while, clones containing UL131A-128 transcripts in an HCMV cDNA library of a clinical strain were selected and sequenced. It was demonstrated that UL131A-128 mRNA was expressed with immediately early, early and late kinetics. Sequences obtained by RT-PCR showed that the UL131A gene consisted of two exons and the coding region of the UL130 gene was not interrupted by any intron in the region as reported earlier. However, the transcript of the UL128 gene showed two patterns: one pattern consisted of three exons as reported earlier; the other contained the three exons and also the first intron. Moreover, the above characteristics of UL131A-128 spliced transcripts were confirmed by the sequences of clones selected from the HCMV cDNA library. Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with the 3′-coterminal, although the initiation points of their mRNA may be different. The variation in the transcripts found in our study indicated the complex nature of transcription of UL131A-128 genes in clinical strains of HCMV.

  3. Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein.

    Science.gov (United States)

    Wang, Yanlin; Hacker, Amy; Murray-Stewart, Tracy; Fleischer, Jennifer G; Woster, Patrick M; Casero, Robert A

    2005-03-15

    The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30-90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA.

  4. Studies on P1, P2 Protamine mRNA in Sperms of Human,Rat and Mouse

    Institute of Scientific and Technical Information of China (English)

    吴小芳; 陈晖; 费仁仁; 陈松; 曹坚

    2001-01-01

    Objective To investigate the existence of the protamine Mrna in sperms of human,rat and mouse Methods By means of RT-PCR technique, protamine cDNA fragments were obtained from total RNA of the mature sperms of human, rat and mouse respectively.Results mRNA of protamine gene was found in the mature sperm of human, rat and mouse. The protamine cDNA with an abnormal head obtained by PT-TCR in rat sperm was much less tn number than that in the normal rat sperm.Conclusion mRNA in the sperms might represent the condition of corresponding gene expression during spermatogenesis.

  5. An Investigation of Voice Quality in Individuals with Inherited Elastin Gene Abnormalities

    Science.gov (United States)

    Watts, Christopher R.; Awan, Shaheen N.; Marler, Jeffrey A.

    2008-01-01

    The human elastin gene (ELN) is responsible for the generation of elastic fibres in the extracellular matrix of connective tissue throughout the body, including the vocal folds. Individuals with Supravalvular aortic stenosis (SVAS) and Williams syndrome (WS) lack one normal ELN allele due to heterozygous ELN abnormalities, resulting in a…

  6. Dendritic transport element of human arc mRNA confers RNA degradation activity in a translation-dependent manner.

    Science.gov (United States)

    Ninomiya, Kensuke; Ohno, Mutsuhito; Kataoka, Naoyuki

    2016-11-01

    Localization of mRNA in neuronal cells is a critical process for spatiotemporal regulation of gene expression. Cytoplasmic localization of mRNA is often conferred by transport elements in 3' untranslated region (UTR). Activity-regulated cytoskeleton-associated protein (arc) mRNA is one of the localizing mRNAs in neuronal cells, and its localization is mediated by dendritic targeting element (DTE). As arc mRNA has introns in its 3' UTR, it was thought that arc mRNA is a natural target of nonsense-mediated mRNA decay (NMD). Here, we show that DTE in human arc 3' UTR has destabilizing activity of RNA independent of NMD pathway. DTE alone was able to cause instability of the reporter mRNA and this degradation was dependent on translation. Our results indicate that DTE has dual activity in mRNA transport and degradation, which suggests the novel spatiotemporal regulation mechanism of activity-dependent degradation of the mRNA.

  7. Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering.

    Science.gov (United States)

    Palumbo, Fabio Salvatore; Pitarresi, Giovanna; Fiorica, Calogero; Rigogliuso, Salvatrice; Ghersi, Giulio; Giammona, Gaetano

    2013-07-01

    In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control.

  8. Alterations in the renal elastin-elastase system in type 1 diabetic nephropathy identified by proteomic analysis.

    Science.gov (United States)

    Thongboonkerd, Visith; Barati, Michelle T; McLeish, Kenneth R; Benarafa, Charaf; Remold-O'Donnell, Eileen; Zheng, Shirong; Rovin, Brad H; Pierce, William M; Epstein, Paul N; Klein, Jon B

    2004-03-01

    Diabetes now accounts for >40% of patients with ESRD. Despite significant progress in understanding diabetic nephropathy, the cellular mechanisms that lead to diabetes-induced renal damage are incompletely defined. For defining changes in protein expression that accompany diabetic nephropathy, the renal proteome of 120-d-old OVE26 transgenic mice with hypoinsulinemia, hyperglycemia, hyperlipidemia, and proteinuria were compared with those of background FVB nondiabetic mice (n = 5). Proteins derived from whole-kidney lysate were separated by two-dimensional PAGE and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Forty-one proteins from 300 visualized protein spots were differentially expressed in diabetic kidneys. Among these altered proteins, expression of monocyte/neutrophil elastase inhibitor was increased, whereas elastase IIIB was decreased, leading to the hypothesis that elastin expression would be increased in diabetic kidneys. Renal immunohistochemistry for elastin of 325-d-old FVB and OVE26 mice demonstrated marked accumulation of elastin in the macula densa, collecting ducts, and pelvicalyceal epithelia of diabetic kidneys. Elastin immunohistochemistry of human renal biopsies from patients with type 1 diabetes (n = 3) showed increased elastin expression in renal tubular cells and the interstitium but not glomeruli. These results suggest that coordinated changes in elastase inhibitor and elastase expression result in increased tubulointerstitial deposition of elastin in diabetic nephropathy. The identification of these coordinated changes in protein expression in diabetic nephropathy indicates the potential value of proteomic analysis in defining pathophysiology.

  9. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    Science.gov (United States)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  10. Combined sequencing of mRNA and DNA from human embryonic stem cells.

    Science.gov (United States)

    Mertes, Florian; Kuhl, Heiner; Wruck, Wasco; Lehrach, Hans; Adjaye, James

    2016-06-01

    Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.

  11. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

    Directory of Open Access Journals (Sweden)

    Jordan R Plews

    Full Text Available BACKGROUND: Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. CONCLUSION/SIGNIFICANCE: Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  12. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

    Science.gov (United States)

    Viguerie-Bascands, N; Saulnier-Blache, J S; Dandine, M; Dauzats, M; Daviaud, D; Langin, D

    1999-03-05

    Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans. Copyright 1999 Academic Press.

  13. Heritability in the efficiency of nonsense-mediated mRNA decay in humans

    KAUST Repository

    Seoighe, Cathal

    2010-07-21

    Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

  14. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    LENUS (Irish Health Repository)

    Seoighe, Cathal

    2010-01-01

    BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  15. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    Directory of Open Access Journals (Sweden)

    Cathal Seoighe

    Full Text Available BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  16. Interaction between fatty acid and the elastin network

    OpenAIRE

    Vreeswijk, van, M.

    1995-01-01

    The aim of the present study was to investigate the interaction between salts of fatty acids (FAS) and elastin. Absorption of fatty acids in elastin may affect the elasticity of elastin-containing tissue. Such phenomena could, for instance, be of relevance for the understanding of the formation of atherosclerotic plaque in blood vessel walls.

    Chapter I gives a general introduction on the relevance of this study and an outline of the thesis. Furthermore, it contains informatio...

  17. Two Human ACAT2 mRNA Variants Produced by Alternative Splicing and Coding for Novel Isoenzymes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min YAO; Bo-Liang LI; Can-Hua WANG; Bao-Liang SONG; Xin-Ying YANG; Zhen-Zhen WANG; Wei QI; Zhi-Xin LIN; Catherine C. Y. CHANG; Ta-Yuan CHANG

    2005-01-01

    Acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms,named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA).Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RTPCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.

  18. Inhibin mRNA in Human Corpora Lutea during the Menstrual Cycle

    Institute of Scientific and Technical Information of China (English)

    孙志达; 沈卫雄; 王寒正; 任佩锋; 张永莲; 龚岳亭

    1994-01-01

    The steady state of mRNAs for the α-and βA-subunits of inhibin was investigated in human corpora lutea(CL) during the menstrual cycle by Northern blot analysis with more sensitive cRNA probes. It revealed α 1.6kb mRNA for a subunit and 6.0,4.0, 2.8 and 1.8kb transcripts for βA subunit, Both of α-and βA inhibin mRNAs were detectable in CLs during early, mid and late luteal phases, but not in retrogressive CL after day-27 of the cycle or in tissues of ovarian stroma. An overall profile of the

  19. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  20. A classical brown adipose tissue mRNA signature partly overlaps with brite in the supraclavicular region of adult humans

    DEFF Research Database (Denmark)

    Jespersen, Naja Zenius; Larsen, Therese Juhlin; Peijs, Lone

    2013-01-01

    . A similar mRNA expression profile was observed when comparing isolated human adipocytes from BAT and white adipose tissue (WAT) depots, differentiated in vitro. In conclusion, our data suggest that human BAT might consist of both classical brown and recruitable brite adipocytes, an observation important...

  1. CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

    DEFF Research Database (Denmark)

    Christiansen, M; Kveiborg, Marie; Kassem, M

    2000-01-01

    reverse transcriptase-polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40...

  2. Cross-linking Electrospun Polydioxanone-Soluble Elastin Blends: Material Characterization

    Directory of Open Access Journals (Sweden)

    Michael J. McClure

    2008-03-01

    Full Text Available The purpose of this study was to establish whether material properties of elastin co-electrospun with polydioxanone (PDO would change over time in both the uncross-linked state and the cross-linked state. First, uncross-linked scaffolds were placed in phosphate buffered saline (PBS for three separate time periods: 15 minutes, 1 hour, and 24 hours, and subsequently tested using uniaxial materials testing. Several cross-linking reagents were then investigated to verify their ability to crosslink elastin: 1-ethyl-3-(dimethylaminopropyl-carbodiimide (EDC, ethylene glycol diglycidyl ether (EGDE, and genipin. Uniaxial tensile testing was performed on scaffolds cross-linked with EDC and genipin, yielding results that warranted further investigation for PDO-elastin blends. Material properties of the cross-linked scaffolds were then found within range of both pig femoral artery and human femoral artery. These results demonstrate PDO-elastin blends could potentially be favorable as vascular grafts, thus warranting future in vitro and in vivo studies.

  3. Quantification of human telomerase RNA (Htr) and human telomerase reverse transcriptase (Htert)Mrna in testicular tissue of infertile patients

    Institute of Scientific and Technical Information of China (English)

    Mark Schrader; Markus Miller; Ridiger Heicappell; Bernd Straub; Kurt Miller

    2001-01-01

    Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presenting with non-obstructive azoospemia. Methods: hTR and hTERT mRNA expression were quantified in 38 cryopreserved testicular tissue specimens by fluorescence real-time reverse transcription- polymerase chain reaction (RT-PCR)in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additional semithin sectioning preparation in cases with maturation arrest ( n = 12) and Sertoli-cell-only syndrome ( n = 12). Results: The average normalized hTERT expression (NhTERT) Was 131.9 ± 48.0 copies (mean ± SD) in tissue specimens with full spermatogenesis, NhTERT = 51.2 ± 17.2 copies in those with maturation arrest and NhTERT = 2.7 ± 2.4 copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of NhTERT (NhTR) had a predictive value of 86.8 % (55.3 % ) for correct classification in one of the three histological subgroups.Conclusion: Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue enables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies is thus well suited for supplementing the histopathological evaluation.

  4. Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

    Science.gov (United States)

    Agoston, D V; Colburn, S; Krajniak, K G; Waschek, J A

    1992-05-25

    Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

  5. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    Science.gov (United States)

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  6. Human low molecular weight neurofilament (NFL) mRNA interacts with a predicted p190RhoGEF homologue (RGNEF) in humans.

    Science.gov (United States)

    Volkening, Kathryn; Leystra-Lantz, Cheryl; Strong, Michael J

    2010-01-01

    In the mouse, p190RhoGEF is a low molecular weight neurofilament (NFL) mRNA stability factor that is involved in NF aggregate formation in neurons. A human homologue of this protein has not been described. Our objective was to identify a human homologue of p190RhoGEF, and to determine its interaction with human NFL mRNA. We used sequence homology searches to predict a human homologue (RGNEF), and RT-PCR to determine the expression of mRNA in ALS and neuropathologically normal control tissues. Gel shift assays determined the interaction of RGNEF with human NFL mRNA in vitro, while IP-RT-PCR and gel shift assays were used to confirm the interaction in tissue lysates. We determined that RGNEF is a human homologue of p190RhoGEF, and that its RNA is expressed in both brain and spinal cord. While RGNEF and NFL mRNA interact directly in vitro, interestingly they only appear to interact in ALS lysates and not in controls. These data add another player to the family of NFL mRNA stability regulators, and raise the intriguing possibility that the mechanism by which p190RhoGEF contributes to murine neuronal NF aggregate formation may be important to human ALS NF aggregate formation.

  7. Expression of ET(A) and ET(B) receptor mRNA in human cerebral arteries

    DEFF Research Database (Denmark)

    Hansen-Schwartz, J; Szok, D; Edvinsson, L

    2002-01-01

    The vascular effects of endothelins (ET) are in mammals mediated via two receptor subtypes, endothelin A (ET(A), mainly constrictive) and endothelin B (ET(B), mainly dilating) receptors. We have examined the presence of ET(A) and ET(B) receptor mRNA using the reverse transcription polymerase chain...... reaction (RT-PCR) in both normal human cerebral arteries and cerebral arteries from patients with cerebrovascular disease. Two vessel preparations were studied: macroscopic arteries and microvessels, the latter obtained through a sensitive separation method. In endothelial cells both ET(A) and ET......(B) receptor mRNA was detected. In almost all samples from normal cerebral arteries only ET(A) receptor mRNA was detected, whereas in vessel samples from patients with cerebrovascular disease as well as cerebral neoplasms, additional ET(B) receptor mRNA was detected significantly more frequently...

  8. Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Niedzwiecka, Anna; Stepinski, Janusz; Mazza, Catherine; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2005-09-01

    The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

  9. Ischemic heart disease down-regulates angiotensin type 1 receptor mRNA in human coronary arteries

    DEFF Research Database (Denmark)

    Wackenfors, Angelica; Emilson, Malin; Ingemansson, Richard;

    2004-01-01

    Angiotensin II is important in the development of cardiovascular disease. In the present study, angiotensin II receptor mRNA levels were quantified by real-time polymerase chain reaction (real-time PCR) in human coronary arteries from patients with ischemic heart disease and controls. Furthermore......, the suitability of artery culture for studying angiotensin receptor changes was evaluated by in vitro pharmacology and real-time PCR. The angiotensin type 1 (AT1) receptor mRNA levels were down-regulated in human coronary arteries from patients with ischemic heart disease as compared to controls (P

  10. Acquired Localized Cutis Laxa due to Increased Elastin Turnover

    DEFF Research Database (Denmark)

    Nygaard, Rie Harboe; Maynard, Scott; Schjerling, Peter

    2016-01-01

    Cutis laxa is a rare disease characterized by abnormal skin wrinkling and laxity, due to decreased elastin synthesis or structural extracellular matrix defects. We have explored elastin metabolism in a case of adult onset cutis laxa localized to the upper body of a woman. For this purpose, we...

  11. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-07-22

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  12. Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

    DEFF Research Database (Denmark)

    Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

    2004-01-01

    Nine widely distributed pesticides were recently demonstrated to posses potential estrogenic properties in oestrogen receptor (ER) transactivation and/or E-screen assays. We tested the effect of these nine pesticides on the human ERα and ERβ mRNA steady state levels in the mamma cancer fibroblast...

  13. Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance

    DEFF Research Database (Denmark)

    Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette

    2016-01-01

    In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30...

  14. Determination of the human cardiomyocyte mRNA and miRNA differentiation network by fine-scale profiling.

    Science.gov (United States)

    Babiarz, Joshua E; Ravon, Morgane; Sridhar, Sriram; Ravindran, Palanikumar; Swanson, Brad; Bitter, Hans; Weiser, Thomas; Chiao, Eric; Certa, Ulrich; Kolaja, Kyle L

    2012-07-20

    To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.

  15. Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes : a long and a short form

    NARCIS (Netherlands)

    Molnar, K; van der Steege, G; Jonkman, MF; Nijenhuis, Albertine; Husz, S; van der Meer, JB; Pas, HH

    We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular

  16. mRNA Transcriptomics of Galectins Unveils Heterogeneous Organization in Mouse and Human Brain

    Directory of Open Access Journals (Sweden)

    Sebastian John

    2016-12-01

    Full Text Available Background: Galectins, a family of non-classically secreted, β-galactoside binding proteins is involved in several brain disorders; however no systematic knowledge on the normal neuroanatomical distribution and functions of galectins exits. Hence, the major purpose of this study was to understand spatial distribution and predict functions of galectins in brain and also compare the degree of conservation vs. divergence between mouse and human species. The latter objective was required to determine the relevance and appropriateness of studying galectins in mouse brain which may ultimately enable us to extrapolate the findings to human brain physiology and pathologies.Results: In order to fill this crucial gap in our understanding of brain galectins, we analyzed the in situ hybridization (ISH and microarray data of adult mouse and human brain respectively, from the Allen Brain Atlas, to resolve each galectin-subtype’s spatial distribution across brain distinct cytoarchitecture. Next, transcription factors (TFs that may regulate galectins were identified using TRANSFAC software and the list obtained was further curated to sort TFs on their confirmed transcript expression in the adult brain. Galectin-TF cluster analysis, gene-ontology annotations and co-expression networks were then extrapolated to predict distinct functional relevance of each galectin in the neuronal processes. Data shows that galectins have highly heterogeneous expression within and across brain sub-structures and are predicted to be the crucial targets of brain enriched TFs. Lgals9 had maximal spatial distribution across mouse brain with inferred predominant roles in neurogenesis while LGALS1 was ubiquitously expressed in human. Limbic region associated with learning, memory and emotions and substantia nigra associated with motor movements showed strikingly high expression of LGALS1 and LGALS8 in human vs. mouse brain. The overall expression profile of galectin-8 was most

  17. Quantifiable mRNA transcripts for tamoxifen-metabolising enzymes in human endometrium.

    Science.gov (United States)

    Singh, Maneesh N; Stringfellow, Helen F; Walsh, Michael J; Ashton, Kate M; Paraskevaidis, Evangelos; Abdo, Khalil R; Martin-Hirsch, Pierre L; Phillips, David H; Martin, Francis L

    2008-07-10

    Tamoxifen has been used in the management of receptor-positive breast cancer for >20 years. Usage confers an elevated risk of developing endometrial carcinoma. Its mechanism of carcinogenicity remains unresolved with controversy as to whether or not this is mediated through a genotoxic mechanism. Usage is not only associated with an elevated occurrence of endometrioid endometrial carcinoma, but also type 2 and mixed epithelial-stromal tumours (MESTs) that have a poorer prognosis. Following hysterectomy, endometrial tissues (n=18) classified as benign (n=6), non-tamoxifen-associated carcinoma (n=6) and tamoxifen-associated carcinoma (n=6) were obtained; quantitative gene expression was performed. Employing real-time RT-PCR, the relative gene expressions of phase I/II metabolic enzymes CYP1A2, CYP1B1 and CYP3A4, cathechol-O-methyltransferase (COMT) and SULT2A1 were ascertained. Measurable mRNA transcripts, especially for those genes associated with tamoxifen bioactivation, were quantifiable in all the tissues examined. Whether this is evidence that generation of genotoxic tamoxifen metabolites may occur in human endometrial tissue remains to be ascertained.

  18. The mRNA expression of SATB1 and SATB2 in human breast cancer

    Directory of Open Access Journals (Sweden)

    Mansel Robert

    2009-07-01

    increasing tumour grade (grade 1 vs. grade 3 p = 0.035. SATB2 was associated with ER positivity (ER(- vs. ER(+ p = 0.0283 within ductal carcinomas. Higher transcript levels showed a significant association with poorer OS (p = 0.0433. Conclusion SATB1 mRNA expression is significantly associated with poor prognostic parameters in breast cancer, including increasing tumour grade, TNM stage and NPI. SATB2 mRNA expression is significantly associated with increasing tumour grade and poorer OS. These results are consistent with the notion that SATB1 acts as a 'master genome organizer' in human breast carcinogenesis.

  19. Changes in elastin, elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Wight Thomas N

    2008-05-01

    Full Text Available Abstract Background COPD is characterised by loss of alveolar elastic fibers and by lack of effective repair. Elastic fibers are assembled at cell surfaces by elastin binding protein (EBP, a molecular chaperone whose function can be reversibility inhibited by chondroitin sulphate of matrix proteoglycans such as versican. This study aimed to determine if alveoli of patients with mild to moderate COPD contained increased amounts of versican and a corresponding decrease in EBP, and if these changes were correlated with decreases in elastin and FEV1. Methods Lung samples were obtained from 26 control (FEV1 ≥ 80% predicted, FEV1/VC >0.7 and 17 COPD patients (FEV1 ≥ 40% – 1/VC ≤ 0.7 who had undergone a lobectomy for bronchial carcinoma. Samples were processed for histological and immuno-staining. Volume fractions (Vv of elastin in alveolar walls and alveolar rims were determined by point counting, and versican and EBP assessed by grading of staining intensities. Results Elastin Vv was positively correlated with FEV1 for both the alveolar walls (r = 0.66, p 1 in both regions (r = 0.30 and 0.32 respectively, p 1. Conversely, staining intensities for EBP in alveolar walls and rims and were positively correlated with FEV1 (r = 0.43 and 0.46, p Conclusion Patients with mild to moderate COPD show progressively increased immuno-staining for versican and correspondingly decreased immuno-staining for EBP, with decreasing values of FEV1. These findings may explain the lack of repair of elastic fibers in the lungs of patients with moderate COPD. Removal of versican may offer a strategy for effective repair.

  20. FUCOIDIN INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN FROM INDUCING HUMAN PERIPHERAL BLOOD MONOCYTE EXPRESSION OF PROINFLAMMATORY CYTOKINES mRNA

    Institute of Scientific and Technical Information of China (English)

    雷新军; 马爱群; 任冰稳; 耿涛; 张葳; 白玲

    2003-01-01

    Objective To study the significance of scavenger receptor class A(SR-A)in mediating human peripheral blood monocyte to uptake oxidized low density lipoprotein(OxLDL) and promoting the atherosclerotic immuno-pathological lesion in the local blood vessel. Methods With the Digoxenin-labeled Oligonucleotide-probes In situ Hybridization, this research investigated the effects of OxLDL on the mRNA expression of proinflammatory cytokines including MCP-1, bFGF, PDGF and IL-10 in the human peripheral blood monocyte and whether fucoidin, a peculiarly inhibitory ligand for SR-A, would influence this process. Results Monocyte was significantly increased the mRNA expression of MCP-1, bFGF, PDGF and IL-10 in a dose-dependent manner after incubating with OxLDL (10,15,20,25,30·mg·L-1, respectively)for 24 hours(P<0.001). Fucoidin(50,100,150,200,250·mg·mL-1, respectively)completely inhibited OxLDL(20·mg·L-1)from inducing monocyte the mRNA expression of above proinflammatory cytokines(P<0.001). Conclusion OxLDL can stimulate human peripheral blood monocyte to give expression to proinflammatory cytokines mRNA in a dose-dependent manner, while a peculiarly inhibitory ligand for SR-A-fucoidin has an obviously opposed role.

  1. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

    Directory of Open Access Journals (Sweden)

    David G Hendrickson

    2009-11-01

    Full Text Available MicroRNAs (miRNAs regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes and ribosome density (the average number of ribosomes bound per unit length of coding sequence were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the

  2. Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

    Institute of Scientific and Technical Information of China (English)

    Kui-Sheng Chen; Lan Zhang; Lin Tang; Yun-Han Zhang; Dong-Ling Gao; Liang Yan; Lei Zhang

    2005-01-01

    AIM: To investigate the effects of anti-sense oligonucleotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells.METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied byin situ hybridization.RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25±0.25, ASODN2: 2.21±0.23, ASODN3:2.23±0.23, ASODN4:2.25±0.24 vs N-ODN: 3.47±2.80 or non- transfected group: 3.51±2.93 respectively, P<0.05).CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.

  3. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    Science.gov (United States)

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  4. Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15 mRNA in Various Human Cell Types

    Directory of Open Access Journals (Sweden)

    Sang Mee Hwang

    2013-01-01

    Full Text Available CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC, two peripheral bloods (PB, 12 granulocyte products, natural killer (NK-92, B-lymphocyte (CO88BV59-1, K-562 leukemia cell line, human embryonic stem cell (hESC, and human fibroblasts (HF. HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs.

  5. Gene Manipulation of Human Embryonic Stem Cells by In Vitro-Synthesized mRNA for Gene Therapy.

    Science.gov (United States)

    Wang, Xiao Li; Yu, Li; Ding, Yan; Guo, Xing Rong; Yuan, Ya Hong; Li, Dong Sheng

    2015-01-01

    The difficulty in producing genetically modified human embryonic stem cells (hESCs) limits research on their applications. Virus-based gene transfer is not safe for clinical use, whereas DNAbased non-viral methods are not efficient or safe, and mRNA-based methods are useful for genetic manipulation. In this study, we easily obtained multiple types and large amounts of in vitro-synthesized mRNA by PCR. The efficiency of different transfection methods was studied by flow cytometry. The effect of different mRNA modifications on protein translation efficiency and dynamics of luciferase mRNA expression in hESCs were studied using a bioluminescence imaging system. The pluripotency of hESCs after transfection was studied by immunofluorescence. In vitro-synthesized pancreatic-duodenal homeobox 1 (PDX1) mRNA was used to induce the differentiation of hESCs into insulin-producing cells. We found that electroporation is the most efficient transfection method, and it produces more than 95% transgene expression in multiple hESC lines. Synthesized mRNA with a combination of a polyA tail, cap and base analogues is more efficiently translated into protein in hESCs compared with single-modified mRNA. Transfection of mRNA into hESCs by trypsinizing the cells into single-cell suspensions did not affect their pluripotency, and multiple types of mRNAs can be transfected into hESCs efficiently. We found that PDX-1 mRNA transfection significantly improved the expression level of genes related to beta cells and differentiated cells that express insulin and C-peptide. ELISA analysis validate the insulin secretion of islet-like cell clusters in response to glucose stimulation. Our results indicate that electroporation of in vitro-synthesized mRNA is useful for genetic manipulation of hESCs and differentiation of hESCs into particular cell types, and this finding will pave the way for clinical applications of this method.

  6. [The expression of human telomerase reverse transcriptase mRNA and its significance in acute leukemia].

    Science.gov (United States)

    Meng, Xiao-Li; Lin, Mao-Fang; Jin, Jie

    2003-02-01

    To investigate the expression of hTERT mRNA in bone marrow mononuclear cells (MNCs) from acute leukemia patients, the method of semi-quntitative RT-PCR was used to examine the expression of hTERT mRNA in marrow MNCs, and the telomerase activity of marrow MNCs was determined with the method of TRAP-PCR-ELISA by using a commercial kit. The results indicated that the expression of hTERT mRNA of marrow MNCs in 30 untreated AL patients was markedly higher than that in 12 CR cases (0.71 +/- 0.34 vs 0.43 +/- 0.25, P < 0.05) and 6 normal volunteers (0.71 +/- 0.34 vs 0.22 +/- 0.21, P < 0.01), respectively. Telomerase activity of marrow MNCs in 30 untreated AL patients was significantly higher than that in 12 CR cases (0.235 +/- 0.395 vs 0.012 +/- 0.015, P = 0.007). Moreover, there was a positive correlation between the hTERT mRNA synthesis and telomerase activity in AL cells (r = 0.421, P < 0.01). The pencentage of blast cells in marrow smear of the untreated AL patients was positively correlated with both the expression of hTERT mRNA and the telomerase activity of bone marrow MNCs (r = 0.457, P < 0.05 and r = 0.411, P < 0.05), respectively. It is concluded that the expression of hTERT mRNA in bone marrow MNCs from untreated AL patients was correlated with their telomerase activity. It is suggested that the expression of hTERT mRNA leukemic cells indicates their higher proliferation ability.

  7. Oxidative stress induced Interleukin-32 mRNA expression in human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Kudo Megumi

    2012-03-01

    Full Text Available Abstract Background Chronic obstructive pulmonary disease (COPD is characterized by airflow obstruction and persistent inflammation in the airways and lung parenchyma. Oxidative stress contributes to the pathogenesis of COPD. Interleukin (IL-32 expression has been reported to increase in the lung tissue of patients with COPD. Here, we show that IFNγ upregulated IL-32 expression and that oxidative stress augmented IFNγ-induced-IL-32 expression in airway epithelial cells. We further investigated transcriptional regulation responsible for IFNγ induced IL-32 expression in human airway epithelial cells. Methods Human bronchial epithelial (HBE cells were stimulated with H2O2 and IFNγ, and IL-32 expression was evaluated. The cell viability was confirmed by MTT assay. The intracellular signaling pathways regulating IL-32 expression were investigated by examining the regulatory effects of MAPK inhibitors and JAK inhibitor after treatment with H2O2 and IFNγ, and by using a ChIP assay to identify transcription factors (i.e. c-Jun, CREB binding to the IL-32 promoter. Promoter activity assays were conducted after mutations were introduced into binding sites of c-Jun and CREB in the IL-32 promoter. IL-32 expression was also examined in HBE cells in which the expression of either c-Jun or CREB was knocked out by siRNA of indicated transcription factors. Results There were no significant differences of cell viability among groups. After stimulation with H2O2 or IFNγ for 48 hours, IL-32 expression in HBE cells was increased by IFNγ and synergistically upregulated by the addition of H2O2. The H2O2 augmented IFNγ induced IL-32 mRNA expression was suppressed by a JNK inhibitor, but not by MEK inhibitor, p38 inhibitor, and JAK inhibitor I. Significant binding of c-Jun and CREB to the IL-32 promoter was observed in the IFNγ + H2O2 stimulated HBE cells. Introducing mutations into the c-Jun/CREB binding sites in the IL-32 promoter prominently suppressed its

  8. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles.

    Science.gov (United States)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte; Pedersen, Bente K; Saltin, Bengt; Pilegaard, Henriette

    2006-09-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

  9. Deletions of the elastin gene in Williams Syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Greenberg, F.; Nickerson, E.; McCaskill, C. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    To investigate deletions in the elastin gene in patients with Williams Syndrome (WS), we screened 37 patients and their parents for deletions in the elastin gene by both fluorescence in situ hybridization (FISH) using cosmid cELN272 containing the 5{prime} end of the elastin gene and by polymerase chain reaction (PCR) using a primer pair which amplifies intron 17 in the elastin gene, producing a polymorphic amplification product. Thirty-two patients have been investigated by both the FISH and PCR techniques, one patient was studied only by PCR, and 4 patients were studied only by FISH. Overall, 34 of 37 patients (92%) were deleted for the elastin gene. Using the PCR marker, 14 patients were informative and 12 were shown to be deleted [maternal (n=5) and paternal (n=7)]. Using cosmid cELN272, 33 of 36 patients demonstrated a deletion of chromosome 7q11.23. In one family, both the mother and daughter were deleted due to an apparently de novo deletion arising in the mother. Three patients were not deleted using the elastin cosmid; 2 of these patients have classic WS. Another non-deleted patient has the typical facial features and hypercalcemia but normal intelligence. These three patients will be important in delineating the critical region(s) responsible for the facial features, hypercalcemia, mental retardation and supravalvular aortic stenosis (SVAS). There was not an absolute correlation between deletions in elastin and SVAS, although these individuals may be at risk for other cardiovascular complications such as hypertention. Since the majority of WS patients are deleted for a portion of the elastin gene, most likely this marker will be an important diagnostic tool, although more patients will need to be studied. Those patients who are not deleted but clinically have WS will be missed using only this one marker. Expansion of the critical region to other loci and identification of additional markers will be essential for identifying all patients with WS.

  10. Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

    DEFF Research Database (Denmark)

    Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren

    2006-01-01

    Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression...... in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4.......5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied...

  11. How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

    Institute of Scientific and Technical Information of China (English)

    Yanfang Guo; Yuan Chen; Hui Jiang; Lijun Tan; Jingyun Xie; Xuezhen Zhu; Songping Liang; Hongwen Deng; Peng Xiao; Shufeng Lei; Feiyan Deng; Gary Guishan Xiao; Yaozhong Liu; Xiangding Chen; Liming Li; Shan Wu

    2008-01-01

    A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However,only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation (r=0.235, P<0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component (r=0.643, P<0.0001) and the lowest correlation was obtained for genes of regulation (r=0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples (r=0.296, P<0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels.However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.

  12. Extraction and characterization of elastin from poultry skin

    Science.gov (United States)

    Nadalian, Mehdi; Yusop, Salma Mohamad; Mustapha, Wan Aida Wan; Azman, Mohd Azri; Babji, Abdul Salam

    2013-11-01

    Poultry by-products have a great economic potential that need to be exploited. Poultry skin could be utilized to produce elastin, which is often incorporated in the production of functional food or medicine due to its antioxidative properties. This study was conducted to determine the physicochemical and microstructural characteristics of elastins isolated from broiler and spent hen skin. Analyses including proximate and amino acid composition along with transmission electron microscopy (TEM) were carried out. In this study, elastin was successfully extracted from broiler and spent hen skin using three successive solvents extract of NaCl, acetone and NaOH respectively. It was apparent that the fat content of extracted elastin from broiler skin was higher (P < 0.05) than spent hen's, with both samples recording less than 1% fat. Moreover, broiler skin elastin also had a higher protein content (68.3%) than spent hen's (67.8%). Both skin sources contained glycine as the major amino acid (19-20%), followed by glutamic acid, proline, alanine and arginine. The results of TEM indicated that the use of collagenase enzyme or further purification efforts should be incorporated along with the extraction methods used because of the presence of collagen and other debris in the resultant elastin.

  13. Extraction and characterization of elastin from poultry skin

    Energy Technology Data Exchange (ETDEWEB)

    Nadalian, Mehdi; Yusop, Salma Mohamad; Mustapha, Wan Aida Wan; Babji, Abdul Salam [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia); Azman, Mohd Azri [Strategic Livestock Research Centre, Malaysian Agricultural Research and Development Institute, MARDI Headquarters, 43400, Serdang, Selangor (Malaysia)

    2013-11-27

    Poultry by-products have a great economic potential that need to be exploited. Poultry skin could be utilized to produce elastin, which is often incorporated in the production of functional food or medicine due to its antioxidative properties. This study was conducted to determine the physicochemical and microstructural characteristics of elastins isolated from broiler and spent hen skin. Analyses including proximate and amino acid composition along with transmission electron microscopy (TEM) were carried out. In this study, elastin was successfully extracted from broiler and spent hen skin using three successive solvents extract of NaCl, acetone and NaOH respectively. It was apparent that the fat content of extracted elastin from broiler skin was higher (P < 0.05) than spent hen’s, with both samples recording less than 1% fat. Moreover, broiler skin elastin also had a higher protein content (68.3%) than spent hen’s (67.8%). Both skin sources contained glycine as the major amino acid (19–20%), followed by glutamic acid, proline, alanine and arginine. The results of TEM indicated that the use of collagenase enzyme or further purification efforts should be incorporated along with the extraction methods used because of the presence of collagen and other debris in the resultant elastin.

  14. Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

    Science.gov (United States)

    Soreq, H; Zevin-Sonkin, D; Razon, N

    1984-06-01

    To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.

  15. Caffeine Bitterness is Related to Daily Caffeine Intake and Bitter Receptor mRNA Abundance in Human Taste Tissue.

    Science.gov (United States)

    Lipchock, Sarah V; Spielman, Andrew I; Mennella, Julie A; Mansfield, Corrine J; Hwang, Liang-Dar; Douglas, Jennifer E; Reed, Danielle R

    2017-01-01

    We investigated whether the abundance of bitter receptor mRNA expression from human taste papillae is related to an individual's perceptual ratings of bitter intensity and habitual intake of bitter drinks. Ratings of the bitterness of caffeine and quinine and three other bitter stimuli (urea, propylthiouracil, and denatonium benzoate) were compared with relative taste papilla mRNA abundance of bitter receptors that respond to the corresponding bitter stimuli in cell-based assays ( TAS2R4, TAS2R10, TAS2R38, TAS2R43, and TAS2R46). We calculated caffeine and quinine intake from a food frequency questionnaire. The bitterness of caffeine was related to the abundance of the combined mRNA expression of these known receptors, r = 0.47, p = .05, and self-reported daily caffeine intake, t(18) = 2.78, p = .012. The results of linear modeling indicated that 47% of the variance among subjects in the rating of caffeine bitterness was accounted for by these two factors (habitual caffeine intake and taste receptor mRNA abundance). We observed no such relationships for quinine but consumption of its primary dietary form (tonic water) was uncommon. Overall, diet and TAS2R gene expression in taste papillae are related to individual differences in caffeine perception.

  16. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    Science.gov (United States)

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin

    2016-07-21

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

  17. Ammonium Chloride Ingestion Attenuates Exercise-Induced mRNA Levels in Human Muscle.

    Directory of Open Access Journals (Sweden)

    Johann Edge

    Full Text Available Minimizing the decrease in intracellular pH during high-intensity exercise training promotes greater improvements in mitochondrial respiration. This raises the intriguing hypothesis that pH may affect the exercise-induced transcription of genes that regulate mitochondrial biogenesis. Eight males performed 10x2-min cycle intervals at 80% VO2speak intensity on two occasions separated by ~2 weeks. Participants ingested either ammonium chloride (ACID or calcium carbonate (PLA the day before and on the day of the exercise trial in a randomized, counterbalanced order, using a crossover design. Biopsies were taken from the vastus lateralis muscle before and after exercise. The mRNA level of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α, citrate synthase, cytochome c and FOXO1 was elevated at rest following ACID (P0.05; the difference in PGC-1α mRNA content 2 h post-exercise between ACID and PLA was not significant (P = 0.08. Thus, metabolic acidosis abolished the early post-exercise increase of PGC-1α mRNA and the mRNA of downstream mitochondrial and glucose-regulating proteins. These findings indicate that metabolic acidosis may affect mitochondrial biogenesis, with divergent responses in resting and post-exercise skeletal muscle.

  18. Sex differences in global mRNA content of human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Amy C Maher

    Full Text Available Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, beta-hydroxyacyl-CoA dehydrogenase, CD36. One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2, trifunctional protein beta (HADHB, catalase, lipoprotein lipase (LPL, and uncoupling protein-2 (UCP-2 were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI, peroxisome proliferator-activated receptor (PPARdelta were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARdelta, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally "primed" for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined.

  19. mRNA expression levels in failing human hearts predict cellular electrophysiological remodeling: a population-based simulation study.

    Directory of Open Access Journals (Sweden)

    John Walmsley

    Full Text Available Differences in mRNA expression levels have been observed in failing versus non-failing human hearts for several membrane channel proteins and accessory subunits. These differences may play a causal role in electrophysiological changes observed in human heart failure and atrial fibrillation, such as action potential (AP prolongation, increased AP triangulation, decreased intracellular calcium transient (CaT magnitude and decreased CaT triangulation. Our goal is to investigate whether the information contained in mRNA measurements can be used to predict cardiac electrophysiological remodeling in heart failure using computational modeling. Using mRNA data recently obtained from failing and non-failing human hearts, we construct failing and non-failing cell populations incorporating natural variability and up/down regulation of channel conductivities. Six biomarkers are calculated for each cell in each population, at cycle lengths between 1500 ms and 300 ms. Regression analysis is performed to determine which ion channels drive biomarker variability in failing versus non-failing cardiomyocytes. Our models suggest that reported mRNA expression changes are consistent with AP prolongation, increased AP triangulation, increased CaT duration, decreased CaT triangulation and amplitude, and increased delay between AP and CaT upstrokes in the failing population. Regression analysis reveals that changes in AP biomarkers are driven primarily by reduction in I[Formula: see text], and changes in CaT biomarkers are driven predominantly by reduction in I(Kr and SERCA. In particular, the role of I(CaL is pacing rate dependent. Additionally, alternans developed at fast pacing rates for both failing and non-failing cardiomyocytes, but the underlying mechanisms are different in control and heart failure.

  20. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    Science.gov (United States)

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  1. Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

    Science.gov (United States)

    Exline, C M; Feng, Z; Stoltzfus, C M

    2008-04-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

  2. Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

    Science.gov (United States)

    Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

    2011-07-01

    RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

  3. Enzymatically stable 5' mRNA cap analogs: synthesis and binding studies with human DcpS decapping enzyme.

    Science.gov (United States)

    Kalek, Marcin; Jemielity, Jacek; Darzynkiewicz, Zbigniew M; Bojarska, Elzbieta; Stepinski, Janusz; Stolarski, Ryszard; Davis, Richard E; Darzynkiewicz, Edward

    2006-05-01

    Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.

  4. Effect of acute resistance exercise and sex on human patellar tendon structural and regulatory mRNA expression

    DEFF Research Database (Denmark)

    Sullivan, B.E.; Carroll, C.C.; Jemiolo, B.;

    2009-01-01

    (6 men and 6 women). Collagen type I, collagen type III, and MMP-2 were downregulated (P 0.05) 24 h after RE. All other genes remained unchanged (P > 0.05) after RE. Women had higher resting mRNA expression (P ... = 0.08) toward lower resting expression of MMP-3 than men. All other genes were not influenced (P > 0.05) by sex. Acute RE appears to stimulate a change in collagen type I, collagen type III, and MMP-2 gene regulation in the human patellar tendon. Sex influences the structural and regulatory m...... and mechanical properties, it is uncertain what structural and regulatory components contribute to these responses. We measured the mRNA expression of tendon's main fibrillar collagens (type I and type III) and the main proteoglycans (decorin, biglycan, fibromodulin, and versican) and the regulatory enzymes MMP...

  5. Expression of LL-37, Human beta Defensin-2, and CCR6 mRNA in Patients with Psoriasis Vulgaris

    Institute of Scientific and Technical Information of China (English)

    李东升; 李家文; 段逸群; 周小勇

    2004-01-01

    To investigate whether LL-37 and human beta defensin-2 (hBD-2) is related to the patients with psoriasis seldom having skin infections and explore the role of the two peptides and CCR6 (the receptor of hBD-2) in the pathogenesis of psoriasis, the expression levels of mRNA of LL-37, hBD-2, and CCR6 in skin lesions of patients with psoriasis vulgaris were detected by using RT-PCR. The results showed that the mRNA expression levels of the two peptides and CCR6 in psoriatic lesions all increased compared with the normal skin (P<0. 001). It was suggested that upregulated expression of LL-37 and hBD-2 might be the main reason that result in the the skin of patients with psoriasis being seldom infected, and the two peptides and CCR6 might play crucial roles in the pathogenesis of psoriasis.

