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Sample records for aminopropionitrile

  1. 21 CFR 522.84 - Beta-aminopropionitrile fumarate.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Beta-aminopropionitrile fumarate. 522.84 Section 522.84 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....84 Beta-aminopropionitrile fumarate. (a) Specifications. Each vial contains 7.0 milligrams of beta...

  2. Rotational spectrum of 3-aminopropionitrile and searches for it in Sagittarius B2(N)

    Science.gov (United States)

    Richard, C.; Belloche, A.; Margulès, L.; Motiyenko, R. A.; Menten, K. M.; Garrod, R. T.; Müller, H. S. P.

    2018-03-01

    Aminoacetonitrile (NH2CH2CN) was detected in the interstellar medium (ISM) one decade ago in the course of a spectral survey of the hot molecular core Sagittarius (Sgr) B2(N) with the IRAM 30 m telescope. With the advent of sensitive telescopes such as the Atacama Large Millimeter/submillimeter Array (ALMA), the degree of chemical complexity in the ISM can be further explored. In the family of aminoacetonitrile, the next stage in complexity with one additional CH2 group is aminopropionitrile. This molecule has two structural isomers, a chain-like (3-aminopropionitrile, NH2CH2CH2CN) and a branched, chiral one (2-aminopropionitrile, CH3CH(NH2) CN). The latter was studied in the laboratory a few years ago and was not detected in the IRAM 30 m survey. We present the search for both isomers in the EMoCA (Exploring Molecular Complexity with ALMA) survey, a sensitive spectral survey of Sgr B2(N) performed with ALMA. The rotational spectrum of 3-aminopropionitrile has been recorded in laboratory with a submillimeter spectrometer using solid-state sources. Helped by ab initio calculations performed for the different possible conformations, we present here the analysis based on a Watson Hamiltonian for an asymmetric one-top rotor in A-reduction performed in the frequency range 150-500 GHz for two conformers. More than 6200 lines of the ground-state and lowest excited vibrational states, corresponding to more than 8200 transitions, were assigned in the experimental spectrum. Partition functions, including the vibrational contribution of these states, and predictions in the JPL-CDMS catalog format were determined in order to search for both conformers of 3-aminopropionitrile in Sgr B2(N). Neither 3-aminopropionitrile, nor 2-aminopropionitrile are detected. The derived upper limits imply that they are at least 12 and 5 times less abundant than aminoacetonitrile, respectively. A comparison to ethyl cyanide and n-propyl cyanide detected in this source suggests that an improvement

  3. Effects of ascorbate and B-aminopropionitrile on tendon fibroblast migration in vitro

    International Nuclear Information System (INIS)

    Nelson, J.M.; Cohen, I.K.; Diegelmann, R.F.

    1986-01-01

    Ascorbate (Asc) stimulates collagen synthesis and hydroxylation whereas beta-aminopropionitrile (BAPN) inhibits collagen cross-link formation. In this study, an in vitro model employing isolated chicken tendon biopsies (2 mm in dia.) in a fibrin clot has been used to examine the effect of Asc and BAPN on tendon fibroblast migration and proliferation. After 5 days in culture with either Asc (0.1 mM), or BAPN (0.5, 1.0, 2.0 mM), cell migration was measured using an automatic planimeter and cell proliferation was quantitated by 125 IUDR incorporation into DNA. In cultures treated with Asc alone, cell migration was enhanced by approximately 33% compared to controls without ascorbate (5.9 mm 2 vs. 4.4 mm 2 ; p < 0.05) with no significant effect on cell proliferation. In contrast, cultures incubated with increasing concentrations of BAPN (0.5 - 2.0 mM) displayed a dose-dependent decrease (up to 9.6-fold at 2 mM) in fibroblast migration into the clot. The inhibitory effect of BAPN on cell migration was not due to a corresponding inhibition of fibroblast proliferation. These observations suggest that Asc enhanced collagen formation and thus allowed greater cell migration into the fibrin matrix. In contrast, in the absence of a mature, cross-linked collagen matrix following exposure to BAPN, cell migration was sub-optimal. These in vitro studies support the hypothesis that modulation of the collagen matrix may be a useful means of regulating tissue repair in vivo

  4. Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

    Science.gov (United States)

    Wright, G C; Wei, X Q; McDevitt, C A; Lane, B P; Sokoloff, L

    1988-01-01

    The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.