  6. Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

    Science.gov (United States)

    Rubtsova, Maria P; Sizova, Daria V; Dmitriev, Sergei E; Ivanov, Dmitri S; Prassolov, Vladimir S; Shatsky, Ivan N

    2003-06-20

    A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

  7. Induction of mRNA expression of osteogenesis-related genes by guaiacol in human dental pulp cells.

    Science.gov (United States)

    Kato, Takashi; Shirayama, Kumiko; Tsutsui, Takeo W; Tsutsui, Takeki

    2010-07-01

    To investigate the stimulating effect of endodontic medications on the mRNA expression of some osteogenesis-related genes associated with reparative dentinogenesis and hard-tissue formation, human dental pulp cells (D824 cells) were treated with calcium hydroxide (Ca (OH)(2)), formocresol, or guaiacol. The effect on growth was determined by growth curves of D824 cells treated for 1-3 days with 0.03-0.3 mM Ca (OH)(2), 0.0007%-0.0014% formocresol, or 0.24-2.43 mM guaiacol. The mitotic activity of individual cells and the mRNA expression of the osteogenesis-related genes for alkaline phosphatase (ALP), type I collagen (COL-1), and bone sialoprotein (BSP) in the cells treated for 24 h with the same concentrations of the medications as described above were determined by colony-forming efficiency and by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, respectively. Cellular growth and mitotic activity were scarcely affected by Ca (OH)(2), but were significantly reduced by formocresol or guaiacol. The mRNA expression of the osteogenesis-related genes was little affected by Ca (OH)(2) or formocresol, but was significantly enhanced by guaiacol. The results indicate that guaiacol may stimulate the mRNA expression of genes associated with reparative dentinogenesis and hard-tissue formation in human dental pulp cells, suggesting that the novel property of guaiacol provides new insights into the utilization of guaiacol in endodontic therapy.

  8. Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes.

    Directory of Open Access Journals (Sweden)

    Anna A Shvedova

    Full Text Available As the application of carbon nanotubes (CNT in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans.In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8, were compared with expression profiles of non-exposed (n = 7 workers (e.g., professional and/or technical staff from the same manufacturing facility.Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs.This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers

  9. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  10. Molecular modeling of protein materials: case study of elastin

    Science.gov (United States)

    Tarakanova, Anna; Buehler, Markus J.

    2013-09-01

    Molecular modeling of protein materials is a quickly growing area of research that has produced numerous contributions in fields ranging from structural engineering to medicine and biology. We review here the history and methods commonly employed in molecular modeling of protein materials, emphasizing the advantages for using modeling as a complement to experimental work. We then consider a case study of the protein elastin, a critically important ‘mechanical protein’ to exemplify the approach in an area where molecular modeling has made a significant impact. We outline the progression of computational modeling studies that have considerably enhanced our understanding of this important protein which endows elasticity and recoil to the tissues it is found in, including the skin, lungs, arteries and the heart. A vast collection of literature has been directed at studying the structure and function of this protein for over half a century, the first molecular dynamics study of elastin being reported in the 1980s. We review the pivotal computational works that have considerably enhanced our fundamental understanding of elastin's atomistic structure and its extraordinary qualities—focusing on two in particular: elastin's superb elasticity and the inverse temperature transition—the remarkable ability of elastin to take on a more structured conformation at higher temperatures, suggesting its effectiveness as a biomolecular switch. Our hope is to showcase these methods as both complementary and enriching to experimental approaches that have thus far dominated the study of most protein-based materials.

  11. Monoclonal anti-elastin antibody labelled with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Lab. de Radiofarmacia; Porto, Luis Cristovao M.S.; Gutfilen, Bianca [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes; Souza, J.E.Q. [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Frier, Malcolm [University Hospital, Nottingham (United Kingdom). Dept. of Medical Physics

    1999-11-01

    Technetium-99m ({sup 99m} Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with {sup 99m} Tc and stannous chloride (Sn C L{sub 2}) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the {sup 99m} Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with {sup 99m} Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the {sup 99m} Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author) 4 refs., 7 figs., 6 tabs.

  12. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

    Science.gov (United States)

    Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

    1997-01-01

    Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

  13. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

    Science.gov (United States)

    Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

    1997-01-01

    Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

  14. CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells▿

    Science.gov (United States)

    Piao, Xianghua; Zhang, Xue; Wu, Ligang; Belasco, Joel G.

    2010-01-01

    MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal. PMID:20065043

  15. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Directory of Open Access Journals (Sweden)

    Dong Zhao

    2017-01-01

    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  16. Epigenetic Regulation of Dopamine Transporter mRNA Expression in Human Neuroblastoma Cells

    Science.gov (United States)

    Green, Ashley L.; Hossain, Muhammad M.; Tee, Siew C.; Zarbl, Helmut; Guo, Grace L.; Richardson, Jason R.

    2016-01-01

    The dopamine transporter (DAT) is a key regulator of dopaminergic neurotransmission. As such, proper regulation of DAT expression is important to maintain homeostasis, and disruption of DAT expression can lead to neurobehavioral dysfunction. Based on genomic features within the promoter of the DAT gene, there is potential for DAT expression to be regulated through epigenetic mechanisms, including DNA methylation and histone acetylation. However, the relative contribution of these mechanisms to DAT expression has not been empirically determined. Using pharmacologic and genetic approaches, we demonstrate that inhibition of DNA methyltransferase (DNMT) activity increased DAT mRNA approximately 1.5–2 fold. This effect was confirmed by siRNA knockdown of DNMT1. Likewise, the histone deacetylase (HDAC) inhibitors valproate and butyrate also increased DAT mRNA expression, but the response was much more robust with expression increasing over tenfold. Genetic knockdown of HDAC1 by siRNA also increased DAT expression, but not to the extent seen with pharmacological inhibition, suggesting additional isoforms of HDAC or other targets may contribute to the observed effect. Together, these data identify the relative contribution of DNMTs and HDACs in regulating expression. These finding may aid in understanding the mechanistic basis for changes in DAT expression in normal and pathophysiological states. PMID:25963949

  17. Regulation of CD163 mRNA and soluble CD163 protein in human adipose tissue in vitro.

    Science.gov (United States)

    Fjeldborg, Karen; Møller, Holger J; Richelsen, Bjørn; Pedersen, Steen B

    2014-10-01

    CD163-positive macrophages are highly expressed in the human adipose tissue (AT) particularly from obese individuals. However, little is known about the regulation of CD163 mRNA and the protein level of sCD163 in human AT. We aimed to examine the regulation of CD163 and sCD163 in AT. Human s.c. AT samples (n=5) were stimulated with dexamethasone (DEX; 200  nmol/l), lipopolysaccharide (LPS; 100  ng/ml), or DEX+LPS for various time periods up to 24  h. Gene expressions of CD163, ADAM17, IL10, and TNFA (TNF) were measured by RT-PCR. Protein levels of sCD163, IL10, and TNFα (TNF) were measured by ELISA. Furthermore, AT was separated into stromal and adipocyte fraction. We found that CD163 mRNA was strongly expressed in the stromal vascular fraction but hardly detectable in the isolated adipocytes. Incubating whole AT with DEX significantly up-regulated CD163 (PCD163 (P>0.05). By contrast, the protein level of sCD163 was not affected by DEX (P>0.05), but LPS significantly increased the level of sCD163 and TNFα (PCD163 and the release of sCD163 are differentially regulated in human AT. Moreover, similar to studies on differentiated blood monocytes, TNFα and sCD163 are concomitantly released in human AT by LPS, which also up-regulate ADAM17. © 2014 Society for Endocrinology.

  18. Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression▿

    Science.gov (United States)

    Exline, C. M.; Feng, Z.; Stoltzfus, C. M.

    2008-01-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication. PMID:18272582

  19. Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

    Energy Technology Data Exchange (ETDEWEB)

    Grzybowska, Ewa A., E-mail: ewag@coi.waw.pl [Cancer Center Institute, Roentgena 5, 02-781 Warsaw (Poland)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Functional characteristics of intronless genes (IGs). Black-Right-Pointing-Pointer Diseases associated with IGs. Black-Right-Pointing-Pointer Origin and evolution of IGs. Black-Right-Pointing-Pointer mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associated diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.

  20. Prevalence of Human Papillomavirus Infection in Unselected SurePath Samples Using the APTIMA HPV mRNA Assay

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte M;

    2013-01-01

    The APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA...... agreement between APTIMA and HC2. This is the first APTIMA study using SurePath samples on the PANTHER platform. The trends in positivity rates on SurePath samples for APTIMA, HC2, and LBC were consistent with studies based on PreservCyt samples, and the agreement between the two HPV assays was substantial....... The high proportions of women testing positive suggest that in countries with a high HPV prevalence, caution will be needed if HPV tests, including mRNA-based tests, are to replace LBC....

  1. Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ying-Nian Yu

    2004-01-01

    AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing.METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced.Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA.Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced.RESULTS: One of the CYP2D6cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4),and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples,only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6gene were detected. The third variant was the skipped exon 3, and 153 bp was lost.CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

  2. Human 8-oxoguanine DNA glycosylase 1 mRNA expression as an oxidative stress exposure biomarker of cooking oil fumes.

    Science.gov (United States)

    Cherng, Shur-Hueih; Huang, Kuo Hao; Yang, Sen-Chih; Wu, Tzu-Chin; Yang, Jia-Ling; Lee, Huei

    2002-02-01

    Epidemiological studies have indicated that the exposure to carcinogenic components formed during the cooking of food might be associated with lung cancer risk of Chinese women. Previous studies have confirmed that cooking oil fumes from frying fish (COF) contained relatively high amount of benzo[a]pyrene, 2-methyl-3,8-dimethylimidazo[4,5-f] qunoxaline, benzene, and 1,3-butadiene, reported in fumes from heated soybean oil. Thus, we consider that oxidative stress induced by COF may play a role in lung cancer development among Chinese women. To verify whether the oxidative DNA damage was induced by COF, high-performance liquid chromatography (HPLC) analysis data showed that the levels of 8-hydroxydeoxyguanine (8-OH dG) were increased in a dose-dependent manner when calf thymus DNA reacted with various concentrations of COF. Since human 8-oxoguanine DNA glycosylase 1 (hOGG1) was a repair enzyme for removing 8- OH dG from damaged DNA, we hypothesized that hOGG1 mRNA may be used to assess the risk of oxidative damage induced by the exposure of COF. The results from reverse-transcription polymerase chain reaction showed that the hOGG1 mRNA expression was induced by hydrogen peroxide (H2O2) and COF in human lung adenocarcinoma CL-3 cells. To elucidate whether hOGG1 mRNA expression was an exposure biomarker of COF, a cross-sectional study of 238 subjects including 94 professional cooks, 43 housewives, and 101 COF-nonexposed control subjects was conducted. The hOGG1 mRNA expression frequencies of COF-exposed cooks (27 of 94, 28.7%) and housewives (6 of 43, 14%) were significantly higher than those of control subjects (4 of 101, 4%). After adjusting for age, sex, and smoking and drinking status, the odds risks (ORs) of housewives versus control and cooks versus control were 3.94 (95% confidence interval [CI] = 0.95-16.62) and 10.12 (95% CI = 2.83-36.15), respectively. These results indicated that hOGG1 may be adequate to act as an exposure biomarker to assess the oxidative

  3. Hepatic lipase mRNA is expressed in rat and human steroidogenic organs

    NARCIS (Netherlands)

    A.J.M. Verhoeven (Adrie); H. Jansen (Hans)

    1994-01-01

    textabstractRat and human steroidogenic organs contain an enzyme activity that is indistinguishable from hepatic lipase present in liver. Using primers that recognize exons 5 and 8 of the rat and human HL gene, a 596-bp product was found by RT-PCR in rat liver, adrenal, ovaries and testes, but not i

  4. Weak binding affinity of human 4EHP for mRNA cap analogs.

    Science.gov (United States)

    Zuberek, Joanna; Kubacka, Dorota; Jablonowska, Agnieszka; Jemielity, Jacek; Stepinski, Janusz; Sonenberg, Nahum; Darzynkiewicz, Edward

    2007-05-01

    Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

  5. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  6. Matrix ageing and vascular impacts: focus on elastin fragmentation.

    Science.gov (United States)

    Duca, Laurent; Blaise, Sébastien; Romier, Béatrice; Laffargue, Muriel; Gayral, Stéphanie; El Btaouri, Hassan; Kawecki, Charlotte; Guillot, Alexandre; Martiny, Laurent; Debelle, Laurent; Maurice, Pascal

    2016-06-01

    Cardiovascular diseases (CVDs) are the leading cause of death worldwide and represent a major problem of public health. Over the years, life expectancy has considerably increased throughout the world, and the prevalence of CVD is inevitably rising with the growing ageing of the population. The normal process of ageing is associated with progressive deterioration in structure and function of the vasculature, commonly called vascular ageing. At the vascular level, extracellular matrix (ECM) ageing leads to molecular alterations in long half-life proteins, such as elastin and collagen, and have critical effects on vascular diseases. This review highlights ECM alterations occurring during vascular ageing with a specific focus on elastin fragmentation and also the contribution of elastin-derived peptides (EDP) in age-related vascular complications. Moreover, current and new pharmacological strategies aiming at minimizing elastin degradation, EDP generation, and associated biological effects are discussed. These strategies may be of major relevance for preventing and/or delaying vascular ageing and its complications. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  7. Electrospinning of collagen and elastin for tissue engineering applications

    NARCIS (Netherlands)

    Buttafoco, L.; Kolkman, N.G.; Engbers-Buijtenhuijs, P.; Poot, A.A.; Dijkstra, P.J.; Vermes, I.; Feijen, J.

    2006-01-01

    Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (Mw=8×106) and NaCl was

  8. Interaction of human decapping scavenger with 5' mRNA cap analogues: structural requirements for catalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Darzynkiewicz, Zbigniew M [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Bojarska, Elzbieta [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kowalska, Joanna [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Lewdorowicz, Magdalena [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Jemielity, Jacek [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kalek, Marcin [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Stepinski, Janusz [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Davis, Richard E [Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 12801 East 17th Avenue, Aurora, CO 80045 (United States); Darzynkiewicz, Edward [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland)

    2007-07-18

    The cap structure is a specific feature of the 5' end of mRNA which plays an important role in the post-transcriptional control in gene expression. A major step of gene regulation occurs at the level of mRNA turnover. Degradation of most eukaryotic mRNAs entails the removal of the cap structure in the various pathways. A human scavenger decapping enzyme (hDcpS) catalyses the cleavage of the residual cap structure m{sup 7}GpppN and/or short oligonucleotides after the 3' to 5' exosom mediated digestion. In this paper we report a fluorescence study of association process of hDcpS with m{sup 7}GMP, m{sup 7}GDP and selected dinucleotide cap analogues resistant to enzymatic hydrolysis. The calculated values of association constants (K{sub as}) and corresponding Gibbs free energies ({delta}G{sup 0}) depend on the type of substituents and their positions in the cap molecule, indicating which structural modifications are crucial for the catalysis.

  9. Tunable elastin-mimetic multiblock hybrid copolymers for biomedical applications

    Science.gov (United States)

    Grieshaber, Sarah Elizabeth

    Elastin-mimetic hybrid polymers (EMHPs) have been developed to capture the multiblock molecular architecture of tropoelastin, allowing tunability in chemical, structural, biological, and mechanical properties. Multiblock EMHPs containing flexible synthetic segments were first synthesized via step growth polymerization of diazido-poly(ethylene glycol) (PEG) and alkyne-terminated AKA3KA (K = lysine, A = alanine) (AK2) peptide employing copper (I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC, or orthogonal click chemistry). Covalent crosslinking of the EMHPs with hexamethylene diisocyanate (HMDI) through the lysine residues in the peptide domain afforded an elastomeric hydrogel (xEMHP) with a compressive modulus of 0.12 +/- 0.018 MPa when hydrated. xEMHPs exhibited minimal cytotoxicity to primary porcine vocal fold fibroblasts. The modular nature of the synthesis allowed facile adjustment of the peptide sequence to modulate the structural and the biological properties of EMHPs. Thus, EMHPs containing integrin-binding peptides were constructed using di-azido-PEG and an alkyne-terminated AK2 peptide with a terminal, integrin-binding GRGDSP domain via the step growth click coupling reaction. Hydrogels formed by covalent crosslinking of the RGD-containing EMHPs had a compressive modulus of 1.06 +/- 0.1MPa when hydrated. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h, and to spread and develop F-actin filaments 24 h post seeding. NHDF proliferation was only observed on hydrogels containing RGD domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. The tunability of the EMHP system was further investigated by development of self-assembling, pH-responsive multiblock polymers composed of alternating domains of poly(acrylic acid) (PAA) and a peptide derived from the hydrophobic domains of elastin with the sequence (VPGVG)2 (VG2). The

  10. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  11. Presence of. beta. hCG mRNA in the human pituitary

    Energy Technology Data Exchange (ETDEWEB)

    van Strien, A.; van Wezenbeek, P. (Organon International BV, Oss (Netherlands))

    1989-07-11

    A strong structural and biological similarity exist between human chorionic gonadotropin (hCG) and luteinizing hormone (LH). They share the same {alpha} subunit and have evolutionary related {beta} subunits. hCG is synthesized in first trimester placenta, whereas LH is synthesized in the pituitary. Substances with immunological, physical and biological properties of hCG have, however, been observed in postmenopausal urinary gonadotropin preparations as well as in pituitary extracts. Despite this finding it has not yet been proven that the hCG-like material is identical to placental hCG. This report describes the presence of a minor messenger RNA species in the human pituitary. RNA was isolated from five human pituitary glands and an amplified cDNA library was prepared.

  12. Analysis of Several PLA2 mRNA in Human Meningiomas

    OpenAIRE

    Yves Denizot; Rafael De Armas; Karine Durand; Sandrine Robert; Jean-Jacques Moreau; François Caire; Nicolas Weinbreck; François Labrousse

    2009-01-01

    In view of the important oncogenic action of phospholipase A2(PLA2) we investigated PLA2 transcripts in human meningiomas. Real-time PCR was used to investigate PLA2 transcripts in 26 human meningioma tumors. Results indicated that three Ca2+-dependent high molecular weight PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC), one Ca2+-independent high molecular weight PLA2 (PLA2-VI) and five low molecular weight secreted forms of PLA2 (PLA2-IB, PLA2-IIA, PLA2-III, PLA2-V, and PLA2-XII) are expressed with PLA...

  13. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils.

    Science.gov (United States)

    Gibson, M A; Hatzinikolas, G; Davis, E C; Baker, E; Sutherland, G R; Mecham, R P

    1995-12-01

    Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of

  14. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhen-Liang Qu; Sheng-Quan Zou; Nai-Qiang Cui; Xian-Zhong Wu; Ming-Fang Qin; Di Kong; Zhen-Li Zhou

    2005-01-01

    AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

  15. Cloning, characterization, and mRNA expression analysis of novel human fetal cochlear cDNAs.

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Pol, T.J.R. van de; Duijnhoven, G.C.F. van; Hollander, A.I. den; Caat, J. ten; Limpt, V. van; Brunner, H.G.; Kremer, J.M.J.; Cremers, F.P.M.

    2003-01-01

    To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fift

  16. Increased levels of endothelin ETB receptor mRNA in human omental arteries after organ culture

    DEFF Research Database (Denmark)

    Möller, S; Adner, M; Edvinsson, L

    1998-01-01

    1. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) and in vitro pharmacology, smooth muscle endothelin ETB receptor expression was studied in segments of human omental artery, fresh and after organ culture for 1 and 5 days. 2. The competitive RT-PCR assay used in the pr...

  17. Vascular nanomedicine: Site specific delivery of elastin stabilizing therapeutics to damaged arteries

    Science.gov (United States)

    Sinha, Aditi

    Elastin, a structural protein in the extra-cellular matrix, plays a critical role in the normal functioning of blood vessels. Apart from performing its primary function of providing resilience to arteries, it also plays major role in regulating cell-cell and cell-matrix interactions, response to injury, and morphogenesis. Medial arterial calcification (MAC) and abdominal aortic aneurysm (AAA) are two diseases where the structural and functional integrity of elastin is severely compromised. Although the clinical presentation of MAC and AAA differ, they have one common underlying causative mechanism---pathological degradation of elastin. Hence prevention of elastin degradation in the early stages of MAC and AAA can mitigate, partially if not wholly, the fatal consequences of both the diseases. The work presented here is motivated by the overwhelming statistics of people afflicted by elastin associated cardiovascular diseases and the unavailability of cure for the same. Overall goal of our research is to understand role of elastin degradation in cardiovascular diseases and to develop a targeted vascular drug delivery system that is minimally invasive, biodegradable, and non-toxic, that prevents elastin from degradation. Our hope is that such treatment will also help regenerate elastin, thereby providing a multi-fold treatment option for elasto-degenerative vascular diseases. For this purpose, we have first confirmed the combined role of degraded elastin and hyperglycemia in the pathogenesis of MAC. We have shown that in the absence of degraded elastin and TGF-beta1 (abundantly present in diabetic arteries) vascular smooth muscle cells maintain their homeostatic state, regardless of environmental glucose concentrations. However simultaneous exposure to glucose, elastin peptides and TGF-beta1 causes the pathological transgenesis of vascular cells to osteoblast-like cells. We show that plant derived polyphenols bind to vascular elastin with great affinity resulting in

  18. Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic β-cells

    DEFF Research Database (Denmark)

    Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel;

    2011-01-01

    of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic β-cells, and to examine this in relation to β-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP......Fatty acid-induced damage in pancreatic β-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve β-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim...

  19. Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic ß-cells

    DEFF Research Database (Denmark)

    Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel;

    2011-01-01

    of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP......Fatty acid-induced damage in pancreatic ß-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve ß-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim...

  20. Bed rest reduces metabolic protein content and abolishes exercise-induced mRNA responses in human skeletal muscle

    DEFF Research Database (Denmark)

    Jørgensen, Stine Ringholm; Biensø, Rasmus S; Kiilerich, Kristian

    2011-01-01

    Background: The aim was to test the hypothesis that one week of bed rest will reduce mitochondrial number and expression and activity of oxidative proteins in human skeletal muscle, but that exercise-induced intracellular signaling as well as mRNA and microRNA (miR) responses are maintained after......-legged knee extensor exercise performed before and after bed rest. Results: Maximal oxygen uptake decreased 5% and exercise endurance decreased non-significantly 25% by bed rest. Bed rest reduced skeletal muscle mitochondrial DNA/nuclear DNA content 15%, hexokinase II and sirtuin 1 protein content ~45%, 3...... bed rest. Research Design and Methods: Twelve young, healthy, male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies taken before and after bed rest. In addition, muscle biopsies were obtained from 6 of the subjects prior to, immediately after and 3h after 45 min one...

  1. Study on the immune responses against pancreatic cancer induced by mucin 4 and human telomerase reverse transcriptase mRNA co-transfected dendritic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    陈江

    2014-01-01

    Objective To investigate the anti-tumor immune response induced by human pancreatic cancer mucin 4mRNA and human telomerase reverse transcriptase(hTERT)mRNA cotransfected dendritic cells(DC),and to provide the experimental evidences for the treatment of pancreatic cancer with multi-epitope loaded DC vaccine.Methods DC were isolated from peripheral DC.

  2. Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Damgaard, Christian Kroun; Tomecki, Rafal

    2013-01-01

    Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes a...

  3. Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

    Directory of Open Access Journals (Sweden)

    Zainuddin Azalina

    2010-08-01

    Full Text Available Abstract Background Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs can be validated. HDFs in 4 different treatment groups viz; young (passage 4, senescent (passage 30, H2O2-induced oxidative stress and γ-tocotrienol (GTT-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene. Results HDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal activity. However the expression level of GAPDH was consistent in all treatment groups. Conclusion The study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.

  4. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    OpenAIRE

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen)...

  5. Expression of a novel pyridoxal kinase mRNA splice variant, PKH-T, in human testis

    Institute of Scientific and Technical Information of China (English)

    XingFang; Zuo-MinZhou; LiLu; Lan-LanYin; Jian-MinLi; YinZhen; HuiWang; Jia-HaoSha

    2004-01-01

    Aim: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa.Methods: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template.The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results:A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene,HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different develop-mental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients.Conclusion: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.

  6. BCFA suppresses LPS induced IL-8 mRNA expression in human intestinal epithelial cells.

    Science.gov (United States)

    Yan, Y; Wang, Z; Greenwald, J; Kothapalli, K S D; Park, H G; Liu, R; Mendralla, E; Lawrence, P; Wang, X; Brenna, J T

    2017-01-01

    Branched chain fatty acids (BCFA) are components of common food fats and are major constituents of the normal term human newborn GI tract. Polyunsaturated fatty acids (PUFA) have been suggested to reduce the risk and development of inflammatory bowel diseases (IBD); however, little is known about the influence of BCFA on inflammation. We investigated the effect of BCFA on interleukin (IL)-8 and NF-κB production in a human intestinal epithelial cell line (Caco-2). Cells were pre-treated with specific BCFA, or DHA, or EPA, and then activated with lipopolysaccharide (LPS). Both anteiso- and iso- BCFA reduce IL-8. Anteiso-BCFA more effectively suppressed IL-8 than iso-BCFA in LPS stimulated Caco-2 cells. However BCFA in general were less effective than DHA or EPA. Activated BCFA-treated cells expressed less of the cell surface Toll-like receptor 4 (TLR-4) compared to controls. These are the first data to show the reduction of pro-inflammatory markers in human cells mediated by BCFA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Analysis of Several PLA2 mRNA in Human Meningiomas

    Directory of Open Access Journals (Sweden)

    Yves Denizot

    2009-01-01

    Full Text Available In view of the important oncogenic action of phospholipase A2(PLA2 we investigated PLA2 transcripts in human meningiomas. Real-time PCR was used to investigate PLA2 transcripts in 26 human meningioma tumors. Results indicated that three Ca2+-dependent high molecular weight PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC, one Ca2+-independent high molecular weight PLA2 (PLA2-VI and five low molecular weight secreted forms of PLA2 (PLA2-IB, PLA2-IIA, PLA2-III, PLA2-V, and PLA2-XII are expressed with PLA2-IVA, PLA2-IVB, PLA2-VI, and PLA2-XIIA as the major expressed forms. PLA2-IIE, PLA2-IIF, PLA2-IVD, and PLA2-XIIB are not detected. Plasma (PLA2-VIIA and intracellular (PLA2-VIIB platelet-activating factor acetylhydrolase transcripts are expressed in human meningiomas. However no difference was found for PLA2 transcript amounts in relation to the tumor grade, the subtype of meningiomas, the presence of inflammatory infiltrated cells, of an associated edema, mitosis, brain invasion, vascularisation or necrosis. In conclusion numerous genes encoding multiples forms of PLA2 are expressed in meningiomas where they might act on the phospholipid remodeling and on the local eicosanoid and/or cytokine networks.

  8. Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Stepinski, Janusz; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Mazza, Catherine; Cusack, Stephen; Stolarski, Ryszard; Darzynkiewicz, Edward

    2005-01-01

    Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.

  9. Clinical performance of human papillomavirus E6, E7 mRNA flow cytometric assay compared to human papillomavirus DNA typing.

    Science.gov (United States)

    Kottaridi, Christine; Tsiodras, Sotirios; Spathis, Aris; Chranioti, Aikaterini; Pappas, Asimakis; Kassanos, Dimitrios; Panayiotides, Ioannis; Karakitsos, Petros

    2011-12-01

    To use flow cytometry to screen cervical samples for the overexpression of human papillomavirus (HPV) E6 and E7 mRNA and compare the performance of this assay with an HPV DNA array for the detection of high-grade cervical lesions. Cervical samples were analyzed for HPV DNA by clinical arrays, and the overexpression of E6 and E7 viral oncogenes was monitored using an HPV mRNA detection kit that quantifies the intracellular HPV E6 and E7 mRNA on a cell-by-cell basis. HPV positivity increased with severity of histologic lesions. On the basis of histology-confirmed CIN 2+ cases the specificity of HPV assay was 73.9% (95% CI 66.07, 80.88), whereas it was 39.3% (95% CI 31.85, 47.1) for the DNA assay. The HPV assay provides an early predictor of persistent HPV infection and may improve cervical cancer screening by increasing the specificity of detecting high-grade lesions.

  10. Direct RNA sequencing mediated identification of mRNA localized in protrusions of human MDA-MB-231 metastatic breast cancer cells

    DEFF Research Database (Denmark)

    Jakobsen, Kristine Raaby; Sørensen, Emilie; Brøndum, Karin Kathrine;

    2013-01-01

    Background Protrusions of cancer cells conferrers a vital function for cell migration and metastasis. Protein and RNA localization mechanisms have been extensively examined and shown to play pivotal roles for the functional presence of specific protein components in cancer cell protrusions. Methods...... To describe genome wide RNA localized in protrusions of the metastatic human breast cancer cell line MDA-MB-231 we used Boyden chamber based methodology followed by direct mRNA sequencing. Results In the hereby identified group of protrusion localized mRNA some previously were described to be localized...... exemplified by mRNA for Ras-Related protein 13 (RAB13) and p0071 (Plakophilin-4/PKP4). For other transcripts, exemplified by mRNA for SH3PXD2A/TKS5 and PPFIA1/Liprin-α1, only the corresponding proteins previously were described to have protrusion localization. Finally, a cohort of MDA-MB-231 protrusion...

  11. Expression of a novel beta adaptin subunit mRNA splice variant in human testes

    Institute of Scientific and Technical Information of China (English)

    Xin-Dong Zhang; Lan-Lan Yin; Ying Zheng; Li Lu; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis. Methods: Using a human testis cDNA microarray, a clone (Ap2β-NY),which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed.Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2β-NY were determined.Results: Ap2β-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2β-NY was restrictively expressed in germ cells. Conclusion: Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.

  12. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  13. Expression of Heat Shock Protein 70 mRNA in Epithelial Cells of Human Lens

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective:To try to find out the pathogenesis of the cataract , effects of beat and oxidative stresson heat shock proteins of tissue cultured human lens epithelial cells (LEC-B3) were investigate& Methods:Cells were exposed to heat shock (45℃) and oxidative stress(5OmMH2O2 for 30 min, and then allowed to recoverat different intervals (Oh, 2h, 4h, 6h, 16h, 24h) in physiological medium Reverse transcription polymerasechain reaction (RT-PCR) were used to determined the level of HSP70. Results: HSPs existed in both physiologicaland stressful situation. The level of HSP7OmRNA increased 2h later after both stresses. The expression of HSP70got to the summit during 2h to 6h in each group. Subsequently it decreased gradually in each group, maintaininga high level at 16h. Conclusion: HSP70 exists in lens epithelial cells and can be induced after stress. Thedata suggested it may play an important protective role in lens epithelial cells in respond to cellular stress.

  14. IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

    Institute of Scientific and Technical Information of China (English)

    Yang Xiang; Lei Li; Fang Tian; Xiu-yu Yang

    2005-01-01

    Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1 (MDR1) mRNA.Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region ofmdrl cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5' and 3' untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdrl mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump functon of P-gp were measured with flow cytometry.Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P< 0.01).And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference.The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P <0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA.And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

  15. Contribution of collagen and elastin fibers to the mechanical behavior of an abdominal connective tissue.

    Science.gov (United States)

    Levillain, A; Orhant, M; Turquier, F; Hoc, T

    2016-08-01

    The linea alba is a complex structure commonly involved in hernia formation. Knowledge of its mechanical behavior is essential to design suitable meshes and reduce the risk of recurrence. The aim of this study was to investigate the relationships between the mechanical properties of the linea alba and the organization of collagen and elastin fibers. For that purpose, longitudinal and transversal samples were removed from four porcine and three human linea alba, to perform tensile tests under a biphotonic confocal microscope, in each direction. Microscopic observation revealed a tissue composed of two layers, made of transversal collagen fibers in the dorsal side and oblique collagen fibers in the ventral side. This particular architecture led to an anisotropic mechanical behavior, with higher stress in the transversal direction. During loading, oblique fibers of the ventral layer reoriented toward the tensile axis in both directions, while fibers of the dorsal layer remained in the transversal direction. This rotation of oblique fibers progressively increased the stiffness of the tissue and induced a non-linear stress-stretch relation. Elastin fibers formed a layer covering the collagen fibers and followed their movement, suggesting that they ensure their elastic recoil. All of these results demonstrated the strong relationships between the microstructure and the mechanical behavior of the linea alba.

  16. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    Science.gov (United States)

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or

  17. EMILIN2 (Elastin microfibril interface located protein, potential modifier of thrombosis

    Directory of Open Access Journals (Sweden)

    Hoover-Plow Jane L

    2011-05-01

    Full Text Available Abstract Background Elastin microfibril interface located protein 2 (EMILIN2 is an extracellular glycoprotein associated with cardiovascular development. While other EMILIN proteins are reported to play a role in elastogenesis and coagulation, little is known about EMILIN2 function in the cardiovascular system. The objective of this study was to determine whether EMILIN2 could play a role in thrombosis. Results EMILIN2 mRNA was expressed in 8 wk old C57BL/6J mice in lung, heart, aorta and bone marrow, with the highest expression in bone marrow. In mouse cells, EMILIN2 mRNA expression in macrophages was higher than expression in endothelial cells and fibroblasts. EMILIN2 was identified with cells and extracellular matrix by immunohistochemistry in the carotid and aorta. After carotid ferric chloride injury, EMILIN2 was abundantly expressed in the thrombus and inhibition of EMILIN2 increased platelet de-aggregation after ADP-stimulated platelet aggregation. Conclusions These results suggest EMILIN2 could play a role in thrombosis as a constituent of the vessel wall and/or a component of the thrombus.

  18. Granulocyte colony-stimulating factor ameliorates coronary artery elastin breakdown in a mouse model of Kawasaki disease

    Institute of Scientific and Technical Information of China (English)

    Liu Junfeng; Chen Zhi; Du Zhongdong; Lu Dunxiang

    2014-01-01

    Background Coronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of the endothelial progenitor cells (EPCs).The aim of the present study was to evaluate the modulatory effect of granulocyte colony stimulating factor (G-CSF) on EPCs and elastin breakdown of coronary arteries in a KD mouse model.Methods A Lactobacillus casei cell wall extract (LCWE)-induced KD model was established in C57BL/6 mice that were subsequently administrated with recombinant human G-CSF (rhG-CSF).Nω-nitro-L-arginine methyl ester (L-NAME) was administrated for the negative intervention.Evaluations included coronary artery lesions,EPC number and functions,and the plasma concentration of nitric oxide (NO).Results Elastin breakdown was found in the coronary arteries of model mice 56 days after injection of LCWE.The number of circulating EPCs,plasma concentration of NO,and functions of bone marrow EPCs,including proliferation,adhesion,and migration abilities,were all lower in the KD model group compared with those in the control group.After administration of rhG-CSF,the number of circulating EPCs and plasma concentration of NO were increased significantly compared with those in the KD model group.There were also increases in the functional indexes of EPCs.Furthermore,rhG-CSF administration improved the elastin breakdown effectively.However,these protective effects of rhG-CSF on coronary arteries were attenuated by L-NAME.Conclusion The present study indicated that the administration of G-CSF prevents elastin breakdown of the coronary arteries by enhancing the number and functions of EPCs via the NO system,and then accelerates the repair of coronary artery lesions in the KD.

  19. Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Lin Wang; Bao-En Wang; Jian Wang; Pei-Gen Xiao; Xue-Hai Tan

    2008-01-01

    AIM: To identify the role of herbal compound 861 (Cpd 861) in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells (HSCs).METHODS: mRNA levels of collagen types I and III, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and transforming growth factor β1 (TGF-βi) in cultured-activated HSCs treated with Cpd 861 or interferon-γ (IFN-γ) were determined by real-time PCR.RESULTS: Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-βl. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to (6.3 + 0.3)-fold compared to the control group.CONCLUSION: The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type in and TGF-pi and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

  20. mRNA Expression of Vimentin Gene in Lens of Transgenic Mouse and DNA Amplification in Human Cataracts

    Institute of Scientific and Technical Information of China (English)

    YanLi; XienpingLiu; 等

    1995-01-01

    Purpose:To investigate the role of vimentin gene in cataractogenesis.Methods:The12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs.841injected embryos were transferred into oviducts of pseudopregnant recipient females.of which 12pregnant mice gave birth to 49offsping mice.The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot byhridizations,In the human senile cataract,the lens vimentin gene was analyzed with the chicken vi-mentin gene probe.Results:It showed that four of F1offspring were transgenic mice in which the chicken vimenttin gene was integrated in their genomes.The transgenic band was12kb,similar to the12.7kb chicken vimentin fragment injected.One2kbvi-mentin mRNAwas visualized on E2 mouse lens blot.which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse.In the humansenile cataract lens,12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot anal-ysis,It implies that the Formation of human senile cataract may be associated with the amplification of vimentin gene.Conclusions:We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study.It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice.The cause of the gene ampli-fication in human ctaract remains for further investigation.Eye Science 1995;11:113-116.

  1. The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

    Directory of Open Access Journals (Sweden)

    Smiths Lueong

    Full Text Available Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II and without (stage I brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II, 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

  2. Effect of c-fos antisense probe on prostaglandin E2-induced upregulation of vascular endothelial growth factor mRNA in human liver cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Qi Li; Kai-Shan Tao; Ning Ren; Yi-Hu Wang

    2005-01-01

    AIM: To examine the effect of prostaglandin E2 (PGE2) on the expression of vascular endothelial growth factor (VEGF) mRNA in the human hepatocellular carcinoma (HCC) HepG2 cells and the possible involvement of c-fos protein in this process.METHODS: Human HCC HepG2 cells were divided into three groups treated respectively with PGE2, a combination of PGE2 and c-fos antisense oligodeoxynucleotide (ASO),and PGE2 plus c-fos sense oligodeoxynudeotide (SO). The expression of VEGF mRNA in HepG2 cells after different treatments was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The relative expression level of VEGF mRNA in HepG2 cells in each group was measured.RESULTS: Administration of PGE2 resulted in an increased expression of c-fosand VEGF mRNA in HepG2 cells. The relative expression level of c-fos mRNA reached the peak at 3 h (68.4±4.7%) after PGE2 treatment, which was significantly higher than that at 0 h (20.6±1.7%, P<0.01).Whereas, the highest expression level of VEGF mRNA was observed at 6 h (100.5±6.1%) after PGE2 treatment, which was significantly higher than that at 0 h (33.2±2.4%,P<0.01). C-fos ASO significantly reduced PGE2-induced VEGF mRNA expression in HepG2 cells.CONCLUSION: PGE2 increases the expression and secretion of VEGF in HCC cells by activating the transcription factor c-fos, promotes the angiogenesis of HCC and plays an important role in the pathogenesis of liver cancer.

  3. Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Clausen, Rikke E; Nguyen, Quoc Hai Trieu;

    2012-01-01

    Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part...... are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases....

  4. Impact of adrenaline and metabolic stress on exercise-induced intracellular signaling and PGC-1α mRNA response in human skeletal muscle

    DEFF Research Database (Denmark)

    Brandt, Nina; Gunnarsson, Thomas Gunnar Petursson; Hostrup, Morten

    2016-01-01

    This study tested the hypothesis that elevated plasma adrenaline or metabolic stress enhances exercise-induced PGC-1α mRNA and intracellular signaling in human muscle. Trained (VO2-max: 53.8 ± 1.8 mL min(-1) kg(-1)) male subjects completed four different exercise protocols (work load of the legs...

  5. Potential Osteoinductive Effects of Calcitriol on the m-RNA of Mesenchymal Stem Cells Derived from Human Alveolar Periosteum

    Directory of Open Access Journals (Sweden)

    Hsiang-Hsi Hong

    2016-01-01

    Full Text Available This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs and examined the hypothesis that 1,25-(OH2D3 (calcitriol exerts osteoinductive effects on P-MSCs. The mRNA expressions of alkaline phosphatase (ALP, bone sialoprotein (BSP, core-binding factor alpha-1 (CBFA1, collagen-1 (Col-1, osteocalcin (OCN, and vitamin D3 receptor (VDR were assessed after incubation with calcitriol for 2 weeks. Vitamin C as positive control (Vit. C-p increased ALP and CBFA1 mRNA expression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week. A concentration of 10−8 M calcitriol enhanced ALP, CBFA1, Col-1, and OCN mRNA expression at both weeks and BSP mRNA expression at the first week. Furthermore, 10−7 M calcitriol increased the mRNA expressions of all compounds at both weeks, except that of CBFA1 at the first week. 10−8 M calcitriol and Vit. C-p enhanced ALP activity at the second and third weeks. The results revealed that 10−9, 10−8, and 10−7 M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP, CBFA1, Col-1, and OCN mRNA expression. A 10−7 M calcitriol yielded a higher mRNA expression than Vit. Cp on VDR and OCN mRNA expression at both weeks and on Col-1 mRNA at the second week.

  6. Potential Osteoinductive Effects of Calcitriol on the m-RNA of Mesenchymal Stem Cells Derived from Human Alveolar Periosteum

    Science.gov (United States)

    Hong, Hsiang-Hsi; Hong, Adrienne

    2016-01-01

    This study characterized alveolar periosteum-derived mesenchymal stem cells (P-MSCs) and examined the hypothesis that 1,25-(OH)2D3 (calcitriol) exerts osteoinductive effects on P-MSCs. The mRNA expressions of alkaline phosphatase (ALP), bone sialoprotein (BSP), core-binding factor alpha-1 (CBFA1), collagen-1 (Col-1), osteocalcin (OCN), and vitamin D3 receptor (VDR) were assessed after incubation with calcitriol for 2 weeks. Vitamin C as positive control (Vit. C-p) increased ALP and CBFA1 mRNA expression at both 1 and 2 weeks and increased BSP and Col-1 mRNA expression only at the first week. A concentration of 10−8 M calcitriol enhanced ALP, CBFA1, Col-1, and OCN mRNA expression at both weeks and BSP mRNA expression at the first week. Furthermore, 10−7 M calcitriol increased the mRNA expressions of all compounds at both weeks, except that of CBFA1 at the first week. 10−8 M calcitriol and Vit. C-p enhanced ALP activity at the second and third weeks. The results revealed that 10−9, 10−8, and 10−7 M calcitriol induced osteoinduction in alveolar P-MSCs by increasing ALP, CBFA1, Col-1, and OCN mRNA expression. A 10−7 M calcitriol yielded a higher mRNA expression than Vit. Cp on VDR and OCN mRNA expression at both weeks and on Col-1 mRNA at the second week. PMID:28105418

  7. Acute simulated soccer-specific training increases PGC-1α mRNA expression in human skeletal muscle.

    Science.gov (United States)

    Jeong, Tae-Seok; Bartlett, Jonathan D; Joo, Chang-Hwa; Louhelainen, Jari; Close, Graeme L; Morton, James P; Drust, Barry

    2015-01-01

    The aim of the current study was to quantify oxygen uptake, heart rate and molecular responses of human skeletal muscle associated with mitochondrial biogenesis following an acute bout of simulated soccer training. Muscle biopsies (vastus lateralis) were obtained from nine active men immediately pre-completion, post-completion and 3 h post-completion of a laboratory-based soccer-specific training simulation on a motorised treadmill. The soccer-specific simulation was a similar intensity (55 ± 6% [Formula: see text]) and duration (60 min) as that observed in professional soccer training (e.g. standing 41%, walking 37%, jogging 11%, high-speed running 9% and sprinting 2%). Post-exercise, muscle glycogen decreased (Pre; 397 ± 86 mmol∙kg(-1) dw, Post; 344 ± 64 mmol∙kg(-1) dw; P = 0.03), plasma lactate increased (P soccer-specific training is associated with increases in PGC-1α mRNA. These data may have implications for practitioners in better understanding the metabolic and muscle responses to soccer-specific training protocols in the field.

  8. X-ray irradiation affects Gadd45 mRNA expression in human peripheral blood lymphocytes%X 射线对人周围血淋巴细胞 Gadd45 mRNA 表达影响

    Institute of Scientific and Technical Information of China (English)

    王恰; 李明芳; 杨爱初; 杨宇华; 梁晓阳; 严茂胜

    2015-01-01

    Objective To analyze the relative expression level of Growth arrest and DNA damage gene (Gadd)45 mRNA of human peripheral blood lymphocytes after X-ray irradiation with different doses and time,and to explore the possibility of Gadd45 gene as a biological dosimeter.Methods i)Dose-effect experiment.The healthy human peripheral blood were irradiated by 0.00,0.10,0.25,0.50,1.00,2.00,3.00 and 5.00 Gy X-ray and the lymphocytes were separated for measurement.ii)Time-effect experiment.The healthy human peripheral blood were irradiated by 2.00 Gy X-ray and the lymphocytes were separated and then cultivate for measurement after cultivation time of 0,3,6,12,24 and 48 hours.The real-time fluorescent quantitative polymerase chain reaction method was used to detect Gadd45 mRNA expression level. Results i)The relative expression level of Gadd45 mRNA increased with the increasing dose from 0.00 to 0.50 Gy.The linear regression equation was ^y =1.056 9 +7.132 2 x (determination coefficient was 0.992,P <0.01 ).The relative expression level of Gadd45 mRNA achieved the peak value when the irradiation dose was 0.50 Gy,which was 4.53 times of that with 0.00 Gy dose irradiation.The relative expression level of Gadd45 mRNA gradually decreased in 0.50-5.00 Gy.ii)In the period of 3-12 hours after 2.00 Gy of X-ray irradiation,the relative expression level of Gadd45 mRNA increased with the increasing exposure time.The linear regression equation was ^y =-6.366 0 +4.965 0 x (determination coefficient was 0.932,P <0.05).It achieved the peak value at the time of 12 hours,which was 57.68 times of that with 0-hour time group.During the time of 24-48 hours after irradiation,the relative expression level of Gadd45 mRNA decreased sharply,the 48-hour group was 2.08 times of 0-hour time group.Conclusion X-ray irradiation up-regulated the expression level of Gadd45 mRNA.There was a good dose-effect and time-effect relation in specific dose and time range.Further study is needed to explore if Gadd45

  9. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    Science.gov (United States)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  10. Regulation and function of FTO mRNA expression in human skeletal muscle and subcutaneous adipose tissue

    DEFF Research Database (Denmark)

    Grunnet, Louise G; Nilsson, Emma; Ling, Charlotte

    2009-01-01

    and adipose tissue, and their influence on in vivo glucose and fat metabolism. Research Design and Methods. The FTO rs9939609 polymorphism was genotyped in two twin cohorts: 1) 298 elderly twins aged 62-83 years with glucose tolerance ranging from normal to type 2 diabetes and 2) 196 young (25-32 years...... expression was not influenced by FTO rs9939609 genotype. FTO mRNA expression in skeletal muscle was regulated by age and sex, whereas age and BMI were predictors of adipose tissue FTO mRNA expression. FTO mRNA expression in adipose tissue was associated with an atherogenic lipid profile. In skeletal muscle...

  11. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles

    DEFF Research Database (Denmark)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte;

    2006-01-01

    was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content...... of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly...

  12. Confocal microscopy for automatic measurement of the density and distance between elastin fibers of histologic preparations of normotensive and hypertensive patients

    Science.gov (United States)

    Vieira, G.; Ferro, D. P.; Adam, R. L.; de Thomaz, A. A.; Cesar, C. L.; Metze, K.

    2010-02-01

    Elastic fibers are essential components of the human aorta, and there is an association between elastin fibers remodeling and several diseases. Hypertension is one such example of a disease leading to elastin fibers remodeling. These fibers can be easily seen in eosin-hematoxilin (HE) stained histologic sections when observed by UV-excited fluorescence microscopy or by a much more precise Laser Scanning Confocal Microscope (LSCM). In order to study the effect of the hypertension on the elastin fibers pattern we developed an automatic system (software and hardware) to count the number of elastin fibers and to measure the distance between them in a LSCM and used it compare the statistical distribution of the distance between these fibers in normotensive and hypertensive patients. The full image of the whole sample (2 or 3mm long) was composed by several 220×220μm frames with 512×512 pixels. The software counters fiber and distance between fibers. We compared the elastic fiber texture in routinely HE-stained histologic slides of the aorta ascendens in 24 normotensive and 30 hypertensive adult patients of both sexes and of similar age from our autopsy files. Our results show that the average number of fibers is the same for both cases but the distance between the fibers are larger for hypertensive patients than for normotensive ones.

  13. Comparative expression profile of miRNA and mRNA in primary peripheral blood mononuclear cells infected with human immunodeficiency virus (HIV-1.

    Directory of Open Access Journals (Sweden)

    Ankit Gupta

    Full Text Available Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.

  14. Human papillomavirus (HPV) E6/E7 mRNA detection in cervical exfoliated cells: a potential triage for HPV-positive women.

    Science.gov (United States)

    Yao, Ye-Li; Tian, Qi-Fang; Cheng, Bei; Cheng, Yi-Fan; Ye, Jing; Lu, Wei-Guo

    Cytology triage has been generally recommended for human papillomavirus (HPV)-positive women, but is highly dependent on well-trained cytologists. The present study was designed to explore whether HPV E6/E7 mRNA detection in cervical exfoliated cells can be a potential triage for HPV-positive women from a clinic-based population. Both the primary HPV testing and Papanicolaou (Pap) test were performed on all eligible HPV-positive women. HPV E6/E7 mRNA was detected by QuantiVirus(®) HPV E6/E7 mRNA assay in cervical exfoliated cells. All HPV-positive women underwent colposcopy and further biopsy if indicated. The data were assessed by Pearson's Chi-squared test and the receiver operating characteristic curve. A total of 404 eligible HPV-positive women were enrolled. Positive rate of E6/E7 mRNA in high-grade squamous intraepithelial lesion (HSIL) cases was higher than that in low-grade squamous intraepithelial lesion (LSIL) or normal cases. There was no statistical difference found between mRNA and cytological testing with sensitivity (89.52% vs. 86.67%, P=0.671), specificity (48.96% vs. 48.96%, P=1.000), positive predictive value (39.00% vs. 38.24%, P=1.000), and negative predictive value (92.76% vs. 90.97%, P=0.678) for detecting ≥HSIL. HPV E6/E7 mRNA detection in cervical exfoliated cells shows the same performance as Pap triage for HSIL identification for HPV-positive women. Detection of HPV E6/E7 mRNA may be used as a new triage option for HPV-positive women.

  15. Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Zhang, Xiaodong; Cai, Hui; Niewiesk, Stefan

    2014-01-01

    ABSTRACT The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2′-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2′-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCE Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine

  16. The human insulin mRNA is partly translated via a cap- and eIF4A-independent mechanism.

    Science.gov (United States)

    Fred, Rikard G; Sandberg, Monica; Pelletier, Jerry; Welsh, Nils

    2011-09-09

    The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data

  17. Elastins from patients with Williams-Beuren syndrome and healthy individuals differ on the molecular level.

    Science.gov (United States)

    Heinz, Andrea; Huertas, Angela C Mora; Schräder, Christoph U; Pankau, Rainer; Gosch, Angela; Schmelzer, Christian E H

    2016-07-01

    Williams-Beuren syndrome (WBS) is a congenital disorder, which involves the heterozygous deletion of the elastin gene and other genes on chromosome 7. Clinical symptoms that are associated with hemizygosity of the essential extracellular matrix protein elastin include premature aging of the skin and supravalvular aortic stenosis. However, only little is known about the molecular basis of structural abnormalities in the connective tissue of WBS patients. Therefore, for the first time this study aimed to systematically characterize and compare the structure and amount of elastin present in skin and aortic tissue from WBS patients and healthy individuals. Elastin fibers were isolated from tissue biopsies, and it was found that skin of WBS patients contains significantly less elastin compared to skin of healthy individuals. Scanning electron microscopy and mass spectrometric measurements combined with bioinformatics data analysis were used to investigate the molecular-level structure of elastin. Scanning electron microscopy revealed clear differences between WBS and healthy elastin. With respect to the molecular-level structure, it was found that the proline hydroxylation degree differed between WBS and healthy elastin, while the tropoelastin isoform appeared to be the same. In terms of cross-linking, no differences in the content of the tetrafunctional cross-links desmosine and isodesmosine were found between WBS and healthy elastin. However, principal component analysis revealed differences between enzymatic digests of elastin from healthy probands and WBS patients, which indicates differing susceptibility toward enzymatic cleavage. Overall, the study contributes to a better understanding of the correlation between genotypic and elastin-related phenotypic features of WBS patients. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

    Science.gov (United States)

    Lu, Sizhao; Mott, Justin L; Harrison-Findik, Duygu Dee

    2015-10-02

    Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

  19. mRNA and DNA detection of human papillomaviruses in women of all ages attending two colposcopy clinics.

    Directory of Open Access Journals (Sweden)

    Aris Spathis

    Full Text Available OBJECTIVE: HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals. METHODS: 1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results. RESULTS: HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests' positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used. CONCLUSIONS: Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results.

  20. Morphine and endomorphins differentially regulate micro-opioid receptor mRNA in SHSY-5Y human neuroblastoma cells.

    Science.gov (United States)

    Yu, Xin; Mao, Xin; Blake, Allan D; Li, Wen Xin; Chang, Sulie L

    2003-08-01

    A sensitive quantitative-competitive reverse transcriptase-polymerase chain reaction method was developed to measure micro-opioid receptor (MOR) mRNA expression in SHSY-5Y neuroblastoma cells. Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased MOR mRNA levels. Morphine treatment (10 microM) for 24 h decreased MOR mRNA levels in control, as well as RA- and TPA-differentiated cells. In contrast, chronic exposure to the opioid peptides endomorphin-1 or endomorphin-2 significantly increased MOR mRNA levels in undifferentiated and RA-differentiated cells. An opioid antagonist, naloxone, reversed the morphine and endomorphin-1 and -2 effects on MOR mRNA levels in undifferentiated SHSY-5Y cells, but naloxone had differential reversing effects on the agonists' regulation of MOR mRNA in RA- or TPA-differentiated cells. To investigate whether the changes in MOR mRNA expression paralleled changes in MOR receptor function, intracellular cAMP accumulation in SHSY-5Y cells was measured. After chronic treatment with morphine, forskolin-induced cAMP levels in SHSY-5Y cells were significantly higher than those of untreated control cells. In contrast, forskolin-induced cAMP accumulation levels were lower in cells treated with endomorphin-1 or -2 than in untreated control cells. Together, our studies indicate that the opioid alkaloid morphine and the opioid peptides endomorphin-1 and -2 differentially regulate MOR mRNA expression and MOR function in SHSY-5Y cells.

  1. Human IFIT1 Inhibits mRNA Translation of Rubulaviruses but Not Other Members of the Paramyxoviridae Family

    Science.gov (United States)

    Young, D. F.; Andrejeva, J.; Li, X.; Inesta-Vaquera, F.; Dong, C.; Cowling, V. H.; Goodbourn, S.

    2016-01-01

    ABSTRACT We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. The IFIT1 sensitivity of PIV5 was not rescued by coinfection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. While the translations of PIV2, PIV5, and MuV mRNAs were directly inhibited by IFIT1, the translations of PIV3, SeV, and CDV mRNAs were not. Using purified human mRNA-capping enzymes, we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5′ guanosine nucleoside cap (which need not be N7 methylated) and that this sensitivity is partly abrogated by 2′O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2′O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2′-O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. IMPORTANCE Paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. Thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. One factor that will influence

  2. Elastin-like polypeptides: biomedical applications of tunable biopolymers.

    Science.gov (United States)

    MacEwan, Sarah R; Chilkoti, Ashutosh

    2010-01-01

    Artificial repetitive polypeptides have grown in popularity as a bioinspired alternative to synthetic polymers. The genetically encoded synthesis, monodispersity, potential lack of toxicity, and biocompatibility are attractive features of these biopolymers for biological applications. Elastin-like polypeptides (ELPs) are one such class of biopolymers that are of particular interest because of their "smart"-stimuli responsive-properties. Herein, we discuss the genetically encoded design and recombinant synthesis of ELPs that enable precise control of their physicochemical properties and which have led to a wide range of biomedical applications of these biopolymers in the last decade.

  3. Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter

    Institute of Scientific and Technical Information of China (English)

    VerenaNordhoff; JorgGromoll; LucaFoppiani; C.MarcLuetjens; StefanSchlatt; ElenaKostova; IlpoHuhtaniemi; EberhardNieschlag; ManuelaSimoni

    2003-01-01

    Aim:To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.Methods:We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5’-flanking region fragment of its promoter.Results: Mice were phenotypically normal and fertile.In males,mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells,spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5"-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

  4. Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling

    DEFF Research Database (Denmark)

    Kristensen, Jacob Hull; Karsdal, Morten A.; Sand, Jannie M. B.;

    2015-01-01

    for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.  Methods: Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra-and inter-assay variation. The NE...

  5. Serum-elastin-peptides as a predictor of expansion of small abdominal aortic aneurysms

    DEFF Research Database (Denmark)

    Lindholt, Jes Sanddal; Heickendorff, Lene; Henneberg, E W

    1997-01-01

    To optimise the indication and time for operation for abdominal aortic aneurysm (AAA), we analysed whether or not serum-elastin-peptides (SEP) could be a potential predictor for expansion.......To optimise the indication and time for operation for abdominal aortic aneurysm (AAA), we analysed whether or not serum-elastin-peptides (SEP) could be a potential predictor for expansion....

  6. Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders

    DEFF Research Database (Denmark)

    Kristensen, J.H.; Larsen, L.; Dasgupta, B.

    2015-01-01

    for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. Design and methods:  Monoclonal antibodies (mABs) were raised against eight carefully selected...... MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n = 123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n = 40......) and compared with age- and sex-matched controls. Results: The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, p cancer (96%, p

  7. Human Epidermal Growth Factor Receptor-3 mRNA Expression as a Prognostic Marker for Invasive Duct Carcinoma not Otherwise Specified

    Science.gov (United States)

    Hammoda, Ghada Ezat; El-Hefnawy, Sally Mohammed; Abdallah, Rania Abdallah

    2017-01-01

    Introduction Breast cancer is the most common cancer in women and the Erythroblastosis Oncogene B(ErbB) receptor family holds crucial role in its pathogenesis. Human Epidermal Growth Factor Receptor 3 (HER-3) gene over expression in breast tissue has been associated with aggressive clinical behaviour and bad prognosis. Aim To evaluate HER-3 mRNA expression level as a prognostic marker for breast cancer and to correlate its level with other established prognostic parameters. Materials and Methods This study was carried out on specimens of 100 cases that were divided into 40 patients presented with fibroadenoma and 60 patients presented with Invasive Ductal Carcinoma (IDC) not otherwise specified and underwent modified radical mastectomy. All specimens were investigated for HER-2/neu, ER and PR expression by Immunohistochemistry (IHC) and quantitative assay of HER-3 mRNA expression using real time PCR technique. Results There was a significant high HER3 mRNA level in carcinoma cases compared to fibroadenoma. In malignant cases, HER3 mRNA level was significantly associated with advanced T stage, advanced N stage, number of positive lymph nodes, large tumour size and cases associated with an adjacent in situ component. Moreover, HER-3 mRNA level was of highest values in Her-2/neu positive group followed by triple negative cases with the lowest level in luminal group (p<0.05). Conclusion HER-3 gene is upregulated in IDC especially those carrying poor prognostic features. HER-3 mRNA level may identify a subset of patients with a poor prognosis, and who could undergo further evaluation for the efficacy of HER3 targeted anticancer therapy. PMID:28384967

  8. mRNA levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue.

    Science.gov (United States)

    Beyer, N; Coulson, D T R; Quinn, J G; Brockbank, S; Hellemans, J; Irvine, G B; Ravid, R; Johnston, J A

    2014-08-22

    β-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aβ peptides, proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (β-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbase(PLUS), employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aβ production.

  9. Collagen/elastin hydrogels cross-linked by squaric acid.

    Science.gov (United States)

    Skopinska-Wisniewska, J; Kuderko, J; Bajek, A; Maj, M; Sionkowska, A; Ziegler-Borowska, M

    2016-03-01

    Hydrogels based on collagen and elastin are very valuable materials for medicine and tissue engineering. They are biocompatible; however their mechanical properties and resistance for enzymatic degradation need to be improved by cross-linking. Up to this point many reagents have been tested but more secure reactants are still sought. Squaric acid (SqAc), 3,4-dihydroxy 3-cyclobutene 1,2-dione, is a strong, cyclic acid, which reacts easily with amine groups. The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5%, 10% and 20% of SqAc and neutralized via dialysis against deionized water were tested. Cross-linked, 3-D, transparent hydrogels were created. The cross-linked materials are stiffer and more resistant to enzymatic degradation than those that are unmodified. The pore size, swelling ability and surface polarity are reduced due to 5% and 10% of SqAc addition. At the same time, the cellular response is not significantly affected by the cross-linking. Therefore, squaric acid would be regarded as a safe, effective cross-linking agent.

  10. Elastin-like recombinamers as smart drug delivery systems.

    Science.gov (United States)

    Javier Arias, F; Santos, Mercedes; Ibáñez-Fonseca, Arturo; Piña, Maria Jesús; Serrano, Sofía

    2016-01-31

    Drug delivery systems that are able to control site and rate release of bioactive molecules are of particular interest for tissue therapy. Systems comprising biocompatible materials that can respond to environmental stimuli include elastin-like recombinamers (ELRs), a class of proteinaceous polymers bioinspired by natural elastin, which are especially useful as advanced drug delivery systems in the biomedical field. This review brings together information concerning different versions of ELR-based delivery systems that allow targeted delivery. ELR-drug systems in their monomeric form as well as drug encapsulation by nanoparticle-forming ELRs will be reviewed, focusing later on these drug carriers in which smart release is triggered by pH or temperature with a particular interest on cancer treatments. Systems for controlled drug release based on depots and hydrogels that act both as a support and reservoir in which drugs can be stored will be described, and their applications in drug delivery discussed. Finally, smart drug-delivery systems not based on ELRs, including those comprising proteins, synthetic polymers and non-polymeric systems, will also be briefly discussed.

  11. Inhibitory effect of theobromine on induction of angiogenesis and VEGF mRNA expression in v-raf transfectants of human urothelial cells HCV-29.

    Science.gov (United States)

    Skopinska-Rózewska, E; Janik, P; Przybyszewska, M; Sommer, E; Bialas-Chromiec, B

    1998-12-01

    Neovascularisation plays a crucial role in solid tumor growth and metastasis formation. Our previous studies showed that theophylline and theobromine suppressed cutaneous neovascular reaction induced in mice by human blood leukocytes, and lung as well as ovarian cancer cells. Here, we investigated the in vivo effect of theobromine on angiogenic activity of human urothelial cell line HCV-29, v-raf transfected (mouse cutaneous assay), and the in vitro effect of this drug on VEGF, tPA, uPA and PAI mRNA expression in these cells (RT-PCR method). Theobromine suppressed angiogenesis induced in mice by HCV-29-v-raf cells, inhibited VEGF mRNA expression, and had no effect on transcription of uPA and tPA in these cells. HCV-29-v-raf transfectants do not display transcripts of PAI, in the presence or the absence of theobromine.

  12. Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

    Science.gov (United States)

    Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

    2017-01-01

    Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

  13. Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

    Directory of Open Access Journals (Sweden)

    Ryan Aideen E

    2006-02-01

    Full Text Available Abstract Background During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat and three mouse (CMT93, CT26, B16F10 cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

  14. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  15. Human retina-specific amine oxidase: genomic structure of the gene (AOC2), alternatively spliced variant, and mRNA expression in retina.

    Science.gov (United States)

    Imamura, Y; Noda, S; Mashima, Y; Kudoh, J; Oguchi, Y; Shimizu, N

    1998-07-15

    Previously, we reported the isolation of cDNA for human retina-specific amine oxidase (RAO) and the expression of RAO exclusively in retina. Bacterial artificial chromosome clones containing the human RAO gene (AOC2) were mapped to human chromosome 17q21 (Imamura et al., 1997, Genomics 40: 277-283). Here, we report the complete genomic structure of the RAO gene, including 5' flanking sequence, and mRNA expression in retina. The human RAO gene spans 6 kb and is composed of four exons corresponding to the amino acid sequence 1-530, 530-598, 598-641, and 642-729 separated by three introns of 3000, 310, and 351 bp. Screening of a human retina cDNA library revealed the existence of an alternatively spliced cDNA variant with an additional 81 bp at the end of exon 2. The sizes of exons and the locations of exon/intron boundaries in the human RAO gene showed remarkable similarity to those of the human kidney diamine oxidase gene (AOC1). In situ hybridization revealed that mRNA coding for RAO is expressed preferentially in the ganglion cell layer of the mouse retina. We designed four sets of PCR primers to amplify four exons, which will be valuable for analyzing mutations in patients with ocular diseases affecting the retinal ganglion cell layer.

  16. Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.

    Science.gov (United States)

    Adams, Rebecca L; Mason, Aaron C; Glass, Laura; Aditi; Wente, Susan R

    2017-09-04

    The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA-dependent DEAD-box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP6 )-bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure-function analysis in S. cerevisiae and human (h) cells, and find that the high-affinity Nup42-Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy-terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5/hDDX19B interaction site. A nup42-CTD/gle1-CTD/Dbp5 trimeric complex forms in the presence of IP6 . Deletion of NUP42 abrogates Gle1-Dbp5 interaction, and disruption of the Nup42 or IP6 binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, non-additive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42-hGle1B interaction. This article is protected by copyright. All rights reserved.

  17. Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test.

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Giorgi Rossi, Paolo

    2011-07-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  18. Molecular Detection of Human Telomerase mRNA (hTERT-mRNA in Egyptian Patients with Hepatocellular Carcinoma

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    Gahan Kamal El-Saeed

    2009-06-01

    Full Text Available Background and Aims: Diagnostic modalities for hepatocellular carcinoma (HCC as markers, sonography, and CT have contributed to the early detection of HCC but are still not sensitive enough. Human telomerase RNA subunit (hTERT-mRNA has been identified in many cancers and claimed to be reactivated in HCC. To investigate hTERT-mRNA in the peripheral blood of HCC and chronic liver disease (CLD patients and correlate its level with alpha feto protein (AFP, the serological marker for HCC.Methods: The study was conducted on 44 patients selected from the National Liver Institute. Patients included Group I (22 patients diagnosed to have HCC, Group II (22 patients with CLD, and 12 apparently healthy volunteers as controls (Group III. All selected individuals were subjected to history taking, a clinical examination, abdominal sonography and laboratory investigations as liver function tests (LFTs, cell blood count (CBC, hepatitis viral markers, AFP, and real-time polymerase chain reaction (PCR Quantitative detection of -mRNA expression, encoding for telomerase catalytic subunit.Results: There was a significant elevation of AFP levels in the HCC group compared to both the CLD and control groups (P < 0.00, P < 0.001. The mean hTERT-mRNA expression in HCC patients was significantly higher than both CLD patients and controls (P < 0.001, P < 0.001. hTERT-mRNA was correlated with AFP and tumor size (P < 0.05, P < 0.001. The AFP cutoff level (185 ng/ml resulted in a 63.6% sensitivity, a 85.3% specificity; a 89.3% positive predictive value (PPV level, a 76.2 % negative predictive value (NPV level and a 83.4% accuracy for HCC prediction. The hTERT-mRNA cutoff level (112.5 copies/ml showed a 77.3% sensitivity, a 97.1% specificity, a 98% PPV level, a 79.2 % NPV level, and an accuracy of 84% for HCC prediction. Combining hTERT-mRNA and AFP increased diagnostic accuracy to 90.5%. Both markers had a 84.1% sensitivity, a 86.4% specificity, a 86.4% PPV level, and a 88

  19. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A L;

    1991-01-01

    We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold...... enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...... show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates...

  20. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

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    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  1. Endurance exercise induces mRNA expression of oxidative enzymes in human skeletal muscle late in recovery

    DEFF Research Database (Denmark)

    Leick, Lotte; Plomgaard, Peter S.; Grønløkke, L.

    2010-01-01

    exercise. To test the hypothesis that mRNA expression of many oxidative enzymes is up-regulated late in recovery (10-24 h) after exercise, male subjects (n=8) performed a 90-min cycling exercise (70% VO(2-max)), with muscle biopsies obtained before exercise (pre), and after 10, 18 and 24 h of recovery...

  2. OIL FLY ASH AND VANADIUM DIMINISH NRAMP-2MRNA AND PROTEIN EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELLS

    Science.gov (United States)

    The capacity of Nramp2 to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. Airway epithelial cells increase both mRNA and expression of that isoform of Nramp-2 without an iron response ele...

  3. Comparative mRNA Expression Profiles of Riboflavin Biosynthesis Genes in Lactobacilli Isolated from Human Feces and Fermented Bamboo Shoots

    Science.gov (United States)

    Thakur, Kiran; Tomar, Sudhir K.; Wei, Zhao-Jun

    2017-01-01

    With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods. PMID:28367143

  4. The optional long 5'-untranslated region of human ACAT1 mRNAs impairs the production of ACAT1 protein by promoting its mRNA decay

    Institute of Scientific and Technical Information of China (English)

    Xiaonan Zhao; Baoliang Song; Tayuan Chang; Boliang Li; Jia Chen; Lei Lei; Guangjing Hu; Ying Xiong; Jiajia Xu; Qin Li; Xinying Yang; Catherine C.Y.Chang

    2009-01-01

    We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG1397-1399 initiation codon,and also a minor 56 kDa isoform using the upstream in-frame GGC1274-1276initiation codon.The GGC1274-1276 codon is located at the optional long 5'-untranslated region(5'-UTR,nt 1-1396)of the mRNAs.The DNA sequences corresponding to this 5'-UTR are located in two different chromosomes,7 and 1.In the current work,we report that the optional long 5'-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG1397-1399 codon,mainly by promoting its mRNA decay.The western blot analyses indicated that the optional long 5'-UTR potently impaired the production of different proteins initiated from the AUG1397-1399codon,meaning that this impairing effect was not influenced by the 3'-UTR or the coding sequence of ACAT1 mRNA.The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5'-UTR dramatically reduced the contents of its linked mRNAs.Analyses of the protein to mRNA ratios showed that this 5'-UTR mainly decreased its mRNA stability rather than altering its translational efficiency.We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription.The results showed that this 5'-UTR promoted its mRNA decay.Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that,in both the cytoplasm and the nucleus of cells,the optional long 5'-UTR-linked mRNAs decayed faster than those without the link.Overall,our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.

  5. On the mechanical role of de novo synthesized elastin in the urinary bladder wall.

    Science.gov (United States)

    Wognum, Silvia; Schmidt, David E; Sacks, Michael S

    2009-10-01

    The urinary bladder wall (UBW), which is composed of smooth muscle, collagen, and elastin, undergoes profound remodeling in response to changes in mechanical loading resulting from various pathologies. In our laboratory, we have observed the production of fibrillar elastin in the extracellular matrix (ECM), which makes the UBW a particularly attractive tissue to investigate smooth muscle tissue remodeling. In the present study, we explored the mechanical role that de novo elastin fibers play in altering UBW ECM mechanical behavior using a structural constitutive modeling approach. The mechanical behavior of the collagen fiber component of the UBW ECM was determined from the biaxial stress-stretch response of normal UBW ECM, based on bimodal fiber recruitment that was motivated by the UBW's unique collagen fiber structure. The resulting fiber ensemble model was then combined with an experimentally derived fiber angular distribution to predict the biaxial mechanical behavior of normal and the elastin-rich UBW ECM to elucidate the underlying mechanisms of elastin production. Results indicated that UBW ECM exhibited a distinct structure with highly coiled collagen fiber bundles and visible elastic fibers in the pathological situation. Elastin-rich UBW ECM had a distinct mechanical behavior with higher compliance, attributable to the indirect effect of elastin fibers contracting the collagen fiber network, resulting in a retracted unloaded reference state of the tissue. In conclusion, our results suggest that the urinary bladder responds to prolonged periods of high strain by increasing its effective compliance through the interaction between collagen and de novo synthesized elastic fibers.

  6. Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    Dian-chao WU

    2010-01-01

    OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues,and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed.METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers.RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues (P = 0.020).The expression level of Tob at Dukes C + D phase was higher than Dukes A + B phase, and the difference was signifi cant (P < 0.05).However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer.CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

  7. Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance: A prospective cohort study.

    Science.gov (United States)

    Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette; Waldstrøm, Marianne; Schledermann, Doris; Frederiksen, Kirsten; Kjaer, Susanne K

    2016-10-15

    In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30-65 years with ASC-US during 2005-2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV-Proofer (n = 3,226) or for high-risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV-Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13,729) were followed for up to 9.5 years for high-grade cervical intraepithelial neoplasia (CIN) or cancer. After 3 years' follow-up, mRNA testing had higher specificity for CIN3 or worse (CIN3+) than HC2 testing (88.1% [95% confidence interval (CI): 86.8-89.6%] versus 59.3% [95% CI: 58.1-60.4%]) and higher positive predictive value (PPV) (38.2% [95% CI: 33.8%-43.1%] versus 19.5% [95% CI: 17.8-20.9%]). However, the sensitivity of mRNA testing was lower than that of HC2 testing (66.7% [95% CI: 59.3-74.5%] versus 97.0% [95% CI: 95.5-98.4%]), and women testing mRNA negative had higher 3-year risk for CIN3+ than those testing HC2 negative (3.2% [95% CI: 2.2-4.2%] versus 0.5% [95% CI: 0.3-0.7%]). Patterns were similar after 18 months and 5 years'; follow-up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16/18/31/33/45 mRNA test is not optimal for ASC-US triage due to its low sensitivity and the substantial risk for precancer following a negative test.

  8. Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

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    Rossie Sandra

    2004-06-01

    Full Text Available Abstract Background Intermediate-conductance, calcium-activated potassium channels (IKs modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. Methods Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1 expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. Results hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D. Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. Conclusion Human keratinocyte differentiation is

  9. Myogenic, matrix and growth factor mRNA expression in human skeletal muscle: effect of contraction intensity and feeding

    DEFF Research Database (Denmark)

    Agergaard, Jakob; Reitelseder, Søren; Pedersen, T.G.

    2013-01-01

    exercise. No major differences were seen in atrophy-related genes between HL and LL resistance exercise. No changes were seen over 12-week training for any of the targets. CONCLUSIONS: Resistance exercise at LL and HL elevated the expression of genes involved in skeletal muscle hypertrophy, although......INTRODUCTION: We examined short-term (3-hour) and long-term (12-week) training effects after heavy load [HL; 70% 1RM] and light load (LL; 16% 1RM) exercise. METHODS: mRNA expression of genes involved in skeletal muscle remodeling were analyzed and muscle activity (EMG measurements) was measured....... RESULTS: Relative muscle activity differed between HL and LL resistance exercise, whereas median power frequency was even, suggesting an equal muscle-fiber-type recruitment distribution. mRNA expression of Myf6, myogenin, and p21 was mostly increased, and myostatin was mostly depressed by HL resistance...

  10. Lorenzo Gotte (1926-1991: a pioneer of elastin

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    M. Onisto

    2016-09-01

    Full Text Available Lorenzo Gotte (1926-1991 was an outstanding histologist at the School of Medicine of Padua. This year marks the 25th anniversary of his passing away – commemorated during the recent congress of the Italian Society for the Connective Tissue (SISC, held in Padua (September 30 - October 1, 2016. This brief note recalls this outstanding figure: indeed, forthose who knew him, Lorenzo Gotte was an exceptional scientist and at the same time, an unparalleled teacher – and, for many, a great friend. It is still difficult to separate these aspects of his personality, so intertwined in his life: studying elastin and elastic tissue was a passion central to Gotte’s life.

  11. MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells

    OpenAIRE

    Kai Xu; Jing Lin; Roza Zandi; Jack A Roth; Lin Ji

    2016-01-01

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endon...

  12. mRNA expression and localization of bNOS, eNOS and iNOS in human cervix at preterm and term labour

    Directory of Open Access Journals (Sweden)

    Byström Birgitta

    2005-08-01

    Full Text Available Abstract Background Preterm birth is the primary cause of the neonatal mortality and morbidity. There will be no preterm birth without a cervical softening. Nitric oxide (NO is shown to be a mediator of term cervical ripening. The aim of this study was to investigate mRNA expression of the three isomers of NO synthases (NOS and to identify them by immunohistochemistry in the human cervix at preterm birth compared to term. Methods The three isomers of NOS- inducible (iNOS, endothelial (eNOS and neuronal (bNOS – were investigated in the human cervix. The expression of mRNA was determined using Real-Time Multiplex RT-PCR. The localisation of synthases in the cervical tissue was analysed using immunohistochemistry. Cervical biopsies were obtained from 4 groups of women without clinical signs of infection: preterm (PTL, term labour (TL, preterm not in labour (PTnotL and term not in labour (TnotL patients. One-Way ANOVA, Kruskal-Wallis, Student t-test or Mann-Whitney test were applied as appropriate to determine statistically significant differences among the groups. Results Patients in preterm labour had significantly (p Conclusion The mRNA levels were higher in the preterm labour group compared to the women at term labour. The significant increase of the eNOS mRNA expression, from the unripe to the favourable cervical state during labour, may indicate a role of eNOS and supports the role of NO in the cervical ripening process. All the three synthases were identified by immunohistochemistry in all the groups of study.

  13. Effect of hepatitis C virus infection on the mRNA expression of drug transporters and cytochrome p450 enzymes in chimeric mice with humanized liver.

    Science.gov (United States)

    Kikuchi, Ryota; McCown, Matthew; Olson, Pamela; Tateno, Chise; Morikawa, Yoshio; Katoh, Yumiko; Bourdet, David L; Monshouwer, Mario; Fretland, Adrian J

    2010-11-01

    The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.

  14. Effect of Nuclear Factor-kappa B on Vascular Endothelial Growth Factor mRNA Expression of Human Pulmonary Artery Smooth Muscle Cells in Hypoxia

    Institute of Scientific and Technical Information of China (English)

    张焕萍; 徐永健; 张珍祥; 许淑云; 倪望; 陈士新

    2004-01-01

    Summary: In order to investigate the effect of nuclear factor-kappa B (NF-κB) on vascular endothelial growth factor (VEGF) mRNA expression of human pulmonary artery smooth muscle cells (HPASMCs) in hypoxia, the cultured HPASMCs in vitro were stimulated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. The NF-κB p65 nuclei positive expression was detected by immunocytochemical technique. The IκBa protein expression was measured by Western blot.RT-PCR was used to detect the VEGF mRNA expression of HPASMCs. The results showed that no significant change was observed in the NF-κB p65 nuclei positive expression of cultured HPASMCs during 6 h-24 h in normoxia, but the levels of NF-κB p65 nuclei positive expression of cultured HPASMCs were significantly increased in hypoxia groups as compared with those in all normoxia groups (P<0.05). The IκBα protein expression of cultured HPASMCs showed no significant change during 6 h-24 h in normoxia, but significantly decreased in hypoxia as comapred with that in normoxia groups (P<0.05). PDTC (1 to 100 μmol/L) could inhibit the VEGF mRNA expression of HPASMCs in a concentration-dependent manner in hypoxia. In conclusion, NF-κB can be partly translocation activated from cytoplasm into nuclei in the cultured HPASMCs under hypoxia. The inhibition of NF-κB activation can decrease the VEGF mRNA expression. h is suggested that the activation of NF-κB is involved in the VEGFmRNA expression of HPASMCs under hypoxia.

  15. Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

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    Joensuu Jussi J

    2009-08-01

    Full Text Available Abstract Background Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants. Results The effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system. Conclusion An endoplasmic reticulum-targeted elastin-like polypeptide fusion approach

  16. Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

    DEFF Research Database (Denmark)

    Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette

    2005-01-01

    Regular exercise induces a variety of adaptive responses that enhance the oxidative and metabolic capacity of human skeletal muscle. Although the physiological adjustments of regular exercise have been known for decades, the underlying mechanisms are still unclear. The hypoxia inducible factors 1...... with a single exercise bout, and that this response is blunted with training. We obtained muscle biopsies from a trained (5 days/week during 4 weeks) and untrained leg from the same human subject before, immediately after, and during the recovery from a 3 h two-legged knee extensor exercise bout, where the two......alpha and HIF-2alpha mRNA levels are transiently increased in untrained human skeletal muscle in response to an acute exercise bout, but this response is blunted after exercise training. We propose that HIFs expression is upregulated with exercise and that it may be an important transcription factor...

  17. Comprehensive Analysis of MicroRNA and mRNA Expression in Normal and Tumorous Human Esophageal Squamous Cell Lines Using Microarray Datasets

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    Ichiro Akagi

    2014-01-01

    Full Text Available Despite the undisputed importance of altered microRNA (miRNA expression in various cancers, there is limited information on the clinicopathologic significance of cancer-related miRNAs in esophageal squamous cell carcinoma (ESCC. Previously, it was reported that the expression of several miRNAs was dysregulated in ESCC. However, the target genes of these miRNAs have not been identified. Furthermore, additional miRNAs in humans have been discovered recently, indicating that revised miRNA and gene expression profiling for ESCC are necessary. Here, we provide datasets from microarray analyses to identify miRNA and mRNA expression comprehensively in Het-1A, a normal human esophageal squamous cell line, and three human ESCC cell lines.

  18. Characteristic interaction of Ca2+ ions with elastin coacervate: ion transport study across coacervate layers of alpha-elastin and elastin model polypeptide, (Val-Pro-Gly-Val-Gly)n.

    Science.gov (United States)

    Kaibara, K; Akinari, Y; Okamoto, K; Uemura, Y; Yamamoto, S; Kodama, H; Kondo, M

    1996-08-01

    Ion transport characteristics across a macrocoacervate layer membrane composed of aqueous elastin model polypeptides with a specific repeating pentapeptide sequence, H-(Val-Pro-Gly-Val-Gly)n-Val-OMe (n > or = 40), were investigated. Transmembrane potential responses for NaCl. MgCl2, and CaCl2 concentration-cell systems were measured and examined systematically by comparing with those across a coacervate membrane composed of bovine neck ligamental alpha-elastin. In the case of the NaCl and MgCl2 systems, potential responses across these protein liquid membranes were different noticeably from each other depending upon the molecular structure with and without charged peptide side chains, whereas in the CaCl2 systems the transmembrane potential responses across the noncharged polypentapeptide coacervate membrane were comparable with those across the alpha-elastin coacervate membrane carrying both the positively and negatively charged amino acid residues as an amphoteric ion-exchange membrane. These results indicated that mechanisms of major Ca2+ ion transport are based on the specific and selective interactions with electrically neutral sites of elastin, such as the polypentapeptide backbone chain.

  19. Downregulation of VEGF mRNA expression by triamcinolone acetonide acetate-loaded chitosan derivative nanoparticles in human retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Zhou H

    2012-08-01

    Full Text Available Huaisheng Zhou,1 Liqun Yang,2,* Huajie Li,2 Haijun Gong,1 Liangzheng Cheng,2 Haisheng Zheng,1 Li-Ming Zhang,2 Yuqing Lan1,*1Department of Ophthalmology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 2Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-sen University, Guangzhou, China*Both corresponding authors contributed equally to this workBackground: The purpose of this study was to investigate the downregulation of mRNA expression of vascular endothelial growth factor (VEGF by triamcinolone acetonide acetate (TAA-loaded chitosan nanoparticles in human retinal pigment epithelial cells.Methods: TAA-loaded deoxycholic acid-modified chitosan (TAA/DA-Chit nanoparticles were prepared via a self-assembly mechanism, and their morphology and zeta potential were examined by transmission electron microscopy and zeta potential analysis, respectively. DA-Chit and TAA/DA-Chit nanoparticle toxicity was evaluated using a Cell Counting Kit-8 assay. The efficiency of cellular uptake was determined using fluorescein isothiocyanate-labeled DA-Chit nanoparticles, in place of TAA/DA-Chit nanoparticles, assessed by both inverted fluorescence microscopy and flow cytometry. Downregulation of VEGF mRNA expression by TAA/DA-Chit nanoparticles was further investigated by real-time reverse transcription polymerase chain reaction (RT-PCR assay of the treated human retinal pigment epithelial cells.Results: TAA/DA-Chit nanoparticles were prepared with a TAA-loading capacity in the range of 12%–82%, which increased the water solubility of TAA from 0.3 mg/mL to 2.1 mg/mL. These nanoparticles showed oblate shapes 100–550 nm in size in transmission electron microscopic images and had positive zeta potentials. The Cell Counting Kit-8 assay indicated that the DA-Chit and

  20. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion

    Science.gov (United States)

    Haque, Shabirul; Yan, Xiao Jie; Rosen, Lisa; McCormick, Steven; Chiorazzi, Nicholas; Mongini, Patricia K. A.

    2014-01-01

    Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM+IgD+CD27− B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID+ cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5×10−4) greater than the baseline error rate (<0.8×10−4). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.—Haque, S., Yan, X. J., Rosen, L., McCormick, S., Chiorazzi, N., Mongini, P. K. A. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion. PMID:24145719

  1. Elastin-insufficient mice show normal cardiovascular remodeling in 2K1C hypertension despite higher baseline pressure and unique cardiovascular architecture.

    Science.gov (United States)

    Wagenseil, Jessica E; Knutsen, Russell H; Li, Dean Y; Mecham, Robert P

    2007-07-01

    Mice heterozygous for the elastin gene (ELN(+/-)) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN(+/-) mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN(+/-) cardiovascular system, we increased blood pressure in adult ELN(+/+) and ELN(+/-) mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN(+/+) and ELN(+/-)-clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN(+/+) and ELN(+/-) hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN(+/-) mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels.

  2. Differential effects of Nd-YAG laser on collagen and elastin production by chick embryo aortae in vitro. Relevance to laser angioplasty for removal of atherosclerotic plaques

    Energy Technology Data Exchange (ETDEWEB)

    Abergel, R.P.; Zaragoza, E.J.; Dwyer, R.M.; Uitto, J.

    1985-08-30

    Aortae from 17-day old chick embryos were subjected to irradiation with a Nd:YAG laser at energy densities varying from 1.2 - 4.7 X 10(3) J/cm2. The aortae were pulse-labeled in vitro with (/sup 3/H)proline or (/sup 14/C)valine, and the synthesis of collagenous polypeptides and soluble elastin was examined by SDS-polyacrylamide gel electrophoresis, followed by fluorography and quantitative scanning densitometry. Irradiation of the aortae with Nd:YAG laser resulted in inhibition of the synthesis of the extracellular matrix proteins. The production of collagen was inhibited to a considerably larger degree than the production of elastin. Thus, the biosynthetic pathway for collagen production appears to be more susceptible to laser inhibition than the corresponding pathway for elastin production. These observations may have relevance to laser angioplasty which has been proposed to be applicable for removal of atherosclerotic plaques in human vessels. Specifically, the results suggest that inhibition of the extracellular matrix production may result in weakening of the vessel wall with subsequent aneurysm formation and rupture.