  5. Mechanisms for the inversion of chirality: global reaction route mapping of stereochemical pathways in a probable chiral extraterrestrial molecule, 2-aminopropionitrile.

    Science.gov (United States)

    Kaur, Ramanpreet; Vikas

    2015-02-21

    2-Aminopropionitrile (APN), a probable candidate as a chiral astrophysical molecule, is a precursor to amino-acid alanine. Stereochemical pathways in 2-APN are explored using Global Reaction Route Mapping (GRRM) method employing high-level quantum-mechanical computations. Besides predicting the conventional mechanism for chiral inversion that proceeds through an achiral intermediate, a counterintuitive flipping mechanism is revealed for 2-APN through chiral intermediates explored using the GRRM. The feasibility of the proposed stereochemical pathways, in terms of the Gibbs free-energy change, is analyzed at the temperature conditions akin to the interstellar medium. Notably, the stereoinversion in 2-APN is observed to be more feasible than the dissociation of 2-APN and intermediates involved along the stereochemical pathways, and the flipping barrier is observed to be as low as 3.68 kJ/mol along one of the pathways. The pathways proposed for the inversion of chirality in 2-APN may provide significant insight into the extraterrestrial origin of life.

  6. The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats

    Directory of Open Access Journals (Sweden)

    María Miana

    2015-06-01

    Full Text Available Extracellular matrix (ECM remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX family of amine oxidases, including LOX and LOX-like (LOXL isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD. Interestingly, treatment with β-aminopropionitrile (BAPN, a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters – it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4, as well as the increase in suppressor of cytokine signaling 3 (SOCS3 and dipeptidyl peptidase 4 (DPP4 levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX

  7. The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats.

    Science.gov (United States)

    Miana, María; Galán, María; Martínez-Martínez, Ernesto; Varona, Saray; Jurado-López, Raquel; Bausa-Miranda, Belén; Antequera, Alfonso; Luaces, María; Martínez-González, José; Rodríguez, Cristina; Cachofeiro, Victoria

    2015-06-01

    Extracellular matrix (ECM) remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX) family of amine oxidases, including LOX and LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD). Interestingly, treatment with β-aminopropionitrile (BAPN), a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters - it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA) index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4), as well as the increase in suppressor of cytokine signaling 3 (SOCS3) and dipeptidyl peptidase 4 (DPP4) levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX activity for

  8. The lysyl oxidase inhibitor ?-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats

    OpenAIRE

    Miana, Mar?a; Gal?n, Mar?a; Mart?nez-Mart?nez, Ernesto; Varona, Saray; Jurado-L?pez, Raquel; Bausa-Miranda, Bel?n; Antequera, Alfonso; Luaces, Mar?a; Mart?nez-Gonz?lez, Jos?; Rodr?guez, Cristina; Cachofeiro, Victoria

    2015-01-01

    ABSTRACT Extracellular matrix (ECM) remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX) family of amine oxidases, including LOX and LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expressi...

  9. Effect of lysyl oxidase (LOX) on corpus cavernous fibrosis caused by ischaemic priapism.

    Science.gov (United States)

    Gao, Liang; Wu, Changjing; Fu, Fudong; You, Xuanhe; Ma, Xue; Qin, Feng; Li, Tao; Wang, Run; Yuan, Jiuhong

    2018-03-01

    Penile fibrosis caused by ischemic priapism (IP) adversely affects patients' erectile function. We explored the role of lysyl oxidase (LOX) in rat and human penes after ischemic priapism (IP) to verify the effects of anti-LOX in relieving penile fibrosis and preventing erectile dysfunction caused by IP in rats. Seventy-two rats were randomly divided into six groups: control group, control + β-aminopropionitrile (BAPN) group, 9 hrs group, 9 hrs + BAPN group, 24 hrs group, and 24 hrs + BAPN group. β-aminopropionitrile (BAPN), a specific inhibitor of LOX, was administered in the drinking water. At 1 week and 4 weeks, half of the rats in each group were randomly selected for the experiment. Compared to the control group, the erectile function of IP rats was significantly decreased while the expression of LOX in the corpus cavernosum was significantly up-regulated in both 9 and 24 hrs group. Proliferated fibroblasts, decreased corpus cavernosum smooth muscle cells/collagen ratios, destroyed endothelial continuity, deposited abnormal collagen and disorganized fibers were observed in IP rats. The relative content of collage I and III was not obviously different among the groups. β-aminopropionitrile (BAPN) could effectively improve the structure and erectile function of the penis, and enhance recovery. The data in this study suggests that LOX may play an important role in the fibrosis of corpus cavernosum after IP and anti-LOX may be a novel target for patients suffering with IP. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  10. Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction

    DEFF Research Database (Denmark)

    González-Santamaría, José; Villalba, María; Busnadiego, Oscar

    2016-01-01

    resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION: LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery....... contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor β-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also......AIMS: After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular...

  11. The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of alpha 1 type-XI and alpha 2 type-V collagen chains.

    Science.gov (United States)

    Kleman, J P; Hartmann, D J; Ramirez, F; van der Rest, M

    1992-11-15

    The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.

  12. Osteitis fibrosa is mediated by Platelet-Derived Growth Factor-A via a phosphoinositide 3-kinase-dependent signaling pathway in a rat model for chronic hyperparathyroidism.

    Science.gov (United States)

    Lowry, Malcolm B; Lotinun, Sutada; Leontovich, Alexey A; Zhang, Minzhi; Maran, Avudaiappan; Shogren, Kristen L; Palama, Brett K; Marley, Kevin; Iwaniec, Urszula T; Turner, Russell T

    2008-11-01

    Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with beta-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.

  13. Impacts on prenatal development of the human cerebellum: a systematic review.

    Science.gov (United States)

    Koning, Irene V; Tielemans, Myrte J; Hoebeek, Freek E; Ecury-Goossen, Ginette M; Reiss, Irwin K M; Steegers-Theunissen, Regine P M; Dudink, Jeroen

    2017-10-01

    The cerebellum is essential for normal neurodevelopment and is particularly susceptible for intra-uterine disruptions. Although some causal prenatal exposures have been identified, the origin of neurodevelopmental disorders remains mostly unclear. Therefore, a systematic literature search was conducted to provide an overview of parental environmental exposures and intrinsic factors influencing prenatal cerebellar growth and development in humans. The literature search was limited to human studies in the English language and was conducted in Embase, Medline, Cochrane, Web of Science, Pubmed and GoogleScholar. Eligible studies were selected by three independent reviewers and study quality was scored by two independent reviewers. The search yielded 3872 articles. We found 15 eligible studies reporting associations between cerebellar development and maternal smoking (4), use of alcohol (3), in vitro fertilization mediums (1), mercury (1), mifepristone (2), aminopropionitriles (1), ethnicity (2) and cortisol levels (1). No studies reported on paternal factors. Current literature on associations between parental environmental exposures, intrinsic factors and human cerebellar development is scarce. Yet, this systematic review provided an essential overview of human studies demonstrating the vulnerability of the cerebellum to the intra-uterine environment.

  14. Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation.

    Science.gov (United States)

    Sato, Fumiaki; Wachi, Hiroshi; Ishida, Marie; Nonaka, Risa; Onoue, Satoshi; Urban, Zsolt; Starcher, Barry C; Seyama, Yoshiyuki

    2007-06-08

    Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.