  3. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    Science.gov (United States)

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  4. MHC2TA mRNA levels and human herpesvirus 6 in multiple sclerosis patients treated with interferon beta along two-year follow-up

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    Dominguez-Mozo Maria Inmaculada

    2012-09-01

    Full Text Available Abstract Background In previous studies we found that MHC2TA +1614 genotype frequency was very different when MS patients with and without human herpesvirus 6 (HHV-6 in serum samples were compared; a different clinical behavior was also described. The purpose of the study was: 1. To evaluate if MHC2TA expression in MS patients was influenced by interferon beta (IFN-beta treatment. 2. To study MHC2TA expression in MS patients with and without minor allele C. 3. To analyze the relation between MHC2TA mRNA levels and HHV-6 active infection in MS patients. Methods Blood and serum samples of 154 MS patients were collected in five programmed visits: basal (prior to beginning IFN-beta treatment, six, twelve, eighteen and twenty-four months later. HHV-6 in serum and MHC2TA mRNA levels were evaluated by PCR and RT-PCR, respectively. Neutralizing antibodies (NAbs against IFN-beta were analyzed by the cytopathic effect assay. Results We found that MHC2TA mRNA levels were significantly lower among MS patients with HHV-6 active infection at the basal visit (without treatment than in those MS patients without HHV-6 active infection at the basal visit (p = 0.012; in all the positive samples we only found variant A. Furthermore, 58/99 (58.6% MS patients without HHV-6 along the five programmed visits and an increase of MHC2TA expression after two-years of IFN-beta treatment were clinical responders vs. 5/21 (23.8% among those MS patients with HHV-6 and a decrease of MHC2TA mRNA levels along the two-years with IFN-beta treatment (p = 0.004; no differences were found between patients with and without NAbs. Conclusions MHC2TA mRNA levels could be decreased by the active replication of HHV-6; the absence of HHV-6 in serum and the increase of MHC2TA expression could be further studied as markers of good clinical response to IFN-beta treatment.

  5. The interaction of serum and arterial lipoproteins with elastin of the arterial intima and its role in the lipid accumulation in atherosclerotic plaques.

    Science.gov (United States)

    Kramsch, D M; Hollander, W

    1973-02-01

    Arterial elastin appears to be a proteinlipid complex with the lipid component being bound to elastin peptide groups. In atherosclerotic lesions the lipid content of elastin increases progressively with increasing severity of atherosclerosis. The increases in the lipid content of plaque elastin are mainly due to large increases in cholesterol with about 80% of the cholesterol being cholesterol ester. This deposition of cholesterol in elastin accounts for a substantial part of the total cholesterol accumulation in atherosclerotic lesions of all stages. The present in vitro study suggests that the mechanism involved in the deposition of lipids in arterial elastin may be an interaction of the elastin protein with serum or arterial low density or very low density lipoproteins (LDL and VLDL) resulting in a transfer of lipids, but not of lipoprotein protein to the elastin. No significant lipid transfer occurred from the high density lipoproteins or chylomicrons. The amount of lipid taken up by plaque elastin was strikingly higher than by normal elastin and consisted mainly of cholesterol with over 80% of the cholesterol being cholesterol ester. The precondition for the lipid accumulation in plaque elastin appeared to be an altered amino acid composition of the elastin protein consisting of an increase in polar amino acids and a reduction in cross-linking amino acids. Subsequent treatment of lipoprotein-incubated arterial elastin with hot alkali and apolipoproteins did not reverse the binding of lipoprotein lipid to diseased elastin.

  6. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  7. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    Science.gov (United States)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  8. Elastin-Coated Biodegradable Photopolymer Scaffolds for Tissue Engineering Applications

    Directory of Open Access Journals (Sweden)

    Rossella Barenghi

    2014-01-01

    Full Text Available One of the main open issues in modern vascular surgery is the nonbiodegradability of implants used for stent interventions, which can lead to small caliber-related thrombosis and neointimal hyperplasia. Some new, resorbable polymeric materials have been proposed to substitute traditional stainless-steel stents, but so far they were affected by poor mechanical properties and low biocompatibility. In this respect, a new material, polypropylene fumarate (PPF, may be considered as a promising candidate to implement the development of next generation stents, due to its complete biodegradability, and excellent mechanical properties and the ease to be precisely patterned. Besides all these benefits, PPF has not been tested yet for vascular prosthesis, mainly because it proved to be almost inert, while the ability to elicit a specific biological function would be of paramount importance in such critical surgery applications. Here, we propose a biomimetic functionalization process, aimed at obtaining specific bioactivation and thus improved cell-polymer interaction. Porous PPF-based scaffolds produced by deep-UV photocuring were coated by elastin and the functionalized scaffolds were extensively characterized, revealing a stable bound between the protein and the polymer surface. Both 3T3 and HUVEC cell lines were used for in vitro tests displaying an enhancement of cells adhesion and proliferation on the functionalized scaffolds.

  9. Relationships between hepatic stearoyl-CoA desaturase-1 activity and mRNA expression with liver fat content in humans.

    Science.gov (United States)

    Peter, Andreas; Cegan, Alexander; Wagner, Silvia; Elcnerova, Michaela; Königsrainer, Alfred; Königsrainer, Ingmar; Häring, Hans-Ulrich; Schleicher, Erwin D; Stefan, Norbert

    2011-02-01

    Stearoyl-CoA desaturase-1 (SCD1) has gained much interest as a future drug target to treat fatty liver and its consequences. However, there are few and inconsistent human data about expression and activity of this important enzyme. We investigated activity and expression of SCD1 and their relationships with liver fat (LF) content in human liver samples. Fifty subjects undergoing liver surgery were studied. SCD1 activity was estimated from the ratio of oleate (C18:1) to stearate (C18:0) within lipid subfractions. Furthermore, SCD1 mRNA expression and LF content were measured. Similarly to previous studies, we observed a strong positive correlation between LF content and the C18:1/C18:0 ratio in the combined fatty acid (FA) fractions (r = 0.96, P < 0.0001), which could be interpreted as higher SCD1 activity with increasing LF. However, hepatic SCD1 mRNA expression did not correlate with LF (r = 0.16, P = 0.13). To solve these conflicting data, we analyzed the FA composition of hepatic lipid subfractions. With increasing LF content the amount of FAs from the triglyceride (TG) fraction increased (r = 0.96, P < 0.0001), whereas the FAs from the phospholipid (PL) fraction remained unchanged (r = -0.17, P = 0.19). Of these two major lipid fractions, the C18:1/C18:0 ratio in TG was 16-fold higher than in PL. Supporting the SCD1 mRNA expression data, the C18:1/C18:0 ratio of the TG or PL fraction did not correlate with LF (r = 0.26, P = 0.12 and r = 0.08, P = 0.29). We provide novel information that SCD1 activity and mRNA expression appear not to be elevated in subjects with high LF content. We suggest that the FA composition of lipid subclasses, rather than of mixed lipids, should be analyzed to estimate SCD1 activity.

  10. Biomimetic collagen/elastin meshes for ventral hernia repair in a rat model.

    Science.gov (United States)

    Minardi, Silvia; Taraballi, Francesca; Wang, Xin; Cabrera, Fernando J; Van Eps, Jeffrey L; Robbins, Andrew B; Sandri, Monica; Moreno, Michael R; Weiner, Bradley K; Tasciotti, Ennio

    2017-03-01

    Ventral hernia repair remains a major clinical need. Herein, we formulated a type I collagen/elastin crosslinked blend (CollE) for the fabrication of biomimetic meshes for ventral hernia repair. To evaluate the effect of architecture on the performance of the implants, CollE was formulated both as flat sheets (CollE Sheets) and porous scaffolds (CollE Scaffolds). The morphology, hydrophylicity and in vitro degradation were assessed by SEM, water contact angle and differential scanning calorimetry, respectively. The stiffness of the meshes was determined using a constant stretch rate uniaxial tensile test, and compared to that of native tissue. CollE Sheets and Scaffolds were tested in vitro with human bone marrow-derived mesenchymal stem cells (h-BM-MSC), and finally implanted in a rat ventral hernia model. Neovascularization and tissue regeneration within the implants was evaluated at 6weeks, by histology, immunofluorescence, and q-PCR. It was found that CollE Sheets and Scaffolds were not only biomechanically sturdy enough to provide immediate repair of the hernia defect, but also promoted tissue restoration in only 6weeks. In fact, the presence of elastin enhanced the neovascularization in both sheets and scaffolds. Overall, CollE Scaffolds displayed mechanical properties more closely resembling those of native tissue, and induced higher gene expression of the entire marker genes tested, associated with de novo matrix deposition, angiogenesis, adipogenesis and skeletal muscles, compared to CollE Sheets. Altogether, this data suggests that the improved mechanical properties and bioactivity of CollE Sheets and Scaffolds make them valuable candidates for applications of ventral hernia repair. Due to the elevated annual number of ventral hernia repair in the US, the lack of successful grafts, the design of innovative biomimetic meshes has become a prime focus in tissue engineering, to promote the repair of the abdominal wall, avoid recurrence. Our meshes (Coll

  11. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  12. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Science.gov (United States)

    Lai, Ming-Chih; Wang, Shainn-Wei; Cheng, Lie; Tarn, Woan-Yuh; Tsai, Shaw-Jenq; Sun, H Sunny

    2013-01-01

    Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  13. EXPRESSION OF mRNA FOR MEMBRANE-TYPE 1, 2, AND 3 MATRIX METALLOPROTEINASES IN HUMAN LARYNGEAL CANCER

    Institute of Scientific and Technical Information of China (English)

    Ya-nan Sun; Yuan Li

    2004-01-01

    Objective To investigate correlation of expressions of membrane-type 1, 2, and 3 matrix metalloproteinases (MT1, MT2,and MT3-MMP) to the invasion and metastases in laryngeal cancer.Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the mRNA level of MT1,MT2, and MT3-MMP in 24 patients with laryngeal cancer. The relationships of these three MT-MMP expressions to clinicopathology were analyzed by statistics.Results The expressions of MT1, MT2, and MT3-MMP were significantly higher in laryngeal cancer tissues than those in para-tumorous tissues (P < 0.01) and had a close relationship with invasive depth (P < 0.05). But no significantly different expressions of these three MT-MMPs were found in different primary location and different histological grade of laryngeal cancer (P > 0.05). The expression of MT1-MMP was obviously higher in patients with metastatic lymph nodes than that in patients without metastatic lymph nodes (P < 0.05).Conclusion MT1, MT2, and MT3-MMP play an important role in the progression of laryngeal cancer, and MT1-MMP may serve as a reliable marker in estimating invasive and metastatic potency of laryngeal cancer. Suppressing expressions of MT 1, MT2, and MT3-MMP early may inhibit the invasion and metastases of laryngeal cancer.

  14. Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

    OpenAIRE

    Fong, Y; M. Rosenbaum; Tracey, K J; Raman, G.; Hesse, D G; Matthews, D. E.; Leibel, R. L.; Gertner, J M; Fischman, D. A.; Lowry, S F

    1989-01-01

    A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous ...

  15. Asymmetric cell-matrix and biomechanical abnormalities in elastin insufficiency induced aortopathy.

    Science.gov (United States)

    Krishnamurthy, Varun K; Evans, Ashlie N; Wansapura, Janaka P; Osinska, Hanna; Maddy, Kelsey E; Biechler, Stefanie V; Narmoneva, Daria A; Goodwin, Richard L; Hinton, Robert B

    2014-10-01

    Aortopathy is characterized by vascular smooth muscle cell (VSMC) abnormalities and elastic fiber fragmentation. Elastin insufficient (Eln (+/-)) mice demonstrate latent aortopathy similar to human disease. We hypothesized that aortopathy manifests primarily in the aorto-pulmonary septal (APS) side of the thoracic aorta due to asymmetric cardiac neural crest (CNC) distribution. Anatomic (aortic root vs. ascending aorta) and molecular (APS vs. non-APS) regions of proximal aorta tissue were examined in adult and aged wild type (WT) and mutant (Eln (+/-)) mice. CNC, VSMCs, elastic fiber architecture, proteoglycan expression, morphometrics and biomechanical properties were examined using histology, 3D reconstruction, micropipette aspiration and in vivo magnetic resonance imaging (MRI). In the APS side of Eln (+/-) aorta, Sonic Hedgehog (SHH) is decreased while SM22 is increased. Elastic fiber architecture abnormalities are present in the Eln (+/-) aortic root and APS ascending aorta, and biglycan is increased in the aortic root while aggrecan is increased in the APS aorta. The Eln (+/-) ascending aorta is stiffer than the aortic root, the APS side is thicker and stiffer than the non-APS side, and significant differences in the individual aortic root sinuses are observed. Asymmetric structure-function abnormalities implicate regional CNC dysregulation in the development and progression of aortopathy.

  16. Application of elastin-mimetic recombinant proteins in chemotherapeutics delivery, cellular engineering, and regenerative medicine.

    Science.gov (United States)

    Jeon, Won Bae

    2013-01-01

    With the remarkable increase in the fields of biomedical engineering and regenerative medicine, biomaterial design has become an indispensable approach for developing the biocompatible carriers for drug or gene cargo and extracellular matrix (ECM) for cell survival, proliferation and differentiation. Native ECM materials derived from animal tissues were believed to be the best choices for tissue engineering. However, possible pathogen contamination by cellular remnants from foreign animal tissues is an unavoidable issue that has limited the use of native ECM for human benefit. Some synthetic polymers have been used as alternative materials for manufacturing native ECM because of the biodegradability and ease of large-scale production of the polymers. However, the inherent polydispersity of the polymers causes batch-to-batch variation in polymer composition and possible cytotoxic interactions between chemical matrices and neighboring cells or tissues have not yet been fully resolved. Elastin-like proteins (ELPs) are genetically engineered biopolymers modeled after the naturally occurring tropoelastin and have emerged as promising materials for biomedical applications because they are biocompatible, non-immunogenic and biodegradable, and their composition, mechanical stiffness and even fate within the cell can be controlled at the gene level. This commentary highlights the recent progresses in the development of the ELP-based recombinant proteins that are being increasingly used for the delivery of chemotherapeutics and to provide a cell-friendly ECM environment.

  17. Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

    Directory of Open Access Journals (Sweden)

    Maciej Lech

    2013-09-01

    Full Text Available The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring.

  18. Increased Levels of Cell-Free Human Placental Lactogen mRNA at 28-32 Gestational Weeks in Plasma of Pregnant Women With Placenta Previa and Invasive Placenta

    OpenAIRE

    2014-01-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive place...

  19. Manganese superoxide dismutase: effect of the ala16val polymorphism on protein, activity, and mRNA levels in human breast cancer cell lines and stably transfected mouse embryonic fibroblasts.

    Science.gov (United States)

    McAtee, Britt L; Yager, James D

    2010-02-01

    The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.

  20. Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization.

    Science.gov (United States)

    Foraida, Zahraa I; Kamaldinov, Tim; Nelson, Deirdre A; Larsen, Melinda; Castracane, James

    2017-08-08

    Development of electrospun nanofibers that mimic the structural, mechanical and biochemical properties of natural extracellular matrices (ECMs) is a promising approach for tissue regeneration. Electrospun fibers of synthetic polymers partially mimic the topography of the ECM, however, their high stiffness, poor hydrophilicity and lack of in vivo-like biochemical cues is not optimal for epithelial cell self-organization and function. In search of a biomimetic scaffold for salivary gland tissue regeneration, we investigated the potential of elastin, an ECM protein, to generate elastin hybrid nanofibers that have favorable physical and biochemical properties for regeneration of the salivary glands. Elastin was introduced to our previously developed poly-lactic-co-glycolic acid (PLGA) nanofiber scaffolds by two methods, blend electrospinning (EP-blend) and covalent conjugation (EP-covalent). Both methods for elastin incorporation into the nanofibers improved the wettability of the scaffolds while only blend electrospinning of elastin-PLGA nanofibers and not surface conjugation of elastin to PLGA fibers, conferred increased elasticity to the nanofibers measured by Young's modulus. After two days, only the blend electrospun nanofiber scaffolds facilitated epithelial cell self-organization into cell clusters, assessed with nuclear area and nearest neighbor distance measurements, leading to the apicobasal polarization of salivary gland epithelial cells after six days, which is vital for cell function. This study suggests that elastin electrospun nanofiber scaffolds have potential application in regenerative therapies for salivary glands and other epithelial organs. Regenerating the salivary glands by mimicking the extracellular matrix (ECM) is a promising approach for long term treatment of salivary gland damage. Despite their topographic similarity to the ECM, electrospun fibers of synthetic polymers lack the biochemical complexity, elasticity and hydrophilicity of the

  1. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  2. Expression of Cyclooxygenase-2 mRNA and Identification of Its Splice Variant in Human Myometrium Obtained from Women in Labor

    Institute of Scientific and Technical Information of China (English)

    HUANG Yinping; WAN Jingyuan; YE Duyun; WU Ping; HUANG Yanjun; ZHANG Li; ZHOU Xiaoyan; HUANG Yunfeng; YUAN Ping; ZHANG Daijuan

    2005-01-01

    In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.

  3. Comparison of three human papillomavirus DNA assays and one mRNA assay in women with abnormal cytology

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Lynge, Elsebeth; Ejegod, Ditte;

    2014-01-01

    OBJECTIVE: To compare the clinical characteristics of four human papillomavirus (HPV) assays: hybrid capture 2 (HC2), cobas, CLART, and APTIMA in Danish women with abnormal cytology. METHODS: SurePath samples from 367 consecutive women from Copenhagen, with atypical squamous cells of undetermined...

  4. Integration Analysis of MicroRNA and mRNA Expression Profiles in Human Peripheral Blood Lymphocytes Cultured in Modeled Microgravity

    Directory of Open Access Journals (Sweden)

    C. Girardi

    2014-01-01

    Full Text Available We analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs incubated in microgravity condition, simulated by a ground-based rotating wall vessel (RWV bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1 g incubated ones. Among these, miR-9-5p, miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs, we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichment in the biological processes of immune/inflammatory response, signal transduction, regulation of response to stress, regulation of programmed cell death, and regulation of cell proliferation. We identified the correlation of miR-9-3p, miR-155-5p, miR-150-3p, and miR-378-3p expression with that of genes involved in immune/inflammatory response (e.g., IFNG and IL17F, apoptosis (e.g., PDCD4 and PTEN, and cell proliferation (e.g., NKX3-1 and GADD45A. Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation.

  5. Structural analysis of human 2'-O-ribose methyltransferases involved in mRNA cap structure formation

    Science.gov (United States)

    Smietanski, Miroslaw; Werner, Maria; Purta, Elzbieta; Kaminska, Katarzyna H.; Stepinski, Janusz; Darzynkiewicz, Edward; Nowotny, Marcin; Bujnicki, Janusz M.

    2014-01-01

    The 5' cap of human messenger RNA contains 2'-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.

  6. Elastin and Mechanics of Pig Pericardial Resistance Arteries (pPRA)

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Leurgans, Thomas; Rosenstand, Kristoffer

    Resistance arteries are remodeled in hypertension and diabetes. Elastin was reported to play a role herein. The parietal pericardium is opened during cardio-thoracic surgeries and might be a valuable biopsy for research in cardio-vascular diseases. We tested the hypothesis that resistance arteries...... can be isolated from the pericardium to study the micro-architecture of elastin and vascular wall mechanics. The pericardium of pigs served to test the hypothesis. pPRAs were microdissected. Their structure was examined using multiphoton excitation fluorescence microscopy. Diameter-tension...... and pressure-diameter-length relationships were recorded in myographs. Findings are compared to rodent mesenteric resistance arteries and –basilar arteries (rMRA, rBA) with comparable lumen diameter (±300µm at 100mmHg). pPRA have no clear external elastic lamina (present in rMRA, but not rBA), scant elastin...

  7. Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level

    Science.gov (United States)

    SERRANO, A. L.; PÉREZ, MARGARITA; LUCÍA, A.; CHICHARRO, J. L.; QUIROZ-ROTHE, E.; RIVERO, J. L. L.

    2001-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC β-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies. PMID:11554510

  8. VHL Frameshift Mutation as Target of Nonsense-Mediated mRNA Decay in Drosophila melanogaster and Human HEK293 Cell Line

    Directory of Open Access Journals (Sweden)

    Lucia Micale

    2009-01-01

    Full Text Available There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD, a process that typically degrades transcripts containing premature termination codons (PTCs in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1D45B line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45B line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.

  9. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions

    DEFF Research Database (Denmark)

    Awan, Aashir; Oliveri, Roberto S; Jensen, Pernille L

    2010-01-01

    This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging...... onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell...

  10. Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

    Science.gov (United States)

    MacEwan, Sarah R; Hassouneh, Wafa; Chilkoti, Ashutosh

    2014-06-09

    Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

  11. Elastin and Mechanics of Pig Pericardial Resistance Arteries (pPRA)

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Leurgans, Thomas; Rosenstand, Kristoffer

    Resistance arteries are remodeled in hypertension and diabetes. Elastin was reported to play a role herein. The parietal pericardium is opened during cardio-thoracic surgeries and might be a valuable biopsy for research in cardio-vascular diseases. We tested the hypothesis that resistance arteries...... can be isolated from the pericardium to study the micro-architecture of elastin and vascular wall mechanics. The pericardium of pigs served to test the hypothesis. pPRAs were microdissected. Their structure was examined using multiphoton excitation fluorescence microscopy. Diameter...

  12. Regulation of the stability of poly(I)xpoly(C)-induced human fibroblast interferon mRNA: selective inactivation of interferon mRNA and lack of involvement of 2',5'-oligo(A) synthetase activation during the shutoff of interferon production.

    Science.gov (United States)

    Sehgal, P B; Gupta, S L

    1980-06-01

    The inactivation of interferon mRNA during the shutoff phase of interferon production in poly(I)xpoly(C)-induced human fibroblast cultures is selective. We have determined that the shutoff of interferon production, which takes place from 3 to 8 hr after the beginning of induction, is not associated with an appreciable declined in the rate of bulk cellular protein synthesis or of cellular protein secretion. While the amount of translatable interferon mRNA declined markedly during the shutoff phase, the level of translatable bulk cellular mRNA and the stability of [3H]uridine-labeled mRNA were unaffected. Superinduction with actinomycin D selectively stabilized interferon mRNA with no apparent effect on the stability of bulk cellular mRNA. Furthermore, an activation of the 2',5'-oligo(A) synthetase/endonuclease system does not appear to be involved in the shutoff phenomenon. Uninduced FS-4 cells contained a low basal level of 2'5'-oligo(A) synthetase activity, which was unchanged in poly(I)xpoly(C)-induced cells during the shutoff phase. Treatment of FS-4 cells with interferon for 16-18 hr prior to induction increased the enzyme activity by approximately 200-fold. However, this did not inhibit interferon production after induction with poly(I)xpoly(C) alone or after superinduction with cycloheximide or actinomycin D or both. Furthermore, the rates of decay of interferon production were comparable in cells with either a basal or an increased level of 2',5'-oligo(A) synthetase. Thus a 200-fold increase in 2',5'-oligo(A) synthetase level did not affect either the stability of interferon mRNA or the efficacy of interferon superinduction by metabolic inhibitors.

  13. TIME- AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer (NSCLC), little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods: Two human NSCLC cell lines (A549: squamous; NCI-H596: adenosquamous) were investigated for their TNF-α mRNA (real-time RT-PCR) after exposure to different irradiation doses (2, 5, 10, 20, 30, 40 Gy) and time intervals (1, 3, 6, 12, 24, 48 or 72 h). The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results: Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1~12 h (maximum 6h: 568fold increase relative to unirradiated cells) in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion: NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.

  14. Lipopolysaccharides and the Enhanced Expression of TACE Mrna in HL-60 Cells and Adhesive Cells from Human Spleen

    Institute of Scientific and Technical Information of China (English)

    DING; Tingbo(

    2001-01-01

    [1]Flad H D Loppnow H.Agonists and antagonists for LPS-induced cytokines.Immunology 1993 187:303[2]Robache-Gallea S Morand V Bruneau J H et al.In vitro processing of human tumor necrosis factor-α.J Bio Chem 1995 270(40):23 688[3]Kim K U Kwon O J Jue D M.Pro-tumor necrosis factor cleaving enzyme in macrophage membrane/particulate.Immunology 1993 80(2) :134[4]Moss M L.Cloning of a disintegrin metalloproteinase that processes precursor tumor necrosis factor-α.Nature 1997 385 (6):733[5]Flick D A Gilford G E.The detection of biological activity of tumor necrosis factor-α.J Immunol Methods 1984 68:167[6]Sambrook J Fritsch E F Maniatis F.In Vitro Amplification of DNA by the Polymerase Chain Reaction.In:Molecular Cloning A Laboratory Manual.2nd edition.New York:Cold Spring Harbor Laboratory Press 1989.10.1-10.70[7]吴朝栋 李明珍 张艳萍等.中药热毒清对内毒素DIC家兔血浆肿瘤坏死因子α白细胞介素水平影响的研究.中国中西医结合杂志 1995 15:365

  15. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are re-quired to produce the 56-kDa isoform

    Institute of Scientific and Technical Information of China (English)

    Jia Chen; Ta-Yuan Chang; Bo-Liang Li; Xiao-Nan Zhao; Li Yang; Guang-Jing Hu; Ming Lu; Ying Xiong; Xin-Ying Yang; Catherine CY Chang; Bao-Liang Song

    2008-01-01

    We have previously reported that the human ACAT1 gene produces a chimeric mRNAthrough the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACATI and a novel enzymatically active 56-kDa isoform,respectively,with the latter being authentically present in human cells,including human monocyte derived macrophages. In this work,we report that RNA secondary structures located in the vicinity,of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268,1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type,deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to theORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome I were needed for production of the 56-kDa isoform,whereas the last stem-ioop1355-1384 from chromosome I was dispensable. The results of experi ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACATI transcript.

  16. Towards a unified biological hypothesis for the BDNF Val66Met-associated memory deficits in humans: a model of impaired dendritic mRNA trafficking

    Directory of Open Access Journals (Sweden)

    Gabriele eBaj

    2013-10-01

    Full Text Available Brain-derived neurotrophic factor (BDNF represents promotesa key molecule for the survival and differentiation of specific populations of neurons in the central nervous system. BDNF also regulates plasticity-related processes underlying memory and learning. A common single nucleotide polymorphism (SNP rs6265 has been identified on the coding sequence of human BDNF located at 11p13. The SNP rs6265 is a single base mutation with an adenine instead of a guanine at position 196 (G196A, resulting in the amino acid substitution Val66Met. This polymorphism only exists in humans and has been associated with a plethora of effects ranging from molecular, cellular and brain structural modifications in association with deficits in social and cognitive functions. To date, the literature on Val66Met polymorphism describes a complex and often conflicting pattern of effects. In this review, we attempt to provide a unifying model of the Val66Met effects. We discuss the clinical evidence of the association between Val66Met and memory deficits, as well as the molecular mechanisms involved including the reduced transport of BDNF mRNA to the dendrites as well as the reduced processing and secretion of BDNF protein through the regulated secretory pathway.

  17. Primary cervical cancer screening and triage using an mRNA human papillomavirus assay and visual inspection.

    Science.gov (United States)

    Nieves, Lucybeth; Enerson, Christine L; Belinson, Suzanne; Brainard, Jennifer; Chiesa-Vottero, Andres; Nagore, Norma; Booth, Christine; Pérez, Areli Gutierrez; Chávez-Avilés, Mauricio N; Belinson, Jerome

    2013-03-01

    Mexican Cervical Cancer Screening Study II (MECCS II) seeks to develop a highly sensitive and highly specific screening program able to be adapted to all socioeconomic levels in Mexico. The objectives of MECCS II are (1) to compare the sensitivity and specificity for cervical intraepithelial neoplasia (CIN) 3 or cancer of self-collected vaginal specimens tested for high-risk types of the human papillomavirus (HR-HPV) by APTIMA with those tested for HR-HPV by Hybrid Capture 2 (HC2); and (2) determine the efficacy of cryotherapy in the treatment of HR-HPV-positive and acetic acid-aided visual inspection (VIA)-positive and -negative women after VIA triage. The study was conducted in rural Mexico. Women aged 30 to 50 years, nonpregnant, with no history of hysterectomy or pelvic irradiation and varied histories of screening, participated. A direct endocervical sample was tested for cytology, HC2, and APTIMA assay (AHPV). Subjects positive on any test were recalled for triage VIA, biopsies, and immediate cryotherapy. Tests were compared using McNemar test. Two thousand forty-nine patients have complete results. Mean age of the patients was 39.2 years; 7.7% presented with ≥atypical squamous cells of uncertain significance (ASCUS), 1.8% ≥low-grade squamous intraepithelial neoplasia, and 0.5% ≥high-grade squamous intraepithelial neoplasia. Two percent of patients had ≥CIN2, and 0.78% had ≥CIN3 (including 2 with invasive disease). The sensitivity of ThinPrep (>ASCUS), HC2, and AHPV for >CIN3 for direct endocervical collection was 87.5%, 100%, and 100%, respectively. The specificity of ThinPrep (>ASCUS), HC2, and AHPV for >CIN3 was 94.1%, 92.2%, and 93.5%, respectively. Specificities of HC2 and AHPV differed significantly. The overall percentage of agreement among HPV assays (HC2 vs APTIMA) is 97%. Four hundred sixty-nine women returned for VIA. Two hundred ninety-one women were treated with cryotherapy. The specificity of the APTIMA assay along with high

  18. Studies on SSTR2 mRNA expression and its correlation to steroid receptors in human benign and malignant breast lesions

    Institute of Scientific and Technical Information of China (English)

    ZENG Xizhi(曾希志); YAO Zhenxiang(姚榛祥)

    2002-01-01

    Objective:This sudy was designed to observe somatostatin receptor subtype 2 (SSTR2) Mrna expression, and investigate the correlations between SSTR2 Mrna expression and steroid receptors in benign and malignant lesions of the breast. Methods: A total of 23 breast carcinomas,16 mammary hyperplasia and 9 mammary adenofibroma samples were analysed. The SSTR2 Mrna expression was examined by in situ hybridization using multiphase oligoprobes.The ER and PR were detected by immunohistochemical staining. A computerized image analysis system was utilized to estimate the relative contents of SSTR2 Mrna. Results: The positive rates of expression (87.0%) and relative contents (0.47) of SSTR2 Mrna in breast cancer were higher than those in benign breast lesions(64%,0.26) respectively( P<0.05). SSTR2 Mrna expression was closely correlated with ER and PR in breast cancer( P<0. 05), A positive correlation between SSTR2 Mrna expression and ER was also found in benign breast lesions. Conclusions: SSTR2 Mrna expressed both in benign and in malignant breast lesions, but higher in malignant than in benign ones. There was a significant positive correlation of SSTR2 Mrna expression with ER or PR. The results suggest that conbined treatment with an antiestrogen and a somatostatin analogue for ER-positive breast cancer is feasible.

  19. Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide.

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    Vijaya Sarangthem

    Full Text Available Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R. The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.

  20. Quantification of human telomerase reverse transcriptase mRNA in testicular germ cell tumors by quantitative fluorescence real-time RT-PCR.

    Science.gov (United States)

    Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Smith, Gilian L; Newlands, Eward S; Miller, Kurt

    2002-01-01

    Telomerase is a ribonucleoprotein enzyme which is endogenously expressed in germ, stem and tumor cells, but absent in benign somatic cells. The two major telomerase components are human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). It has been shown that hTERT is rate-limiting for telomerase activity and that it plays a central role in human carcinogenesis. Here, we investigated the potential of hTERT and hTR gene expression as diagnostic markers in testicular germ cell tumors (TGCT). hTERT mRNA and hTR expression were quantified in 55 testicular germ cell tumors comprising 36 primary and 19 germ cell tumors from retroperitonal sides by fluorescence real-time RT-PCR using the LightCycler technology. Porphobilinogen deaminase (PBGD) was used as housekeeping gene and to enable relative quantification. For comparison to TGCTs, 38 benign testicular biopsies from patients with fertility disorders were assayed. hTERT expression was detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content (N(hTERT) 38-127). In contrast, mature teratomas from primary and post-chemotherapy masses, which are characterized by well-differentiated tissue components showed a nearly complete downregulation of hTERT expression (N(hTERT) 2-4, pmRNA is expressed in all undifferentiated TGCTs but repressed in mature teratomas. This suggests an inverse correlation between the differentiation status of germ cell tumors and hTERT expression. Thus, detection of hTERT expression in tumors histopathologically classified as mature teratomas enables a molecular-diagnostic confirmation and might aid decision making for treatment of patients presenting with this tumor subtype.

  1. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

    Science.gov (United States)

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

  2. Epithelial-Derived Inflammation Disrupts Elastin Assembly and Alters Saccular Stage Lung Development.

    Science.gov (United States)

    Benjamin, John T; van der Meer, Riet; Im, Amanda M; Plosa, Erin J; Zaynagetdinov, Rinat; Burman, Ankita; Havrilla, Madeline E; Gleaves, Linda A; Polosukhin, Vasiliy V; Deutsch, Gail H; Yanagisawa, Hiromi; Davidson, Jeffrey M; Prince, Lawrence S; Young, Lisa R; Blackwell, Timothy S

    2016-07-01

    The highly orchestrated interactions between the epithelium and mesenchyme required for normal lung development can be disrupted by perinatal inflammation in preterm infants, although the mechanisms are incompletely understood. We used transgenic (inhibitory κB kinase β transactivated) mice that conditionally express an activator of the NF-κB pathway in airway epithelium to investigate the impact of epithelial-derived inflammation during lung development. Epithelial NF-κB activation selectively impaired saccular stage lung development, with a phenotype comprising rapidly progressive distal airspace dilation, impaired gas exchange, and perinatal lethality. Epithelial-derived inflammation resulted in disrupted elastic fiber organization and down-regulation of elastin assembly components, including fibulins 4 and 5, lysyl oxidase like-1, and fibrillin-1. Fibulin-5 expression by saccular stage lung fibroblasts was consistently inhibited by treatment with bronchoalveolar lavage fluid from inhibitory κB kinase β transactivated mice, Escherichia coli lipopolysaccharide, or tracheal aspirates from preterm infants exposed to chorioamnionitis. Expression of a dominant NF-κB inhibitor in fibroblasts restored fibulin-5 expression after lipopolysaccharide treatment, whereas reconstitution of fibulin-5 rescued extracellular elastin assembly by saccular stage lung fibroblasts. Elastin organization was disrupted in saccular stage lungs of preterm infants exposed to systemic inflammation. Our study reveals a critical window for elastin assembly during the saccular stage that is disrupted by inflammatory signaling and could be amenable to interventions that restore elastic fiber assembly in the developing lung.

  3. The coupled bio-chemo-electro-mechanical behavior of glucose exposed arterial elastin

    Science.gov (United States)

    Zhang, Yanhang; Li, Jiangyu; Boutis, Gregory S.

    2017-04-01

    Elastin, the principle protein component of the elastic fiber, is a critical extracellular matrix (ECM) component of the arterial wall providing structural resilience and biological signaling essential in vascular morphogenesis and maintenance of mechanical homeostasis. Pathogenesis of many cardiovascular diseases have been associated with alterations of elastin. As a long-lived ECM protein that is deposited and organized before adulthood, elastic fibers can suffer from cumulative effects of biochemical exposure encountered during aging and/or disease, which greatly compromise their mechanical function. This review article covers findings from recent studies of the mechanical and structural contribution of elastin to vascular function, and the effects of biochemical degradation. Results from diverse experimental methods including tissue-level mechanical characterization, fiber-level nonlinear optical imaging, piezoelectric force microscopy, and nuclear magnetic resonance are reviewed. The intriguing coupled bio-chemo-electro-mechanical behavior of elastin calls for a multi-scale and multi-physical understanding of ECM mechanics and mechanobiology in vascular remodeling.

  4. Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy.

    Science.gov (United States)

    Cui, Jason Z; Tehrani, Arash Y; Jett, Kimberly A; Bernatchez, Pascal; van Breemen, Cornelis; Esfandiarei, Mitra

    2014-09-01

    In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.

  5. Analytic and Diagnostic Performances of Human Papillomavirus E6/E7 mRNA Test on up-to 11-Year-Old Liquid-Based Cervical Samples. A Biobank-Based Longitudinal Study

    Directory of Open Access Journals (Sweden)

    Roberta Zappacosta

    2017-07-01

    Full Text Available Human Papillomavirus (HPV E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001–2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/μL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A “one sample for all” approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.

  6. Effect of D-penicillamine on rat lung elastin cross-linking during the perinatal period.

    Science.gov (United States)

    Koçtürk, Semra; Oktay, Gülgün; Güner, Gül; Pekçetin, Cetin; Güre, Ataman

    2006-01-01

    This study was designed to clarify the effects of D-penicillamine (DPA), a drug used for treatment of various pathological events, on lung elastin formation and maturation of the newborn in the perinatal period. The investigation was conducted on 20 newborn rats bred from 40 female and six male rats. DPA doses 400 mg kg(-1) day(-1) and physiological saline were given intraperitoneally (i.p) to experimental and control groups. To assess newborn maturation, their body and lung weights were determined. Serum Cu levels were measured by atomic absorption spectroscopy and ceruloplasmin (Cp) activities were measured spectrophotometrically. Newborn lung tissue elastin, desmosine (DES) and isodesmosine (IDES) levels were measured by HPLC. The results showed that DPA treatment caused loss of skin elasticity and reduction in body and lung weight in newborns of the experimental group. The serum Cu levels and Cp activity were found to be significantly lower in both maternal and newborn of the experimental groups compared with the control group. The lung DES, IDES and elastin values of newborns in the experimental group were decreased compared with the control group. In conclusion, our results indicate that 400 mg kg(-1) day(-1) DPA, a dose that is used in the treatment of Wilson's disease, rheumatoid arthritis and cystinuria, caused the retardation of newborn maturation, a decrease in DES-IDES cross-links and levels of lung elastin of offspring in the perinatal period. Another conclusion to be drawn from this study is that even low levels of Cu depletion due to DPA administration induces a change in cross-linking in lung elastin during the perinatal period.

  7. Diversity in the organization of elastin bundles and intramembranous muscles in bat wings.

    Science.gov (United States)

    Cheney, Jorn A; Allen, Justine J; Swartz, Sharon M

    2017-04-01

    Unlike birds and insects, bats fly with wings composed of thin skin that envelops the bones of the forelimb and spans the area between the limbs, digits, and sometimes the tail. This skin is complex and unusual; it is thinner than typical mammalian skin and contains organized bundles of elastin and embedded skeletal muscles. These elements are likely responsible for controlling the shape of the wing during flight and contributing to the aerodynamic capabilities of bats. We examined the arrangement of two macroscopic architectural elements in bat wings, elastin bundles and wing membrane muscles, to assess the diversity in bat wing skin morphology. We characterized the plagiopatagium and dactylopatagium of 130 species from 17 families of bats using cross-polarized light imaging. This method revealed structures with distinctive relative birefringence, heterogeneity of birefringence, variation in size, and degree of branching. We used previously published anatomical studies and tissue histology to identify birefringent structures, and we analyzed their architecture across taxa. Elastin bundles, muscles, neurovasculature, and collagenous fibers are present in all species. Elastin bundles are oriented in a predominantly spanwise or proximodistal direction, and there are five characteristic muscle arrays that occur within the plagiopatagium, far more muscle than typically recognized. These results inform recent functional studies of wing membrane architecture, support the functional hypothesis that elastin bundles aid wing folding and unfolding, and further suggest that all bats may use these architectural elements for flight. All species also possess numerous muscles within the wing membrane, but the architecture of muscle arrays within the plagiopatagium varies among families. To facilitate present and future discussion of these muscle arrays, we refine wing membrane muscle nomenclature in a manner that reflects this morphological diversity. The architecture of the

  8. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marino, M.W.; Guidon, P.T. Jr.; Donner, D.B. (Cornell Univ. Graduate School of Medical Sciences, New York, NY (USA)); Pfeffer, L.M. (Rockefeller Univ., New York, NY (USA))

    1989-11-01

    Tumor necrosis factor {alpha} (TNF-{alpha}) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-{alpha} on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of {sup 125}I-labeled TNF-{alpha} to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-{alpha}, and the inhibition of cell proliferation by TNF-{alpha} occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-{alpha}-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5{prime}-triposphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-{alpha}-promoted phosphorylation, p28. Thus, TNF-{alpha} stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-{alpha}-receptor binding into cellular responses.

  9. Tumor necrosis factor induces phosphorylation of a 28-kDa mRNA cap-binding protein in human cervical carcinoma cells.

    Science.gov (United States)

    Marino, M W; Pfeffer, L M; Guidon, P T; Donner, D B

    1989-11-01

    Tumor necrosis factor alpha (TNF-alpha) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-alpha on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of 125I-labeled TNF-alpha to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-alpha, and the inhibition of cell proliferation by TNF-alpha occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-alpha-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5'-triphosphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-alpha-promoted phosphorylation, p28. Thus, TNF-alpha stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-alpha-receptor binding into cellular responses.

  10. Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    Hai-rong Jiang; Peng Wang; Yong Li; Tao Ning; Xiao-song Rao; Bao-guoLiu

    2010-01-01

    Objective:Laryngeal squamous cell carcinoma(LSCC)is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus(HPV)is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods:In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6mRNA.Results:The positive rate of HPV16 E6 mRNA was 36.36%(36/99)in laryngeal squamous cell carcinoma(LSCC),whereas only 3 of 50(6%)specimens of the normal laryngeal mucosa as a control group showed positive results(P0.05).Conclusion:The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types(especially type 16)were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

  11. Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    Hai-rong Jiang; Peng Wang; Yong Li; Tao Ning; Xiao-song Rao; Bao-guoLiu

    2011-01-01

    Objective: Laryngeal squamous cell carcinoma(LSCC) is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus (HPV) is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods: In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 mRNA.Results: The positive rate of HPV 16 E6 mRNA was 36.36%(36/99) in laryngeal squamous cell carcinoma (LSCC),whereas only 3 of 50(6%) specimens of the normal laryngeal mucosa as a control group showed positive results(P0.05).Conclusion: The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types(especially type 16) were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

  12. mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

    Science.gov (United States)

    Bryant, Dean; Onions, Tiffany; Raybould, Rachel; Flynn, Áine; Tristram, Amanda; Meyrick, Sian; Giles, Peter; Ashelford, Kevin; Hibbitts, Samantha; Fiander, Alison; Powell, Ned

    2014-09-01

    Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

  13. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)~+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 袁直; 宓怀风; 元云峰; 何炳林; 王亦农

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPlase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  14. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 元云峰; 王亦农; 袁直; 宓怀风; 何炳林

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPIase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  15. The stoichiometric production of IL-2 and IFN-γ mRNA defines memory T cells that can self-renew after adoptive transfer in humans.

    Science.gov (United States)

    Wang, Anran; Chandran, Smita; Shah, Syed A; Chiu, Yu; Paria, Biman C; Aghamolla, Tamara; Alvarez-Downing, Melissa M; Lee, Chyi-Chia Richard; Singh, Sanmeet; Li, Thomas; Dudley, Mark E; Restifo, Nicholas P; Rosenberg, Steven A; Kammula, Udai S

    2012-08-29

    Adoptive immunotherapy using ex vivo-expanded tumor-reactive lymphocytes can mediate durable cancer regression in selected melanoma patients. Analyses of these trials have associated the in vivo engraftment ability of the transferred cells with their antitumor efficacy. Thus, there is intensive clinical interest in the prospective isolation of tumor-specific T cells that can reliably persist after transfer. Animal studies have suggested that central memory CD8(+) T cells (T(CM)) have divergent capabilities including effector differentiation to target antigen and stem cell-like self-renewal that enable long-term survival after adoptive transfer. We sought to isolate human melanoma-specific T(CM) to define their in vivo fate and function after autologous therapeutic transfer to metastatic patients. To facilitate the high-throughput identification of these rare cells from patients, we report that T(CM) have a defined stoichiometric production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) mRNA after antigen stimulation. Melanoma-specific T cells screened for high relative IL-2 production had a T(CM) phenotype and superior in vitro proliferative capacity compared to cells with low IL-2 production. To investigate in vivo effector function and self-renewal capability, we allowed melanoma-specific T(CM) to undergo in vitro expansion and differentiation into lytic effector clones and then adoptively transferred them back into their hosts. These clones targeted skin melanocytes in all five patients and persisted long term and reacquired parental T(CM) attributes in four patients after transfer. These findings demonstrate the favorable engraftment fitness for human T(CM)-derived clones, but further efforts to improve their antitumor efficacy are still necessary.

  16. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.

    Directory of Open Access Journals (Sweden)

    Alessia Galgano

    Full Text Available Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

  17. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system.

    Science.gov (United States)

    Galgano, Alessia; Forrer, Michael; Jaskiewicz, Lukasz; Kanitz, Alexander; Zavolan, Mihaela; Gerber, André P

    2008-09-08

    Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

  18. Increased levels of cell-free human placental lactogen mRNA at 28-32 gestational weeks in plasma of pregnant women with placenta previa and invasive placenta.

    Science.gov (United States)

    Kawashima, Akihiro; Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

    2014-02-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.

  19. Aryl hydrocarbon hydroxylase represents CYP1B1, and not CYP1A1, in human freshly isolated white cells: trimodal distribution of Japanese population according to induction of CYP1B1 mRNA by environmental dioxins.

    Science.gov (United States)

    Toide, Kenji; Yamazaki, Hiroshi; Nagashima, Rikako; Itoh, Keisuke; Iwano, Shunsuke; Takahashi, Yoshiki; Watanabe, Shaw; Kamataki, Tetsuya

    2003-03-01

    The expression level of mRNAs for cytochrome P450 (CYP) 1A1 and 1B1 in freshly prepared white cells from 72 subjects exposed to dioxins at waste incinerators was investigated. The amounts of CYP1B1 mRNA ranged from 0.16 to 671 molecules/10(7) molecules of 18S rRNA, whereas the amounts of CYP1A1 mRNA were dioxins. The inducibility of CYP1B1 mRNA in leukocytes, defined as the ratio of CYP1B1 mRNA to the plasma concentration of dioxins, varied among the subjects. It was found that the subjects showed trimodal distribution according to inducibility: 39 (54.2%), 25 (34.7%), and 8 (11.1%) of 72 subjects were judged as poor, intermediate, and high responders to environmental dioxins, respectively. The amounts of CYP1B1 mRNA in leukocytes of the intermediate and high responders were highly correlated with the plasma concentrations of dioxins (P dioxins is involved in aromatic hydrocarbon hydroxylase activities in human lymphocytes.

  20. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    Science.gov (United States)

    2016-09-07

    modification or cleavages of transcripts through various RNA editing mechanisms , including siRNA targeted mRNA cleavages. Currently, 5′-RACE (rapid ampli... transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the siRNA- mediated target mRNA cleavage sites by determining precise...Reverse Transcription kit (Life Technologies) in combination with an array of stem-loop RT primers. The 20 ml of RT reaction con- tained 50 ng of total RNA

  1. Diagnostic and prognostic validity of the human papillomavirus E6/E7 mRNA test in cervical cytological samples of HC2-positive patients.

    Science.gov (United States)

    Benevolo, Maria; Terrenato, Irene; Mottolese, Marcella; Marandino, Ferdinando; Carosi, Mariantonia; Rollo, Francesca; Ronchetti, Livia; Muti, Paola; Mariani, Luciano; Sindico, Stefano; Vocaturo, Giuseppe; Vocaturo, Amina

    2011-06-01

    The study aimed to assess the clinical utility in identifying CIN2 or worse (CIN2+), of the Pretect HPV-Proofer test for E6/E7 mRNA detection in Hybrid Capture 2 (HC2)-positive patients, who underwent colposcopy. In particular, the study analyzed the mRNA test performance as the third test in a subgroup of HC2+ patients with less severe than high-grade squamous intraepithelial lesions (HSIL-). We analyzed 464 cervico-vaginal samples by liquid-based cytology (LBC) and PreTect HPV-Proofer. Moreover 231 patients also had a biopsy at baseline and 75, with HSIL-, were followed up within 2 years by LBC, colposcopy, and histology when indicated. The highest sensitivity for CIN2+ belonged to the mRNA compared to LBC, at the HSIL+ threshold (72% vs. 58%), whereas the LBC showed the highest specificity and positive predictive value (PPV) (99 and 93% vs. 73 and 39%, respectively). Focusing on the 408 HSIL- patients, the mRNA positivity was significantly more associated with CIN2+ than CIN2- lesions (p < 0.0001). Moreover, among the 75 HSIL- followed up patients, the mRNA displayed high longitudinal Specificity (89%), even if the sensitivity and the PPV were low (50 and 20%, respectively). The present data suggest that the mRNA test may have a diagnostic and a potentially prognostic role in HC2+/HSIL- patients.

  2. Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

    Science.gov (United States)

    Adinolfi, Barbara; Pellegrino, Mario; Giannetti, Ambra; Tombelli, Sara; Trono, Cosimo; Sotgiu, Giovanna; Varchi, Greta; Ballestri, Marco; Posati, Tamara; Carpi, Sara; Nieri, Paola; Baldini, Francesco

    2017-02-15

    One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm(2)), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm(2)). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Performance of the Aptima High-Risk Human Papillomavirus mRNA Assay in a Referral Population in Comparison with Hybrid Capture 2 and Cytology▿

    Science.gov (United States)

    Clad, Andreas; Reuschenbach, Miriam; Weinschenk, Johanna; Grote, Ruth; Rahmsdorf, Janina; Freudenberg, Nikolaus

    2011-01-01

    This study compared the Aptima human papillomavirus (HPV) (AHPV; Gen-Probe Incorporated) assay, which detects E6/E7 mRNA from 14 high-risk types, the Hybrid Capture 2 HPV DNA (HC2; Qiagen Incorporated) test, and repeat cytology for their ability to detect high-grade cervical lesions (cervical intraepithelial neoplasia grade 2+ [CIN2+]) in women referred to colposcopy due to an abnormal Papanicolaou (Pap) smear. A total of 424 clinical specimens, stored in liquid-based cytology (LBC) vials at room temperature for up to 3 years, were tested by repeat cytology, the AHPV assay, and the HC2 test. Assay results were compared to each other and to histology results. The overall agreement between the AHPV assay and the HC2 test was 88.4%. The sensitivity (specificity) of cytology, the HC2 test, and the AHPV assay for the detection of CIN2+ was 84.9% (66.3%), 91.3% (61.0%), and 91.7% (75.0%) and for the detection of CIN3+ was 93.9% (54.4%), 95.7% (46.0%), and 98.2% (56.3%), respectively. Of the disease-positive specimens containing high-risk HPV (HR HPV) DNA as determined by Linear Array (Roche Diagnostics), the AHPV assay missed 3 CIN2 and 1 microfocal CIN3 specimen, while the HC2 test missed 6 CIN2, 4 CIN3, and 1 cervical carcinoma specimen. The AHPV assay had a sensitivity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detection of CIN2+. The AHPV assay was significantly more sensitive (P = 0.0041) and significantly more specific (P = 0.0163) than cytology for the detection of disease (CIN2+). PMID:21191046

  4. Performance of the Aptima high-risk human papillomavirus mRNA assay in a referral population in comparison with Hybrid Capture 2 and cytology.

    Science.gov (United States)

    Clad, Andreas; Reuschenbach, Miriam; Weinschenk, Johanna; Grote, Ruth; Rahmsdorf, Janina; Freudenberg, Nikolaus

    2011-03-01

    This study compared the Aptima human papillomavirus (HPV) (AHPV; Gen-Probe Incorporated) assay, which detects E6/E7 mRNA from 14 high-risk types, the Hybrid Capture 2 HPV DNA (HC2; Qiagen Incorporated) test, and repeat cytology for their ability to detect high-grade cervical lesions (cervical intraepithelial neoplasia grade 2+ [CIN2+]) in women referred to colposcopy due to an abnormal Papanicolaou (Pap) smear. A total of 424 clinical specimens, stored in liquid-based cytology (LBC) vials at room temperature for up to 3 years, were tested by repeat cytology, the AHPV assay, and the HC2 test. Assay results were compared to each other and to histology results. The overall agreement between the AHPV assay and the HC2 test was 88.4%. The sensitivity (specificity) of cytology, the HC2 test, and the AHPV assay for the detection of CIN2+ was 84.9% (66.3%), 91.3% (61.0%), and 91.7% (75.0%) and for the detection of CIN3+ was 93.9% (54.4%), 95.7% (46.0%), and 98.2% (56.3%), respectively. Of the disease-positive specimens containing high-risk HPV (HR HPV) DNA as determined by Linear Array (Roche Diagnostics), the AHPV assay missed 3 CIN2 and 1 microfocal CIN3 specimen, while the HC2 test missed 6 CIN2, 4 CIN3, and 1 cervical carcinoma specimen. The AHPV assay had a sensitivity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detection of CIN2+. The AHPV assay was significantly more sensitive (P = 0.0041) and significantly more specific (P = 0.0163) than cytology for the detection of disease (CIN2+).

  5. Prevalence of type-specific oncogenic human papillomavirus infection assessed by HPV E6/E7 mRNA among women with high-grade cervical lesions.

    Science.gov (United States)

    Wang, Hye-Young; Park, Sunyoung; Lee, Dongsup; Kim, Sunghyun; Kim, Geehyuk; Park, Kwang Hwa; Lee, Hyeyoung

    2015-08-01

    Human papillomavirus (HPV) infection is a major cause of premalignant dysplasia and cervical cancer. There are no data on the prevalence of genotype-specific HPV infection assessed by HPV E6/E7 mRNA in women representative of the Korean population across a broad age range. A total of 630 women aged 17-90 years were enrolled in this study. ThinPrep liquid-based cytology samples were evaluated using the CervicGen HPV RT-qDx assay, which detects 16 high-risk (HR) HPV genotypes (set 1: HPV 16, 31, 33, 35, 52, and 58; set 2: HPV 18, 39, 45, 51, 59, and 68; and set 3: HPV 53, 56, 66, and 69). The overall prevalence of HPV infection was 33.2% (n=209), and oncogenic high-risk HPV was detected in 75.9% (n=107) of 141 women with high-grade cervical lesions. HPV 16 was the most common HPV genotype among women with high-grade cervical lesions and histologically confirmed cervical intraepithelial neoplasia grade 2 and above (CIN2+) in the Republic of Korea (41.6%). Among women aged over 30 years, 182/329 (55%) had invasive cervical cancer and 135 (74%) of these were infected with oncogenic HR-HPV types (in particular 25% with HPV 16). Among patients diagnosed with CIN2+, the positivity rate of HR-HPV was the highest in women aged 40-49 years. These results suggest that the determination of specific HPV genotypes is very important for evaluating the potential impact of preventive measures, including the use of prophylactic vaccines, on reducing the burden of cervical cancer. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Beaded nanofibers assembled from double-hydrophobic elastin-like block polypeptides: Effects of trifluoroethanol.

    Science.gov (United States)

    Le, Duc H T; Okubo, Tatsuya; Sugawara-Narutaki, Ayae

    2015-03-01

    A "double-hydrophobic" elastin-like triblock polypeptide GPG has been constructed by mimicking the localization of proline- and glycine-rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self-assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG, where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one-dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration-dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG. Well-defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering.

  7. Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin.

    Science.gov (United States)

    Muiznieks, Lisa D; Cirulis, Judith T; van der Horst, Astrid; Reinhardt, Dieter P; Wuite, Gijs J L; Pomès, Régis; Keeley, Fred W

    2014-06-01

    Elastin self-assembles from monomers into polymer networks that display elasticity and resilience. The first major step in assembly is a liquid-liquid phase separation known as coacervation. This process represents a continuum of stages from initial phase separation to early growth of droplets by coalescence and later "maturation" leading to fiber formation. Assembly of tropoelastin-rich globules is on pathway for fiber formation in vivo. However, little is known about these intermediates beyond their size distribution. Here we investigate the contribution of sequence and structural motifs from full-length tropoelastin and a set of elastin-like polypeptides to the maturation of coacervate assemblies, observing their growth, stability and interaction behavior, and polypeptide alignment within matured globules. We conclude that maturation is driven by surface properties, leading to stabilization of the interface between the hydrophobic interior and aqueous solvent, potentially through structural motifs, and discuss implications for droplet interactions in fiber formation.

  8. Development and initial characterization of a chemically stabilized elastin-glycosaminoglycan-collagen composite shape-memory hydrogel for nucleus pulposus regeneration.

    Science.gov (United States)

    Mercuri, Jeremy; Addington, Caroline; Pascal, Richard; Gill, Sanjitpal; Simionescu, Dan

    2014-12-01

    Nucleus pulposus (NP) is a resilient and hydrophilic tissue which plays a significant role in the biomechanical function of the intervertebral disc (IVD). Destruction of the NP extracellular matrix (ECM) is observed during the early stages of IVD degeneration. Herein, we describe the development and initial characterization of a novel biomaterial which attempts to recreate the resilient and hydrophilic nature of the NP via the construction of a chemically stabilized elastin-glycosaminoglycan-collagen (EGC) composite hydrogel. Results demonstrated that a resilient, hydrophilic hydrogel which displays a unique "shape-memory" sponge characteristic could be formed from a blend of soluble elastin aggregates, chondroitin-6-sulfate, hyaluronic acid and collagen following freeze-drying, stabilization with a carbodiimide and penta-galloyl glucose-based fixative, and subsequent partial degradation with glycosaminoglycan degrading enzymes. The resultant material exhibited the ability to restore its original dimensions and water content following multi-cycle mechanical compression and illustrated resistance to accelerated enzymatic degradation. Preliminary in vitro studies utilizing human adipose derived stem cells (hADSCs) demonstrated that the material was cytocompatible and supported differentiation towards an NP cell-like phenotype. In vivo biocompatibility studies illustrated host cell infiltration and evidence of active remodeling following 4 weeks of implantation. Feasibility studies demonstrated that the EGC hydrogel could be delivered via minimally invasive methods.

  9. The Elastin Receptor Complex: a unique matricellular receptor with high anti-tumoral potential

    Directory of Open Access Journals (Sweden)

    Amandine eScandolera

    2016-03-01

    Full Text Available Elastin, one of the longest-lived proteins, confers elasticity to tissues with high mechanical constraints. During aging or pathophysiological conditions such as cancer progression, this insoluble polymer of tropoelastin undergoes an important degradation leading to the release of bioactive elastin-derived peptides (EDP, named elastokines. EDP exhibit several biological functions able to drive tumor development by regulating cell proliferation, invasion, survival, angiogenesis, and matrix metalloproteinase expression in various tumor and stromal cells. Although several receptors have been suggested to bind elastokines (αvβ3 and αvβ5 integrins, galectin-3, their main receptor remains the Elastin Receptor Complex (ERC. This heterotrimer comprises a peripheral subunit, named Elastin Binding Protein (EBP, associated to the Protective Protein/Cathepsin A (PPCA. The latter is bound to a membrane-associated protein called Neuraminidase-1 (Neu-1. The pro-tumoral effects of elastokines have been linked to their binding onto EBP. Additionally, Neu-1 sialidase activity is essential for their signal transduction. Consistently, EDP-EBP interaction and Neu-1 activity emerge as original anti-tumoral targets. Interestingly, besides its direct involvement in cancer progression, the ERC also regulates diabetes outcome and thrombosis, an important risk factor for cancer development and a vascular process highly increased in patients suffering from cancer. In this review, we will describe ERC and elastokines involvement in cancer development suggesting that this unique receptor would be a promising therapeutic target. We will also discuss the pharmacological concepts aiming at blocking its pro-tumoral activities. Finally, its emerging role in cancer-associated complications and pathologies such as diabetes and thrombotic events will be also considered.

  10. Effects of Arg-Gly-Asp-modified elastin-like polypeptide on pseudoislet formation via up-regulation of cell adhesion molecules and extracellular matrix proteins.

    Science.gov (United States)

    Lee, Kyeong-Min; Jung, Gwon-Soo; Park, Jin-Kyu; Choi, Seong-Kyoon; Jeon, Won Bae

    2013-03-01

    Extracellular matrix (ECM) plays an important role in controlling the β-cell morphology, survival and insulin secretary functions. An RGD-modified elastin-like polypeptide (RGD-ELP), TGPG[VGRGD(VGVPG)(6)](20)WPC, has been reported previously as a bioactive matrix. In this study, to investigate whether RGD-ELP affects β-cell growth characteristics and insulin secretion, β-TC6 cells were cultured on the RGD-ELP coatings prepared via thermally induced phase transition. On RGD-ELP, β-TC6 cells clustered into an islet-like architecture with high cell viability. Throughout 7days' culture, the proliferation rate of the cells within a pseudoislet was similar to that of monolayer culture. Under high glucose (25mM), β-TC6 pseudoislets showed up-regulated insulin gene expression and exhibited glucose-stimulated insulin secretion. Importantly, the mRNA and protein abundances of cell adhesion molecules (CAM) E-cadherin and connexin-36 were much higher in pseudoislets than in monolayer cells. The siRNA-mediated inhibition of E-cadherin or connexin-36 expression severely limited pseudoislet formation. In addition, the mRNA levels of collagen types I and IV, fibronectin and laminin were significantly elevated in pseudoislets. The results suggest that RGD-ELP promotes pseudoislet formation via up-regulation of the CAM and ECM components. The functional roles of RGD-ELP are discussed in respect of its molecular composition.

  11. Elastin Cables Define the Axial Connective Tissue System in the Murine Lung.

    Science.gov (United States)

    Wagner, Willi; Bennett, Robert D; Ackermann, Maximilian; Ysasi, Alexandra B; Belle, Janeil; Valenzuela, Cristian D; Pabst, Andreas; Tsuda, Akira; Konerding, Moritz A; Mentzer, Steven J

    2015-11-01

    The axial connective tissue system is a fiber continuum of the lung that maintains alveolar surface area during changes in lung volume. Although the molecular anatomy of the axial system remains undefined, the fiber continuum of the lung is central to contemporary models of lung micromechanics and alveolar regeneration. To provide a detailed molecular structure of the axial connective tissue system, we examined the extracellular matrix of murine lungs. The lungs were decellularized using a 24 hr detergent treatment protocol. Systematic evaluation of the decellularized lungs demonstrated no residual cellular debris; morphometry demonstrated a mean 39 ± 7% reduction in lung dimensions. Scanning electron microscopy (SEM) demonstrated an intact structural hierarchy within the decellularized lung. Light, fluorescence, and SEM of precision-cut lung slices demonstrated that alveolar duct structure was defined by a cable line element encased in basement membrane. The cable line element arose in the distal airways, passed through septal tips and inserted into neighboring blood vessels and visceral pleura. The ropelike appearance, collagenase resistance and anti-elastin immunostaining indicated that the cable was an elastin macromolecule. Our results indicate that the helical line element of the axial connective tissue system is composed of an elastin cable that not only defines the structure of the alveolar duct, but also integrates the axial connective tissue system into visceral pleura and peripheral blood vessels.

  12. Is dialdehyde starch a valuable cross-linking agent for collagen/elastin based materials?

    Science.gov (United States)

    Skopinska-Wisniewska, J; Wegrzynowska-Drzymalska, K; Bajek, A; Maj, M; Sionkowska, A

    2016-04-01

    Collagen and elastin are the main structural proteins in mammal bodies. They provide mechanical support, strength, and elasticity to various organs and tissues, e.g. skin, tendons, arteries, and bones. They are readily available, biodegradable, biocompatible and they stimulate cell growth. The physicochemical properties of collagen and elastin-based materials can be modified by cross-linking. Glutaraldehyde is one of the most efficient cross-linking agents. However, the unreacted molecules can be released from the material and cause cytotoxic reactions. Thus, the aim of our work was to investigate the influence of a safer, macromolecular cross-linking agent--dialdehyde starch (DAS). The properties of hydrogels based on collagen/elastin mixtures (95/5, 90/10) containing 5 and 10% of DAS and neutralized via dialysis against deionized water were tested. The homogenous, transparent, stiff hydrogels were obtained. The DAS addition causes the formation of intermolecular cross-linking bonds but does not affect the secondary structure of the proteins. As a result, the thermal stability, mechanical strength, and, surprisingly, swelling ability increased. At the same time, the surface properties test and in vitro study show that the materials are attractive for 3T3 cells. Moreover, the materials containing 10% of DAS are more resistant to enzymatic degradation.

  13. Refining Disordered Peptide Ensembles with Computational Amide I Spectroscopy: Application to Elastin-Like Peptides

    Science.gov (United States)

    Reppert, Mike; Roy, Anish R.; Tempkin, Jeremy O. B.; Dinner, Aaron R.; Tokmakoff, Andrei

    2017-01-01

    The characterization of intrinsically disordered protein (IDP) ensembles is complicated both by inherent heterogeneity and by the fact that many common experimental techniques function poorly when applied to IDPs. For this reason, the development of alternative structural tools for probing IDP ensembles has attracted considerable attention. Here we describe our recent work in developing experimental and computational tools for characterizing IDP ensembles using Amide I (backbone carbonyl stretch) vibrational spectroscopy. In this approach, the infrared (IR) absorption frequencies of isotope-labeled amide bonds probe their local electrostatic environments and structures. Empirical frequency maps allow us to use this spectroscopic data as a direct experimental test of atomistic structural models. We apply these methods to a family of short elastin-like peptides (ELPs), fragments of the elastin protein based around the Pro-Gly turn motif characteristic of the elastomeric segments of the full protein. Using a maximum entropy analysis of experimental spectra on the basis of predicted spectra from molecular dynamics (MD) ensembles, we find that peptides with Ala or Val sidechains preceding the Pro-Gly turn unit exhibit a stronger tendency toward extended structures than do Gly-Pro-Gly motifs, suggesting an important role for steric interactions in tuning the molecular properties of elastin. PMID:27736076

  14. MMP-12、Timp-1、Elastin在女性压力性尿失禁患者阴道壁组织中的表达%Expressions of matrix metalloproteinase-12,tissue inhibitor of matrix metallopro-teinase-1 and Elastin in vaginal wall tissues of women with stress urinary incontinence

    Institute of Scientific and Technical Information of China (English)

    张晓丹; 李晓果; 许雅娟; 张彩霞; 王鲁文

    2011-01-01

    Objective: To explore the expressions of matrix metalloproteinase - 12 ( MMP - 12), tissue inhibitor of matrix metalloproteinase- 1 (Timp- 1 ) and Elastin in vaginal wall tissues of women with stress urinary incontinence (SUI), and their relationship with occurrence of SUI. Methods: 30 samples of vaginal wall connective tissues of women with SUI were selected as study group, and 30 samples of vaginal wall connective tissues of women with pelvic organ prolapse (POP) were selected as POP group, 30 women without SUI and POP were selected as control group, immunohistochemical staining was used in the study, the expression levels of MMP - 12, Timp - 1 and Elastin were detected, PCR was used to detect the expression of Elastin mRNA in vaginal wall tissues of the three groups. Results: The expression level of MMP - 12 in SUI group was significantly higher than that in control group (P < 0. 05 ), the expression levels of Timp - 1 and Elastin in SUI group was significantly lower than those in control group ( P < 0. 05 ), but there was no significant difference in expression levels of MMP - 12, Timp - 1 and Elastin between SUI group and POP group, the expression results of Elastin detected by immanohistochemical staining and PCR were coincident. Conclusion: The expression level of MMP - 12 is high in vaginal wall tissues of women with SUI, Timp -1 is an inhibitor of MMP - 12; the expression level of Timp - 1 is low, which increases the degradation of Elastin, reduces the Elastin fibers,then the elasticity of pelvic floor tissues decreases.%目的:探讨基质金属蛋白酶(MMP-12)、基质金属蛋白酶抑制剂(Timp-1)、弹性蛋白(Elastin)在女性压力性尿失擎(SUI)患者阴道壁巾的表达及三者与SUI发病的关系.方法:研究组(SUI组)为30例有完整随访资料的女性SUI患者阴迫壁结缔组织标本,盆底器官脱垂组(POP组)为30例有完整随访资料的女性盆底器官脱垂(POP)患者的阴道壁结缔组织标本,对照组为30

  15. Nitinol-based Nanotubular and Nanowell Coatings for the Modulation of Human Vascular Cell Functions

    Science.gov (United States)

    Lee, Phin Peng

    Current approaches to reducing restenosis do not balance the reduction of vascular smooth muscle cell proliferation with the increase in the healing of the endothelium. Here, I present my study on the synthesis and characterization of a nanotubular coating on Nitinol substrates. I found that the coating demonstrated 'pro-healing' properties by increasing primary human aortic endothelial cell spreading, migration and collagen and elastin production. Certain cellular functions such as collagen and elastin production were also found to be affected by changes in nanotube diameter. The coating also reduced the proliferation and mRNA expression of collagen I and MMP2 for primary human aortic smooth muscle cells. I will also demonstrate the synthesis of a nanowell coating on Nitinol stents as well as an additional poly(lactic-co-glycolic acid) coating on top of the nanowells that has the potential for controlling drug release. These findings demonstrate the potential for the coatings to aid in the prevention of restenosis and sets up future explorations of ex vivo and in vivo studies.

  16. Unaltered mRNA expression of calcitonin-like receptor and receptor activity modifying proteins in human arteries in stroke and myocardial infarction

    DEFF Research Database (Denmark)

    Eskesen, Karen; János, Tajti; Tibor, Hortobágyi;

    2007-01-01

    -dependent change in total RNA and level of mRNA for p-actin or GAPDH could be detected in vessels removed from 1 and 5 days post mortem. The expression of beta-actin appears lower in coronary artery than in pulmonary artery and middle cerebral artery with no significant difference for GAPDH; both worked well......-R and RAMPs in arteries from patients with hemorrhagic stroke, arteriosclerosis and acute myocardial infarction when compared to patients without these diagnoses. Thus the mRNA expression seems to be unaltered in these disorders....

  17. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  18. Human immunodeficiency virus type 1 Vif functionally interacts with diverse APOBEC3 cytidine deaminases and moves with them between cytoplasmic sites of mRNA metabolism.

    Science.gov (United States)

    Marin, Mariana; Golem, Sheetal; Rose, Kristine M; Kozak, Susan L; Kabat, David

    2008-01-01

    Vif(IIIB), which has been a standard model for the viral infectivity factor of human immunodeficiency virus type 1 (HIV-1), binds the cytidine deaminase APOBEC3G (A3G) and induces its degradation, thereby precluding its lethal incorporation into assembling virions. Additionally, Vif(IIIB) less efficiently degrades A3F, another potent anti-HIV-1 cytidine deaminase. Although the APOBEC3 paralogs A3A, A3B, and A3C have weaker anti-HIV-1 activities and are only partially degraded by Vif(IIIB), we found that Vif(IIIB) induces their emigration from the nucleus to the cytosol and thereby causes net increases in the cytosolic concentrations and anti-HIV-1 activities of A3A and A3B. In contrast, some other Vifs, exemplified by Vif(HXB2) and Vif(ELI-1), much more efficiently degrade and thereby neutralize all APOBEC3s. Studies focused mainly on A3F imply that it occurs associated with mRNA-PABP1 in translationally active polysomes and to a lesser extent in mRNA processing bodies (P-bodies). A3F appears to stabilize the P-bodies with which it is associated. A correspondingly small proportion of Vif(IIIB) also localizes in P-bodies in an A3F-dependent manner. Stress causes A3A, A3B, A3C, and A3F to colocalize efficiently with Vif(IIIB) and mRNA-PABP1 complexes in stress granules in a manner that is prevented by cycloheximide, an inhibitor of translational elongation. Coimmunoprecipitation studies suggest that Vifs from different HIV-1 isolates associate with all tested APOBEC3s. Thus, Vifs interact closely with structurally diverse APOBEC3s, with effects on their subcellular localization, degradation rates, and antiviral activities. Cytosolic APOBEC3-Vif complexes are predominantly bound to mRNAs that dynamically move between translationally active and storage or processing pools.

  19. Repression of microRNA-768-3p by MEK/ERK signalling contributes to enhanced mRNA translation in human melanoma.

    Science.gov (United States)

    Jiang, C C; Croft, A; Tseng, H-Y; Guo, S T; Jin, L; Hersey, P; Zhang, X D

    2014-05-15

    Increased global protein synthesis and selective translation of mRNAs encoding proteins contributing to malignancy is common in cancer cells. This is often associated with elevated expression of eukaryotic translation initiation factor 4 (eIF4E), the rate-limiting factor of cap-dependent translation initiation. We report here that in human melanoma downregulation of miR-768-3p as a result of activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway has an important role in the upregulation of eIF4E and enhancement in protein synthesis. Melanoma cells displayed increased nascent protein production and elevated eIF4E expression, which was associated with the downregulation of miR-768-3p that was predicted to target the 3'-untranslated region of the eIF4E mRNA. Overexpression of miR-768-3p led to the downregulation of the endogenous eIF4E protein, reduction in nascent protein synthesis and inhibition of cell survival and proliferation. These effects were efficiently reversed when eIF4E was co-overexpressed in melanoma cells. On the other hand, introduction of anti-miR-768-3p into melanocytes upregulated endogenous eIF4E protein expression and increased global protein synthesis. Downregulation of miR-768-3p appeared to be mediated by activation of the MEK/ERK pathway, in that treatment of BRAF(V600E) melanoma cells with the mutant BRAF inhibitor PLX4720 or exposure of either BRAF(V600E) or wild-type BRAF melanoma cells to the MEK inhibitor U0126 resulted in the upregulation of miR-768-3p and inhibition of nascent protein synthesis. This inhibition was partially blocked in cells cointroduced with anti-miR-768-3p. Significantly, miR-768-3p was similarly downregulated, which was inversely associated with the expression levels of eIF4E in fresh melanoma isolates. Taken together, these results identify downregulation of miR-768-3p and subsequent upregulation of eIF4E as an important mechanism in addition to

  20. 14-3-3 isoforms bind directly exon B of the 5'-UTR of human surfactant protein A2 mRNA.

    Science.gov (United States)

    Noutsios, Georgios T; Ghattas, Paul; Bennett, Stephanie; Floros, Joanna

    2015-07-15

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5'-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722-L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'-UTR mRNA.

  1. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    DEFF Research Database (Denmark)

    Lund, L R; Rønne, E; Roldan, A.L.

    1991-01-01

    enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...

  2. Passive leg movement enhances interstitial VEGF protein, endothelial cell proliferation, and eNOS mRNA content in human skeletal muscle

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Rufener, Nora; Nielsen, Jens J

    2008-01-01

    were analyzed for mRNA content of VEGF, endothelial nitric oxide synthase (eNOS), and matrix metalloproteinase-2 (MMP-2). The passive leg movement caused an increase (P dialysate...... to cultured endothelial cells revealed that dialysate obtained during leg movement induced a 3.2-fold higher proliferation rate (P dialysate obtained at rest. Passive movement also enhanced (P

  3. Impact of elastin incorporation into electrochemically aligned collagen fibers on mechanical properties and smooth muscle cell phenotype.

    Science.gov (United States)

    Nguyen, Thuy-Uyen; Bashur, Chris A; Kishore, Vipuil

    2016-03-17

    Application of tissue-engineered vascular grafts (TEVGs) for the replacement of small-diameter arteries is limited due to thrombosis and intimal hyperplasia. Previous studies have attempted to address the limitations of TEVGs by developing scaffolds that mimic the composition (collagen and elastin) of native arteries to better match the mechanical properties of the graft with the native tissue. However, most existing scaffolds do not recapitulate the aligned topography of the collagen fibers found in native vessels. In the current study, based on the principles of isoelectric focusing, two different types of elastin (soluble and insoluble) were incorporated into highly oriented electrochemically aligned collagen (ELAC) fibers and the effect of elastin incorporation on the mechanical properties of the ELAC fibers and smooth muscle cell (SMC) phenotype was investigated. The results indicate that elastin incorporation significantly decreased the modulus of ELAC fibers to converge upon that of native vessels. Further, a significant increase in yield strain and decrease in Young's modulus was observed on all fibers post SMC culture compared with before the culture. Real-time polymerase chain reaction results showed a significant increase in the expression of α-smooth muscle actin and calponin on ELAC fibers with insoluble elastin, suggesting that incorporation of insoluble elastin induces a contractile phenotype in SMCs after two weeks of culture on ELAC fibers. Immunofluorescence results showed that calponin expression increased with time on all fibers. In conclusion, insoluble elastin incorporated ELAC fibers have the potential to be used for the development of functional TEVGs for the repair and replacement of small-diameter arteries.

  4. Human papillomavirus mRNA testing for the detection of anal high-grade squamous intraepithelial lesions in men who have sex with men infected with HIV.

    Science.gov (United States)

    Sendagorta, Elena; Romero, Maria P; Bernardino, Jose I; Beato, María J; Alvarez-Gallego, Mario; Herranz, Pedro

    2015-08-01

    Currently, screening for anal high-grade squamous intraepithelial lesions (anal HSIL) relies on anal cytology and high-resolution anoscopy. Since this approach has limited sensitivity and specificity for detecting anal HSIL, there is increasing interest in the role of biomarkers for predicting anal HSIL. The aim of this study is to evaluate the diagnostic accuracy of HPV E6/E7-mRNA expression for the detection of anal HSIL in MSM infected with HIV, in comparison to DNA-HR-HPV and anal cytology. This cross-sectional screening study included 101 MSM followed at the HIV-unit of La Paz University Hospital. Intra-anal swabs from patients participating in a screening program including cytology, high-resolution anoscopy and histology were analyzed. HR-HPV-DNA detection was performed by means of the CLART® HPV2 assay (GENOMICA S.A.U., Madrid, Spain). E6/E7-mRNA detection of HR-HPV-types 16, 18, 31, 33, and 45 was performed using the NucliSENS-EasyQ assay (BioMérieux, Marcy ĺEtoile, France). HR-HPV DNA and HPVE6/E7 mRNA were detected in 82% and 57% of the anal smears respectively. Anal cytology screening was abnormal in 70.3%. For the detection of HSIL sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 71.7%, 55.6%, 57.9%, and 69.8% for E6/E7-mRNA testing, respectively, compared to 100%, 31.5%, 55.4%, and 100% for HR-HPV-DNA testing and to 83%, 40.7%, 54.9%, 73.3% of cytology testing. In comparison with the other tests, HPVE6/E7 mRNA testing yielded a lower clinical sensitivity but a higher clinical specificity and PPV for the detection of anal HSIL in MSM infected with HIV.

  5. Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

    Science.gov (United States)

    Pescador, David; Ibáñez-Fonseca, Arturo; Sánchez-Guijo, Fermín; Briñón, Jesús G; Arias, Francisco Javier; Muntión, Sandra; Hernández, Cristina; Girotti, Alessandra; Alonso, Matilde; Del Cañizo, María Consuelo; Rodríguez-Cabello, José Carlos; Blanco, Juan Francisco

    2017-08-01

    Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p hyaline cartilage in osteochondral lesions.

  6. Vimentin is a component of a complex that binds to the 5'-UTR of human heme-regulated eIF2α kinase mRNA and regulates its translation.

    Science.gov (United States)

    Chatterjee, Sangeeta; Panda, Amaresh C; Berwal, Sunil K; Sreejith, R K; Ritvika, Charu; Seshadri, Vasudevan; Pal, Jayanta K

    2013-03-01

    The human heme-regulated eIF2α kinase, also called the human heme-regulated inhibitor (hHRI) is significantly up-regulated particularly at the level of translation during stress. In this report we show that during lead-stress, the regulation of hHRI mRNA translation is mediated through its 5'-untranslated region (UTR) that interacts with specific trans-acting factors. Further, vimentin has been identified as one of the trans-acting factors that contribute to this regulation.

  7. In humans IL-6 is released from the brain during and after exercise and paralleled by enhanced IL-6 mRNA expression in the hippocampus of mice

    DEFF Research Database (Denmark)

    Rasmussen, Per; Vedel, J-C; Olesen, J

    2011-01-01

    . Additionally, it was evaluated in mice whether brain release of IL-6 reflected enhanced IL-6 mRNA expression in the brain as modulated by brain glycogen levels. Methods: Nine healthy male subjects completed 4 h of ergometer rowing while the arterio-jugular venous difference (a-v diff) for IL-6 was determined....... The IL-6 mRNA and the glycogen content were determined in mouse hippocampus, cerebellum and cortex before and after 2 h treadmill running (N = 8). Results: At rest, the IL-6 a-v diff was negligible but decreased to -2.2 ± 1.9 pg ml(-1) at the end of exercise and remained low (-2.1 ± 2.1 pg ml(-1) ) 1 h...

  8. 吗啡对人胃癌MGC-803细胞p53 mRNA和E2F-1 mRNA表达的影响%Effect of morphine on expression of p53 mRNA and E2F-1 mRNA in human gastric carcinoma cell line MGC-803

    Institute of Scientific and Technical Information of China (English)

    覃怡; 唐小曼; 廖淳杰; 谢玉波

    2010-01-01

    Objective To investigate the effect of morphine on the expression of p53 mRNA and E2F-1 mRNA in human gastric carcinoma cell line MGC-803 .Methods The human gastric cancer cell line MGC-803 was purchased from Cell Biology Research Institute, Chinese Academy of Sciences, and cultured in DMEM liquid culture mediun. The cells were seeded in 6-well plates (1 × 103/ml or 2 × 105/ml, 1 ml/well) and divided into 2 groups (n = 18 wells each):group Ⅰ normal control (group C); group Ⅱ was exposed to 10 μmol/L morphine (group M). The proliferation of the cells was determined by colony formation assay at 7 day of incubation with morphine. The expression of p53 mRNA and E2F-1 mRNA was detected and the ulrastructure of the cells examined with transmission electron microscope after being incubated with morphine for 24 h. Results The proliferation of the cells and E2F-1 mRNA expression were significantly lower and p53 mRNA expression was significantly higher in group M than in group C (P < 0.05). The nuclear evelope was intact and the nucleolus and chromosomes were clearly visible in group C, while in group M fragmentation of nuclear envelope and nucleolus and apoptotic bodies were observed. Conclusion Morphine can inhibit the proliferation of the cells and accelerate the cell apoptosis through up-regulating the expression of p53 gene and down-regulating the expression of E2F-1gene in human gastric carcinoma cell line MGC-803.%目的 探讨吗啡对人胃癌MGC-803细胞p53 mRNA和E2F-1 mRNA表达的影响.方法 人胃癌MGC-803细胞以1×103/ml或2×105/ml密度接种于6孔培养板中,1 ml/孔,随机分为2组(n=18),正常对照组(C组)不作任何处理,吗啡组(M组)加入吗啡,使其终浓度为10μmol/L.于吗啡孵育7 d时应用克隆形成实验测定细胞增殖情况,于吗啡孵育24 h时测定细胞中p53 mRNA、E2F-1 mRNA的表达情况,并采用透射电子显微镜观察细胞超微结构.结果 与C组比较,M组克隆形成率降低,p53 m

  9. Direct inhibition of elastase and matrixmetalloproteinases and stimulation of biosynthesis of fibrillar collagens, elastin, and fibrillins by xanthohumol.

    Science.gov (United States)

    Philips, Neena; Samuel, Mathew; Arena, Rosemarie; Chen, Yu-Jun; Conte, Jennifer; Natarajan, Prashanthi; Natrajan, Prashanti; Haas, Gerhard; Gonzalez, Salvador

    2010-01-01

    In skin aging there is deterioration of the extracellular matrix's collagen and elastin fibers, from its reduced biosynthesis and increased degradation by elastase and matrixmetalloproteinases (MMPs). Xanthohumol is a flavonoid isolated from the hop plant Humulus lupulus L., with anti-microbial, antioxidant, anti-inflammatory, and anti-carcinogenic properties. The goal of this research was to investigate xanthohumol as an anti-skinaging agent via its beneficial regulation of the extracellular matrix. To this purpose, we examined the direct effect of xanthohumol on the activities of elastase and MMPs (MMPs 1, 2, and 9) and its effect on the expression (protein and/or transcription levels) of collagens (types I, III, and V), elastin, and fibrillins (1 and 2) in dermal fibroblasts. Xanthohumol significantly inhibited elastase and MMP-9 activities from its lowest concentration, and MMP-1 and MMP-2 at its higher concentrations, which implies a greater protective effect on elastin. It dramatically increased the expression of types I, III, and V collagens, and elastin, fibrillin-1, and fibrillin-2 in dermal fibroblasts. The effects were similar to those of ascorbic acid. This is the first report identifying xanthohumol's potential to improve skin structure and firmness: it simultaneously inhibits the activities of elastase/MMPs and stimulates the biosynthesis of fibrillar collagens, elastin, and fibrillins.

  10. A possible role for elastin ligands in the proteolytic degradation of arterial elastic lamellae in the rabbit.

    Science.gov (United States)

    Kagan, H M; Milbury, P E; Kramsch, D M

    1979-01-01

    Thoracic aortae of normal rabbits were perfused with pancreatic elastase in vitro at 37 degrees C and 70 mm Hg pressure in the presence or absence of elastin ligands previously shown to stimulate or inhibit the enzymatic degradation of elastin. Perfusion with elastase results in an average of 3.6 lamellae degraded, whereas addition of sodium linoleate before and during the perfusion with elastase increases this value to 7.9 (P less than 0.001). Conversely, perfusion with the cationic detergent, dodecyltrimethylammonium chloride, completely prevents the degradation of elastic lamellae by elastase. These effects do not reflect alterations of the intrinsic catalytic activity of elastase, but apparently indicate the formation of complexes between the elastin ligands and arterial elastic lamellae, as is consistent with prior studies indicating such interactions between fatty acids or detergents and purified elastin. These studies suggest that agents such as fatty acids may significantly alter the metabolic susceptibility of elastin in vivo and possibly contribute to the degradation of elastic lamellae seen in arteries with advanced atherosclerosis.

  11. EFFECTS OF ENZYMATIC HYDROLYSIS ON THE ANTIOXIDANT ACTIVITY OF WATER- SOLUBLE ELASTIN EXTRACTED FROM BROILER AND SPENT HEN SKIN

    Directory of Open Access Journals (Sweden)

    Mehdi Nadalian

    2015-12-01

    Full Text Available Poultry by-products are great economic sources that need to be exploited. Poultry skin could be utilized to extract protein particularly elastin, which is often incorporated in the production of functional food, cosmetic industry or medicine due to its antioxidative properties. In this study, water-soluble elastin was successfully extracted from broiler and spent hen skin and analysed for antioxidant activities including DPPH (1,1-diphenyl-2-picryl hydrazyl, ABTS and metal chelating activity. Antioxidant activity of elastin extracted from broiler skin hydrolysed by Alcalase (EBA and Elastase (EBE also elastin extracted from spent hen skin hydrolyzed by Alcalase (ESA and Elastease (ESE. The EBE, EBA, ESE and ESA had higher DPPH (16-30, 19-35, 29-48 and 31-50%, respectively, ABTS activity ( 73-79, 60-79, 67-79 and 72-79 %, respectively, and Fe2+chelating activity ( 65-69, 50-56, 71-77 and 62-70 %, respectively. This concluded that water-soluble elastin is a bioactive component that could potentially be used in the formulation of functional foods, nutraceuticals, cosmetic and pharmaceutical industry

  12. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A in the Human Schizophrenic Brain

    Directory of Open Access Journals (Sweden)

    Sern-Yih Cheah

    2017-01-01

    Full Text Available Serotonin receptor 2A (HTR2A is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006 in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017 and HTR2A polymorphisms rs6314 (p = 0.008 and rs6313 (p = 0.026 showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  13. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A) in the Human Schizophrenic Brain.

    Science.gov (United States)

    Cheah, Sern-Yih; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

    2017-01-04

    Serotonin receptor 2A (HTR2A) is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006) in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017) and HTR2A polymorphisms rs6314 (p = 0.008) and rs6313 (p = 0.026) showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  14. Studies on microsatellite instability in p16 gene and expression of hMSH2 mRNA in human gastric cancer tissues

    Institute of Scientific and Technical Information of China (English)

    Qin-Xian Zhang; Yi Ding; Xiao-Ping Le; Peng Du

    2003-01-01

    AIM: To detect the loss of heterozygosity (LOH) frequency of microsatellite sites D9s171, D9s1604 of p16 gene and expression of hMSH2 mRNA in various differentiated types of gastric cancer, adjacent cancer tissues and normal gastric mucosa.METHODS: LOH was detected by polymerase chain reaction (PCR)-denaturing polyacrylamide gel electrophoresis-silver staining. The expression of hMSH2 mRNA was examined with in situ hybridization.RESULTS: The frequency rate of LOH was significantly higher in gastric cancers than that in adjacent cancer tissues (P=0.032). No significant difference was noted among various differentiated types and various clinical stages of gastric cancers. The significantly reduced expression of hMSH2mRNA positive signal cells exhibited in gastric cancers, in comparison with that in the adjacent cancer tissues and normal gastric mucosa, respectively (P=0.001). No significant difference was noted among various clinical stages of gastric cancers (P>0.05). The difference of positive signal cells in poorly differentiated cancers and those in well and moderately differentiated cancers were significant (P<0.001).CONCLUSION: The frequencies of LOH in two microsatellite sites, D9s171 and D9s1604, in p16 genome were associated with development of gastric cancer and no significant correlation was demonstrated between the LOH frequency and the cell differentiated types of tumor cells or clinical stages. There was a positive relationship between the expression of hMSH2 mRNA and the differentiated types of gastric cancer.

  15. Dose-Dependent Pattern of Inducible mRNA Expression of PIG3 Gene in Normal Human Lymphoblastoid Cells by Thermal Neutron

    Institute of Scientific and Technical Information of China (English)

    MA; Nan-ru; SUI; Li; WANG; Xiao; KONG; Fu-quan; LIU; Xiao-dan; ZHOU; Ping-kun

    2012-01-01

    <正>Using the thermal neutron produced by the hospital neutron irradiator, AHH-1 cell was irradiated at the various dose, 0, 0.5, 2, 4, 6 and 8 Gy, respectively. After irradiation, cells were collected at 2, 6, 12, 24 and 48 h post-irradiation, and then cell cycle distribution was tested using flow cytometry, as well as PIG3 mRNA expression level was detected using real-time fluorescent quantitative PCR detection.

  16. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    Science.gov (United States)

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  17. Impaired elastin deposition in Fstl1-/- lung allograft under the renal capsule.

    Directory of Open Access Journals (Sweden)

    Yan Geng

    Full Text Available Lung alveolar development in late gestation is a process important to postnatal survival. Follistatin-like 1 (Fstl1 is a matricellular protein of the Bmp antagonist class, which is involved in the differentiation/maturation of alveolar epithelial cells during saccular stage of lung development. This study investigates the role of Fstl1 on elastin deposition in mesenchyme and subsequent secondary septation in the late gestation stage of terminal saccular formation. To this aim, we modified the renal capsule allograft model for lung organ culture by grafting diced E15.5 distal lung underneath the renal capsule of syngeneic host and cultured up to 7 days. The saccular development of the diced lung allografts, as indicated by the morphology, epithelial and vascular developments, occurred in a manner similar to that in utero. Fstl1 deficiency caused atelectatic phenotype companied by impaired epithelial differentiation in D3 Fstl1(-/- lung allografts, which is similar to that of E18.5 Fstl1(-/- lungs, supporting the role of Fstl1 during saccular stage. Inhibition of Bmp signaling by intraperitoneal injection of dorsomorphin in the host mice rescued the pulmonary atelectasis of D3 Fstl1(-/- allografts. Furthermore, a marked reduction in elastin expression and deposition was observed in walls of air sacs of E18.5 Fstl1(-/- lungs and at the tips of the developing alveolar septae of D7 Fstl1(-/- allografts. Thus, in addition to its role on alveolar epithelium, Fstl1 is crucial for elastin expression and deposition in mesenchyme during lung alveologenesis. Our data demonstrates that the modified renal capsule allograft model for lung organ culture is a robust and efficient technique to increase our understanding of saccular stage of lung development.

  18. Expression of COLLAGEN 1 and ELASTIN Genes in Mitral Valvular Interstitial Cells within Microfiber Reinforced Hydrogel

    Directory of Open Access Journals (Sweden)

    Eslami Maryam

    2015-10-01

    Full Text Available Objective The incidence of heart valve disease is increasing worldwide and the number of heart valve replacements is expected to increase in the future. By mimicking the main tissue structures and properties of heart valve, tissue engineering offers new options for the replacements. Applying an appropriate scaffold in fabricating tissue-engineered heart valves (TEHVs is of importance since it affects the secretion of the main extracellular matrix (ECM components, collagen 1 and elastin, which are crucial in providing the proper mechanical properties of TEHVs. Materials and Methods Using real-time polymerase chain reaction (PCR in this experi- mental study, the relative expression levels of COLLAGEN 1 and ELASTIN were obtained for three samples of each examined sheep mitral valvular interstitial cells (MVICs-seeded onto electrospun poly (glycerol sebacate (PGS-poly (ε-caprolactone (PCL microfibrous, gelatin and hyaluronic acid based hydrogel-only and composite (PGS-PCL/hydrogel scaffolds. This composite has been shown to create a synthetic three-dimensional (3D microenvironment with appropriate mechanical and biological properties for MVICs. Results Cell viability and metabolic activity were similar among all scaffold types. Our results showed that the level of relative expression of COLLAGEN 1 and ELASTIN genes was higher in the encapsulated composite scaffolds compared to PGS-PCL-only and hydrogel-only scaffolds with the difference being statistically significant (P<0.05. Conclusion The encapsulated composite scaffolds are more conducive to ECM secretion over the PGS-PCL-only and hydrogel-only scaffolds. This composite scaffold can serve as a model scaffold for heart valve tissue engineering.

  19. A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

    Institute of Scientific and Technical Information of China (English)

    Li YANG; Jiang CHEN; Catherine C. Y. CHANG; Xin-Ying YANG; Zhen-Zhen WANG; Ta-Yuan CHANG; Bo-Liang LI

    2004-01-01

    Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397-1399 and AUG1415-1417, at 5′-terminus of the open reading frame (ORF,nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5 ′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397-1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5 ′-ORF-AUG1397-1399 as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.

  20. Simultaneous observation of collagen and elastin based on the combined nonlinear optical imaging technique coupled with two-channel synchronized detection method

    Science.gov (United States)

    Chen, Jianxin; Zhuo, Shuangmu; Luo, Tianshu; Liu, Dingzhong; Zhao, Jingjun

    2008-08-01

    Collagen and elastin are the most important proteins of the connective tissues in higher vertebrates. In this paper, we present a combined nonlinear optical imaging technique of second-harmonic generation and two-photon excited fluorescence to simultaneously observe the collagen and elastic fiber of dermis in a freshly excised human skin and rabbit aorta using a two-channel synchronized detection method. The obtained two-channel overlay image in the backward direction can clearly distinguish the morphological structure and distribution of collagen and elastic fibers. Tissue spectrum further confirms the obtained structural information. These results suggest that the combined nonlinear optical imaging technique coupled with two-channel synchronized detection method can be an effective tool for detecting collage and elastic fibers without any invasive tissue procedure of slicing, embedding, fixation and staining when two structural proteins are simultaneously present in the biological tissue.

  1. Elastin organization in pig and cardiovascular disease patients' pericardial resistance arteries

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Leurgans, Thomas; Nissen, Inger;

    2015-01-01

    coronary artery bypass grafting or cardiac valve replacement surgeries, can serve as a source of resistance arteries for structural research in cardiovascular disease patients. We applied two-photon excitation fluorescence microscopy to study the parietal pericardium and isolated pericardial resistance...... arteries with a focus on the collagen and elastin components of the extracellular matrix. Initial findings in pig tissue were confirmed in patient biopsies. The microarchitecture of the internal elastic lamina in both the pig and patient pericardial resistance arteries (studied at a transmural pressure...

  2. Effect of phytic acid ketone on expression of HPV16/18 E6/E7 mRNA and protein in human cervical cancer cell line%植酸酮对宫颈癌细胞株中 HPV16/18 E6/E7 mRNA 及蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    王侃; 高晓丽; 田文艳; 岳天孚

    2016-01-01

    Objective To observe the effects of phytic acid ketone on expression of HPV 16/18 E6/E7 mRNA and protein in human cervical cancer cells.Methods The Caski cells ( containing HPV16 ) and Hela cells ( containing HPV18) were cultivated in vitro and then divided into the experimental groups 1, 2, 3, 4 and the control group ( each group contained Caski cell line and Hela cell line).The experimental groups 1, 2, 3, 4 were respectively treated with dif-ferent concentrations of phytic acid ketone (58.6 mg/L, 117 mg/L, 586 mg/L, 5 860 mg/L) for 72 h.The levels of HPV16/18 E6/E7 mRNA were determined with real-time PCR.Western blotting was used to measure the expression levels of E6 and E7 proteins.Results E6/E7 mRNA and protein expression in the same type of cells of the experimental groups 1, 2, 3 and 4 was lower than that of the control group, and the E6/E7 mRNA and protein expression showed a decreasing tendency in the experimental groups 1, 2, 3 and 4 (all P<0.05).In each experimental group, E6/E7 mRNA expression in Caski cells was lower than that in Hela cells (all P<0.05).In the same type of cells of the experiential groups, the E6 mRNA expression was lower than that of E7 mRNA (all P<0.05).Conclusions Phytic acid ketone could down-regulate the expression of E6/E7 mRNA and protein in human cervical cancer lines, which was in a dose-dependent manner.The inhibitory effect of phytic acid ketone on HPV16 E6/E7 mRNA was stronger than that on HPV18 E6/E7 mRNA and the suppression of E6 probably played a leading role.%目的:观察植酸酮对宫颈癌细胞株中HPV16/18 E6/E7 mRNA及蛋白表达的影响。方法体外培养Caski细胞株(含HPV16)与Hela细胞株(含HPV18),将所有细胞分成实验1、2、3、4组和对照组,每组均包含Caski细胞和Hela细胞。实验1、2、3、4组分别加入含58.6、117、586、5860 mg/L植酸酮的培养液,对照组加入不含植酸酮的培养液。各组培养72 h后,采用real-time PCR

  3. Acute Myocardial Infarction and Pulmonary Diseases Result in Two Different Degradation Profiles of Elastin as Quantified by Two Novel ELISAs

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Clausen, Rikke E; Rasmussen, Lars M

    2013-01-01

    BACKGROUND: Elastin is a signature protein of the arteries and lungs, thus it was hypothesized that elastin is subject to enzymatic degradation during cardiovascular and pulmonary diseases. The aim was to investigate if different fragments of the same protein entail different information associated......: ELM and ELM-2 neoepitopes were both localized in diseased carotid arteries and fibrotic lungs. In the cardiovascular cohort, ELM-2 levels were 66% higher in serum from AMI patients compared to patients with no AMI (p... to two different diseases and if these fragments have the potential of being diagnostic biomarkers. METHODS: Monoclonal antibodies were raised against an identified fragment (the ELM-2 neoepitope) generated at the amino acid position '552 in elastin by matrix metalloproteinase (MMP) -9/-12. A newly...

  4. Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

    Science.gov (United States)

    Biela, Ewa; Galas, Jerzy; Lee, Brian; Johnson, Gary L; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

    2013-06-01

    A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials.

  5. First steps towards tissue engineering of small-diameter blood vessels: preparation of flat scaffolds of collagen and elastin by means of freeze drying

    NARCIS (Netherlands)

    Buttafoco, L.; Engbers-Buijtenhuijs, P.; Poot, Andreas A.; Dijkstra, Pieter J.; Daamen, W.F.; van Kuppevelt, T.H.; Vermes, I.; Feijen, Jan

    2006-01-01

    Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196°C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18°C afforded collagen and collagen/elastin matrices with average

  6. Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hans Prochnow

    Full Text Available Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU, which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.

  7. Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach.

    Science.gov (United States)

    Tikole, Suhas; Sankararamakrishnan, Ramasubbu

    2008-05-16

    Translation of eukaryotic mRNAs is often regulated by nucleotides around the start codon. A purine at position -3 and a guanine at position +4 contribute significantly to enhance the translation efficiency. Algorithms to predict the translation initiation site often fail to predict the start site if the sequence context is not present. We have developed a neural network method to predict the initiation site of mRNA sequences that lack the preferred nucleotides at the positions -3 and +4 surrounding the translation initiation site. Neural networks of various architectures comprising different number of hidden layers were designed and tested for various sizes of windows of nucleotides surrounding translation initiation sites. We found that the neural network with two hidden layers showed a sensitivity of 83% and specificity of 73% indicating a vastly improved performance in successfully predicting the translation initiation site of mRNA sequences with weak Kozak context. WeakAUG server is freely available at http://bioinfo.iitk.ac.in/AUGPred/.

  8. EFFECTS OF ALVEOLAR MACROPHAGE CONDITIONED MEDIA FROM INTERSTITIAL LUNG DISEASEPATIENTS ON THE PROCOLLAGEN mRNA EXPRESSION IN HUMAN LUNG FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    郭子健; 朱元珏; 刘秉慈; 朱亚玲; 赵文理; 陈勇

    1996-01-01

    Progressive inflammation and fibrosis are the central processez in the pathogenesis of pulmonary fibrosis. It is believed that macrophages in areas of chronically inflamed lung play a key role in fibrotic response. Therefore, we investigated the effects of alveolar macrophage (Amφ) conditioned media from interstitial lung disease (ILD) patients on lung fibroblast proliferation and procollagen mRNA expression, After stimulating with Amφ conditioned media from ILD pasients, the fibroblast proliferation increased 71.4% compared with the control, but for media from bronchial carcinoma (BC) patients, it just increased 14.3%. There is a significant dffference between the two groups (P<0. 05). The procollagen αl(I) mRNA in fibroblasts stimulated with Amφ conditioned media from ILD patients was increased 21.3% α1(Ⅲ)was 37.2 higher than control (P<0. 05). It increased 6. 8% and 12.8% fof media from BC patients respectively, but there was no difference when compared to the control. We considered that Amφ from ILD patients might be in an activated state and could release some growth factors to stimulate fibroblast proliferation and promote collagen DNA expression,

  9. 军团菌体外刺激对人PBMC TLR2 mRNA表达影响%Effects of Legionella on expression of TLR2 mRNA in human peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    高向东; 张帆; 王素萍; 史晓红; 王伟刚; 王芳芳; 董洁敏; 雷智

    2013-01-01

    目的 探讨嗜肺军团菌活菌刺激对人外周血单个核细胞(PBMC)表达TLR2 mRNA的影响.方法 采用不同浓度的嗜肺军团菌活菌悬液刺激体外培养的健康人PBMC,收集细胞运用RT-PCR方法测定TLR2mRNA的表达水平.结果 析因分析结果显示,时间的主效应有统计学意义(F=26.06,P<0.05);时间与浓度的交互作用有统计学意义(F=11.39,P <0.05);24 h时,MOI 1 、MOI 10刺激组TLR2 mRNA的表达量均高于对照组(P<0.05),呈现随菌液浓度升高表达量也逐渐升高,48 h时,各组之间TLR2 mRNA的表达量差异虽无统计学意义(P>0.05),但表达量随浓度升高呈现下降趋势,72 h时,各组之间TLR2 mRNA的表达量差异有统计学意义(P<0.05),但随菌液浓度升高表达量反而降低.结论 用军团菌体外刺激人PBMC,其TLR2mRNA的表达在一定范围内呈一定的时效与量效关系,可为今后的研究提供有益的时间和剂量参考.%Objective To examine the expression of TLR2 in human peripheral blood mononuclear cells (PBMCs) stimulatedby Legionella. Methods Human peripheral blood mononuclear cells ( POMCs) were treated with different concentrations of Legionella(2×106/ml,2×107/ml) ,and real timel reverse transcriptase PCR(RT-PCR) was used to detect TLR2 mRNA expression. Results Factorial analyses showed that leading effect of time had statistical significance (F = 26. 06,P < 0. 05) and there was an interaction between the time and concentration ( F = 11. 39, P < 0. 05). In 24 hours,the expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml and 2×107/ml Legionella were obviously higher than TLR2 mRNA expression in control group. In 48 hours,there was no difference between stimulated group and control group. In 72 hours,the expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml and 2×107/ml Legionella , were obviously lower than TLR2 mRNA expression in the control group. When the PBMCs were stimulated at the level of 2×106/ml and 2×107/ml

  10. Antiageing Mechanisms of a Standardized Supercritical CO2 Preparation of Black Jack (Bidens pilosa L. in Human Fibroblasts and Skin Fragments

    Directory of Open Access Journals (Sweden)

    Gustavo Dieamant

    2015-01-01

    Full Text Available The use of topical retinoids to treat skin disorders and ageing can induce local reactions, while oral retinoids are potent teratogens and produce several unwanted effects. This way, efforts to explore complementary care resources should be supported. Based on this, we evaluate the antiageing effects of a supercritical CO2 extract from Bidens pilosa L. (BPE-CO2A containing a standardized multicomponent mixture of phytol, linolenic, palmitic, linoleic, and oleic acids. BPE-CO2A was assessed for its effects on human dermal fibroblasts (TGF-β1 and FGF levels using ELISA; collagen, elastin, and glycosaminoglycan by colorimetric assays, and mRNA expression of RXR, RAR, and EGFr by qRT-PCR and human skin fragments (RAR, RXR, collagen, elastin, and glycosaminoglycan by immunohistochemical analysis. Levels of extracellular matrix elements, TGF-β1 and FGF, and EGFr gene expression were significantly increased by BPE-CO2A. The modulation of RXR and RAR was positively demonstrated after the treatment with BPE-CO2A or phytol, a component of BPE-CO2A. The effects produced by BPE-CO2A were similar to or better than those produced by retinol and retinoic acid. The ability to stimulate extracellular matrix elements, increase growth factors, and modulate retinoid and rexinoid receptors provides a basis for the development of preparation containing BPE-CO2A as an antiageing/skin-repair agent.

  11. Aqueous-Based Coaxial Electrospinning of Genetically Engineered Silk Elastin Core-Shell Nanofibers

    Directory of Open Access Journals (Sweden)

    Jingxin Zhu

    2016-03-01

    Full Text Available A nanofabrication method for the production of flexible core-shell structured silk elastin nanofibers is presented, based on an all-aqueous coaxial electrospinning process. In this process, silk fibroin (SF and silk-elastin-like protein polymer (SELP, both in aqueous solution, with high and low viscosity, respectively, were used as the inner (core and outer (shell layers of the nanofibers. The electrospinnable SF core solution served as a spinning aid for the nonelectrospinnable SELP shell solution. Uniform nanofibers with average diameter from 301 ± 108 nm to 408 ± 150 nm were obtained through adjusting the processing parameters. The core-shell structures of the nanofibers were confirmed by fluorescence and electron microscopy. In order to modulate the mechanical properties and provide stability in water, the as-spun SF-SELP nanofiber mats were treated with methanol vapor to induce β-sheet physical crosslinks. FTIR confirmed the conversion of the secondary structure from a random coil to β-sheets after the methanol treatment. Tensile tests of SF-SELP core-shell structured nanofibers showed good flexibility with elongation at break of 5.20% ± 0.57%, compared with SF nanofibers with an elongation at break of 1.38% ± 0.22%. The SF-SELP core-shell structured nanofibers should provide useful options to explore in the field of biomaterials due to the improved flexibility of the fibrous mats and the presence of a dynamic SELP layer on the outer surface.

  12. Architecturally defined scaffolds from synthetic collagen and elastin analogues for the fabrication of bioengineered tissues

    Science.gov (United States)

    Caves, Jeffrey Morris

    The microstructure and mechanics of collagen and elastin protein fiber networks dictate the mechanical responses of all soft tissues and related organ systems. In this project, we endeavored to meet or exceed native tissue biomechanical properties through mimicry of these extracellular matrix components with synthetic collagen fiber and a recombinant elastin-like protein polymer. Significantly, this work led to the development of a framework for the design and fabrication of protein-based tissue substitutes with enhanced strength, resilience, anisotropy, and more. We began with the development of a spinning process for scalable production of synthetic collagen fiber. Fiber with an elliptical cross-section of 53 +/- 14 by 21 +/- 3 mum and an ultimate tensile strength of 90 +/- 19 MPa was continuously produced at 60 meters per hour from an ultrafiltered collagen solution. The starting collagen concentration, flowrate, and needle size could be adjusted to control fiber size. The fiber was characterized with mechanical analysis, micro-differential scanning calorimetry, transmission electron microscopy, second harmonic generation analysis, and subcutaneous murine implant. We subsequently describe the scalable, semi-automated fabrication of elastin-like protein sheets reinforced with synthetic collagen fibers that can be positioned in a precisely defined three-dimensional hierarchical pattern. Multilamellar, fiber-reinforced elastic protein sheets were constructed with controlled fiber orientation and volume fraction. Structures were analyzed with scanning electron microscopy, transmission electron microscopy, and digital volumetric imaging. The effect of fiber orientation and volume fraction on Young's Modulus, yield stress, ultimate tensile stress, strain-to-failure, and resilience was evaluated in uniaxial tension. Increased fiber volume fraction and alignment with applied deformation significantly increased Young's Modulus, resilience, and yield stress. Highly

  13. Suppression of testosterone does not blunt mRNA expression of myoD, myogenin, IGF, myostatin or androgen receptor post strength training in humans

    DEFF Research Database (Denmark)

    Kvorning, Thue; Andersen, Marianne; Brixen, Kim

    2007-01-01

    We hypothesized that suppression of endogenous testosterone blunts mRNA expression post strength training (ST). Twenty-two young men were randomized for treatment with the GnRH analogue goserelin (3.6 mg every 4 weeks) or placebo for a period of 12 weeks. The ST period of 8 weeks started at week ...... events were the same, despite divergent muscle hypertrophy and strength gains........ Strength test, blood sampling, muscle biopsies, and whole-body dual-energy X-ray absorptiometry (DXA) scan were performed at weeks 4 and 12. Muscle biopsies were taken during the final ST session (pre, post 4 h, and post 24 h). Resting serum testosterone decreased significantly (P ... group (P Muscle strength and muscle mass showed a tendency to increase more in the placebo group than in the goserelin group (P = 0...

  14. A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Hansen, Christina N; Vinther, Jeppe

    2003-01-01

    RNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic m...... from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream...... of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells....

  15. Calciotrophic hormones and hyperglycemia modulate vitamin D receptor and 25 hydroxyy vitamin D 1-α hydroxylase mRNA expression in human vascular smooth muscle cells.

    Science.gov (United States)

    Somjen, D; Knoll, E; Sharon, O; Many, A; Stern, N

    2015-04-01

    Estrogen receptors (ERα and ERβ), the vitamin D receptor (VDR) and 25 hydroxyy vitamin D 1-α hydroxylase (1OHase) mRNA are expressed in vascular smooth muscle cells (VSMC). In these cells estrogenic hormones modulate cell proliferation as measured by DNA synthesis (DNA). In the present study we determined whether or not the calciotrophic hormones PTH 1-34 (PTH) and less- calcemic vitamin D analog QW as well as hyperglycemia can regulate DNA synthesis and CK. E2 had a bimodal effect on VSMC DNA synthesis, such that proliferation was inhibited at 30nM but stimulated at 0.3nM. PTH at 50nM increased, whereas QW at 10nM inhibited DNA synthesis. Hyperglycemia inhibited the effects on high E2, QW and PTH on DNA only. Both QW and PTH increased ERα mRNA expression, but only PTH increased ERβ expression. Likewise, both PTH and QW stimulated VDR and 1OHase expression and activity. ERβ, VDR and 1OHase expression and activity were inhibited by hyperglycemia, but ERα expression was unaffected by hyperglycemia. In conclusion, calcitrophic hormones modify VSMC growth and concomitantly affect ER expression in these cells as well as the endogenous VSMC vitamin D system elements, including VDR and 1OHase. Some of the later changes may likely participate in growth effects. Of importance in the observation is that several regulatory effects are deranged in the presence of hyperglycemia, particularly the PTH- and vitamin D-dependent up regulation of VDR and 1OHase in these cells. The implications of these effects require further studies. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  16. Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Allameh, Abdolamir; Esmaeli, Shahnaz; Kazemnejad, Somaieh; Soleimani, Masoud

    2009-06-01

    The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.

  17. Preparation of Photocrosslinked Fish Elastin Polypeptide/Microfibrillated Cellulose Composite Gels with Elastic Properties for Biomaterial Applications

    Science.gov (United States)

    Yano, Shinya; Mori, Megumi; Teramoto, Naozumi; Iisaka, Makoto; Suzuki, Natsumi; Noto, Masanari; Kaimoto, Yasuko; Kakimoto, Masashi; Yamada, Michio; Shiratsuchi, Eri; Shimasaki, Toshiaki; Shibata, Mitsuhiro

    2015-01-01

    Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity. PMID:25584682

  18. Preparation of Photocrosslinked Fish Elastin Polypeptide/Microfibrillated Cellulose Composite Gels with Elastic Properties for Biomaterial Applications

    Directory of Open Access Journals (Sweden)

    Shinya Yano

    2015-01-01

    Full Text Available Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO. First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC, a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.

  19. Neomycin and pentagalloyl glucose enhanced cross-linking for elastin and glycosaminoglycans preservation in bioprosthetic heart valves.

    Science.gov (United States)

    Tripi, Daniel R; Vyavahare, Naren R

    2014-01-01

    Glutaraldehyde cross-linked bioprosthetic heart valves fail within 12-15 years of implantation due to limited durability. Glutaraldehyde does not adequately stabilize extracellular matrix components such as glycosaminoglycans and elastin, and loss of these components could be a major cause of degeneration of valve after implantation. We have shown earlier that neomycin-based cross-linking stabilizes glycosaminoglycans in the tissue but fails to stabilize elastin component. Here, we report a new treatment where neomycin and pentagalloyl glucose (PGG) were incorporated into glutaraldehyde cross-linking neomycin-PGG-Glutaraldehyde (NPG) to stabilize both glycosaminoglycans and elastin in porcine aortic valves. In vitro studies demonstrated a marked increase in extracellular matrix stability against enzymatic degradation after cross-linking and 10 month storage in NPG group when compared to glutaraldehyde controls. Tensile properties showed increased lower elastic modulus in both radial and circumferential directions in NPG group as compared to glutaraldehyde, probably due to increased elastin stabilization with no changes in upper elastic modulus and extensibility. The enhanced extracellular matrix stability was further maintained in NPG-treated tissues after rat subdermal implantation for three weeks. NPG group also showed reduced calcification when compared to glutaraldehyde controls. We conclude that NPG cross-linking would be an excellent alternative to glutaraldehyde cross-linking of bioprosthetic heart valves to improve its durability.

  20. Preparation of photocrosslinked fish elastin polypeptide/microfibrillated cellulose composite gels with elastic properties for biomaterial applications.

    Science.gov (United States)

    Yano, Shinya; Mori, Megumi; Teramoto, Naozumi; Iisaka, Makoto; Suzuki, Natsumi; Noto, Masanari; Kaimoto, Yasuko; Kakimoto, Masashi; Yamada, Michio; Shiratsuchi, Eri; Shimasaki, Toshiaki; Shibata, Mitsuhiro

    2015-01-09

    Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N'-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress-strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.

  1. Deposition of insoluble elastin by pulmonary fibroblasts from patients with COPD is increased by treatment with versican siRNA

    Science.gov (United States)

    Wu, Lian; Zhang, Jing; Qu, Jie Ming; Bai, Chun-xue; Merrilees, Mervyn J

    2017-01-01

    A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA) against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients (P<0.01) and significantly increased deposition of insoluble elastin in the COPD cell cultures (P<0.05). The treatment, however, did not significantly affect production of soluble elastin (tropoelastin) in either the COPD or non-COPD cell cultures, supporting a role for versican in inhibiting assembly but not synthesis of tropoelastin. These results suggest that removal or knockdown of versican may be a possible therapeutic strategy for increasing deposition of insoluble elastin and stimulating repair of elastic fibers in COPD lung. PMID:28138236

  2. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Deposition of insoluble elastin by pulmonary fibroblasts from patients with COPD is increased by treatment with versican siRNA

    Directory of Open Access Journals (Sweden)

    Wu L

    2017-01-01

    Full Text Available Lian Wu,1,2 Jing Zhang,3 Jie Ming Qu,4 Chun-xue Bai,3 Mervyn J Merrilees5 1Department of Community and Health Services, Unitec, 2Department of Pharmacology & Clinical Pharmacology, University of Auckland, Auckland, New Zealand; 3Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 4Department of Pulmonary Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 5Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand Abstract: A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients (P<0.01 and significantly increased deposition of insoluble elastin in the COPD cell cultures (P<0.05. The treatment, however, did not significantly affect production of soluble elastin (tropoelastin in either the COPD or non-COPD cell cultures, supporting a role for versican in inhibiting assembly but not synthesis of tropoelastin. These results suggest that removal or knockdown of versican may be a possible therapeutic strategy for increasing deposition of insoluble elastin and stimulating repair of elastic fibers in COPD lung. Keywords: pulmonary fibroblasts, COPD, elastin, versican

  4. The contribution of vascular smooth muscle, elastin and collagen on the passive mechanics of porcine carotid arteries.

    Science.gov (United States)

    Kochová, P; Kuncová, J; Svíglerová, J; Cimrman, R; Miklíková, M; Liška, V; Tonar, Z

    2012-08-01

    The main components responsible for the mechanical behavior of the arterial wall are collagen, elastin, and smooth muscle cells (SMCs) in the medial layer. We determined the structural and mechanical changes in porcine carotid arteries after administration of Triton® X-100, elastase, and collagenase using the inflation-deflation test. The arteries were intraluminarly pressurized from 0 to 200 mmHg, and the outer diameter of the artery was measured. The pressure-strain elastic modulus was determined based on the pressure/diameter ratio. The intima-media thickness, wall thickness, thickness of the tunica adventitia layer, and the area fractions of SMCs, elastin, and collagen within the arterial wall (A(A)(SMC/elastin/collagen, wall)) were measured using stereological methods. The relative changes in the relevant components of the treated samples were as follows: the decrease in A(A)(SMC, wall) after administration of Triton® X-100 was 11% ± 7%, the decrease in A(A)(elastin, wall) after administration of elastase was 40% ± 22%, and the decrease in A(A)(collagen, wall) after the application of collagenase was 51% ± 22%. The Triton® X-100 treatment led to a decrease in the SMC content that was associated with enlargement of the arterial wall (outer diameter) for pressures up to 120 mmHg, and with mechanical stiffening of the arterial wall at higher pressures. Elastase led to a decrease in the elastin content that was associated with enlargement of the arterial wall, but not with stiffening or softening. Collagenase led to a decrease in collagen content that was associated with a change in the stiffness of the arterial wall, although the exact contribution of mechanical loading and the duration of treatment (enlargement) could not be quantified.

  5. A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons.

    Science.gov (United States)

    Klimeš, Vladimír; Gentekaki, Eleni; Roger, Andrew J; Eliáš, Marek

    2014-07-10

    Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3'-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3'-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  6. Hydrophobic interactions are involved in the inhibition of human leukocyte elastase by alkyltrimethylammonium salts.

    Science.gov (United States)

    Kouadri-Boudjelthia, A; Wallach, J M

    1997-02-01

    Electrostatic forces and hydrophobic interactions had been suggested to modify the adsorption of elastases onto insoluble fibrous elastin, which is the initial stage of elastolysis, but conflicting results had been obtained, and comparison between compounds with different structures was difficult. In order to explore these observations, we have studied the effect of six alkyltrimethylammonium bromides, with alkyl chain length ranging from six to 16 carbon atoms, on human leucocyte elastase activities, either with a synthetic substrate or with insoluble elastin. The enzymatic studies were performed either spectrophotometrically or using conductimetry, and direct binding on to elastin was conductimetrically measured. Binding of the alkyltrimethylammonium salts is increasing with alkyl chain length and we could demonstrate a cooperative binding for tetra- and hexadecyl chains. No effect of the six compounds could be evidenced on hydrolysis of a specific synthetic substrate. With insoluble elastin, elastolysis inhibition could be demonstrated for alkyl chain longer than ten carbon atoms, the effect increasing with chain length. A similar inhibition was observed with the soluble kappa-elastin, but it was less effective. The study shows that the interaction between the alkyltrimethylammonium salts and elastin plays a major role in the inhibitory potency of these molecules. As this effect is enhanced with alkyl chain length, it was concluded that hydrophobic interactions favour their binding, protecting elastin against elastase adsorption.

  7. Elastin-Like Protein, with Statherin Derived Peptide, Controls Fluorapatite Formation and Morphology

    Directory of Open Access Journals (Sweden)

    Kseniya Shuturminska

    2017-06-01

    Full Text Available The process of enamel biomineralization is multi-step, complex and mediated by organic molecules. The lack of cells in mature enamel leaves it unable to regenerate and hence novel ways of growing enamel-like structures are currently being investigated. Recently, elastin-like protein (ELP with the analog N-terminal sequence of statherin (STNA15-ELP has been used to regenerate mineralized tissue. Here, the STNA15-ELP has been mineralized in constrained and unconstrained conditions in a fluoridated solution. We demonstrate that the control of STNA15-ELP delivery to the mineralizing solution can form layered ordered fluorapatite mineral, via a brushite precursor. We propose that the use of a constrained STNA15-ELP system can lead to the development of novel, bioinspired enamel therapeutics.

  8. Inhibition of Mahkota Dewa (Phaleria macrocarpa bioactive fraction on proliferation of human retinoblastoma tumor cells Y-79 through suppression of mRNA level of cyclin E

    Directory of Open Access Journals (Sweden)

    Nugroho Trilaksana

    2017-04-01

    Conclusions: Our results suggest that DLBS1425 is a potential anticancer agent which targets genes involved in cell proliferation in human retinoblastoma cells which make it pharmacologically ideal for the prevention and/or treatment of retinoblastoma cancer.

  9. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

    DEFF Research Database (Denmark)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj

    2010-01-01

    -life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non...... using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates....

  10. Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes that Cause Thoracic Aortic Aneurysms and Dissections Disrupt this Structure

    OpenAIRE

    Karimi, Ashkan; Milewicz, Dianna M.

    2015-01-01

    The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibers. The SMC elastin-contractile unit is a structural unit that links the elastin fibers to the SMCs and is characterized by the following: 1. Layers of elastin fibers that are surrounded by microfibrils. 2. Microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs. 3. SMC contractile filam...

  11. 3D silicon doped hydroxyapatite scaffolds decorated with Elastin-like Recombinamers for bone regenerative medicine.

    Science.gov (United States)

    Vila, Mercedes; García, Ana; Girotti, Alessandra; Alonso, Matilde; Rodríguez-Cabello, Jose Carlos; González-Vázquez, Arlyng; Planell, Josep A; Engel, Elisabeth; Buján, Julia; García-Honduvilla, Natalio; Vallet-Regí, María

    2016-11-01

    The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) functionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca10(PO4)5.7(SiO4)0.3(OH)1.7h0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineering: ELR-RGD that contain cell attachment specific sequences and ELR-SNA15/RGD with both hydroxyapatite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts. Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights

  12. Nuclear phosphoinositide-specific phospholipase C β1 controls cytoplasmic CCL2 mRNA levels in HIV-1 gp120-stimulated primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Francesca Spadaro

    Full Text Available HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of CCL2 from macrophages. In turn, this chemokine acts as an autocrine factor enhancing viral replication. In this study, we show for the first time that phosphoinositide-specific phospholipase C (PI-PLC is required for the production of CCL2 triggered by gp120 in macrophages. Using a combination of confocal laser-scanner microscopy, pharmacologic inhibition, western blotting and fluorescence-activated cell sorter analysis, we demonstrate that gp120 interaction with CCR5 leads to nuclear localization of the PI-PLC β1 isozyme mediated by mitogen-activated protein kinase ERK-1/2. Notably, phosphatidylcholine-specific phospholipase C (PC-PLC, previously reported to be required for NF-kB-mediated CCL2 production induced by gp120 in macrophages, drives both ERK1/2 activation and PI-PLC β1 nuclear localization induced by gp120. PI-PLC β1 activation through CCR5 is also triggered by the natural chemokine ligand CCL4, but independently of ERK1/2. Finally, PI-PLC inhibition neither blocks gp120-mediated NF-kB activation nor overall accumulation of CCL2 mRNA, whereas it decreases CCL2 transcript level in the cytoplasm. These results identify nuclear PI-PLC β1 as a new intermediate in the gp120-triggered PC-PLC-driven signal transduction pathway leading to CCL2 secretion in macrophages. The finding that a concerted gp120-mediated signaling involving both PC- and PI-specific PLCs is required for the expression of CCL2 in macrophages suggests that this signal transduction pathway may also be relevant for the modulation of viral replication in these cells. Thus, this study may contribute to identify novel targets for therapeutic intervention in HIV-1 infection.

  13. Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT: analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues

    Directory of Open Access Journals (Sweden)

    Gaudernack Gustav

    2006-08-01

    Full Text Available Abstract Background Human telomerase reverse transcriptase (hTERT is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. Results By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. Conclusion The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

  14. {sup 68}Ga-DOTATOC PET/CT and somatostatin receptor (sst1-sst5) expression in normal human tissue: correlation of sst2 mRNA and SUV{sub max}

    Energy Technology Data Exchange (ETDEWEB)

    Boy, Christian; Poeppel, Thorsten D.; Jentzen, Walter; Brandau, Wolfgang; Bockisch, Andreas [University Hospital Essen, University of Duisburg-Essen, Department of Nuclear Medicine, Essen (Germany); Heusner, Till A.; Antoch, Gerald [University Hospital Essen, University of Duisburg-Essen, Department of Diagnostic and Interventional Radiology and Neuroradiology, Essen (Germany); Redmann-Bischofs, Anja; Unger, Nicole; Mann, Klaus; Petersenn, Stephan [University of Duisburg-Essen, Department of Endocrinology and Division of Laboratory Research, Essen (Germany)

    2011-07-15

    By targeting somatostatin receptors (sst) radiopeptides have been established for both diagnosis and therapy. For physiologically normal human tissues the study provides a normative database of maximum standardized uptake value (SUV{sub max}) and sst mRNA. A total of 120 patients were subjected to diagnostic {sup 68}Ga-DOTATOC positron emission tomography (PET)/CT (age range 19-83 years). SUV{sub max} values were measured in physiologically normal tissues defined by normal morphology, absence of surgical intervention and absence of metastatic spread during clinical follow-up. Expression of sst subtypes (sst1-sst5) was measured independently in pooled adult normal human tissue by real-time reverse transcriptase polymerase chain reaction (RT-PCR). SUV{sub max} revealed a region-specific pattern (e.g., mean {+-} SD, spleen 31.1 {+-} 10.9, kidney 16.9 {+-} 5.3, liver 12.8 {+-} 3.6, stomach 7.0 {+-} 3.1, head of pancreas 6.2 {+-} 2.3, small bowel 4.8 {+-} 1.8, thyroid 4.7 {+-} 2.2, bone 3.9 {+-} 1.3, large bowel 2.9 {+-} 0.8, muscle 2.1 {+-} 0.5, parotid gland 1.9 {+-} 0.6, axillary lymph node 0.8 {+-} 0.3 and lung 0.7 {+-} 0.3). SUV{sub max} was age independent. Gender differences were evident within the thyroid (female/male: 3.7 {+-} 1.6/5.5 {+-} 2.4, p < 0.001; Mann-Whitney U test) and the pancreatic head (5.5 {+-} 1.9/6.9 {+-} 2.2, p < 0.001). The sst mRNA was widely expressed and heterogeneous, showing sst1 to be most abundant. SUV{sub max} values exclusively correlated with sst2 expression (r = 0.846, p < 0.001; Spearman rank correlation analysis), whereas there was no correlation of SUV{sub max} with the expression of the other four subtypes. In normal human tissues {sup 68}Ga-DOTATOC imaging has been related to the expression of sst2 at the level of mRNA. The novel normative database may improve diagnostics, monitoring and therapy of sst-expressing tumours or inflammation on a molecular basis. (orig.)

  15. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure.

    Science.gov (United States)

    Noutsios, Georgios T; Silveyra, Patricia; Bhatti, Faizah; Floros, Joanna

    2013-06-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5' untranslated (5'UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5'UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA.

  16. The human Ago2 MC region does not contain an eIF4E-like mRNA cap binding motif

    Directory of Open Access Journals (Sweden)

    Grishin Nick V

    2009-01-01

    Full Text Available Abstract Background Argonaute (Ago proteins interact with small regulatory RNAs to mediate gene regulatory pathways. A recent report by Kiriakidou et al. 1 describes an MC sequence region identified in Ago2 that displays similarity to the cap-binding motif in translation initiation factor 4E (eIF4E. In a cap-bound eIF4E structure, two important aromatic residues of the motif stack on either side of a 7-methylguanosine 5'-triphosphate (m7Gppp base. The corresponding Ago2 aromatic residues (F450 and F505 were hypothesized to perform the same cap-binding function. However, the detected similarity between the MC sequence and the eIF4E cap-binding motif was questionable. Results A number of sequence-based and structure-based bioinformatics methods reveal the reported similarity between the Ago2 MC sequence region and the eIF4E cap-binding motif to be spurious. Alternatively, the MC sequence region is confidently assigned to the N-terminus of the Ago piwi module, within the mid domain of experimentally determined prokaryotic Ago structures. Confident mapping of the Ago2 MC sequence region to the piwi mid domain results in a homology-based structure model that positions the identified aromatic residues over 20 Å apart, with one of the aromatic side chains (F450 contributing instead to the hydrophobic core of the domain. Conclusion Correct functional prediction based on weak sequence similarity requires substantial evolutionary and structural support. The evolutionary context of the Ago mid domain suggested by multiple sequence alignment is limited to a conserved hydrophobicity profile required for the fold and a motif following the MC region that binds guide RNA. Mapping of the MC sequence to the mid domain structure reveals Ago2 aromatics that are incompatible with eIF4E-like mRNA cap-binding, yet display some limited local structure similarities that cause the chance sequence match to eIF4E. Reviewers This article was reviewed by Arcady Mushegian

  17. Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells.

    OpenAIRE

    1996-01-01

    FK506 (tacrolimus) is an immunosuppressive drug which interrupts Ca2+-calmodulin-calcineurin signaling pathways in T lymphocytes, thereby blocking antigen activation of T cell early activation genes. Regulation of insulin gene expression in the beta cell may also involve Ca2+-signaling pathways and FK506 has been associated with insulin-requiring diabetes mellitus during clinical use. The purpose of this study was to characterize the effects of FK506 on human insulin gene transcription, insul...

  18. Ascorbic Acid, Ultraviolet C Rays, and Glucose but not Hyperthermia Are Elicitors of Human β-Defensin 1 mRNA in Normal Keratinocytes

    Science.gov (United States)

    Cruz Díaz, Luis Antonio; Flores Miramontes, María Guadalupe; Allen, Kirk; Gonzalez Ávila, Marisela; Prado Montes de Oca, Ernesto

    2015-01-01

    Hosts' innate defense systems are upregulated by antimicrobial peptide elicitors (APEs). Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA), and ultraviolet C rays (UVC) as well as glucose and ascorbic acid (AA) on the regulation of human β-defensin 1 (DEFB1), cathelicidin (CAMP), and interferon-γ (IFNG) genes in normal human keratinocytes (NHK). The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C) and hyperthermia (39°C) are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections. PMID:25815330

  19. Ascorbic Acid, Ultraviolet C Rays, and Glucose but not Hyperthermia Are Elicitors of Human β-Defensin 1 mRNA in Normal Keratinocytes

    Directory of Open Access Journals (Sweden)

    Luis Antonio Cruz Díaz

    2015-01-01

    Full Text Available Hosts’ innate defense systems are upregulated by antimicrobial peptide elicitors (APEs. Our aim was to investigate the effects of hyperthermia, ultraviolet A rays (UVA, and ultraviolet C rays (UVC as well as glucose and ascorbic acid (AA on the regulation of human β-defensin 1 (DEFB1, cathelicidin (CAMP, and interferon-γ (IFNG genes in normal human keratinocytes (NHK. The indirect in vitro antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes of these potential APEs was tested. We found that AA is a more potent APE for DEFB1 than glucose in NHK. Glucose but not AA is an APE for CAMP. Mild hypo- (35°C and hyperthermia (39°C are not APEs in NHK. AA-dependent DEFB1 upregulation below 20 mM predicts in vitro antimicrobial activity as well as glucose- and AA-dependent CAMP and IFNG upregulation. UVC upregulates CAMP and DEFB1 genes but UVA only upregulates the DEFB1 gene. UVC is a previously unrecognized APE in human cells. Our results suggest that glucose upregulates CAMP in an IFN-γ-independent manner. AA is an elicitor of innate immunity that will challenge the current concept of late activation of adaptive immunity of this vitamin. These results could be useful in designing new potential drugs and devices to combat skin infections.

  20. Elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo.

    Science.gov (United States)

    Shi, Pu; Aluri, Suhaas; Lin, Yi-An; Shah, Mihir; Edman, Maria; Dhandhukia, Jugal; Cui, Honggang; MacKay, J Andrew

    2013-11-10

    Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug

  1. Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

    Science.gov (United States)

    Karimi, Ashkan; Milewicz, Dianna M

    2016-01-01

    The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta.

  2. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates.

    Science.gov (United States)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj; Lindholm, Marie W; Rosenbohm, Christoph; Aarup, Vibeke; Hansen, Henrik Frydenlund; Ørum, Henrik; Hansen, Jens B Rode; Koch, Troels

    2010-11-01

    The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

  3. Identification of a novel splice variant of human PD-L1 Mrna encoding an isoform-lacking Igv-like domain

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2005-01-01

    Aim: To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). Methods: The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. Results: A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon 2 encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intmcellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. Conclusion: PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

  4. The anti-inflammatory pharmacology of Pycnogenol in humans involves COX-2 and 5-LOX mRNA expression in leukocytes.

    Science.gov (United States)

    Canali, Raffaella; Comitato, Raffaella; Schonlau, Frank; Virgili, Fabio

    2009-09-01

    We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.

  5. Direct measurement of the correlated dynamics of the protein-backbone and proximal waters of hydration in mechanically strained elastin

    CERN Document Server

    Sun, Cheng; Huang, Jiaxin; Boutis, Gregory S

    2011-01-01

    We report on the direct measurement of the correlation times of the protein backbone carbons and proximal waters of hydration in mechanically strained elastin by nuclear magnetic resonance methods. The experimental data indicate a decrease in the correlation times of the carbonyl carbons as the strain on the biopolymer is increased. These observations are in good agreement with short 4ns molecular dynamics simulations of (VPGVG)3, a well studied mimetic peptide of elastin. The experimental results also indicate a reduction in the correlation time of proximal waters of hydration with increasing strain applied to the elastomer. A simple model is suggested that correlates the increase in the motion of proximal waters of hydration to the increase in frequency of libration of the protein backbone that develops with increasing strain. Together, the reduction in the protein entropy accompanied with the increase in entropy of the proximal waters of hydration with increasing strain, support the notion that the source ...

  6. Role of Matrix Metalloproteinases-1 and -2 in Interleukin-13-Suppressed Elastin in Airway Fibroblasts in Asthma.

    Science.gov (United States)

    Ingram, Jennifer L; Slade, David; Church, Tony D; Francisco, Dave; Heck, Karissa; Sigmon, R Wesley; Ghio, Michael; Murillo, Anays; Firszt, Rafael; Lugogo, Njira L; Que, Loretta; Sunday, Mary E; Kraft, Monica

    2016-01-01

    Elastin synthesis and degradation in the airway and lung parenchyma contribute to airway mechanics, including airway patency and elastic recoil. IL-13 mediates many features of asthma pathobiology, including airway remodeling, but the effects of IL-13 on elastin architecture in the airway wall are not known. We hypothesized that IL-13 modulates elastin expression in airway fibroblasts from subjects with allergic asthma. Twenty-five subjects with mild asthma (FEV1, 89 ± 3% predicted) and 30 normal control subjects (FEV1, 102 ± 2% predicted) underwent bronchoscopy with endobronchial biopsy. Elastic fibers were visualized in airway biopsy specimens using Weigert's resorcin-fuchsin elastic stain. Airway fibroblasts were exposed to IL-13; a pan-matrix metalloproteinase (MMP) inhibitor (GM6001); specific inhibitors to MMP-1, -2, -3, and -8; and combinations of IL-13 with MMP inhibitors in separate conditions in serum-free media for 48 hours. Elastin (ELN) expression as well as MMP secretion and activity were quantified. Results of this study show that elastic fiber staining of airway biopsy tissue was significantly associated with methacholine PC20 (i.e., the provocative concentration of methacholine resulting in a 20% fall in FEV1 levels) in patients with asthma. IL-13 significantly suppressed ELN expression in asthmatic airway fibroblasts as compared with normal control fibroblasts. The effect of IL-13 on ELN expression was significantly correlated with postbronchodilator FEV1/FVC in patients with asthma. MMP inhibition significantly stimulated ELN expression in patients with asthma as compared with normal control subjects. Specific inhibition of MMP-1 and MMP-2, but not MMP-3 or MMP-8, reversed the IL-13-induced suppression of ELN expression. In asthma, MMP-1 and MMP-2 mediate IL-13-induced suppression of ELN expression in airway fibroblasts.

  7. Changes of collagen, elastin, and tryptophan contents in laser welded porcine aorta tissues studied using fluorescence spectroscopy

    Science.gov (United States)

    Liu, C.-H.; Wang, W. B.; Kartazaev, V.; Savage, H.; Alfano, R. R.

    2010-02-01

    The emission spectra from welded and un-welded (normal) porcine aorta tissues were measured on both sides of intima and adventitia layers. A tunable Forsterite laser and a Cr4+: YAG laser with wavelengths of 1250nm, 1455nm and 1460nm were used to weld porcine aorta tissues. Three emission bands emitted from three key fluorophores were studied under different welding and excitation conditions. With excitation wavelength of 340nm, the 395nm band is associated with the emission from the structural proteins of collagen type III and type I. The 445nm band obtained is associated with the emission of the structural protein of elastin. The 350nm band recorded with excitation wavelength of 300nm is associated with the amino acid of tryptophan. The relative emission intensities of collagen, elastin and tryptophan at their fluorescence peaks changes with laser tissue welding wavelengths indicate the change of contents of those tissue molecules. The ratio of emission peak intensities of collagen to elastin with welding laser wavelength of 1250nm increases by 0.13 as compared to the normal aorta tissue at the intimal side. For the adventitial side of aorta tissue, this ratio decreases by 0.38 in comparison with the normal tissue. These results indicate that content of collagen changes relative to elastin due to laser tissue welding. The peak fluorescence intensity of tryptophan for both sides of welded tunica intima and adventitia increases significantly in comparison with the normal tissue when the optimum laser welding wavelength of 1455 nm was used.

  8. IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM.

    Science.gov (United States)

    Henness, Sheridan; van Thoor, Eveline; Ge, Qi; Armour, Carol L; Hughes, J Margaret; Ammit, Alaina J

    2006-06-01

    Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells.

  9. One-stage reconstruction of soft tissue defects with the sandwich technique: Collagen-elastin dermal template and skin grafts

    Directory of Open Access Journals (Sweden)

    Uwe Wollina

    2011-01-01

    Full Text Available Background : A full-thickness soft tissue defect closure often needs complex procedures. The use of dermal templates can be helpful in improving the outcome. Objective : The objective was to evaluate a sandwich technique combining the dermal collagen-elastin matrix with skin grafts in a one-stage procedure. Materials and Methods : Twenty-three patients with 27 wounds were enrolled in this prospective single-centre observational study. The mean age was 74.8 ± 17.2 years. Included were full-thickness defects with exposed bone, cartilage and/ or tendons. The dermal collagen-elastin matrix was applied onto the wound bed accomplished by skin transplants, i.e. ′sandwich′ transplantation. In six wounds, the transplants were treated with intermittent negative pressure therapy. Results : The size of defects was ≤875 cm 2 . The use of the dermal template resulted in a complete and stable granulation in 100% of wounds. Seventeen defects showed a complete closure and 19 achieved a complete granulation with an incomplete closure. There was a marked pain relief. No adverse events were noted due to the dermal template usage. Conclusions : Sandwich transplantation with the collagen-elastin matrix is a useful tool when dealing with full-thickness soft tissue defects with exposed bone, cartilage or tendons.

  10. Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7.

    Science.gov (United States)

    Ravon, Morgane; Berrera, Marco; Ebeling, Martin; Certa, Ulrich

    2012-01-01

    We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3'-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3'-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts.

  11. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    Science.gov (United States)

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-07-14

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

  12. Microarray study reveals that HIV-1 induces rapid type-I interferon-dependent p53 mRNA up-regulation in human primary CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Tremblay Michel J

    2009-01-01

    Full Text Available Abstract Background Infection with HIV-1 has been shown to alter expression of a large array of host cell genes. However, previous studies aimed at investigating the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines. To better approximate natural conditions, we monitored gene expression changes in a cell population highly enriched in human primary CD4+ T lymphocytes exposed to HIV-1 using commercial oligonucleotide microarrays from Affymetrix. Results We report here that HIV-1 influences expression of genes related to many important biological processes such as DNA repair, cellular cycle, RNA metabolism and apoptosis. Notably, expression of the p53 tumor suppressor and genes involved in p53 homeostasis such as GADD34 were up-regulated by HIV-1 at the mRNA level. This observation is distinct from the previously reported p53 phosphorylation and stabilization at the protein level, which precedes HIV-1-induced apoptosis. We present evidence that the HIV-1-mediated increase in p53 gene expression is associated with virus-mediated induction of type-I interferon (i.e. IFN-α and IFN-β. Conclusion These observations have important implications for our understanding of HIV-1 pathogenesis, particularly in respect to the virus-induced depletion of CD4+ T cells.

  13. Polypentapeptide of elastin: temperature dependence of ellipticity and correlation with elastomeric force.

    Science.gov (United States)

    Urry, D W; Shaw, R G; Prasad, K U

    1985-07-16

    Circular dichroism (CD) is used to follow the conformational changes that attend temperature dependent aggregation leading to the viscoelastic (coacervate) state of the polypentapeptide of elastin in water. Two concentrations are used, 2.3 mg/ml and 0.023 mg/ml. The former results in aggregates of a size that exhibit much particulate distortion of the CD spectra whereas the latter results in spectra that are relatively free of distortions. Given the CD spectra of the temperature dependent aggregation of the lower concentration, it is possible to show that the same conformational change is occurring at high concentration. The structure of the polypentapeptide is one of limited order below 20 degrees C which undergoes an inverse temperature transition to a conformation characterized by a regularly recurring beta-turn at 40 degrees C. The temperature profile for the conformational change is compared to the temperature dependence of elastomeric force of gamma-irradiation cross-linked polypentapeptide coacervate. The curves virtually superimpose. When there is little order, there is little elastomeric force and elastomeric force develops to a near maximal value as the repeating Type II beta-turn conformation develops. Not only is the elastomeric state non-random, the more nearly random state has very little elasticity. These results are the inverse of expectations based on the classical theory of rubber elasticity.

  14. Preparation and characterization of novel elastin-like polypeptide-collagen composites.

    Science.gov (United States)

    Amruthwar, Shruti S; Puckett, Aaron D; Janorkar, Amol V

    2013-08-01

    Collagen-based biomaterials suffer from poor mechanical properties and rapid degradation. Elastin-like polypeptides (ELPs) possess good biocompatibility and have unique solution properties that allow them to coacervate above a critical temperature. The objective of this research was to prepare a series of freeze dried ELP-collagen composite scaffolds as a proof of concept. Combination of ELP and collagen has the potential to produce composite structures with varying strengths. Four different composite structures were prepared by varying the ratio of ELP to collagen. Increased ELP content in the scaffolds appears to have reduced the residual water content based on Fourier transformed infrared spectroscopy and differential scanning calorimetry. Scanning electron microscopy images of ELP-collagen composites showed a three-dimensional, open porous structure with the formation of characteristic aggregates of ELP. The mechanical testing experiments showed that the elastic modulus, tensile strength, and toughness of ELP-collagen scaffolds were significantly greater than neat collagen scaffolds. The improved mechanical properties were attributed to a homogeneous network structure with additional reinforcement coming from the ELP aggregates. Our study confirms that ELP-collagen composites with superior physical and mechanical properties compared to collagen scaffolds can be produced. Further optimization of design parameters will allow producing ELP-collagen composites for specific biomedical applications.

  15. Subarachnoid hemorrhage: tests of association with apolipoprotein E and elastin genes

    Directory of Open Access Journals (Sweden)

    Sauerbeck Laura

    2007-07-01

    Full Text Available Abstract Background Apolipoprotein E (APOE and elastin (ELN are plausible candidate genes involved in the pathogenesis of stroke. We tested for association of variants in APOE and ELN with subarachnoid hemorrhage (SAH in a population-based study. We genotyped 12 single nucleotide polymorphisms (SNPs on APOE and 10 SNPs on ELN in a sample of 309 Caucasian individuals, of whom 107 are SAH cases and 202 are age-, race-, and gender-matched controls from the Greater Cincinnati/Northern Kentucky region. Associations were tested at genotype, allele, and haplotype levels. A genomic control analysis was performed to check for spurious associations resulting from population substructure. Results At the APOE locus, no individual SNP was associated with SAH after correction for multiple comparisons. Haplotype analysis revealed significant association of the major haplotype (Hap1 in APOE with SAH (p = 0.001. The association stemmed from both the 5' promoter and the 3' region of the APOE gene. APOE ε2 and ε 4 were not significantly associated with SAH. No association was observed for ELN at genotype, allele, or haplotype level and our study failed to confirm previous reports of ELN association with aneurysmal SAH. Conclusion This study suggests a role of the APOE gene in the etiology of aneurysmal SAH.

  16. Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides

    Directory of Open Access Journals (Sweden)

    Koushik Ghosh

    2014-02-01

    Full Text Available We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca2+ or Cu2+, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon the temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.

  17. Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

    Science.gov (United States)

    Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

    2017-09-01

    Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent Kd of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

  18. Synthesis and characterization of an elastin-mimetic amphiphilic block copolymer protein

    Science.gov (United States)

    Lee, Terrence Anita-Talley

    2000-10-01

    The overall goal in material science is to be able to control the molecular architecture of a material and thus its end properties. There is no method that offers greater control than the biological synthesis of proteins. From the DNA sequence to the final synthesized protein, the entire process is finitely controlled. This present work describes methods developed and used to synthesize protein polymers by manipulating this process. From the initial DNA sequence chosen, the end properties that the protein polymer will have are dictated. An amphiphilic diblock copolymer was designed based on environmentally responsive elastin-mimetic peptide sequences [(Val/Ile)-Pro-Gly-Xaa-Gly] (Xaa = Ala or Glu for the hydrophilic block, Val or Phe for the hydrophobic block) and synthesized using a genetic engineering approach. Differential scanning calorimetry measurements in aqueous solution revealed that reversible hydrophobic folding and assembly of the copolymer occurs above the inverse temperature transition, Tt, of the hydrophobic block. This process results in the formation of 50 nm protein-based micellar aggregates, which were characterized by electron microscopy and temperature-dependent dynamic light scattering techniques. The distribution of micellar aggregates can be altered reproducibly through variation of environmental conditions including pH and temperature. The uniform and defined macromolecular architecture of this protein copolymer permits greater control over the physical properties of the micelles, which therefore may facilitate applications in controlled release of small molecules.

  19. Endothelial progenitor cell transplantation ameliorates elastin breakdown in a Kawasaki disease mouse model

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhi; DU Zhong-dong; LIU Jun-feng; LU Dun-xiang; LI Li; GUAN Yun-qian; WAN Sui-gui

    2012-01-01

    Background Coronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of endothelial progenitor cells (EPCs).The aim of the present study was to evaluate the therapeutic effect of EPCs transplantation in KD model.Methods Lactobacillus casei cell wall extract (LCWE)-induced KD model in C57BL/6 mice was established.The model mice were injected intravenously with bone marrow-derived in vitro expanded EPCs.Histological evaluation,number of circulating EPCs and the function of bone marrow EPCs were examined at day 56.Results Inflammation was found around the coronary artery of the model mice after 14 days,Elastin breakdown was observed after 56 days.CM-Dil labeled EPCs incorporated into vessel repairing foci was found.At day 56,the number of peripheral EPCs in the KD model group was lower than in EPCs transplanted and control group.The functional index of bone marrow EPCs from the KD model group decreased in proliferation,adhesion and migration.Increased number of circulating EPCs and improved function were observed on the EPCs transplanted group compared with model group.Conclusion Exogenously administered EPCs,which represent a novel strategy could prevent the dysfunction of EPCs,accelerate the repair of coronary artery endothelium lesion and decrease the occurrence of aneurysm.

  20. Effects of short elastin-like peptides on filamentous particles and their transition behavior.

    Science.gov (United States)

    Hathorne, Adam P; Bermudez, Harry

    2013-07-01

    While elastin-like polypeptides and peptides (ELPs) have been used for various stimulus-responsive applications, details of their switching remain unclear. We therefore constructed a novel series of filamentous phage particles displaying a high surface density of short ELPs. The surface display of ELPs did not disrupt either particle shape or dimensions, and the resulting ELP-phage particles were colloidally stable over several weeks. However, in spite of a saturating surface density, macroscopic aggregation of ELP-phages cannot be triggered in water. To investigate the underlying mechanisms we examined conformational changes in the secondary structure of the phage proteins by circular dichroism and tryptophan fluorescence, which indicate partial protein unfolding in ELP-phage particles. To gain further insight into the ELP itself, analogous "free" ELP peptides were synthesized and characterized. Circular dichroism of these peptides shows the onset of β-type conformations with increasing temperature, consistent with the accepted view of the microscopic transition that underlies the inverse phase behavior of ELPs. Increased guest residue hydrophobicity was found to depress the microscopic transition temperature of the peptides, also consistent with a previously proposed intramolecular hydrogen-bonding mechanism. Importantly, our results indicate that although the nanoscale presentation state can suppress macroscopic aggregation of ELPs, microscopic transitions of the ELP can still occur. Given the growing use of ELPs within supra-molecular scaffolds, such effects are important design considerations for future applications.

  1. Blood flow interplays with elastin: collagen and MMP: TIMP ratios to maintain healthy vascular structure and function

    Directory of Open Access Journals (Sweden)

    Poulami Basu

    2010-03-01

    Full Text Available Poulami Basu, Utpal Sen, Neetu Tyagi, Suresh C TyagiDepartment of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky, USAAbstract: Differential vascular remodeling is one of the major mechanisms of heterogeneity in atherosclerosis. The structural and functional heterogeneity between arteries and veins determines the degree of vascular remodeling. Matrix metalloproteases (MMPs and their tissue inhibitors (TIMPs play key roles in vascular structural and functional remodeling. We hypothesized that the level of blood flow in different arteries and veins caused structural and functional heterogeneity that ultimately determined potential vascular remodeling. To test this hypothesis, in vivo blood flow and blood pressure in the aorta, carotid, femoral artery, and femoral vein was measured in male Sprague-Dawley rats (weight 380–400 gm. Arterial and venous pressures were measured by PE-50 catheter cannulation. Blood flow was measured by a transonic ultrasound system. The aortic arch, femoral and carotid arteries, and abdominal vena cava were isolated to determine the expression of MMP-2, -9, -12, and -13 and TIMP-1, -3, and -4 by Western blot and in gelatin gel zymography. Masson trichrome and van Gieson stains were used to stain the histologic tissue sections. The results revealed that blood flow was higher in the aorta and carotid artery than the femoral artery and vein. MMP-9 and MMP-13 were higher in the carotid artery in comparison with the other blood vessels, while TIMP-3 showed higher expression in the aorta than the arteries. Further, the MMP-9 activity was significantly higher in the carotid artery than in the aorta and femoral artery. There was a higher degree of basement membrane collagen in the femoral artery and therefore a low elastin: collagen ratio, while in the carotid artery a higher level of elastin and, therefore, a high elastin: collagen ratio was found. The results suggested that medial

  2. Correlation between high risk type human papillomavirus E6/E7 mRNA and cervical cancer%高危型HPV E6/E7 mRNA与宫颈癌相关性分析

    Institute of Scientific and Technical Information of China (English)

    王小红; 钱艺美; 缪铃; 乐瑶; 杜娟

    2016-01-01

    Objective To investigate the correlation between the positive rate of high risk human papillomavirus (HPV) mRNA E6/E7 and cervical cancer,and provide evidence for the prevention and treatment of cervical cancer.Methods A total of 100 cervical cancer cases and 100 healthy controls were selected in our hospital from January 2015 to December 2015.The fluorescence quantitative PCR and pathological examination on HPV E6/E7 mRNA were carried out.The correlation between HPV E6/E7 mRNA and cervical squamous epithelial lesions were analyzed.Results In case group,the positive rate ofHPV E6/E7 mRNA was 76.0% (76/100).In control group,the positive rate was 13.0% (13/100).The positive rate in case group was significantly higher than that in control group,and the difference was statistically significant (x2=24.522,P<0.001).The positive predictive value and negative predictive value of the two groups were compared,and the difference was not significant (P>0.05).The positive rate of HPV E6/E7 mRNA was significantly higher than high-grade squamous intraepithelial lesion (SIL) rate (26.1%),low-grade SIL rate (17.6%) and atypical squamous cell hyperplasia rate (6.7%),the difference was statistically significant (x2=7.615,P=0.001;x2=9.114,P=0.001;x2=18.241,P<0.001).Conclusions The detection rate ofHPV E6/E7 mRNA in cervical cancer patients was high.And with the increased severity of cervical squamous epithelial lesions,the positive rate of HPV E6/E7 mRNA increased.%目的 探讨高危型HPV E6/E7 mRNA检出率与宫颈癌的相关性,为临床防治宫颈癌提供依据.方法 选择2015年收治的100例宫颈癌患者为A组,同期100例健康体检者为B组,采用荧光定量PCR检测入组患者高危型HPV E6/E7 mRNA和病理学检查,比较两组患者HPV E6/E7感染率和荧光定量PCR检查效率,分析HPV E6/E7感染与宫颈鳞状上皮病变的相关性.结果 A组阳性76例,阳性率为76.0%;B组阳性13例,阳性率为13.0%;A组阳性率高于B

  3. Effects of intracellular zinc depletion on the abundance of VDAC mRNA in cultured human spermatozoa%Zn缺乏对人精子VDAC mRNA的影响

    Institute of Scientific and Technical Information of China (English)

    赵丹; 吴炜; 章轶; 王增军; 王心如

    2012-01-01

    目的:探讨Zn离子缺乏对成熟精子活动能力和电压依赖性离子通道(voltage-dependent anion channel,VDAC)的mRNA丰度的影响.方法:从3个正常生育者捐献所得的精液经非连续Percoll梯度离心获得精子样本,并将其分3组连续培养24h:对照组;加入Zn离子螯合剂的TPEN组(2 μmol/L);同时加入Zn离子螯合剂N,N,N',N'-tetrakis (2-pyridylmethy1)Ethylenediamine (TPEN)和Zn离子(5μmol/L)组.行CASA和荧光定量PCR(qPCR)分别分析不同时间的精子活动能力和24h后各VDAC亚型mRNA丰度.结果:CASA显示:培养3h,TPEN组与对照组比较,精子活力明显下降(P<0.05).qPCR结果显示:培养24h后,TPEN组VDAC3 mRNA丰度明显低于对照组(P<0.05).而同时加入TPEN和Zn离子组与对照组比较,差异无统计学意义.结论:Zn离子缺乏使VDAC3 mRNA丰度降低,可能影响到精子的正常功能.%Objective:To investigate whether intracellular zinc depletion can actually change spermatozoa motiJity and the abundance of voltage-dependent anion channel (VDAC) mRNA in cultured human spermatozoa. Methods; Spermatozoa were separated from ejaculates of three donors using a discontinuous Percoll gradient centrifugation. Each spermatozoa specimen were divided into 3 groups,one was exposed to a cell membrane-permeable zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl) Ethylenediamine (TPEN) (2 μmol/L) for 24 hours,one was to TPEN plus zinc sulfate (5 μmol/L) for 24 hours,and one was as control. CASA and quantitative real-time PCR (qPCR) were performed to detect sperm motility characteristics,mRNA abundance and difference of the three VDAC subtypes between three different treatment groups respectively. Results;The results of CASA demonstrated that exposure of spermatozoa to TPEN for three hours significantly decreased sperm motility compared with the control group (P = 0.049). qPCR demonstrated that VDAC3 mRNA level of TPEN group were significantly lower than that of control group,while no

  4. Hybrid Elastin-like Polypeptide–Polyethylene Glycol (ELP-PEG) Hydrogels with Improved Transparency and Independent Control of Matrix Mechanics and Cell Ligand Density

    Science.gov (United States)

    2015-01-01

    Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP’s lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell–matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics. PMID:25111283

  5. Paradoxical expression of IL-28B mRNA in peripheral blood in human T-cell leukemia virus Type-1 mono-infection and co-infection with hepatitis C Virus

    Directory of Open Access Journals (Sweden)

    Kamihira Shimeru

    2012-02-01

    Full Text Available Abstract Background Human T-cell leukemia virus type-1 (HTLV-1 carriers co-infected with and hepatitis C virus (HCV have been known to be at higher risk of their related diseases than mono-infected individuals. The recent studies clarified that IL-28B polymorphism rs8099917 is associated with not only the HCV therapeutic response by IFN, but also innate immunity and antiviral activity. The aim of our research was to clarify study whether IL-28B gene polymorphism (rs8099917 is associated with HTLV-1/HCV co-infection. Results The genotyping and viral-serological analysis for 340 individuals showed that IL-28B genotype distribution of rs8099917 SNP did not differ significantly by respective viral infection status. However, the IL-28B mRNA expression level was 3.8 fold higher in HTLV-1 mono-infection than HTLV-1/HCV co-infection. The high expression level was associated with TT (OR, 6.25, whiles the low expression was associated with co-infection of the two viruses (OR, 9.5. However, there was no association between down-regulation and ATL development (OR, 0.8. Conclusion HTLV-1 mono-infection up-regulates the expression of IL-28B transcripts in genotype-dependent manner, whiles HTLV-1/HCV co-infection down-regulates regardless of ATL development.

  6. Intraoperative use of enriched collagen and elastin matrices with freshly isolated adipose-derived stem/stromal cells: a potential clinical approach for soft tissue reconstruction.

    Science.gov (United States)

    Alharbi, Ziyad; Almakadi, Sultan; Opländer, Christian; Vogt, Michael; Rennekampff, Hans-Oliver; Pallua, Norbert

    2014-02-20

    Adipose tissue contains a large number of multipotent cells, which are essential for stem cell-based therapies. The combination of this therapy with suitable commercial clinically used matrices, such as collagen and elastin matrices (i.e. dermal matrices), is a promising approach for soft tissue reconstruction. We previously demonstrated that the liposuction method affects the adherence behaviour of freshly isolated adipose-derived stem/stromal cells (ASCs) on collagen and elastin matrices. However, it remains unclear whether freshly isolated and uncultured ASCs could be directly transferred to matrices during a single transplantation operation without additional cell culture steps. After each fat harvesting procedure, ASCs were isolated and directly seeded onto collagen and elastin matrices. Different time intervals (i.e. 1, 3 and 24 h) were investigated to determine the time interval needed for cellular attachment to the collagen and elastin matrices. Resazurin-based vitality assays were performed after seeding the cells onto the collagen and elastin matrices. In addition, the adhesion and migration of ASCs on the collagen and elastin matrices were visualised using histology and two-photon microscopy. A time-dependent increase in the number of viable ASCs attached to the collagen and elastin matrices was observed. This finding was supported by mitochondrial activity and histology results. Importantly, the ASCs attached and adhered to the collagen and elastin matrices after only 1 h of ex vivo enrichment. This finding was also supported by two-photon microscopy, which revealed the presence and attachment of viable cells on the upper layer of the construct. Freshly isolated uncultured ASCs can be safely seeded onto collagen and elastin matrices for ex vivo cellular enrichment of these constructs after liposuction. Although we observed a significant number of seeded cells on the matrices after a 3-h enrichment time, we also observed an adequate number of isolated

  7. Mechanistic Insight into the Elastin Degradation Process by the Metalloprotease Myroilysin from the Deep-Sea Bacterium Myroides profundi D25

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2015-03-01

    Full Text Available Elastases have been widely studied because of their important uses as medicine and meat tenderizers. However, there are relatively few studies on marine elastases. Myroilysin, secreted by Myroides profundi D25 from deep-sea sediment, is a novel elastase. In this study, we examined the elastin degradation mechanism of myroilysin. When mixed with insoluble bovine elastin, myroilysin bound hydrophobically, suggesting that this elastase may interact with the hydrophobic domains of elastin. Consistent with this, analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaves peptide bonds with hydrophobic residues at the P1 and/or P1′ positions. Scanning electron microscopy (SEM of cross-linked recombinant tropoelastin degraded by myroilysin showed preferential damages of spherules over cross-links, as expected for a hydrophobic preference. The degradation process of myroilysin on bovine elastin fibres was followed by light microscopy and SEM, revealing that degradation begins with the formation of crevices and cavities at the fibre surface, with these openings increasing in number and size until the fibre breaks into small pieces, which are subsequently fragmented. Our results are helpful for developing biotechnological applications for myroilysin.

  8. Investigation on Elastin Derived Peptides in Dermatology%弹性蛋白肽在皮肤科应用研究的进展

    Institute of Scientific and Technical Information of China (English)

    田珊; 陈周

    2013-01-01

    Elastin-derived peptides (EDPs) are the degradation products of elastin.EDPs could induce biological activities on numerous cell types through their cell surface receptor named elastin-binding protein (EBP),such as release of proteolytic enzymes in stromal and cancer cells,contribution to tumor progression and involved in skin wound repair and regeneration.Here,we reviewed the origin and the receptor of EDPs and its roles in dermatology.%弹性蛋白肽(elastin-derived peptide,EDP)是弹性蛋白的降解产物,它通过作用于细胞表面的受体——弹性蛋白结合蛋白(elastin-binding protein,EBP)而发挥多种生物学功能,如促进正常细胞和肿瘤细胞蛋白酶的释放、增强肿瘤细胞的侵袭性和促进皮肤损伤的修复等.本文综述了弹性蛋白肽的来源、分类、受体及其在皮肤科领域的研究进展.

  9. Lung collagen and elastin after ozone exposure in vitamin B-6-deficient rats

    Energy Technology Data Exchange (ETDEWEB)

    Myers, B.A.; Dubick, M.A.; Gerriets, J.E.; Reiser, K.M.; Last, J.A.; Rucker, R.B.

    1986-01-01

    The effects of vitamin B-6 deficiency and ozone exposure on selected features of connective tissue metabolism in lung were investigated in groups of weanling male rats fed one of three diets: B-6-supplemented, fed ad lib; B-6-deficient, fed ad lib; or B-6-supplemented, restricted to the food intake of deficient rats for 5 weeks. Also, perinatal rat pups were studied that were nursed from dams fed one of the 3 diets from parturition to day 15 of lactation. During the final week of each experiment, half of the rats in each of the groups were exposed to 0.64 ppm of ozone (23.5 h per day). The collagen and elastin content, collagen synthesis rate, total protein synthesis rate, and lysyloxidase activity of lungs were measured. Perinatal pups rendered vitamin B-6-deficient were particularly sensitive to ozone exposure (65% died as compared to fewer than 5% of the ad lib or food-restricted controls). When L-proline incorporation into collagen and total protein was investigated using lung minces, food restriction and B-6-deficiency resulted in about one-half the incorporation normally observed. Total lung lysyl oxidase activity was also decreased in B-6-deficient and food-restricted rats compared to B-6-supplemented rats fed ad lib. Exposure to ozone resulted in increased lysyl oxidase activity and collagen synthesis in lungs from B-6-supplemented rats, but such responses were not observed in B-6-deficient or food-restricted (FR) rats exposed to ozone.

  10. Regulation of Vif mRNA splicing by human immunodeficiency virus type 1 requires 5' splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G.

    Science.gov (United States)

    Mandal, Dibyakanti; Exline, Colin M; Feng, Zehua; Stoltzfus, C Martin

    2009-06-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of

  11. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  12. Optical detection of structural changes in human carotid atherosclerotic plaque

    Science.gov (United States)

    Korol, R. M.; Canham, P. B.; Finlay, H. M.; Hammond, R. R.; Quantz, M.; Ferguson, G. G.; Liu, L. Y.; Lucas, A. R.

    2005-08-01

    Background: Arterial bifurcations are commonly the sites of developing atherosclerotic plaque that lead to arterial occlusions and plaque rupture (myocardial infarctions and strokes). Laser induced fluorescence (LIF) spectroscopy provides an effective nondestructive method supplying spectral information on extracellular matrix (ECM) protein composition, specifically collagen and elastin. Purpose: To investigate regional differences in the ECM proteins -- collagen I, III and elastin in unstable plaque by analyzing data from laser-induced fluorescence spectroscopy of human carotid endarterectomy specimens. Methods: Gels of ECM protein extracts (elastin, collagen types I & III) were measured as reference spectra and internal thoracic artery segments (extra tissue from bypass surgery) were used as tissue controls. Arterial segments and the endarterectomy specimens (n=21) were cut into 5mm cross-sectional rings. Ten fluorescence spectra per sampling area were then recorded at 5 sites per ring with argon laser excitation (357nm) with a penetration depth of 200 μm. Spectra were normalized to maximum intensity and analyzed using multiple regression analysis. Tissue rings were fixed in formalin (within 3 hours of surgery), sectioned and stained with H&E or Movat's Pentachrome for histological analysis. Spectroscopy data were correlated with immunohistology (staining for elastin, collagen types I, III and IV). Results: Quantitative fluorescence for the thoracic arteries revealed a dominant elastin component on the luminal side -- confirmed with immunohistology and known artery structure. Carotid endarterectomy specimens by comparison had a significant decrease in elastin signature and increased collagen type I and III. Arterial spectra were markedly different between the thoracic and carotid specimens. There was also a significant elevation (pcollagen type I distal to the bifurcation compared to proximal tissue in the carotid specimens. Conclusion: Fluorescence spectroscopy

  13. Flow calorimetry of the sorption of butanols to elastin preparations and comparison with surface areas determined by krypton-85 adsorption.

    Science.gov (United States)

    Robert, L; Robert, B; Houtman, J P; Stack, M V

    1971-12-28

    1. The apparent surface areas of elastin samples as well as of several other fibrous protein preparations (collagen, keratin, polymeric stroma of aorta) were determined using two different approaches: (a) the Brunauer-Emmett-Teller method with 85Kr and (b) microflow calorimetry with n- and tert.-butanol as adsorbents in a heptane stream. 2. Areas of heat signals obtained by flow calorimetry for the adsorption and desorption of n- and tert.-butanol were substantially equivalent; desorption was more protracted than adsorption, the difference between the speed of desorption and adsorption increased with decreasing chain length of the alcohols (methanol, ethanol, n-propanol and n-butanol). 3. An inverse linear relationship was found between the energy change recorded during the adsorption process and the chain length of the alcohols (methanol, ethanol, n-propanol, n-butanol). 4. Heats of adsorption of tert.-butanol were systematically found to be significantly lower than those of n-butanol with all the protein samples investigated. 5. The apparent surface areas of the protein samples determined with tert.-butanol were on the average of the same order or only slightly higher than those obtained with 85Kr. Results obtained with n-butanol were significantly higher. The difference between surface areas obtained with n- and tert.-butanol depended on the nature of the protein sample, on its method of preparation and to some extent on the residual humidity of the sample. 6. The results could be explained on the basis of the hydrophobic theory of elastin structure (see ref. 4) and confirmed our former conclusions (see ref. 3) concerning the significantly higher surface areas of elastin samples purified by different procedures as compared to collagen or to keratin. They also confirmed the accessibility of the surface of elastic fibers to the molecular probes used in the polymeric stroma of aorta.

  14. Elastin-like Polypeptide Enriched Surfaces for Cardiovascular Applications through the use of Bioactive Fluorinated Surface Modifiers

    Science.gov (United States)

    Blit, Patrick Herve

    Currently used small diameter synthetic vascular grafts are prone to high rates of failure related to thrombosis and neointimal hyperplasia. Biomimetic materials, based on the extracellular matrix (ECM) composition of native tissues, represent an attractive solution to address these complications. The inherent low thrombogenicity and cell signalling properties of elastin-like polypeptides (ELPs) make them a suitable option for these applications. In this thesis, ELP surface modification has been achieved through the use of elastin cross-linking peptide bioactive fluorinated surface modifiers (ECP-BFSMs). The synthesis of these low molecular weight fluorinated additives was described and their subsequent blending with a base polycarbonate-urethane (PCNU) was shown to successfully enrich the surface to allow for ELP surface cross-linking. The kinetic surface migration of fluorescent ECP-BFSMs was studied over a 2 week casting period by two-photon confocal microscopy. Contact angle and x-ray photoelectron spectroscopy (XPS) confirmed the surface localization of the ECP-BFSMs. Changes in contact angle and XPS spectrums following ELP surface cross-linking confirmed the success of the surface modification approach. The novel ELP surface modified materials were demonstrated to inhibit fibrinogen surface adsorption and platelet adhesion under physiological flow conditions and inhibit bulk platelet activation following blood-material contact. Moreover, these ELP modified surfaces were shown to promote increased endothelial and smooth muscle cell adhesion, spreading and retention over a 7 day culture period relative to their non-ELP analogs. Endothelial and smooth muscle cells seeded on the elastin-like materials were shown to express endothelial nitric oxide synthase (eNOS) and smooth muscle myosin heavy chain (SM-MHC) cell specific phenotypic markers, respectively. Furthermore, competitive inhibition experiments revealed that initial smooth muscle cell adhesion to ELP

  15. Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability

    Directory of Open Access Journals (Sweden)

    Gorospe Myriam

    2005-05-01

    Full Text Available Abstract Background Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Results In order to investigate the relative contribution of gene transcription and changes in mRNA stability regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell and nuclear run-on (newly transcribed RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. Conclusion We propose that regulation of mRNA stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

  16. Elastin exhibits a distinctive temporal and spatial pattern of distribution in the developing chick limb in association with the establishment of the cartilaginous skeleton.

    Science.gov (United States)

    Hurle, J M; Corson, G; Daniels, K; Reiter, R S; Sakai, L Y; Solursh, M

    1994-09-01

    In this work we have analyzed the presence of elastic components in the extracellular matrices of the developing chick leg bud. The distributions of elastin and fibrillin were studied immunohistochemically in whole-mount preparations using confocal laser microscopy. The association of these constituents of the elastic matrix with other components of the extracellular matrix was also studied, using several additional antibodies. Our results reveal the transient presence of an elastin-rich scaffold of extracellular matrix fibrillar material in association with the establishment of the cartilaginous skeleton of the leg bud. The scaffold consisted of elastin-positive fibers extending from the ectodermal surface of the limb to the central cartilage-forming regions and between adjacent cartilages. Fibrillin immunolabeling was negative in this fibrillar scaffold while other components of the extracellular matrix including: tenascin, laminin and collagens type I, type III and type VI; appeared codistributed with elastin in some regions of the scaffold. Progressive changes in the spatial pattern of distribution of the elastin-positive scaffold were detected in explant cultures in which one expects a modification in the mechanical stresses of the tissues related to growth. A scaffold of elastin comparable to that found in vivo was also observed in high-density micromass cultures of isolated limb mesodermal cells. In this case the elastic fibers are observed filling the spaces located between the cartilaginous nodules. The fibers become reoriented and attach to the ectodermal basal surface when an ectodermal fragment is located at the top of the growing micromass. Our results suggest that the formation of the cartilaginous skeleton of the limb involves the segregation of the undifferentiated limb mesenchyme into chondrogenic and elastogenic cell lineages. Further, a role for the elastic fiber scaffold in coordinating the size and the spatial location of the cartilaginous

  17. Is cardiovascular disease in patients with diabetes associated with serum levels of MMP-2, LOX, and the elastin degradation products ELM and ELM-2?

    DEFF Research Database (Denmark)

    Rørdam Preil, Simone; Faarvang Thorsen, Anne-Sofie; Christiansen, Anne Lindegaard

    2017-01-01

    BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered mRNA-expression i......BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered m...

  18. Multi-species sequence comparison reveals dynamic evolution of the elastin gene that has involved purifying selection and lineage-specific insertions/deletions

    Directory of Open Access Journals (Sweden)

    Green Eric D

    2004-05-01

    Full Text Available Abstract Background The elastin gene (ELN is implicated as a factor in both supravalvular aortic stenosis (SVAS and Williams Beuren Syndrome (WBS, two diseases involving pronounced complications in mental or physical development. Although the complete spectrum of functional roles of the processed gene product remains to be established, these roles are inferred to be analogous in human and mouse. This view is supported by genomic sequence comparison, in which there are no large-scale differences in the ~1.8 Mb sequence block encompassing the common region deleted in WBS, with the exception of an overall reversed physical orientation between human and mouse. Results Conserved synteny around ELN does not translate to a high level of conservation in the gene itself. In fact, ELN orthologs in mammals show more sequence divergence than expected for a gene with a critical role in development. The pattern of divergence is non-conventional due to an unusually high ratio of gaps to substitutions. Specifically, multi-sequence alignments of eight mammalian sequences reveal numerous non-aligning regions caused by species-specific insertions and deletions, in spite of the fact that the vast majority of aligning sites appear to be conserved and undergoing purifying selection. Conclusions The pattern of lineage-specific, in-frame insertions/deletions in the coding exons of ELN orthologous genes is unusual and has led to unique features of the gene in each lineage. These differences may indicate that the gene has a slightly different functional mechanism in mammalian lineages, or that the corresponding regions are functionally inert. Identified regions that undergo purifying selection reflect a functional importance associated with evolutionary pressure to retain those features.

  19. Stromal Cell-Derived Growth Factor-1 Alpha-Elastin Like Peptide Fusion Protein Promotes Cell Migration and Revascularization of Experimental Wounds in Diabetic Mice

    Science.gov (United States)

    Yeboah, Agnes; Maguire, Tim; Schloss, Rene; Berthiaume, Francois; Yarmush, Martin L.

    2017-01-01

    Objective: In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 × 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro. Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro. The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro. SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo, SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds. PMID:28116224

  20. A novel thiol-modified hyaluronan and elastin-like polypetide composite material for tissue engineering of the nucleus pulposus of the intervertebral disc.