  15. Inhibition of Lysyl Oxidases Impairs Migration and Angiogenic Properties of Tumor-Associated Pericytes

    Directory of Open Access Journals (Sweden)

    Aline Lopes Ribeiro

    2017-01-01

    Full Text Available Pericytes are important cellular components of the tumor microenviroment with established roles in angiogenesis and metastasis. These two cancer hallmarks are modulated by enzymes of the LOX family, but thus far, information about LOX relevance in tumor-associated pericytes is lacking. Here, we performed a comparative characterization of normal and tumoral pericytes and report for the first time the modulatory effects of LOX enzymes on activated pericyte properties. Tumoral pericytes isolated from childhood ependymoma and neuroblastoma specimens displayed angiogenic properties in vitro and expressed typical markers, including CD146, NG2, and PDGFRβ. Expression of all LOX family members could be detected in both normal and tumor-associated pericytes. In most pericyte samples, LOXL3 was the family member displaying the highest transcript levels. Inhibition of LOX/LOXL activity with the inhibitor β-aminopropionitrile (βAPN significantly reduced migration of pericytes, while proliferation rates were kept unaltered. Formation of tube-like structures in vitro by pericytes was also significantly impaired upon inhibition of LOX/LOXL activity with βAPN, which induced more prominent effects in tumor-associated pericytes. These findings reveal a novel involvement of the LOX family of enzymes in migration and angiogenic properties of pericytes, with implications in tumor development and in therapeutic targeting tumor microenvironment constituents.

  16. Bone fracture toughness and strength correlate with collagen cross-link maturity in a dose-controlled lathyrism mouse model.

    Science.gov (United States)

    McNerny, Erin M B; Gong, Bo; Morris, Michael D; Kohn, David H

    2015-03-01

    Collagen cross-linking is altered in many diseases of bone, and enzymatic collagen cross-links are important to bone quality, as evidenced by losses of strength after lysyl oxidase inhibition (lathyrism). We hypothesized that cross-links also contribute directly to bone fracture toughness. A mouse model of lathyrism using subcutaneous injection of up to 500 mg/kg β-aminopropionitrile (BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg BAPN treatment in young, growing mice significantly reduced cortical bone fracture toughness, strength, and pyridinoline cross-link content. Ratios reflecting relative cross-link maturity were positive regressors of fracture toughness (HP/[DHLNL + HLNL] r(2)  = 0.208, p toughness and strength. Thus, cross-link profile perturbations associated with bone disease may provide insight into bone mechanical quality and fracture risk. © 2014 American Society for Bone and Mineral Research.

  17. Assaying embryotoxicity in the test tube: current limitations of the embryonic stem cell test (EST) challenging its applicability domain.

    Science.gov (United States)

    Riebeling, Christian; Hayess, Katrin; Peters, Annelieke K; Steemans, Margino; Spielmann, Horst; Luch, Andreas; Seiler, Andrea E M

    2012-05-01

    Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, β-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.

  18. Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases.

    Science.gov (United States)

    Zajoncová, L; Frébort, I; Luhová, L; Sebela, M; Galuszka, P; Pec, P

    1999-01-01

    Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.

  19. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  20. Crosslinking Enzyme Lysyl Oxidase Modulates Scleral Remodeling in Form-Deprivation Myopia.

    Science.gov (United States)

    Yuan, Ying; Li, Min; Chen, Qingzhong; Me, Rao; Yu, Yunjie; Gu, Qing; Shi, Guangsen; Ke, Bilian