    Science.gov (United States)

    Moss, Isaac L; Gordon, Lyle; Woodhouse, Kimberly A; Whyne, Cari M; Yee, Albert J M

    2011-06-01

    Biomechanical, in vitro, and initial in vivo evaluation of a thiol-modified hyaluronan (TM-HA) and elastin-like polypeptide (ELP) composite hydrogel for nucleus pulposus (NP) tissue engineering. To investigate the utility of a TM-HA and ELP composite material as a potential tissue-engineering scaffold to reconstitute the NP in early degenerative disc disease (DDD) on the basis of both biomechanical and biologic parameters. DDD is a common ailment with enormous medical, psychosocial, and economic ramifications. Only end-stage surgical therapies are currently widely available. A less invasive, early stage therapy may provide a clinically relevant treatment option. TM-HA and ELP were combined in various concentrations and cross-linked using poly (ethylene glycol) diacrylate. Resulting materials were evaluated biomechanically using confined compression to determine biphasic material properties. In vitro cell culture with human intervertebral disc (IVD) cells seeded within TM-HA/ELP scaffolds was analyzed for cell viability and phenotype. The hydrogels' materials were evaluated in an established New Zealand White (NZW) rabbit model of DDD. The addition of ELP to TM-HA-based hydrogels resulted in a stiffer construct, which is less stiff than native NP but has time-dependant loading characteristics that may be desirable when injected into the IVD. In vitro experiments demonstrated 70% cell viability at 3 weeks with apparent maintenance of phenotype on the basis of morphologic and immunohistochemical data. The addition of ELP had a positive desirable biomechanical effect but did not have a significant positive or negative biologic effect on cell activity. The in vivo feasibility study demonstrated favorable material characteristics and biocompatibility for application as a minimally invasive injectable NP supplement. TM-HA-based hydrogels provide a hospitable environment for human IVD cells and have material characteristics, particularly when supplemented with ELPs that

  1. In vivo guided vascular regeneration with a non-porous elastin-like polypeptide hydrogel tubular scaffold.

    Science.gov (United States)

    Mahara, Atsushi; Kiick, Kristi L; Yamaoka, Tetsuji

    2017-01-28

    Herein, we demonstrate a new approach for small-caliber vascular reconstruction using a non-porous elastin-like polypeptide hydrogel tubular scaffold, based on the concept of guided vascular regeneration (GVR). The scaffolds are composed of elastin-like polypeptide, (Val-Pro-Gly-Ile-Gly)n , for compliance matching and antithrombogenicity and an Arg-Gly-Asp (RGD) motif for connective tissue regeneration. When the polypeptide was mixed with an aqueous solution of β-[Tris(hydroxymethyl)phosphino]propionic acid at 37°C, the polypeptide hydrogel was rapidly formed. The elastic modulus of the hydrogel was 4.4kPa. The hydrogel tubular scaffold was formed in a mold and reinforced with poly(lactic acid) nanofibers. When tubular scaffolds with an inner diameter of 1 mm and length of 5 mm were implanted into rat abdominal aortae, connective tissue grew along the scaffold luminal surface from the flanking native tissues, resulting in new blood vessel tissue with a thickness of 200 μm in 1 month. In contrast, rats implanted with control scaffolds without the RGD motif died. These results indicate that the non-porous hydrogel tubular scaffold containing the RGD motif effectively induced rapid tissue regeneration and that GVR is a promising strategy for the regeneration of small-diameter blood vessels. This article is protected by copyright. All rights reserved.

  2. Noncovalent Modulation of the Inverse Temperature Transition and Self-Assembly of Elastin-b-Collagen-like Peptide Bioconjugates.

    Science.gov (United States)

    Luo, Tianzhi; Kiick, Kristi L

    2015-12-16

    Stimuli-responsive nanostructures produced with peptide domains from the extracellular matrix offer great opportunities for imaging and drug delivery. Although the individual utility of elastin-like (poly)peptides and collagen-like peptides in such applications has been demonstrated, the synergistic advantages of combining these motifs in short peptide conjugates have surprisingly not been reported. Here, we introduce the conjugation of a thermoresponsive elastin-like peptide (ELP) with a triple-helix-forming collagen-like peptide (CLP) to yield ELP-CLP conjugates that show a remarkable reduction in the inverse transition temperature of the ELP domain upon formation of the CLP triple helix. The lower transition temperature of the conjugate enables the facile formation of well-defined vesicles at physiological temperature and the unexpected resolubilization of the vesicles at elevated temperatures upon unfolding of the CLP domain. Given the demonstrated ability of CLPs to modify collagens, our results not only provide a simple and versatile avenue for controlling the inverse transition behavior of ELPs, but also suggest future opportunities for these thermoresponsive nanostructures in biologically relevant environments.

  3. Biofunctionalization of REDV elastin-like recombinamers improves endothelialization on CoCr alloy surfaces for cardiovascular applications.

    Science.gov (United States)

    Castellanos, Maria Isabel; Zenses, Anne-Sophie; Grau, Anna; Rodríguez-Cabello, Jose Carlos; Gil, Francisco Javier; Manero, Jose María; Pegueroles, Marta

    2015-03-01

    To improve cardiovascular implant success, metal-based stents are designated to modulate endothelial cells adhesion and migration in order to prevent restenosis and late thrombosis diseases. Biomimetic coatings with extra-cellular matrix adhesive biomolecules onto stents surfaces are a strategy to recover a healthy endothelium. However, the appropriate bioactive sequences to selective promote growth of endothelium and the biomolecules surface immobilization strategy remains to be elucidated. In this study, biofunctionalization of cobalt chromium, CoCr, alloy surfaces with elastin-like recombinamers, ELR, genetically modified with an REDV sequence, was performed to enhance metal surfaces endothelialization. Moreover, physical adsorption and covalent bonding were used as biomolecules binding strategies onto CoCr alloy. Surfaces were activated with plasma and etched with sodium hydroxide previous to silanization with 3-chloropropyltriethoxysilane and functionalized with the ELR. CoCr alloy surfaces were successfully biofunctionalized and the use of an ELR with an REDV sequence, allows conferring bioactivity to the biomaterials surface, demonstrating a higher cell adhesion and spreading of HUVEC cells on the different CoCr surfaces. This effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique, covalent bonding, and the increase of surface charge electronegativity. Our strategy of REDV elastin-like recombinamers immobilization onto CoCr alloy surfaces via covalent bonding through organosilanes provides a bioactive surface that promotes endothelial cell adhesion and spreading.

  4. Expression and significance of Cystatin SN mRNA in human gastric cancer tissues%胃癌组织Cystatin SN mRNA表达及其临床意义的探讨

    Institute of Scientific and Technical Information of China (English)

    徐大方; 陈平; 石磊; 赵群; 顾学文

    2012-01-01

    OBJECTIVE! To investigate the expression and clinical significance of Cystatin SN in human gastric cancer. METHODS; The Cystatin SNmRNA expression was examined quantitatively by using real-time quantitative polymer-ase chain reaction on a cohort of human gastric cancer tissues (n=40) and corresponding adjacent tissues (w=40). RESULTS: The expression levels of Cystatin SN mRNA in gastric cancer were 5. 370 0±3. 034 5 and adjacent tissues were 1. 705 0±0. 738 8 respectively; Cystatin SN expression was dramatically increased in gastric cancer tissues compared with the corresponding adjacent tissues (?=8. 297, P = 0. 000). The expression of Cystatin SN was significantly associated with tumor size (t = - 0. 646, P = 0. 001), lymphnode metastasis (t = 3. 393 ,-P = 0. 002), gastric cancer stage (I = - 3. 798,P = 0. 001) and infiltration (£=4. 904,P=0. 000). However.it was not related to gastric differentiation,age and grader of patients (P>0. 05). CONCLUSION: Cystatin SN is overexpressed in gastric cancer tissues and significantly correlates with clinical prognosis.%目的:研究半胱氨酸蛋白酶抑制剂1 (Cystatin SN)在胃癌组织中的表达及临床意义.方法:荧光定量PCR方法检测40例胃癌组织和40例癌旁组织中Cystatin SN的表达.结果:胃癌组织中Cystatin SN mRNA的表达水平为5.370 0±3.034 5,相应的癌旁组织为1.705 0±0.738 8,癌组织显著高于癌旁组织,t=8.297,P=0.000.Cystatin SN的表达与肿瘤大小(t=-0.646,P=0.001)、淋巴转移(t=3.393,P=0.002)、TNM分期(t=-3.798,P=0.001)和浸润程度(t=4.904,P=0.000)相关,与肿瘤的分化程度、患者年龄和性别无关,P>0.05.结论:Cystatin SN在胃癌组织中的高表达与胃癌的发生发展密切相关.

  5. Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

    Science.gov (United States)

    Halper, Jaroslava; Kjaer, Michael

    2014-01-01

    Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non

  6. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    Science.gov (United States)

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

  7. Recent innovations in mRNA vaccines.

    Science.gov (United States)

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

    Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have yet been licensed for human use. Recently, mRNA based vaccines have emerged as an alternative approach. They promise the flexibility of plasmid DNA vaccines, without the need for electroporation, but with enhanced immunogenicity and safety. In addition, they avoid the limitations of anti-vector immunity seen with viral vectors, and can be dosed repeatedly. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

  8. Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.

    Science.gov (United States)

    Liu, Wen-Jun; Wu, Qian; Xu, Bi; Zhang, Xin-Yu; Xia, Xiao-Li; Sun, Huai-Chang

    2014-06-01

    Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human β defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

  9. Expression of heat shock protein mRNA in epithelial cells of human lens%人晶状体上皮细胞内热休克蛋白mRNA的表达

    Institute of Scientific and Technical Information of China (English)

    张雪岩; 贾琳琳; 刘宏伟

    2007-01-01

    BACKGROUND: Special anatomical location makes eye lens expose to stressful situation in a long term, so it needs persistent protection to resist stress. Heat shock proteins (HSPs) exert self-protective defensive effect under the stress.OBJECTIVE: To observe the changes in mRNA expression of HSPs in the lens under the condition of exogenous stress.DESIGN: Repeated measurement, controlled observation trial.SETTING: Department of Ophthalmology, First Hospital Affiliated to Jiamusi University.PARTICIPANTS: Human lens endothelial cells B3 (LEC-B3) were obtained from the Department of Ophthalmology, First Hospital Affiliated to China Medical University. Reverse transcription-polymerase chain reaction (RT-PCR) kit was purchased from Bao Biotechnology Company, Japan.METHODS: This experiment was carried out in the Laboratory of Congenital Malformation, Ministry of Public Health,China Medical University between September 2003 and September 2004. LEC-B3 of the exponential growth was used for the experiment. Thermal or oxidative stress on LEC-B3 cells were produced by 30-minute exposure at 45 ℃ or incubated for 30 minutes in DMEM supplemented with 50 mmol/L H2O2. Some stressful cells were allowed to recuperate after exposure to stress in normal culture medium at 37 ℃ for 0, 2, 4, 6, 16 and 24 hours, separately. The expressions of HSP27 and HSP70 in the LEC-B3 were detected by RT-PCR.MAIN OUTCOME MEASURES: The expressions of HSP27 and HSP70 in LEC-B3.RESULTS: HSPs existed in both physiological and stressful situation. The levels of HSPmRNA were increased 2 hours after both stresses. But the expressions of HSP27 and HSP70 got to the summit at different intervals from 2 hours to 6 hours in each group. Subsequently, they were decreased gradually in each group, but they all could maintain a high level at 16 hours.CONCLUSION: Stressful environment induces the over-expressions of HSP27 mRNA and HSP70 mRNA in LEC-B3. This may play an important protective role in lens epithelial

  10. Activation of the elastin-laminin receptor (S-Gal) induces preconditioning in isolated rat heart submitted to ischemia and reperfusion

    Institute of Scientific and Technical Information of China (English)

    ArnaudROBINET; GeorgesBELLON; WilliamHORNEBECK; HerveMILLART

    2004-01-01

    AIM: Elastin-laminin receptor (S-Gal), was described to belong to G-protein-coupled receptors (GPCRs). Using an isolated nonworking rat heart model, we investigated whether S-Gal stimulation was able to mimic ischemic preconditioning as observed with some other GPCRs. METHODS: Hearts, after 6-hydroxydopamine pretreatment and a 20-min stabilization period,

  11. Vascular replacement using a layered elastin-collagen vascular graft in a porcine model: one week patency versus one month occlusion.

    Science.gov (United States)

    Koens, M J W; Krasznai, A G; Hanssen, A E J; Hendriks, T; Praster, R; Daamen, W F; van der Vliet, J A; van Kuppevelt, T H

    2015-01-01

    A persistent clinical demand exists for a suitable arterial prosthesis. In this study, a vascular conduit mimicking the native 3-layered artery, and constructed from the extracellular matrix proteins type I collagen and elastin, was evaluated for its performance as a blood vessel equivalent. A tubular 3-layered graft (elastin-collagen-collagen) was prepared using highly purified type I collagen fibrils and elastin fibers, resembling the 3-layered native blood vessel architecture. The vascular graft was crosslinked and heparinised (37 ± 4 μg heparin/mg graft), and evaluated as a vascular graft using a porcine bilateral iliac artery model. An intra-animal comparison with clinically-used heparinised ePTFE (Propaten®) was made. Analyses included biochemical characterization, duplex scanning, (immuno)histochemistry and scanning electron microscopy. The tubular graft was easy to handle with adequate suturability. Implantation resulted in pulsating grafts without leakage. One week after implantation, both ePTFE and the natural acellular graft had 100% patencies on duplex scanning. Grafts were partially endothelialised (Von Willebrand-positive endothelium with a laminin-positive basal membrane layer). After one month, layered thrombi were found in the natural (4/4) and ePTFE graft (1/4), resulting in occlusion which in case of the natural graft is likely due to the porosity of the inner elastin layer. In vivo application of a molecularly-defined tubular graft, based on nature's matrix proteins, for vascular surgery is feasible.

  12. Vascular replacement using a layered elastin-collagen vascular graft in a porcine model: one week patency versus one month occlusion

    NARCIS (Netherlands)

    Koens, M.J.W.; Krasznai, A.G.; Hanssen, A.E.J.; Hendriks, T.; Praster, R.; Daamen, W.F.; Vliet, J.A. van der; Kuppevelt, T.H. van

    2015-01-01

    A persistent clinical demand exists for a suitable arterial prosthesis. In this study, a vascular conduit mimicking the native 3-layered artery, and constructed from the extracellular matrix proteins type I collagen and elastin, was evaluated for its performance as a blood vessel equivalent. A tubul

  13. Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Thomassen, Martin; Lundby, Carsten

    2005-01-01

    The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na+-K+-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na+-K+-ATPase subunit a1, a2, a3,...

  14. Splice variants of the human zinc transporter ZnT5 (SLC30A5) are differentially localized and regulated by zinc through transcription and mRNA stability.

    Science.gov (United States)

    Jackson, Kelly A; Helston, Rachel M; McKay, Jill A; O'Neill, Elaine D; Mathers, John C; Ford, Dianne

    2007-04-06

    Maintenance of cellular zinc homeostasis includes regulating the expression of cell membrane zinc transporters. Knowledge about the mechanisms underlying changes in mammalian zinc transporter mRNA abundance is poor. We demonstrated that when expressed in Chinese hamster ovary cells as N-terminal fusions to green fluorescent protein, two splice variants of ZnT5 adopt different subcellular locations (either in the Golgi apparatus or throughout the cell, including at the plasma membrane) indicating discrete roles in cellular zinc homeostasis. We demonstrated, using a beta-galactosidase reporter gene, that both splice variants were expressed from a promoter region that was transcriptionally repressed by increased extracellular zinc (150 microM compared with 3 mum; approximately 40%) and by extracellular zinc depletion, using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine ( approximately 20%). We mapped the zinc-responsive element to the region -154 to +50, relative to the predicted start of transcription, and showed that a consensus metal response element sequence (-410 to -404) was not responsible for these effects. Changes in ZnT5 mRNA abundance in Caco-2 cells at different zinc concentrations were in parallel to the changes in promoter activity ( approximately 40% reduction at 150 microM zinc) but in the presence of actinomycin D, to prevent transcription, we observed a marked stabilization (1.7-2-fold accumulation over 24 h) of ZnT5 mRNA. We conclude that effects of zinc on ZnT5 transcription and mRNA stability act in opposition to balance mRNA abundance for cellular zinc homeostasis. To our knowledge, this is the first report that zinc affects the stability of a transcript with a direct role in cellular zinc homeostasis.

  15. Consequences of nigrostriatal denervation on the functioning of the basal ganglia in human and nonhuman primates: an in situ hybridization study of cytochrome oxidase subunit I mRNA.

    Science.gov (United States)

    Vila, M; Levy, R; Herrero, M T; Ruberg, M; Faucheux, B; Obeso, J A; Agid, Y; Hirsch, E C

    1997-01-15

    To examine the consequences of nigrostriatal denervation and chronic levodopa (L-DOPA) treatment on functional activity of the basal ganglia, we analyzed, using in situ hybridization, the cellular expression of the mRNA encoding for cytochrome oxidase subunit I (COI mRNA), a molecular marker for functional neuronal activity, in the basal ganglia. This analysis was performed in monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Intoxication, some of which had been receiving L-DOPA, and in patients with Parkinson's disease (PD). In MPTP-intoxicated monkeys compared with control animals, COI mRNA expression was increased in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia, i.e., the internal segment of the globus pallidus and the substantia nigra pars reticulata. This increase was partially reversed by L-DOPA treatment. COI mRNA expression remained unchanged in the external segment of the globus pallidus (GPe). In PD patients, all of whom had been treated chronically by L-DOPA, COI mRNA expression in the analyzed basal ganglia structures was similar to that in control subjects. These results are in agreement with the accepted model of basal ganglia organization, to the extent that the output nuclei of the basal ganglia are considered to be overactive after nigrostriatal denervation, partly because of increased activity of excitatory afferents from the STN. Yet, our results would also seem to contradict this model, because the overactivity of the STN does not seem to be attributable to a hypoactivation of the GPe.

  16. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  17. 垂体生长激素腺瘤中gsp癌基因对GHSR-1a、Ghrelin mRNA表达的影响%Effect of gsp oncogene on expression of GHSR - 1a and Ghrelin mRNA in human GH - secreting pituitary adenomas

    Institute of Scientific and Technical Information of China (English)

    徐同江; 叶飞; 田春雷; 谢蕊繁; 舒凯; 郭东生; 雷霆

    2010-01-01

    目的 研究垂体生长激素(GH)腺瘤组织中Ghrelin、生长激素释放剂受体-1a亚型(GHSR-1a)mRNA的表达,并探讨其表达与gsp癌基因的关系.方法 采用PCR-DNA直接序列分析方法,观察43例垂体GH腺瘤组织中gsp癌基因的表达;应用实时荧光定量PCR检测Ghrelin和GHSR-1a mRNA的表达水平;统计学分析Ghrelin和GHSR-1a mRNA表达与gsp癌基因的关系.结果 Ghrelin mRNA表达在gsp癌基因阳性、阴性组织间差异无统计学意义(P>0.05);gsp阳性组织中GHSR-1a mRNA表达明显高于gsp阴性组织(P<0.05);gsp阳性、阴性腺瘤组织中GHSR-1amRNA与Ghrelin mRNA的表达均呈明显正相关(R=0.592或0.544,P<0.05).结论 垂体GH腺瘤中gsp癌基因上调GHSR-1a mRNA的表达;而对Ghrelin mRNA表达无明显影响.gsp阳性、阴性腺瘤中Ghrelin均可正向调控GHSR-1a mRNA表达.%Objective To investigate the relationship between the expression of growth hormone secretagogue receptor type 1a (GHSR- 1a) 、Ghrelin mRNA and gsp oncogene in human GH- secreting pituitary adenomas. Methods The gsp oncogene mutation in 43 cases of GH -secreting pituitary adenomas was detected by PCR - DNA direct sequencing analysis. The expression of GHSR - 1a、Ghrelin mRNA was determined by SYBR green real -time fluorescent quantitative PCR. Then the relationships between the expression of GHSR -1a、Ghrelin mRNA and gsp oncegene were studied with statistical methods. Results There was no significant difference in the expression of ghrelin mRNA between mutation - positive and mutation - negative adenomas. The expression of GHSR - 1a mRNA was significantly higher in gsp - positive adenomas than that in gsp - negative adenomas. There was significant direct correlation between the levels of ghrelin mRNA and GHSR mRNA expression in gsp - positive and gsp - negative adenomas. Conclusions Gsp oncogene may up - regulate the expression of GHSR - 1a mRNA and has no effect on the expression of ghrelin mRNA in human GH

  18. Induction of macrophage chemotaxis by aortic extracts from patients with Marfan syndrome is related to elastin binding protein.

    Directory of Open Access Journals (Sweden)

    Gao Guo

    Full Text Available Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62.

  19. Design and synthesis of elastin-like polypeptides for an ideal nerve conduit in peripheral nerve regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Hsueh, Yu-Sheng [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Savitha, S. [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); Department of Biotechnology, Sree Sastha Institute of Engineering and Technology, Chennai (India); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Sadhasivam, S. [Division of Biomedical Engineering and Nanomedicine Research, National Health Research Institutes, Miaoli 350, Taiwan (China); Lin, Feng-Huei, E-mail: double@ntu.edu.tw [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); Division of Biomedical Engineering and Nanomedicine Research, National Health Research Institutes, Miaoli 350, Taiwan (China); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Shieh, Ming-Jium [Institute of Biomedical Engineering, College of Engineering, National Taiwan University, Taipei 100, Taiwan (China); College of Medicine, National Taiwan University Hospital, Taipei 100, Taiwan (China); Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China)

    2014-05-01

    The study involves design and synthesis of three different elastin like polypeptide (ELP) gene monomers namely ELP1, ELP2 and ELP3 that encode for ELP proteins. The formed ELPs were assessed as an ideal nerve conduit for peripheral nerve regeneration. ELP1 was constructed with a small elongated pentapeptide carrying VPGVG sequence to mimic the natural polypeptide ELP. The ELP2 was designed by the incorporation of 4-penta peptide chains to improve the biocompatibility and mechanical strength. Thus, the third position in unique VPGVG was replaced with alanine to VPAVG and in a similar way modified to VPGKG, VPGEG and VPGIG with the substitution of lysine, glutamic acid and isoleucine. In ELP3, fibronectin C5 domain endowed with REDV sequence was introduced to improve the cell attachment. The ELP1, ELP2 and ELP3 proteins expressed by Escherichia coli were purified by inverse transition cycling (ITC). The purified ELPs were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The Schwann cell (SC) morphology and cell adhesion were assessed by fabrication of ELP membrane cross-linked with glutaraledhyde. The Schwann cell proliferation was measured by WST-1 assay. Immunofluorostaining of Schwann cells was accomplished with SC specific phenotypic marker, S100. - Highlights: • Design and synthesis of three gene monomers of elastin like polypeptides (ELP1, 2 and 3) were reported. • Molecular weight of ITC purified ELP1, ELP2 and ELP3 was in the range of 37–38 kDa. • Schwann cell adhesion was found to be prominent in ELP3 and could be used as nerve conduit for peripheral nerve regeneration.

  20. Main: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 S000069 17-May-1998 (last modified) kehi Cis element in 3' end region ...of wheat (T.a.) histone H3 mRNA; 3' end formation; Also found in histone genes of other plants, yeast, etc; histone H3; mRNA

  1. Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo.

    Science.gov (United States)

    Li, Wen-Hwa; Wong, Heng-Kuan; Serrano, José; Randhawa, Manpreet; Kaur, Simarna; Southall, Michael D; Parsa, Ramine

    2017-05-01

    Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

  2. Influence of the Cyp1B1 L432V gene polymorphism and exposure to tobacco smoke on Cyp1B1 mRNA expression in human leukocytes.

    Science.gov (United States)

    Helmig, Simone; Hadzaad, Bahar; Döhrel, Juliane; Schneider, Joachim

    2009-07-01

    Cytochrome P450 1B1 (CYP1B1), a phase I enzyme, is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 L432V polymorphism with diverse types of cancer, as well as an impact on the catalytic activity of the enzyme, has been described. To show the functional impact of the allelic variant Cyp1B1*3, we investigated the quantitative Cyp1B1 mRNA expression in a population of smokers, nonsmokers, and ex-smokers and determined their genotypes. Detection of the L432V polymorphism in exon 3 of the Cyp1B1 gene was performed by rapid capillary polymerase chain reaction (PCR) with melting curve analysis. For quantitative comparison of Cyp1B1 mRNA levels, real-time PCR was performed using SYBR Green fluorescence in a LightCycler system. Calculations of expression were made with the 2(-DeltaDeltaCT) method. In comparing relative Cyp1B1 mRNA expression, highly significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.185 +/- 0.027, n = 118 versus 0.071 +/- 0.013, n = 56; p = 0.000), as well as between the heterozygote genotype *1/*3 and the homozygote genotype *3/*3 (0.178 +/- 0.025, n = 171 versus 0.071 +/- 0.013, n = 56; p = 0.000), were revealed. Significant differences between the genotypes were also detected within the subgroups of smokers, nonsmokers, and ex-smokers. No significant differences were determined in comparing the relative Cyp1B1 mRNA expression with regard to tobacco smoke exposure. Our results suggest that genotypes carrying the C allele (*1/*1 and *1/*3) at Cyp1B1 L432V polymorphism have a significantly higher Cyp1B1 mRNA expression compared with the genotype without the C allele (*3/*3). Gene expression of Cyp1B1 mRNA cannot be used as a biomarker for exposure of tobacco smoke.

  3. HIF1A and EPAS1 mRNA and protein expression during in vitro culture of human primary term cytotrophoblasts and effect of oxygen tension on their expression.

    Science.gov (United States)

    Depoix, Christophe Louis; Flabat, Olivier; Debiève, Frédéric; Hubinont, Corinne

    2016-09-01

    During the first trimester of pregnancy, placenta formation probably occurs in a low-oxygen environment necessary to protect cytotrophoblasts from oxidative stress and to allow proper gene regulation. Transcription factors involved in gene regulation under low oxygen tension are the hypoxia-inducible factors, mainly HIF1A, EPAS1 and their dimerization partner HIF1B. Little is known about their expression during in vitro culture of cytotrophoblasts under chronic hypoxia. We assessed HIF1A and EPAS1 expression in a 4-day in vitro culture of primary term cytotrophoblasts under 21% O2 and 2.5% O2. Copy numbers and relative mRNA expression were assessed by real-time quantitative polymerase chain reaction. Protein levels were quantified by immunoblot and densitometric analysis. In undifferentiated cytotrophoblasts, EPAS1 transcripts were four times more abundant than HIF1A transcripts (2.14e(7) and 5e(6)copies/μg total RNA, respectively). During cell culture, HIF1A mRNA expression increased after 24h and then decreased to stay stable. The expression was even lower when cells were grown under 2.5% O2. EPAS1 mRNA expression increased during cytotrophoblast differentiation. The expression was higher when cells were under 21% O2 than when they were under 2.5% O2. Interestingly, HIF1A, but not EPAS1, was detected in the nuclei of undifferentiated cytotrophoblasts, and in the nuclei of cytotrophoblasts that grew under 21% O2. During cytotrophoblast differentiation, no variation in HIF1A protein levels was detected. To the contrary, EPAS1 protein level increased during differentiation, and oxygen tension had no effect on EPAS1 protein level. In conclusion, HIF1A and EPAS1 expression was not inhibited by chronic hypoxia during in vitro cytotrophoblast differentiation.

  4. Efficient Translation Initiation Directed by the 900-Nucleotide-Long and GC-Rich 5′ Untranslated Region of the Human Retrotransposon LINE-1 mRNA Is Strictly Cap Dependent Rather than Internal Ribosome Entry Site Mediated▿

    Science.gov (United States)

    Dmitriev, Sergey E.; Andreev, Dmitri E.; Terenin, Ilya M.; Olovnikov, Ivan A.; Prassolov, Vladimir S.; Merrick, William C.; Shatsky, Ivan N.

    2007-01-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event. PMID:17470553

  5. Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.

    Science.gov (United States)

    Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M; Olovnikov, Ivan A; Prassolov, Vladimir S; Merrick, William C; Shatsky, Ivan N

    2007-07-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

  6. Self-organized ECM-mimetic model based on an amphiphilic multiblock silk-elastin-like corecombinamer with a concomitant dual physical gelation process.

    Science.gov (United States)

    Fernández-Colino, Alicia; Arias, F Javier; Alonso, Matilde; Rodríguez-Cabello, J Carlos

    2014-10-13

    Although significant progress has been made in the area of injectable hydrogels for biomedical applications and model cell niches, further improvements are still needed, especially in terms of mechanical performance, stability, and biomimicry of the native fibrillar architecture found in the extracellular matrix (ECM). This work focuses on the design and production of a silk-elastin-based injectable multiblock corecombinamer that spontaneously forms a stable physical nanofibrillar hydrogel under physiological conditions. That differs from previously reported silk-elastin-like polymers on a major content and predominance of the elastin-like part, as well as a more complex structure and behavior of such a part of the molecule, which is aimed to obtain well-defined hydrogels. Rheological and DSC experiments showed that this system displays a coordinated and concomitant dual gelation mechanism. In a first stage, a rapid, thermally driven gelation of the corecombinamer solution takes place once the system reaches body temperature due to the thermal responsiveness of the elastin-like (EL) parts and the amphiphilic multiblock design of the corecombinamer. A bridged micellar structure is the dominant microscopic feature of this stage, as demonstrated by AFM and TEM. Completion of the initial stage triggers the second, which is comprised of a stabilization, reinforcement, and microstructuring of the gel. FTIR analysis shows that these events involve the formation of β-sheets around the silk motifs. The emergence of such β-sheet structures leads to the spontaneous self-organization of the gel into the final fibrous structure. Despite the absence of biological cues, here we set the basis of the minimal structure that is able to display such a set of physical properties and undergo microscopic transformation from a solution to a fibrous hydrogel. The results point to the potential of this system as a basis for the development of injectable fibrillar biomaterial platforms

  7. 人肝癌组织中MCT1,MCT2基因mRNA表达的半定量研究%Semi-quantitative analysis of MCT1 and MCT2 genes mRNA expression in human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    李靖; 韩本立

    2001-01-01

    Objective To study the mRNA expression of MCT1 and MCT2 genes in human hepatocellular carcinoma (HCC) and paracarcinoma liver tissue (PCLT). Methods The semi-quantitative analysis of MCT1 and MCT2 genes mRNA expression in human HCC and PCLT was conducted by RT-PCR method and electrophoresis band opacity density (OD) comparison analysis method in 25 patients with HCC. Results The mRNA expression of MCT1 was significantly higher than MCT2 in HCC and PCLT, in HCC the mRNA expression of MCT1 and MCT2 genes were significant higher than that in PCLT. Conclusions The high expression of mRNA of MCT1 and MCT2 genes in HCC indicates that these genes may take a significant role on lactate and other monocarboxylate transmembrane transportation and on pH regulation in tumor cells.%目的探讨单羧酸转运蛋白-1(MCT1)、单羧酸转运蛋白-2(MCT2) mRNA在人肝癌及癌旁肝组织中的表达及意义。方法应用RT-PCR及电泳条带光密度比较法对25例肝癌病人的肝癌及癌旁肝组织中MCT1,MCT2 mRNA的表达进行半定量分析。结果肝癌及癌旁肝组织的MCT1 mRNA的表达显著高于MCT2。肝癌组织中MCT1,MCT2 mRNA的表达显著高于癌旁肝组织。结论 MCT1,MCT2在肝癌细胞内乳酸等单羧酸的跨膜转运及细胞内pH的调节上可能起重要作用。

  8. Continuous exposure to low amplitude extremely low frequency electrical fields characterizing the vascular streaming potential alters elastin accumulation in vascular smooth muscle cells.

    Science.gov (United States)

    Bergethon, Peter R; Kindler, Dean D; Hallock, Kevin; Blease, Susan; Toselli, Paul

    2013-07-01

    In normal development and pathology, the vascular system depends on complex interactions between cellular elements, biochemical molecules, and physical forces. The electrokinetic vascular streaming potential (EVSP) is an endogenous extremely low frequency (ELF) electrical field resulting from blood flowing past the vessel wall. While generally unrecognized, it is a ubiquitous electrical biophysical force to which the vascular tree is exposed. Extracellular matrix elastin plays a central role in normal blood vessel function and in the development of atherosclerosis. It was hypothesized that ELF fields of low amplitude would alter elastin accumulation, supporting a link between the EVSP and the biology of vascular smooth muscle cells. Neonatal rat aortic smooth muscle cell cultures were exposed chronically to electrical fields characteristic of the EVSP. Extracellular protein accumulation, DNA content, and electron microscopic (EM) evaluation were performed after 2 weeks of exposure. Stimulated cultures showed no significant change in cellular proliferation as measured by the DNA concentration. The per-DNA normalized protein in the extracellular matrix was unchanged while extracellular elastin accumulation decreased 38% on average. EM analysis showed that the stimulated cells had a 2.85-fold increase in mitochondrial number. These results support the formulation that ELF fields are a potential factor in both normal vessel biology and in the pathogenesis of atherosclerotic diseases including heart disease, stroke, and peripheral vascular disease.

  9. Telomere length and DCC gene mRNA expression of human large intestine cancers%大肠癌中端粒长度与DCC基因mRNA表达的研究

    Institute of Scientific and Technical Information of China (English)

    张方信; 谢咏梅; 邓芝云; 贾忠建; 魏亚明; 樊俊杰; 吴汉平

    2001-01-01

    目的分析端粒长度及DCC基因mRNA表达在大肠癌及腺瘤发生发展中的作用。方法采用Sourthern印迹杂交及RT-PCR技术,分别检测46例大肠腺瘤及62例大肠癌组织中的端粒限制性片段(TRF)长度及DCC mRNA表达状态,并观察它们与肿瘤临床病理的关系。结果在大肠癌及大肠腺瘤中,TRF长度较正常组织明显缩短,其缩短者占53.2%和41.3%,而延长者仅占6.5%和4.4%,结肠癌的TRF长度也较直肠癌的TRF长度明显缩短。DCC mRNA表达缺失率在大肠癌及大肠腺瘤中则分别达62.9%和34.8%,显著高于正常组织(1.6%);同时,大肠癌患者的平均TRF长度还随患者年龄的增长而缩短,DCC mRNA表达缺失率则随肿瘤的分化程度下降及临床阶段的进展而升高,但DCC mRNA表达缺失与TRF长度缩短在大肠癌中未表现出明显的相关性。结论端粒缩短与DCC mRNA表达缺失可能是大肠腺瘤恶变及大肠癌形成过程中较具特征表现的生物学异常行为。%Objective To evaluate the role of telomere and DCC in tumor transformation and progression. Methods  Telomere length and DCC gene mRNA expression were examined by southern blot hybridization and RT-PCR analysis in 46 adenomas of large intestine, 62 cancers of large intestine and corresponding normal mucosa. Results  Shortening of the telomere was present in the tissues of 41.3% of the adenomas and 53.2% of the cancers, and their average TRF lengths were significantly shorter than those of corresponding normal mucosa(P<0.05, P<0.01), whereas the telomere elongation was only detected in 4.4% and 6.5% of the adenomas and cancers respectively. In addition, the average telomere length in colon carcinomas was also shorter than that in rectal carcinomas. Moreover, the average telomere lengths of the colorectal cancer mucosa became shorter with age. The rates of DCC mRNA expression deletion were 34.8% and 62.9% in the tissues of adenomas and cancers

  10. A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces.

    Science.gov (United States)

    Salvagni, Emiliano; Berguig, Geoffrey; Engel, Elisabeth; Rodriguez-Cabello, J Carlos; Coullerez, Geraldine; Textor, Marcus; Planell, Josep A; Gil, F Javier; Aparicio, Conrado

    2014-02-01

    We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm(2)) compared to the bare metal surface (310 ng/cm(2)). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications.

  11. A unified model for de novo design of elastin-like polypeptides with tunable inverse transition temperatures.

    Science.gov (United States)

    McDaniel, Jonathan R; Radford, D Christopher; Chilkoti, Ashutosh

    2013-08-12

    Elastin-like polypeptides (ELPs) are stimulus-responsive peptide polymers that exhibit inverse temperature phase transition behavior, causing an ELP to aggregate above its inverse transition temperature (T(t)). Although this property has been exploited in a variety of biotechnological applications, de novo design of ELPs that display a specific T(t) is not trivial because the T(t) of an ELP is a complex function of several variables, including its sequence, chain length, polypeptide concentration, and the type and concentration of cosolutes in solution. This paper provides a quantitative model that predicts the T(t) of a family of ELPs (Val-Pro-Gly-Xaa-Gly, where Xaa = Ala and/or Val) from their composition, chain length, and concentration in phosphate buffered saline. This model will enable de novo prediction of the amino acid sequence and chain length of ELPs that will display a predetermined T(t) in physiological buffer within a specified concentration regime, thereby greatly facilitating the design of new ELPs for applications in medicine and biotechnology.

  12. Fabrication and Pilot In Vivo Study of a Collagen-BDDGE-Elastin Core-Shell Scaffold for Tendon Regeneration.

    Science.gov (United States)

    Sandri, Monica; Filardo, Giuseppe; Kon, Elizaveta; Panseri, Silvia; Montesi, Monica; Iafisco, Michele; Savini, Elisa; Sprio, Simone; Cunha, Carla; Giavaresi, Gianluca; Veronesi, Francesca; Fini, Milena; Salvatore, Luca; Sannino, Alessandro; Marcacci, Maurilio; Tampieri, Anna

    2016-01-01

    The development of bio-devices for complete regeneration of ligament and tendon tissues is presently one of the biggest challenges in tissue engineering. Such device must simultaneously possess optimal mechanical performance, suitable porous structure, and biocompatible microenvironment. This study proposes a novel collagen-BDDGE-elastin (CBE)-based device for tendon tissue engineering, by the combination of two different modules: (i) a load-bearing, non-porous, "core scaffold" developed by braiding CBE membranes fabricated via an evaporative process and (ii) a hollow, highly porous, "shell scaffold" obtained by uniaxial freezing followed by freeze-drying of CBE suspension, designed to function as a physical guide and reservoir of cells to promote the regenerative process. Both core and shell materials demonstrated good cytocompatibility in vitro, and notably, the porous shell architecture directed cell alignment and population within the sample. Finally, a prototype of the core module was implanted in a rat tendon lesion model, and histological analysis demonstrated its safety, biocompatibility, and ability to induce tendon regeneration. Overall, our results indicate that such device may have the potential to support and induce in situ tendon regeneration.

  13. Stage III-IV Uterine Prolapse Risk Factors: Sacrouterine Ligaments High Estrogen Receptor Alpha and Collagen III Expression and Low Elastin Expression

    Directory of Open Access Journals (Sweden)

    I Wayan Megadhana

    2016-08-01

    Full Text Available Background: Uterine prolapse is common, non-life-threatening, but has a negative impact on women psychosocial and economic life. Damage to levator ani muscle is the early onset of uterine prolapse, while the damage of sacrouterine ligaments aggravates the stage. The strength of sacrouterine ligament depends on tissue cellularity, the formation of collagen I/III ratio, and the decreased expression of elastin. The lower the ratio of collagen I/III, the higher the risk of stage III-IV uterine prolapse. The ratio of collagen I/III formation is allegedly influencing through the expression of estrogen receptor alpha, by increasing collagen III synthesis and decreasing the degradation. Objective: We aimed to investigate whether high estrogen receptor alpha and collagen III expression, and the low elastin expression in the sacrouterine ligaments were stage III-IV uterine prolapse risk factors. Method: In March to August 2014, a non-matching case control study was conducted in 3 hospitals in Denpasar, and the materials were processed in the Faculty of Veterinary Medicine Laboratory of Udayana University. The case was uterine prolapse stage III-IV, the control was the non-uterine prolapse. We collected 1.5 cm residual sacrouterine ligaments from the edge of the cervix fixed with 10% buffered formalin from patients who underwent a total hysterectomy. They were examined immunohistochemically to identify estrogen receptor alpha expression, collagen III, and elastin. Results: Our sample was 44, divided equally between the case and control group. Compared to the control, in the case group, the proportion was significantly higher for the high estrogen receptor alpha expression (OR=5.71, 95%CI 1.56-20.93, p=0.007, high collagen III (OR=6.50, 95% CI 1.64- 25.76, p=0.005, and low elastin (OR=5.40, 95%CI 1.37-21.26, p=0.012. Conclusion: the high expression of estrogen receptor alpha and collagen III and low expressions of elastin in sacrouterine ligaments served as

  14. Reference: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 Ohtsubo N, Iwabuchi M The conserved 3'-flanking sequence, AATGGAAATG, ...of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA. Nucleic Acids Res 22:1052-1058 (1994) PubMed: 8152910; ...

  15. First-in-human Phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer.

    Science.gov (United States)

    Bianchini, D; Omlin, A; Pezaro, C; Lorente, D; Ferraldeschi, R; Mukherji, D; Crespo, M; Figueiredo, I; Miranda, S; Riisnaes, R; Zivi, A; Buchbinder, A; Rathkopf, D E; Attard, G; Scher, H I; de Bono, J; Danila, D C

    2013-11-12

    Prostate cancer remains dependent of androgen receptor (AR) signalling, even after emergence of castration resistance. EZN-4176 is a third-generation antisense oligonucleotide that binds to the hinge region (exon 4) of AR mRNA resulting in full-length AR mRNA degradation and decreased AR protein expression. This Phase I study aimed to evaluate EZN-4176 in men with castration-resistant prostate cancer (CRPC). Patients with progressing CRPC were eligible; prior abiraterone and enzalutamide treatment were allowed. EZN-4176 was administered as a weekly (QW) 1-h intravenous infusion. The starting dose was 0.5 mg kg(-1) with a 4-week dose-limiting toxicity (DLT) period and a 3+3 modified Fibonacci dose escalation design. After determination of the DLT for weekly administration, an every 2 weeks schedule was initiated. A total of 22 patients were treated with EZN-4176. At 10 mg kg(-1) QW, two DLTs were observed due to grade 3-4 ALT or AST elevation. No confirmed biochemical or soft tissue responses were observed. Of eight patients with <5 circulating tumour cells at baseline, a conversion to <5 was observed in three (38%) patients. The most common EZN-4176-related toxicities (all grades) were fatigue (59%), reversible abnormalities in liver function tests ALT (41%) and AST (41%) and infusion-related reactions including chills (36%) and pyrexia (14%). Activity of EZN-4176 at the doses and schedules explored was minimal. The highest dose of 10 mg kg(-1) QW was associated with significant but reversible transaminase elevation.

  16. Tenascin-X, collagen, elastin, and the Ehlers-Danlos syndrome

    NARCIS (Netherlands)

    Bristow, J.; Carey, W.F.; Egging, D.; Schalkwijk, J.

    2005-01-01

    Tenascin-X is an extracellular matrix protein initially identified because the gene encoding it overlaps with the human CYP21B gene. Because studies of gene and protein function of other tenascins had been poorly predictive of essential functions in vivo, we used a genetic approach that critically

  17. Systems perspectives on mRNA processing

    Institute of Scientific and Technical Information of China (English)

    Adrienne E McKee; Pamela A Silver

    2007-01-01

    The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses of mRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers of mRNA processing. Here, we review global studies of post-transcriptional processes and discuss the challenges facing our understanding of mRNA regulation in metazoan organisms. In parallel, we examine genome-scale investigations that have expanded our knowledge of RNA-binding proteins and the networks of mRNAs that they regulate.

  18. Influenza virus