    2018-02-01

    Scleral remodeling causes the excessive ocular elongation that underlies myopia. Lysyl oxidase (LOX), a copper-containing amine oxidase, can catalyze collagen and elastin crosslinking. The purpose of this study was to investigate the role of LOX in scleral remodeling in form-deprivation myopia (FDM). Seventy-five guinea pigs were randomly divided into five groups as follows: a normal control group, an FDM group, an FDM plus β-aminopropionitrile (BAPN) group, an FDM plus TGF-β1 (TGF-β1) group, and an FDM plus vehicle group. A translucent diffuser was used to induce FDM, and intravitreal injection was used to administer BAPN, TGF-β1 or vehicle. The scleral LOX and collagen gene and protein levels and the posterior scleral ultrastructure and biomechanics were measured. In the FDM group, both the scleral LOX and collagen gene and protein levels were significantly lower than those in the control eyes. The collagen fibril diameters were significantly decreased in the FDM group compared with the diameters in the control group. A significant decrease in LOX gene and protein expression was observed after BAPN injection, and an increase was observed after TGF-β1 treatment compared with the levels in the FDM group. Additionally, the scleral collagen fibrils were significantly decreased in the BAPN-treated eyes but increased in the TGF-β1-treated eyes compared with the FDM eyes. The ultimate stress and Young's modulus of the sclera were lowest in the BAPN group, followed by the FDM group and the TGF-β1 group. The ultimate strain (%) of the sclera was lowest in the TGF-β1 group, followed by the FDM group and the BAPN group. LOX expression was significantly lowered in myopic sclera. Modulating LOX expression induced a change in both the scleral collagen fibril diameter and the scleral biomechanics. Therefore, LOX may play a key role in the myopia scleral remodeling procedure.

  1. Targeting lysyl oxidase for molecular imaging in breast cancer.

    Science.gov (United States)

    Wuest, Melinda; Kuchar, Manuela; Sharma, Sai Kiran; Richter, Susan; Hamann, Ingrit; Wang, Monica; Vos, Larissa; Mackey, John R; Wuest, Frank; Löser, Reik

    2015-08-13

    Lysyl oxidase (LOX; ExPASy ENZYME entry: EC 1.4.3.13) and members of the LOX-like family, LOXL1-LOXL4, are copper-dependent enzymes that can modify proteins of the extracellular matrix. Expression of LOX is elevated in many human cancers, including breast cancer. LOX expression correlates with the level of tissue hypoxia, and it is known to play a critical role in breast cancer metastasis. The goal of the present study was to target LOX with (1) molecular probe fluorescent labeling to visualize LOX in vitro and (2) a radiolabeled peptide to target LOX in vivo in three different preclinical models of breast cancer. Gene expression of all five members of the LOX family was analyzed at the transcript level via microarray analysis using tissue biopsy samples from 176 patients with breast cancer. An oligopeptide sequence (GGGDPKGGGGG) was selected as a substrate-based, LOX-targeting structure. The peptide was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy experiments with the murine breast cancer cell line EMT-6. In vivo molecular imaging experiments were performed using a C-terminal amidated peptide, GGGDPKGGGGG, labeled with a short-lived positron emitter, fluorine-18 ((18)F), for positron emission tomography (PET) in three different breast cancer models: EMT6, MCF-7 and MDA-MB-231. The PET experiments were carried out in the presence or absence of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX. Immunostaining experiments using a LOX-specific antibody on EMT-6 cells cultured under hypoxic conditions confirmed the elevation of LOX expression in these cells. An FITC-labeled oligopeptide, FITC-Ava-GGGDPKGGGGG-NH2, was found to be localized in different cellular compartments under these conditions. After injection of [(18)F]fluorobenzoate-GGGDPKGGGGG-NH2, radioactivity uptake was visible in all three breast cancer models in vivo. Tumor uptake was reduced by predosing the animals with 2 mg of BAPN 4 h or 24 h before injection of the

  2. Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral

    Science.gov (United States)

    Paschalis, E.P.; Tatakis, D.N.; Robins, S.; Fratzl, P.; Manjubala, I.; Zoehrer, R.; Gamsjaeger, S.; Buchinger, B.; Roschger, A.; Phipps, R.; Boskey, A.L.; Dall'Ara, E.; Varga, P.; Zysset, P.; Klaushofer, K.; Roschger, P.

    2011-01-01

    In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2–L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p < 0.05). Further, compression tests revealed a significant negative impact of β-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, β-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence