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Sample records for lacking asparaginyl endopeptidase

  1. The asparaginyl endopeptidase legumain after experimental stroke.

    PubMed

    Ishizaki, Taku; Erickson, Agnes; Kuric, Enida; Shamloo, Mehrdad; Hara-Nishimura, Ikuko; Inácio, Ana Rita Lourenço; Wieloch, Tadeusz; Ruscher, Karsten

    2010-10-01

    Various proteases in the brain contribute to ischemic brain injury. We investigated the involvement of the asparaginyl endopeptidase legumain after experimental stroke. On the basis of gene array studies and in situ hybridizations, we observed an increase of legumain expression in the peri-infarct area of rats after transient occlusion of the middle cerebral artery (MCAO) for 120 mins with a maximum expression at 24 and 48 h. Immunohistochemical analyses revealed the expression of legumain in Iba1(+) microglial cells and glial fibrillary acidic protein-positive astrocytes of the peri-infarct area in mice after MCAO. Post-stroke recovery was also studied in aged legumain-deficient mice (45 to 58 weeks old). Legumain-deficient mice did not show any differences in physiologic parameters compared with respective littermates before, during MCAO (45 mins), and the subsequent recovery period of 8 days. Moreover, legumain deficiency had no effect on mortality, infarct volume, and the neurologic deficit determined by the rotating pole test, a standardized grip strength test, and the pole test. However, a reduced number of invading CD74(+) cells in the ischemic hemisphere indicates an involvement in post-stroke inflammation. We conclude that legumain is not essential for the functional deficit after MCAO but may be involved in mechanisms of immune cell invasion.

  2. Activation of asparaginyl endopeptidase leads to Tau hyperphosphorylation in Alzheimer disease.

    PubMed

    Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Tung, Yunn Chyn; Liu, Fei; Iqbal, Khalid

    2013-06-14

    Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I2(PP2A). In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I2(PP2A) is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I2NTF) and the C-terminal fragment (I2CTF). Both I2NTF and I2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I2(PP2A) translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I2(PP2A), except when I2(PP2A) was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I2(PP2A), inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I2(PP2A)-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.

  3. Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease*

    PubMed Central

    Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Tung, Yunn Chyn; Liu, Fei; Iqbal, Khalid

    2013-01-01

    Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I2PP2A. In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I2PP2A is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I2NTF) and the C-terminal fragment (I2CTF). Both I2NTF and I2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I2PP2A translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I2PP2A, except when I2PP2A was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I2PP2A, inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I2PP2A-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target. PMID:23640887

  4. Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis

    PubMed Central

    Laha, Thewarach; Sripa, Jittiyawadee; Sripa, Banchob; Pearson, Mark; Tribolet, Leon; Kaewkes, Sasithorn; Sithithaworn, Paiboon; Brindley, Paul J.; Loukas, Alex

    2008-01-01

    Summary Objectives To isolate and characterize an asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and evaluate its expression profile, biochemical activity, and potential as an immunodiagnostic antigen. Methods The full length mRNA encoding an asparaginyl endopeptidase (family C13), Ov-aep-1, was isolated by immunoscreening of a cDNA bacteriophage library of adult O. viverrini using sera from patients infected with O. viverrini. Investigation of Ov-aep-1 transcripts in developmental stages of the parasite, and phylogenetic analysis, immunohistochemical localization, and recombinant protein expression and enzymology were employed to characterize the Ov-AEP-1 protein. Immunoblotting was used to assess the potential of this enzyme for immunodiagnosis of human opisthorchiasis. Results Ov-AEP-1 is characteristic of the C13 cysteine protease family. Ov-aep-1 transcripts were detected in adult and juvenile worms, eggs, and metacercariae. Phylogenetic analysis indicated that Ov-AEP-1 is closely related to homologous proteins in other trematodes. Recombinant Ov-AEP-1 was expressed in bacteria in inclusion bodies and refolded to a soluble form. Excretory–secretory (ES) products derived from adult O. viverrini and refolded recombinant Ov-AEP-1 both displayed catalytic activity against the diagnostic tripeptide substrate, Ala–Ala–Asn-aminomethylcoumarin. Rabbit antiserum raised to recombinant Ov-AEP-1 identified the native AEP-1 protease in both somatic extract and ES products of adult worms. Anti-Ov-AEP-1 IgG immunolocalized the anatomical site of expression to the gut of the fluke, implying a physiological role in digestion of food or activation of other digestive enzymes. Recombinant Ov-AEP-1 was recognized by serum antibodies from patients with opisthorchiasis but not other helminth infections, with a sensitivity and specificity of 85% and 100%, respectively. The positive and negative predictive values are 100% and 67

  5. Development of a smart activity-based probe to detect subcellular activity of asparaginyl endopeptidase in living cells.

    PubMed

    Hong, Jong-Ah; Choi, Na-Eun; La, Yeo-Kyoung; Nam, Ho Yeon; Seo, Jiwon; Lee, Jiyoun

    2017-09-18

    We developed a smart activity-based probe that detects the activity of asparaginyl endopeptidase (AEP) in live cells to monitor the dynamics of enzyme regulation. The newly designed probe generated a turn-on fluorescence signal in response to the activity of AEP in living cells without compromising the labelling efficiency or selectivity. Our probe closely reflected the enzyme activity in its native state, detecting subcellular AEP activity in colon cancer cells and neuronal cells.

  6. Development of a selected reaction monitoring mass spectrometry-based assay to detect asparaginyl endopeptidase activity in biological fluids

    PubMed Central

    Walker, Michael J.; Gray, Oliver J.; Parker, Catriona; Holland, Mark; Williamson, Andrew J.K.; Pierce, Andrew; Unwin, Richard D.; Krishnan, Shekhar

    2016-01-01

    Cancer Biomarkers have the capability to improve patient outcomes. They have potential applications in diagnosis, prognosis, monitoring of disease progression and measuring response to treatment. This type of information is particularly useful in the individualisation of treatment regimens. Biomarkers may take many forms but considerable effort has been made to identify and quantify proteins in biological fluids. However, a major challenge in measuring protein in biological fluids, such as plasma, is the sensitivity of the assay and the complex matrix of proteins present. Furthermore, determining the effect of proteases in disease requires measurement of their activity in biological fluids as quantification of the protein itself may not provide sufficient information. To date little progress has been made towards monitoring activity of proteases in plasma. The protease asparaginyl endopeptidase has been implicated in diseases such as breast cancer, leukaemia and dementia. Here we describe a new approach to sensitively and in a targeted fashion quantify asparaginyl endopeptidase activity in plasma using a synthetic substrate peptide protected from nonspecific hydrolysis using D-amino acids within the structure. Our selected reaction monitoring approach enabled asparaginyl endopeptidase activity to be measured in human plasma with both a high dynamic range and sensitivity. This manuscript describes a paradigm for future development of assays to measure protease activities in biological fluids as biomarkers of disease. PMID:27683124

  7. Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase

    PubMed Central

    Harris, Karen S.; Durek, Thomas; Kaas, Quentin; Poth, Aaron G.; Gilding, Edward K.; Conlan, Brendon F.; Saska, Ivana; Daly, Norelle L.; van der Weerden, Nicole L.; Craik, David J.; Anderson, Marilyn A.

    2015-01-01

    Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired. PMID:26680698

  8. Schistosome asparaginyl endopeptidase (legumain) is not essential for cathepsin B1 activation in vivo.

    PubMed

    Krautz-Peterson, Greice; Skelly, Patrick J

    2008-05-01

    Schistosomes are parasitic platyhelminths that constitute an important public health problem. Adult parasites live in the vasculature of their vertebrate hosts where they consume blood. Ingested blood proteins are degraded by a proteolytic cascade. One of the best characterized schistosome proteases is cathepsin B1 (SmCB1 or Sm31). This protein is synthesized as a large 38 kDa precursor form which is proteolytically cleaved to yield a mature, active 31 kDa enzyme. A second schistosome protease--the asparaginyl endopeptidase SmAE (also known as Sm32, or schistosome legumain), has been proposed to proteolytically convert the 38 kDa precursor SmCB1 into its mature form. Recombinant activated SmAE has been shown to trans-process SmCB1 into the mature, catalytic form in vitro. In the present study, our aim was to test the hypothesis that in vivo SmAE likewise processes SmCB1 into its active form. To do this, expression of the SmAE gene was suppressed in adult Schistosoma mansoni using RNA interference (RNAi). The results of these experiments show that, even in the absence of detectable SmAE protein, SmCB1 is fully processed and active and support the assertion that SmAE is not essential to activate SmCB1 in vivo. The data indicate that our original hypothesis is incorrect and that SmAE is not pivotal in the in vivo conversion of cathepsin B1 into its mature, active form.

  9. Asparaginyl endopeptidase promotes the invasion and metastasis of gastric cancer through modulating epithelial-to-mesenchymal transition

    PubMed Central

    Li, Hong; Li, Qian; Yu, Yiyi; Xu, Xiaojing; Xu, Bei; Liu, Tianshu

    2016-01-01

    Asparaginyl endopeptidase (AEP) is a lysosomal protease often overexpressed in gastric cancer. AEP was expressed higher in peritoneal metastatic loci than in primary gastric cancer. Then we overexpressed AEP or knocked it down with a lentiviral vector in gastric cancer cell lines and detected the cell cycle arrest and the changes of the invasive and metastatic ability in vitro and in vivo. When AEP was knocked-down, the proliferative, invasive and metastatic capacity of gastric cancer cells were inhibited, and the population of sub-G1 cells increased. AEP knockdown led to significant decrease of expression of transcription factor Twist and the mesenchymal markers N-cadherin, ß-catenin and Vimentin and to increased expression of epithelial marker E-cadherin. These results showed that AEP could promote invasion and metastasis by modulating EMT. We used phosphorylation-specific antibody microarrays to investigate the mechanism how AEP promotes gastric cancer invasion and metastasis, and found that the phosphorylation level of AKT and MAPK signaling pathways was decreased significantly if AEP was knocked-down. Therefore, AKT and MAPK signaling pathways took part in the modulation. PMID:27102302

  10. IrAE – an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus

    PubMed Central

    Sojka, Daniel; Hajdušek, Ondřej; Dvořák, Jan; Sajid, Mohammed; Franta, Zdeněk; Schneider, Eric L.; Craik, Charles S.; Vancová, Marie; Burešová, Veronika; Bogyo, Matthew; Sexton, Kelly B.; McKerrow, James H.; Caffrey, Conor R.; Kopáček, Petr

    2008-01-01

    Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite’s ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterized a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which is the first such characterization of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and electron microscopy localized IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH ≥ 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4 kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 – an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites. PMID:17336985

  11. Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase.

    PubMed

    Sajid, Mohammed; McKerrow, James H; Hansell, Elizabeth; Mathieu, Mary A; Lucas, Kimberley D; Hsieh, Ivy; Greenbaum, Doron; Bogyo, Matthew; Salter, Jason P; Lim, Kee C; Franklin, Christopher; Kim, Jea-Hyoun; Caffrey, Conor R

    2003-09-01

    Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.

  12. Aza-peptidyl Michael acceptor and epoxide inhibitors--potent and selective inhibitors of Schistosoma mansoni and Ixodes ricinus legumains (asparaginyl endopeptidases).

    PubMed

    Ovat, Asli; Muindi, Fanuel; Fagan, Crystal; Brouner, Michelle; Hansell, Elizabeth; Dvorák, Jan; Sojka, Daniel; Kopácek, Petr; McKerrow, James H; Caffrey, Conor R; Powers, James C

    2009-11-26

    Aza-peptide Michael acceptors and epoxides with the general structure of YCO-Ala-Ala-AAsn-trans-CH horizontal lineCHCOR and YCO-Ala-Ala-AAsn-EP-COR, respectively, are shown to be potent inhibitors of asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE), and the hard tick, Ixodes ricinus (IrAE). Structure-activity relationships (SARs) were determined for a set of 41 aza-peptide Michael acceptors and eight aza-peptide epoxides. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position, and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues at P2. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors, and for some of these compounds, second-order inhibiton rate constants are the fastest yet discovered. Given the central functions of these enzymes in both parasites, the data presented here may facilitate the eventual design of selective antiparasitic drugs.

  13. Asparaginyl endopeptidase improves the resistance of microtubule-targeting drugs in gastric cancer through IQGAP1 modulating the EGFR/JNK/ERK signaling pathway

    PubMed Central

    Cui, Yuehong; Li, Qian; Li, Hong; Wang, Yan; Wang, Hongshan; Chen, Weidong; Zhang, Shangmin; Cao, Jian; Liu, Tianshu

    2017-01-01

    Purpose In recent years, understanding of the role of asparaginyl endopeptidase (AEP) in tumorigenesis has steadily increased. In this study, we investigated whether AEP expression correlates with sensitivity to chemotherapeutic drugs in gastric cancer and explored the mechanism. Patients and methods AEP expression in the serum of patients’ peripheral blood was measured by enzyme-linked immunosorbent assay. Patient survival time was evaluated using univariate and multivariate analyses. Mass spectrometry and co-immunoprecipitation assays were utilized to discover proteins that interact with AEP. Gastric cancer cell lines were established, in which AEP was overexpressed or knocked out using lentiviral CRISPR. The proliferative abilities of these cell lines in response to chemotherapy agents were evaluated using the Cell Counting Kit-8 method. Gene expression changes in these lines were assessed by real-time polymerase chain reaction and Western blot. Results Patients with low expression of AEP were significantly more likely to have a good prognosis and experience complete response or partial response after treatment with docetaxel/S-1 regimen. Mass spectrum analysis showed that several proteins in the focal adhesion and mitogen-activated protein kinase signaling pathways interacted with AEP. IQGAP1 was confirmed to be one of the proteins interacting with AEP, and its protein level increased when AEP was knocked out. AEP knockout decreased resistance to microtubule inhibitors, including paclitaxel, docetaxel, and T-DM1. The expression levels of MDR1, p-EGFR, p-JNK, p-ERK, and p-Rac1/cdc42 were decreased in AEP knockout gastric cancer cell lines, and inhibitors of both JNK and ERK could block AEP-induced expression of MDR1. Conclusion AEP was not only a prognostic factor but also a predictive marker. AEP knockout could inhibit the activity of the EGFR/JNK/ERK signaling pathway and improve sensitivity to microtubule inhibitors through interacting with IQGAP1. PMID

  14. The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site

    PubMed Central

    Stickler, M M; Reddy, A; Xiong, J M; Hinton, P R; DuBridge, R; Harding, F A

    2011-01-01

    The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ∼40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315−29). CD4+ T-cell proliferative responses to CH315−29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315−29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype. PMID:21326320

  15. Prolyl endopeptidases.

    PubMed

    Gass, J; Khosla, C

    2007-02-01

    This review describes the structure and function of prolyl endopeptidase (PEP) enzymes and how they are being evaluated as drug targets and therapeutic agents. The most well studied PEP family has a two-domain structure whose unique seven-blade beta-propeller domain works with the catalytic domain to hydrolyze the peptide bond on the carboxyl side of internal proline residues of an oligopeptide substrate. Structural and functional studies on this protease family have elucidated the mechanism for peptide entry between the two domains. Other structurally unrelated PEPs have been identified, but have not been studied in detail. Human PEP has been evaluated as a pharmacological target for neurological diseases due to its high brain concentration and ability to cleave neuropeptides in vitro. Recently, microbial PEPs have been studied as potential therapeutics for celiac sprue, an inflammatory disease of the small intestine triggered by proline-rich gluten.

  16. Synthetic Lethality of the lytE cwlO Genotype in Bacillus subtilis Is Caused by Lack of d,l-Endopeptidase Activity at the Lateral Cell Wall

    PubMed Central

    Hashimoto, Masayuki; Ooiwa, Seika

    2012-01-01

    Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two d,l-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the d,l-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these d,l-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of d,l-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed. PMID:22139507

  17. Cyclic Peptides Arising by Evolutionary Parallelism via Asparaginyl-Endopeptidase–Mediated Biosynthesis[C][W

    PubMed Central

    Mylne, Joshua S.; Chan, Lai Yue; Chanson, Aurelie H.; Daly, Norelle L.; Schaefer, Hanno; Bailey, Timothy L.; Nguyencong, Philip; Cascales, Laura; Craik, David J.

    2012-01-01

    The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization. PMID:22822203

  18. Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae.

    PubMed

    DeLuna, Alexander; Quezada, Héctor; Gómez-Puyou, Armando; González, Alicia

    2005-03-25

    The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.

  19. cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

    SciTech Connect

    Rennex, D.; Hemmings, B.A.; Hofsteenge, J.; Stone, S.R. )

    1991-02-26

    Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. ({sup 3}H)Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

  20. Endopeptidase 24.16B. A new variant of endopeptidase 24.16.

    PubMed

    Rodd, D; Hersh, L B

    1995-04-28

    A peptidase, isolated from rat testes, is inhibited by 1 mM o-phenanthroline, 1 microM N-(1-(R,S)-carboxyl-3-phenylpropyl)-Ala-Ala-Phe-p-aminobenzoate, and 6 mM Pro-Ile, properties similar to those ascribed to endopeptidase 24.16. The enzyme hydrolyzes dynorphin A-8, neurotensin 1-13, angiotensin I, and substance P. Kinetic analysis of a series of angiotensin I analogs showed that substitutions at P-1, P-1', or P-2' had little effect on Km or Kcat. Variation of peptide size with a series of dynorphin A peptides showed chain length to be significant. The peptidase cleaved dynorphin A-8 at both Leu5-Arg6 and Arg6-Arg7, and neurotensin 1-13 at Pro10-Tyr11 and Arg8-Arg9. In contrast, rat endopeptidase 24.16 cleaves dynorphin A-8 at Gly4-Leu5 and Leu5-Arg6, and neurotensin 1-13 only at Pro10-Tyr11. These findings, as well as the observation that endopeptidase 24.16 exhibits a considerably higher affinity for Pro-Ile, Ki = 90 microM, indicates the peptidase isolated in this study is related to, but distinct from, rat endopeptidase 24.16. We propose that this new endopeptidase be referred to as endopeptidase 24.16B, while the originally described enzyme be referred to as endopeptidase 24.16A.

  1. The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

    PubMed Central

    Berthet-Colominas, C; Seignovert, L; Härtlein, M; Grotli, M; Cusack, S; Leberman, R

    1998-01-01

    The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor. PMID:9582288

  2. Distinct properties of neuronal and astrocytic endopeptidase 3.4.24.16: a study on differentiation, subcellular distribution, and secretion processes.

    PubMed

    Vincent, B; Beaudet, A; Dauch, P; Vincent, J P; Checler, F

    1996-08-15

    Endopeptidase 3.4.24.16 belongs to the zinc-containing metalloprotease family and likely participates in the physiological inactivation of neurotensin. The peptidase displays distinct features in pure primary cultured neurons and astrocytes. Neuronal maturation leads to a decrease in the proportion of endopeptidase 3.4.24.16-bearing neurons and to a concomitant increase in endopeptidase 3.4.24.16 activity and mRNA content. By contrast, there is no change with time in endopeptidase 3.4.24.16 activity or content in astrocytes. Primary cultured neurons exhibit both soluble and membrane-associated endopeptidase 3.4.24.16 activity. The latter behaves as an ectopeptidase on intact plated neurons and resists treatments with 0.2% digitonin and Na2CO3. Further evidence for an association of the enzyme with plasma membranes was provided by cryoprotection experiments and electron microscopic analysis. The membrane-associated form of endopeptidase 3.4.24.16 increased during neuronal differentiation and appears to be mainly responsible for the overall augmentation of endopeptidase 3.4.24.16 activity observed during neuronal maturation. Unlike neurons, astrocytes only contain soluble endopeptidase 3.4.24.16. Astrocytes secrete the enzyme through monensin, brefeldin A, and forskolin-independent mechanisms. This indicates that endopeptidase 3.4.24.16 is not released by classical regulated or constitutive secreting processes. However, secretion is blocked at 4 degrees C and by 8 bromo cAMP and is enhanced at 42 degrees C, two properties reminiscent of that of other secreted proteins lacking a classical signal peptide. By contrast, neurons appear unable to secrete endopeptidase 3.4.24.16.

  3. Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors

    PubMed Central

    Price, Christopher; Merchant, Michael; Jones, Snake; Best, Ashley; Von Dwingelo, Juanita; Lawrenz, Matthew B.; Alam, Nawsad; Schueler-Furman, Ora; Kwaik, Yousef A.

    2017-01-01

    FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function. PMID:28321389

  4. Anti-Angiogenic Action of Neutral Endopeptidase

    DTIC Science & Technology

    2005-11-30

    production (this is more of an in vitro phenomenon). A number of studies both in vitro and in patient specimens suggest that enhanced expression of...enzymatic site exposed to the external cell surface, Neutral endopeptidase (NEP) is a cell-surface peptidase normally expressed by prostatic epithelial...cells, whose expression is lost in over half of prostate cancers. NEP substrates include small peptides that have been implicated in prostate

  5. Specific inhibition of endopeptidase 24.16 by dipeptides.

    PubMed

    Dauch, P; Vincent, J P; Checler, F

    1991-12-05

    The inhibitory effect of various dipeptides on the neurotensin-degrading metallopeptidase, endopeptidase 24.16, was examined. These dipeptides mimick the Pro10-Tyr11 bond of neurotensin that is hydrolyzed by endopeptidase 24.16. Among a series of Pro-Xaa dipeptides, the most potent inhibitory effect was elicited by Pro-Ile (Ki approximately 90 microM) with Pro-Ile greater than Pro-Met greater than Pro-Phe. All the Xaa-Tyr dipeptides were unable to inhibit endopeptidase 24.16. The effect of Pro-Ile on several purified peptidases was assessed by means of fluorigenic assays and HPLC analysis. A 5 mM concentration of Pro-Ile does not inhibit endopeptidase 24.11, endopeptidase 24.15, angiotensin-converting enzyme, proline endopeptidase, trypsin, leucine aminopeptidase, pyroglutamyl aminopeptidase I and carboxypeptidase B. The only enzyme that was affected by Pro-Ile was carboxypeptidase A, although it was with a 50-fold lower potency (Ki approximately 5 mM) than for endopeptidase 24.16. By means of fluorimetric substrates with a series of hydrolysing activities, we demonstrate that Pro-Ile can be used as a specific inhibitor of endopeptidase 24.16, even in a complex mixture of peptidase activities such as found in whole rat brain homogenate.

  6. Neutral endopeptidase inhibition: could it have a role in the treatment of female sexual arousal disorder?

    PubMed

    Angulo, Javier

    2010-05-01

    Female sexual arousal disorder (FSAD) is the inability to attain or maintain an adequate lubrication-swelling response of sexual excitement. The potentiation of vascular responses leading to increased blood flow in clitoris and vagina has represented the main focus in the pharmacological treatment of FSAD, including the evaluation of the type 5 phosphodiesterase (PDE5) inhibitors. However, due to a lack of clear efficacy, there is no approved pharmacotherapy for FSAD to date. In the present issue of the British Journal of Pharmacology, Wayman et al. show that the administration by intravenous or intravaginal routes of a novel neutral endopeptidase inhibitor, UK-414,445, results in enhanced genital blood flow responses to pelvic nerve stimulation in female rabbits, without significantly affecting blood pressure. Neutral endopeptidase inhibition, by preserving vasoactive peptides such as vasoactive intestinal polypeptide, raises the possibility of a new pharmacological approach to the treatment of FSAD.

  7. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    SciTech Connect

    Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira; Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J.; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2015-03-01

    The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  8. Induced-fit Mechanism for Prolyl Endopeptidase

    SciTech Connect

    Li, Min; Chen, Changqing; Davies, David R.; Chiu, Thang K.

    2010-11-15

    Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the {beta}-propeller and {alpha}/{beta}-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.

  9. Serotype-Selective, Small-Molecule Inhibitors of the Zinc Endopeptidase of Botulinum Neurotoxin Serotype A

    DTIC Science & Technology

    2006-01-01

    domain lacks a deep pocket at the interface of its complex .5,9 Third, the four-ligand coordination of the zinc ion embedded in the active site of the... complex with a substrate has been reported.25 In addition to the use of the active site of the endopeptidase as a target described above, the X- ray...simulations were carried out for each of the eight com- puter-identified compounds in complex with the endo- peptidase. Each of these simulations was

  10. Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

    PubMed

    Barelli, H; Dive, V; Yiotakis, A; Vincent, J P; Checler, F

    1992-10-15

    A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides.

  11. Cell Sorting Enriches Escherichia coli Mutants That Rely on Peptidoglycan Endopeptidases To Suppress Highly Aberrant Morphologies

    PubMed Central

    Laubacher, Mary E.; Melquist, Amy L.; Chandramohan, Lakshmi

    2013-01-01

    Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy. Here, we show that flow cytometry, coupled with the sorting capability of fluorescence-activated cell sorting (FACS), can detect, quantify, and enrich bacteria with morphological alterations. The light scattering properties of several highly aberrant morphological mutants of Escherichia coli were characterized by flow cytometry. Cells from a region that overlapped the distribution of normal rod-shaped cells were collected by FACS and reincubated. After 4 to 15 iterations of this enrichment process, suppressor mutants were isolated that returned almost all the population to a near-normal shape. Suppressors were successfully isolated from strains lacking three or four penicillin binding proteins (PBPs) but not from a mutant lacking a total of seven PBPs. The peptidoglycan endopeptidase, AmpH, was identified as being important for the suppression process, as was a related endopeptidase, MepA. The results validate the use of cell sorting as a means for studying bacterial morphology and identify at least one new class of enzymes required for the suppression of cell shape defects. PMID:23243305

  12. Brugia malayi Asparaginyl - tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis

    PubMed Central

    D, Jeeva Jothi; Dhanraj, Muthu; Solaiappan, Shanmugam; Sivanesan, Sanjana; Kron, Michael; Dhanasekaran, Anuradha

    2016-01-01

    A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis. PMID:26751209

  13. A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence.

    PubMed

    Rico-Pérez, Gadea; Pezza, Alejandro; Pucciarelli, M Graciela; de Pedro, Miguel A; Soncini, Fernando C; García-del Portillo, Francisco

    2016-02-01

    Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ-D-glutamyl-meso-diaminopimelic acid D,L-endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for endopeptidase responding to cessation of growth', is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra- and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation.

  14. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase.

    PubMed

    Wong, Jaslyn E M M; Midtgaard, Søren Roi; Gysel, Kira; Thygesen, Mikkel B; Sørensen, Kasper K; Jensen, Knud J; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2015-03-01

    LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  15. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    PubMed Central

    Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira; Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J.; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2015-01-01

    LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed. PMID:25760608

  16. Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase.

    PubMed

    Dodt, Johannes; Reichwein, Jörg

    2003-09-01

    The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.

  17. A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues.

    PubMed

    Güttler, Bert H-O; Cynis, Holger; Seifert, Franziska; Ludwig, Hans-Henning; Porzel, Andrea; Schilling, Stephan

    2013-04-01

    The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.

  18. Stably transfected human cells overexpressing rat brain endopeptidase 3.4.24.16: biochemical characterization of the activity and expression of soluble and membrane-associated counterparts.

    PubMed

    Vincent, B; Dauch, P; Vincent, J P; Checler, F

    1997-02-01

    We recently cloned endopeptidase-24.16 (neurolysin; EC 3.4.24.16), a neurotensin-degrading peptidase likely involved in the physiological termination of the neurotensinergic signal in the central nervous system and in the gastrointestinal tract. We stably transfected human kidney cells with the pcDNA3-lambda 7aB1 construction bearing the whole open reading frame encoding the rat brain peptidase. Transfectants displayed endopeptidase-24.16 immunoreactivity and exhibited QFS- and neurotensin-hydrolyzing activities, the biochemical and specificity properties of which fully matched those observed with the purified murine enzyme. Cryoprotection experiments and substrate degradation by intact plated cells indicated that transfectants exhibited a membrane-associated form of endopeptidase-24.16, the catalytic site of which clearly faced the extracellular domain. Transfected cells were unable to secrete the enzyme. Overall, our experiments indicate that we have obtained stably transfectant cells that overexpress an enzymatic activity displaying biochemical properties identical to those of purified endopeptidase-24.16. The membrane-associated counterpart and lack of secretion of the enzyme were clearly reminiscent of what was observed with pure cultured neurons, but not with astrocytes. Therefore, the transfected cell model described here could prove useful for establishing, by a mutagenesis approach, the structural elements responsible for the "neuronal" phenotype exhibited by the enzyme in transfected cells.

  19. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-10-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

  20. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed Central

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  1. Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases*

    PubMed Central

    Chan, Mun Chiang; Ilott, Nicholas E.; Schödel, Johannes; Sims, David; Tumber, Anthony; Lippl, Kerstin; Mole, David R.; Pugh, Christopher W.; Ratcliffe, Peter J.; Ponting, Chris P.; Schofield, Christopher J.

    2016-01-01

    The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable. PMID:27502280

  2. Specific catalysis of asparaginyl deamidation by carboxylic acids: kinetic, thermodynamic, and quantitative structure-property relationship analyses.

    PubMed

    Connolly, Brian D; Tran, Benjamin; Moore, Jamie M R; Sharma, Vikas K; Kosky, Andrew

    2014-04-07

    Asparaginyl (Asn) deamidation could lead to altered potency, safety, and/or pharmacokinetics of therapeutic protein drugs. In this study, we investigated the effects of several different carboxylic acids on Asn deamidation rates using an IgG1 monoclonal antibody (mAb1*) and a model hexapeptide (peptide1) with the sequence YGKNGG. Thermodynamic analyses of the kinetics data revealed that higher deamidation rates are associated with predominantly more negative ΔS and, to a lesser extent, more positive ΔH. The observed differences in deamidation rates were attributed to the unique ability of each type of carboxylic acid to stabilize the energetically unfavorable transition-state conformations required for imide formation. Quantitative structure property relationship (QSPR) analysis using kinetic data demonstrated that molecular descriptors encoding for the geometric spatial distribution of atomic properties on various carboxylic acids are effective determinants for the deamidation reaction. Specifically, the number of O-O and O-H atom pairs on carboxyl and hydroxyl groups with interatomic distances of 4-5 Å on a carboxylic acid buffer appears to determine the rate of deamidation. Collectively, the results from structural and thermodynamic analyses indicate that carboxylic acids presumably form multiple hydrogen bonds and charge-charge interactions with the relevant deamidation site and provide alignment between the reactive atoms on the side chain and backbone. We propose that carboxylic acids catalyze deamidation by stabilizing a specific, energetically unfavorable transition-state conformation of l-asparaginyl intermediate II that readily facilitates bond formation between the γ-carbonyl carbon and the deprotonated backbone nitrogen for cyclic imide formation.

  3. Targeting of endopeptidase 24.16 to different subcellular compartments by alternative promoter usage.

    PubMed

    Kato, A; Sugiura, N; Saruta, Y; Hosoiri, T; Yasue, H; Hirose, S

    1997-06-13

    Endopeptidase 24.16 or mitochondrial oligopeptidase, abbreviated here as EP 24.16 (MOP), is a thiol- and metal-dependent oligopeptidase that is found in multiple intracellular compartments in mammalian cells. From an analysis of the corresponding gene, we found that the distribution of the enzyme to appropriate subcellular locations is achieved by the use of alternative sites for the initiation of transcription. The pig EP 24.16 (MOP) gene spans over 100 kilobases and is organized into 16 exons. The core protein sequence is encoded by exons 5-16 which match perfectly with exons 2-13 of the gene for endopeptidase 24.15, another member of the thimet oligopeptidase family. These two sets of 11 exons share the same splice sites, suggesting a common ancestor. Multiple species of mRNA for EP 24.16 (MOP) were detected by the 5'-rapid amplification of cDNA ends and they were shown to have been generated from a single gene by alternative choices of sites for the initiation of transcription and splicing. Two types of transcript were prepared, corresponding to transcription from distal and proximal sites. Their expression in vitro in COS-1 cells indicated that they encoded two isoforms (long and short) which differed only at their amino termini: the long form contained a cleavable mitochondrial targeting sequence and was directed to mitochondria; the short form, lacking such a signal sequence, remained in the cytosol. The complex structure of the EP 24.16 (MOP) gene thus allows, by alternative promoter usage, a fine transcriptional regulation of coordinate expression, in the different subcellular compartments, of the two isoforms arising from a single gene.

  4. Highly active and selective endopeptidases with programmed substrate specificities

    PubMed Central

    Varadarajan, Navin; Rodriguez, Sarah; Hwang, Bum-Yeol; Georgiou, George; Iverson, Brent L

    2009-01-01

    A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (kcat/KM > 104 M−1 s−1). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu↓Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic. PMID:18391948

  5. Processing of Proopiornelanocortin by Insulin Secretory Granule Proinsulin Processing Endopeptidases*

    PubMed Central

    Rhodes, Christopher J.; Thorne, Barbara A.; Lincoln, Beth; Nielsen, Egon; Hutton, John C.; Thomas, Gary

    2015-01-01

    A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93–96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as α-melanocyte-stimulating hormone, corticotropin-like intermediate, γ-lipotropin, β-endorphin-(1–31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and β-lipotropin. This processing had an acidic pH optimum (pH 5–5.5) and was Ca2+-dependent (K0.5 activation = 5–80 µm). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway PMID:8382698

  6. Determination of fluorescence-labeled asparaginyl-oligosaccharide in glycoprotein by reversed-phase ultraperformance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.

    PubMed

    Kurihara, Takamasa; Min, Jun Zhe; Toyo'oka, Toshimasa; Fukushima, Takeshi; Inagaki, Shinsuke

    2007-11-15

    Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.

  7. Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications.

    PubMed

    Sutton, J Mark; Wayne, Jonathan; Scott-Tucker, Anthony; O'Brien, Susan M; Marks, Philip M H; Alexander, Frances C G; Shone, Clifford C; Chaddock, John A

    2005-03-01

    Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.

  8. Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases.

    PubMed

    Rhodes, C J; Thorne, B A; Lincoln, B; Nielsen, E; Hutton, J C; Thomas, G

    1993-02-25

    A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as alpha-melanocyte-stimulating hormone, corticotropin-like intermediate, gamma-lipotropin, beta-endorphin-(1-31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and beta-lipotropin. This processing had an acidic pH optimum (pH 5-5.5) and was Ca(2+)-dependent (K0.5 activation = 5-80 microM). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway (Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. S., Steiner, D. F., and Thomas, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5297-5301).

  9. Two siblings with homozygous pathogenic splice-site variant in mitochondrial asparaginyl-tRNA synthetase (NARS2).

    PubMed

    Vanlander, Arnaud V; Menten, Björn; Smet, Joél; De Meirleir, Linda; Sante, Tom; De Paepe, Boel; Seneca, Sara; Pearce, Sarah F; Powell, Christopher A; Vergult, Sarah; Michotte, Alex; De Latter, Elien; Vantomme, Lies; Minczuk, Michal; Van Coster, Rudy

    2015-02-01

    A homozygous missense mutation (c.822G>C) was found in the gene encoding the mitochondrial asparaginyl-tRNA synthetase (NARS2) in two siblings born to consanguineous parents. These siblings presented with different phenotypes: one had mild intellectual disability and epilepsy in childhood, whereas the other had severe myopathy. Biochemical analysis of the oxidative phosphorylation (OXPHOS) complexes in both siblings revealed a combined complex I and IV deficiency in skeletal muscle. In-gel activity staining after blue native-polyacrylamide gel electrophoresis confirmed the decreased activity of complex I and IV, and, in addition, showed the presence of complex V subcomplexes. Considering the consanguineous descent, homozygosity mapping and whole-exome sequencing were combined revealing the presence of one single missense mutation in the shared homozygous region. The c.822G>C variant affects the 3' splice site of exon 7, leading to skipping of the whole exon 7 and a part of exon 8 in the NARS2 mRNA. In EBV-transformed lymphoblasts, a specific decrease in the amount of charged mt-tRNA(Asn) was demonstrated as compared with controls. This confirmed the pathogenic nature of the variant. To conclude, the reported variant in NARS2 results in a combined OXPHOS complex deficiency involving complex I and IV, making NARS2 a new member of disease-associated aaRS2.

  10. The inhibition of proinsulin-processing endopeptidase activities by active-site-directed peptides.

    PubMed Central

    Rhodes, C J; Zumbrunn, A; Bailyes, E M; Shaw, E; Hutton, J C

    1989-01-01

    Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities. PMID:2649090

  11. Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley.

    PubMed

    Zhang, N; Jones, B L

    1996-01-01

    Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S2 site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1-9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.

  12. Molecular cloning and expression of rat brain endopeptidase 3.4.24.16.

    PubMed

    Dauch, P; Vincent, J P; Checler, F

    1995-11-10

    We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.

  13. Homologous inhibitors from potato tubers of serine endopeptidases and metallocarboxypeptidases.

    PubMed

    Hass, C M; Venkatakrishnan, R; Ryan, C A

    1976-06-01

    A potent polypeptide inhibitor of chymotrypsin has been purified from Russett Burbank potatoes. The inhibitor has no effect on bovine carboxypeptidases A or B but exhibits homology with a carboxypeptidase inhibitor that is also present in potato tubers. The chymotrypsin inhibitor has a molecular weight of approximately 5400 as estimated by gel filtration, amino acid analysis, and titration with chymotrypsin. The polypeptide chain consists of 49 amino acid residues, of which six are half-cystine, forming three disulfide bonds. Its size is similar to that of the carboxypeptidase inhibitor, which contains 39 amino acid residues and also has three disulfide bridges. In immunological double diffusion assays, the chymotrypsin inhibitor and the carboxypeptidase inhibitor do not crossreact; however, automatic Edman degradation of reduced and alkylated derivatives of the chymotrypsin inhibitor, yielding a partial sequence of 18 amino acid residues at the NH2-terminus, reveals a similarity in sequence to that of the carboxypeptidase inhibitor. Thus, inhibitors directed toward two distinct classes of proteases, the serine endopeptidases and the metallocarboxypeptidases, appear to have evolved from a common ancestor.

  14. Protein engineering of improved prolyl endopeptidases for celiac sprue therapy

    PubMed Central

    Ehren, Jennifer; Govindarajan, Sridhar; Morón, Belén; Minshull, Jeremy; Khosla, Chaitan

    2008-01-01

    Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity. PMID:18836204

  15. Asparagine endopeptidase is an innovative therapeutic target for neurodegenerative diseases.

    PubMed

    Zhang, Zhentao; Xie, Manling; Ye, Keqiang

    2016-10-01

    Asparagine endopeptidase (AEP) is a pH-dependent endolysosomal cysteine protease that cleaves its substrates after asparagine residues. Our most recent study identifies that it possesses the delta-secretase activity, and that it is implicated in numerous neurological diseases such as Alzheimer's disease (AD) and stroke. Accumulating evidence supports that the inhibition of AEP exhibits beneficial effects for treating these devastating diseases. Based on recent evidence, it is clear that AEP cleaves its substrate, such as amyloid precursor protein (APP), tau and SET, and plays a critical role in neuronal cell death in various neurodegenerative diseases and stroke. In this article, the basic biology of AEP, its knockout phenotypes in mouse models, its substrates in neurodegenerative diseases, and its small peptidyl inhibitors and prodrugs are discussed. In addition, we discuss the potential of AEP as a novel therapeutic target for neurodegenerative diseases. AEP plays a unique role in numerous biological processes, depending on both pH and context. Most striking is our most recent finding; that AEP is activated in an age-dependent manner and simultaneously cleaves both APP and tau, thereby unifying both major pathological events in AD. Thus, AEP acts as an innovative trigger for neurodegenerative diseases. Inhibition of AEP will provide a disease-modifying treatment for neurodegenerative diseases including AD.

  16. Crystal structure of the archaeal asparagine synthetase: interrelation with aspartyl-tRNA and asparaginyl-tRNA synthetases.

    PubMed

    Blaise, Mickaël; Fréchin, Mathieu; Oliéric, Vincent; Charron, Christophe; Sauter, Claude; Lorber, Bernard; Roy, Hervé; Kern, Daniel

    2011-09-23

    Asparagine synthetase A (AsnA) catalyzes asparagine synthesis using aspartate, ATP, and ammonia as substrates. Asparagine is formed in two steps: the β-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. Interestingly, this mechanism of amino acid activation resembles that used by aminoacyl-tRNA synthetases, which first activate the α-carboxylate group of the amino acid to form also an aminoacyl-AMP before they transfer the activated amino acid onto the cognate tRNA. In a previous investigation, we have shown that the open reading frame of Pyrococcus abyssi annotated as asparaginyl-tRNA synthetase (AsnRS) 2 is, in fact, an archaeal asparagine synthetase A (AS-AR) that evolved from an ancestral aspartyl-tRNA synthetase (AspRS). We present here the crystal structure of this AS-AR. The fold of this protein is similar to that of bacterial AsnA and resembles the catalytic cores of AspRS and AsnRS. The high-resolution structures of AS-AR associated with its substrates and end-products help to understand the reaction mechanism of asparagine formation and release. A comparison of the catalytic core of AS-AR with those of archaeal AspRS and AsnRS and with that of bacterial AsnA reveals a strong conservation. This study uncovers how the active site of the ancestral AspRS rearranged throughout evolution to transform an enzyme activating the α-carboxylate group into an enzyme that is able to activate the β-carboxylate group of aspartate, which can react with ammonia instead of tRNA. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome

    PubMed Central

    Shahzad, Mohsin; Huang, Vincent H.; Qaiser, Tanveer A.; Potluri, Prasanth; Mahl, Sarah E.; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-sheqaih, Nada; Newman, William G.; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N.; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B.; Wu, Doris K.; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C.; Ahmed, Zubair M.; Huang, Taosheng; Riazuddin, Saima

    2015-01-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome. PMID:25807530

  18. Nematode asparaginyl-tRNA synthetase resolves intestinal inflammation in mice with T-cell transfer colitis.

    PubMed

    Kron, Michael A; Metwali, Ahmed; Vodanovic-Jankovic, Sanja; Elliott, David

    2013-02-01

    The therapeutic effects of a controlled parasitic nematode infection on the course of inflammatory bowel disease (IBD) have been demonstrated in both animal and human models. However, the inability of individual well-characterized nematode proteins to recreate these beneficial effects has limited the application of component immunotherapy to human disease. The nematodes that cause chronic human lymphatic filariasis, Brugia malayi and Wuchereria bancrofti, are among the parasites that induce immune suppression. Filarial lymphatic pathology has been shown to involve NF-κB pathway-dependent production of vascular endothelial growth factor (VEGF), and stimulation of VEGF expression has also been reported by interleukin 8 (IL-8) via NF-κB pathways. Previously, we have shown that the filarial asparaginyl-tRNA synthetase (rBmAsnRS) interacts with IL-8 receptors using a combination of extracellular loops that differ from those bound by IL-8. To test the hypothesis that rBmAsnRS might induce an anti-inflammatory effect in vivo, we studied the effects of rBmAsnRS in an established murine colitis model using T-cell transfer mice. T-cell transfer colitis mice treated intraperitoneally with 100 μg of rBmAsnRS four times over 2 weeks showed resolution of cellular infiltration in the colonic mucosa, along with induction of a CD8(+) cellular response. In addition, rBmAsnRS induced a rise in IL-10 production from CD3(+) and lipopolysaccharide (LPS)- and cytosine phosphate guanosine (CPG)-stimulated splenic cells. In summary, this work demonstrates a novel anti-inflammatory nematode protein, supports the hygiene hypothesis, and supports continued refinement of alternative immunotherapies for treatment of IBD.

  19. Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

    PubMed

    Simon, Mariella; Richard, Elodie M; Wang, Xinjian; Shahzad, Mohsin; Huang, Vincent H; Qaiser, Tanveer A; Potluri, Prasanth; Mahl, Sarah E; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-Sheqaih, Nada; Newman, William G; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B; Wu, Doris K; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C; Ahmed, Zubair M; Huang, Taosheng; Riazuddin, Saima

    2015-03-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

  20. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    PubMed Central

    Wünschiers, Röbbe; Batur, Mehtap; Lindblad, Peter

    2003-01-01

    Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation

  1. Endopeptidase 24-16 in murines: tissue distribution, cerebral regionalization, and ontogeny.

    PubMed

    Dauch, P; Masuo, Y; Vincent, J P; Checler, F

    1992-11-01

    The tissue distribution, cerebral regionalization, and ontogeny of endopeptidase 24-16 were established in murines by means of its quenched fluorimetric substrate, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp, and its selective dipeptide blocker, Pro-Ile. Endopeptidase 24-16 was particularly abundant in the liver and kidney, and the lowest specific activity was detected in the heart. In the brain, a 16-fold difference in specific activity was observed between the poorest and the richest cerebral areas. Endopeptidase 24-16 appeared in high concentrations in the olfactory bulb and tubercule, cingulate cortex, medial striatum, and globus pallidus, and was particularly weak in the CA1, CA2, and CA3 parts of the hippocampal formation and in the cerebellum. Endopeptidase 24-16 content in thirteen thalamic nuclei indicated a rather homogeneous distribution. This homogeneity was not observed in the hypothalamus, where pronounced variations occurred between enriched zones such as suprachiasmatic and arcuate nuclei and relatively poor areas such as periventricular and supraoptic nuclei. Endopeptidase 24-16 appeared to be developmentally regulated in the mouse brain; it was already detected at the fetal stage, increased transiently after birth, then regularly declined until adulthood.

  2. Characterization of neutral endopeptidase 24.11 in dog glomeruli.

    PubMed Central

    Landry, C; Santagata, P; Bawab, W; Fournié-Zaluski, M C; Roques, B P; Vinay, P; Crine, P

    1993-01-01

    Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8489505

  3. Neuropeptide specificity and inhibition of recombinant isoforms of the endopeptidase 3.4.24.16 family: comparison with the related recombinant endopeptidase 3.4.24.15.

    PubMed

    Rioli, V; Kato, A; Portaro, F C; Cury, G K; te Kaat, K; Vincent, B; Checler, F; Camargo, A C; Glucksman, M J; Roberts, J L; Hirose, S; Ferro, E S

    1998-09-08

    Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases. Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m). We have overexpressed two porcine EP24.16 isoforms in E. coli and purified the recombinant proteins to homogeneity. We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c. All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16. These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15. The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme.

  4. Altered dipeptidyl peptidase IV and prolyl endopeptidase activities in chronic tonsillitis, tonsillar hyperplasia and adenoid hyperplasia.

    PubMed

    Larrinaga, Gorka; Pérez, Itxaro; Sanz, Begoña; Zarrazquin, Idoia; Casis, Luis; Anta, Jose Antonio; Martínez, Agustin; Santaolalla, Francisco

    2011-03-01

    To analyse peptidase activities in the removed tonsils and adenoids from patients with chronic tonsillitis, tonsillar hyperplasia and adenoid hyperplasia. We have analyzed 48 tissue samples from patients undergoing tonsillectomy and adenoidectomy for chronic tonsillitis, tonsillar hyperplasia or adenoid hyperplasia. Tonsillectomy and adenoidectomy samples were collected and frozen for later enzyme analysis. The catalytic activity of a pool of peptidases (dipeptidyl peptidase IV, prolyl endopeptidase, aminopeptidase A, aminopeptidase N, aspartyl aminopeptidase, aminopeptidase B, neutral endopeptidase, pyroglutamyl peptidase I, puromycin-sensitive aminopeptidase and cystinyl aminopeptidase) was measured fluorometrically. The activity of prolyl endopeptidase was higher in tonsillar hyperplasia and adenoid hyperplasia than in chronic tonsillitis. On the contrary, dipeptidyl peptidase IV activity was higher in chronic tonsillitis than in hypertrophic tissues. When data were stratified by age and gender, dipeptidyl peptidase IV was also found to be more active in adult and male chronic tonsillitis tissues. Inversely, dipeptidyl peptidase IV activity was higher in tissues of females with tonsillar hyperplasia. These data indicate the involvement of dipeptidyl peptidase IV and prolyl endopeptidase in the mechanisms underlying chronic tonsillitis, tonsillar hyperplasia and adenoid hyperplasia. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  5. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

    USDA-ARS?s Scientific Manuscript database

    LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

  6. Early, Real-Time Medical Diagnosis of Botulism by Endopeptidase-Mass Spectrometry.

    PubMed

    Rosen, Osnat; Feldberg, Liron; Gura, Sigalit; Brosh-Nissimov, Tal; Guri, Alex; Zimhony, Oren; Shapiro, Eli; Beth-Din, Adi; Stein, Dana; Ozeri, Eyal; Barnea, Ada; Turgeman, Amram; Ben David, Alon; Schwartz, Arieh; Elhanany, Eytan; Diamant, Eran; Yitzhaki, Shmuel; Zichel, Ran

    2015-12-15

    Botulinum toxin was detected in patient serum using Endopeptidase-mass-spectrometry assay, although all conventional tests provided negative results. Antitoxin was administered, resulting in patient improvement. Implementing this highly sensitive and rapid assay will improve preparedness for foodborne botulism and deliberate exposure.

  7. Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots.

    PubMed Central

    James, F; Brouquisse, R; Suire, C; Pradet, A; Raymond, P

    1996-01-01

    An endopeptidase (designated RSIP, for root-starvation-induced protease) was purified to homogeneity from glucose-starved maize roots. The molecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing conditions and 62 kDa by gel filtration on a Sephacryl S-200 column. The isoelectric point of RSIP was 4.55. The purified enzyme was stable, with no auto-proteolytic activity. The enzyme activity was strongly inhibited by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a serine protease. The maximum proteolytic activity against different protein substrates occurred at pH 6.5. With the exception of succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determination of the cleavage sites in the oxidized B-Chain of insulin showed specificity for hydrophobic residues at the P2 and P3 positions, indicating that RSIP is distinct from other previously characterized maize endopeptidases. Both subcellular fractionation and immuno-detection in situ indicated that RSIP is localized in the vacuole of the root cells. RSIP is the first vacuolar serine endopeptidase to be identified. Glucose starvation induced RSIP: after 4 days of starvation, RSIP was estimated to constitute 80% of total endopeptidase activity in the root tip. These results suggest that RSIP is implicated in vacuolar autophagic processes triggered by carbon limitation. PMID:8947499

  8. Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.

    PubMed Central

    Tan, P S; Pos, K M; Konings, W N

    1991-01-01

    An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus. Images PMID:1785932

  9. Synthesis and evaluation of heteroarylalanine diacids as potent and selective neutral endopeptidase inhibitors.

    PubMed

    Glossop, Melanie S; Bazin, Richard J; Dack, Kevin N; Fox, David N A; MacDonald, Graeme A; Mills, Mark; Owen, Dafydd R; Phillips, Chris; Reeves, Keith A; Ringer, Tracy J; Strang, Ross S; Watson, Christine A L

    2011-06-01

    Heteroarylalanine derivatives 4 were designed as potential inhibitors of neutral endopeptidase (NEP EC 3.4.24.11). Selectivity over other zinc metalloproteinases was explored through occupation of the S2' subsite within NEP. Structural optimisation led to the identification of 5-phenyl oxazole 4f, a potent and selective NEP inhibitor. A crystal structure of the inhibitor bound complex is reported.

  10. Decreased expression of messenger RNAs encoding endothelin receptors and neutral endopeptidase 24.11 in endometrial cancer.

    PubMed Central

    Pekonen, F.; Nyman, T.; Ammälä, M.; Rutanen, E. M.

    1995-01-01

    In this study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to compare the expression of mRNAs encoding endothelin-1 (ET-1), endothelin receptors type A (ETA-R) and type B (ETB-R) and ET-1-degrading enzyme neutral endopeptidase 24.11 (NEP) in 15 endometrial cancer tissues and 13 normal endometrial tissues. The relative levels of ET-1 mRNA in endometrial cancer tissues did not differ from those in normal endometrium. Both ETA-R and ETB-R mRNA levels were significantly lower in endometrial cancer tissue than in normal endometrium (P < 0.001). The complete lack of NEP mRNA in endometrial cancer tissues was in marked contrast to results from normal endometrium (P < 0.001). In conclusion, differential expression of mRNAs encoding ET-R and NEP in normal endometrium and endometrial cancer suggests that ET action is altered in endometrial cancer compared with normal endometrium. Images Figure 2 PMID:7819049

  11. Alpha 2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture.

    PubMed Central

    Slot, L A; Hendil, K B

    1988-01-01

    Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed. Images Fig. 4. Fig. 5. PMID:2462415

  12. The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

    PubMed Central

    Martinelli, Daniel J.

    2016-01-01

    ABSTRACT Mycobacteria possess a series of Rip peptidoglycan endopeptidases that have been characterized in various levels of detail. The RipA and RipB proteins have been extensively studied and are dl-endopeptidases, and RipA has been considered essential to Mycobacterium smegmatis and Mycobacterium tuberculosis. We show here that the ripA and ripB genes are individually dispensable in M. smegmatis and that at least one of the genes must be expressed for viability. We characterized strains carrying in-frame deletion mutations of ripA and ripB and found that both mutant strains exhibited increased susceptibility to a limited number of antibiotics and to detergent but that only the ΔripA mutant displayed hypersusceptibility to lysozyme. We also constructed and characterized ΔripD and ΔripA ΔripD mutants and found that the single mutant had only an intermediate lysozyme hypersusceptibility phenotype compared to that of wild-type cells while loss of ripD in the ΔripA background partially rescued the antibiotic and lysozyme phenotypes of the ΔripA mutant. IMPORTANCE We show that the RipA endopeptidase, which has been considered essential for cell division in certain mycobacteria, is not essential but that at least it or a similar protein, RipB, must be expressed by the bacteria for viability. This work is the first description of strains carrying single deletion mutations of RipA, RipB, and a novel endopeptidase-like protein, RipD. PMID:26977111

  13. Glycyl endopeptidase from papaya latex: partial purification and use for production of fish gelatin hydrolysate.

    PubMed

    Karnjanapratum, Supatra; Benjakul, Soottawat

    2014-12-15

    An aqueous two-phase system (ATPS) in combination with ammonium sulphate ((NH4)2SO4) precipitation was applied to fractionate glycyl endopeptidase from the papaya latex of Red Lady and Khack Dum cultivars. ATPS containing polyethylene glycol (PEG 2000 and 6000) and salts ((NH4)2SO4 and MgSO4) at different concentrations were used. Glycyl endopeptidase with high purification fold (PF) and yield was found in the salt-rich bottom phase of ATPS with 10%PEG 6000-10% (NH4)2SO4. When ATPS fraction from Red Lady cultivar was further precipitated with 40-60% saturation of (NH4)2SO4, PF of 2.1-fold with 80.23% yield was obtained. Almost all offensive odorous compounds, particularly benzyl isothiocyanate, were removed from partially purified glycyl endopeptidase (PPGE). The fish gelatin hydrolysates prepared using PPGE showed higher ABTS radical scavenging activity and less odour, compared with those of crude extract (CE). Thus antioxidative gelatin hydrolysate with negligible undesirable odour could be prepared with the aid of PPGE. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

    PubMed

    Lipman, M L; Panda, D; Bennett, H P; Henderson, J E; Shane, E; Shen, Y; Goltzman, D; Karaplis, A C

    1998-05-29

    Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

  15. Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans.

    PubMed

    Baumgartner, B; Chrispeels, M J

    1976-07-01

    Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.The inhibitory activity against the mung bean endopeptidase is due to the presence of two inhibitors which can be separated by chromatography on Sephadex G-100. The two inhibitors have approximate molecular weights of 12,000 and smaller than 2,000 daltons. The large inhibitor coelutes with trypsin inhibitor on Sephadex G-100, but these two inhibitory activities can be separated by means of a trypsin affinity column.The inhibitory activity disappears slowly from crude extracts incubated at 6 C and more rapidly when the extracts are incubated at 25 C or 37 C. The disappearance of inhibitory activity is accompanied by a rise of the endopeptidase activity, but an examination of the kinetics of these two phenomena suggests that they are not causally related. Fractionation of the cellular organelles on sucrose gradients shows that the inhibitory activity is not associated with the protein bodies, but rather with the cytosol. Our results suggest that the endopeptidase inhibitor(s) does not regulate the increase in endopeptidase activity which accompanies germination or the metabolism of storage protein. We, therefore, postulate that the inhibitor(s) may function in protecting the cytoplasm from accidental rupturing of the protease-containing protein bodies.

  16. A Radiolabeled Fully Human Antibody to Human Aspartyl (Asparaginyl) β-Hydroxylase Is a Promising Agent for Imaging and Therapy of Metastatic Breast Cancer.

    PubMed

    Revskaya, Ekaterina; Jiang, Zewei; Morgenstern, Alfred; Bruchertseifer, Frank; Sesay, Muctarr; Walker, Susan; Fuller, Steven; Lebowitz, Michael S; Gravekamp, Claudia; Ghanbari, Hossein A; Dadachova, Ekaterina

    2017-03-01

    There is a need for novel effective and safe therapies for metastatic breast cancer based on targeting tumor-specific molecular markers of cancer. Human aspartyl (asparaginyl) β-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation-associated proteins and is overexpressed in a variety of cancers, including breast cancer. A fully human monoclonal antibody (mAb) PAN-622 has been developed to HAAH. In this study, they describe the development of PAN-622 mAb as an agent for imaging and radioimmunotherapy of metastatic breast cancer. PAN-622 was conjugated to several ligands such as DOTA, CHXA″, and DTPA to enable subsequent radiolabeling and its immunoreactivity was evaluated by an HAAH-specific enzyme-linked immunosorbent assay and binding to the HAAH-positive cells. As a result, DTPA-PAN-622 was chosen to investigate biodistribution in healthy CD-1 female mice and 4T1 mammary tumor-bearing BALB/c mice. The (111)In-DTPA-pan622 mAb concentrated in the primary tumors and to some degree in lung metastases as shown by SPECT/CT and Cherenkov imaging. A pilot therapy study with (213)Bi-DTPA-PAN-622 demonstrated a significant effect on the primary tumor. The authors concluded that human mAb PAN-622 to HAAH is a promising reagent for development of imaging and possible therapeutic agents for the treatment of metastatic breast cancer.

  17. Purification of substance P endopeptidase activity in the rat ventral tegemental area with the Akta-Purifier chromatographic system.

    PubMed

    Karlsson, K; Nyberg, F

    2000-09-29

    The new chromatographic system Akta-Purifier 10 (Amersham-Pharmacia Biotech), scaled for preparative HPLC, was used for the purification of Substance P (SP) endopeptidase activity in the ventral tegemental area (VTA) of the rat brain. SP endopeptidase previously identified and purified from human cerebrospinal fluid has been found to degrade the neuroactive peptide SP in a specific pattern. In this study we have recovered SP endopeptidase from the rat VTA following a purification scheme involving homogenization (ultrasonication) and extraction of the excised tissue, size-exclusion chromatography (Superdex 75 HR), and ion-exchange chromatography (Resource Q). In this way we were able to achieve a purification factor of almost 7,500, based on specific activity. The obtained SP endopeptidase activity, was then subjected to characterization with regard to inhibition profile. The enzyme activity was monitored by following the conversion of SP to its N-terminal fragment SP(1-7) using a radioimmunoassay, specific for the heptapeptide product. On basis of inhibition profile it was possible to discern two different SP endopeptidase-like activities, one sensitive toward the protease inhibitor phosphoramidon (preparation A), and another non-sensitive to phosphoramidon or captopril (preparation B). The molecular masses of preparations A and B, as derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were found to be 90,000 and 76,000, respectively. Our data suggest that the purified phosphoramidon sensitive endopeptidase activity may be an enzyme that plays a major role in the conversion of SP to its bioactive fragment SP(1-7) in the rat VTA. This is likely to be identical to the previously known neutral endopeptidase (EC 3.4.24.11). However, this study also demonstrates the existence of a distinct endopeptidase activity with properties in agreement with rat spinal cord SP endopeptidase. In the context of previously shown altered levels of SP(1-7) in the VTA

  18. Rat kidney endopeptidase 24.16. Purification, physico-chemical characteristics and differential specificity towards opiates, tachykinins and neurotensin-related peptides.

    PubMed

    Barelli, H; Vincent, J P; Checler, F

    1993-01-15

    Endopeptidase 24.16 was purified from rat kidney homogenate on the basis of its ability to generate the biologically inactive degradation products neurotensin (1-10) and neurotensin (11-13). On SDS gels of the proteins pooled after the last purification step, the enzyme appeared homogeneous and behaved as a 70-kDa monomer. The peptidase was not sensitive to specific inhibitors of aminopeptidases, pyroglutamyl aminopeptidase I, endopeptidase 24.11, endopeptidase 24.15, proline endopeptidase and angiotensin-converting enzyme but was potently inhibited by several metal chelators such as o-phenanthroline and EDTA and was blocked by divalent cations. The specificity of endopeptidase 24.16 towards peptides of the tachykinin, opioid and neurotensin families was examined by competition experiments of tritiated neurotensin hydrolysis as well as HPLC analysis. These results indicated that endopeptidase 24.16 could discriminate between peptides belonging to the same family. Neurotensin, Lys8-Asn9-neurotensin(8-13) and xenopsin were efficiently hydrolysed while neuromedin N and kinetensin underwent little if any proteolysis by the peptidase. Analogously, substance P and dynorphins (1-7) and (1-8) were readily proteolysed by endopeptidase 24.16 while neurokinin A, amphibian tachykinins and leucine or methionine enkephalins totally resisted degradation. By Triton X-114 phase separation, 15-20% of endopeptidase 24.16 partitioned in the detergent phase, indicating that renal endopeptidase 24.16 might exist in a genuine membrane-bound form. The equipotent solubilization of the enzyme by seven detergents of various critical miscellar concentrations confirmed the occurrence of a membrane-bound counterpart of endopeptidase 24.16. Furthermore, the absence of release elicited by phosphatidylinositol-specific phospholipase C suggested that the enzyme was not attached by a glycosyl-phosphatidylinositol anchor in the membrane of renal microvilli. Finally, endopeptidase 24.16 could not be

  19. Irreversible photolabeling of active site of neutral endopeptidase-24. 11 enkephalinase by azidothiorphan and (/sup 14/C)-azidothiorphan

    SciTech Connect

    Beaumont, A.; Hernandez, J.F.; Chaillet, P.; Crine, P.; Roques, B.P.

    1987-11-01

    Azidothiorphan and its (/sup 14/C)-labeled analogue have been developed as photoaffinity ligands for the active site of the neutral endopeptidase 24.11. In in vitro assays azidothiorphan inhibits the endopeptidase activity with a Ki of 0.75 nM. After ultraviolet irradiation the inhibitor binds irreversibly to the enzyme, and many factors suggest that the photolabeling occurs at the active site. The binding is accompanied by a loss of enzymatic activity, and the inclusion of the competitive inhibitor thiorphan protects the endopeptidase from this inactivation. In addition the binding of another competitive inhibitor (/sup 3/H)N-((R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)-glycine to the active site of endopeptidase-24.11 is inhibited after irradiation with azidothiorphan. Experiments with (/sup 14/C)-azidothiorphan have shown that very little nonspecific binding of inhibitor to enzyme occurs and the the labeled probe remains bound under denaturing conditions. Azidothiorphan has also been found to produce a long-lasting naloxone-reversible analgesia after intracerebroventricular administration. The results show that azidothiorphan should prove useful both for structural studies and for investigations on the synthesis and turnover of the neutral endopeptidase-24.11.

  20. Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay.

    PubMed Central

    Bailyes, E M; Hutton, J C

    1992-01-01

    A simple, rapid and sensitive assay for the type-1 endopeptidase (Arg-Arg cleaving) was developed by using an antiproinsulin monoclonal immunoadsorbent to separate reaction products from the substrate. The values obtained by this assay were identical with those obtained by an h.p.l.c.-based procedure and yielded similar values for the pH optimum (5.6) and Ca2+ activation (K0.5 = 2 mM). It was shown that the type-1 endopeptidase was readily solubilized by Triton X-114 (87 +/- 3%, n = 12) and partitioned principally into the aqueous phase at 30 degrees C (90.1 +/- 2.6%, n = 12). Activity was lost on gel filtration, but could be restored by adenosine 5'-[gamma-thio]triphosphate (K0.5 = 6 microM), 50 microM-dithiothreitol or 50 microM-Ca(2+)-trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid (CDTA), indicating that the enzyme was particularly sensitive to heavy metal ions. The Km obtained with proinsulin as substrate (13 +/- 1.7 microM) indicated that the enzyme works at close to its Vmax. in the nascent secretory granule. The Vmax. of the enzyme prepared from insulin granules (0.6% proinsulin converted/min) corresponded closely to the rate measured in vivo in rat islets. The type-1 endopeptidase also appears to be capable of binding to proinsulin in the region of the C-peptide/A-chain junction, since a peptide spanning this region was found to inhibit the 125I-proinsulin processing measured by this assay. PMID:1520272

  1. Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue.

    PubMed

    Shan, Lu; Marti, Thomas; Sollid, Ludvig M; Gray, Gary M; Khosla, Chaitan

    2004-10-15

    Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

  2. The role of neutral endopeptidase in dogs with evolving congestive heart failure.

    PubMed

    Margulies, K B; Barclay, P L; Burnett, J C

    1995-04-01

    Recent studies suggest that neurohumoral mechanisms including decreased renal responses to increases in atrial natriuretic factor (ANF) play a central role in the progression from asymptomatic cardiac dysfunction to advanced congestive heart failure (CHF) with sodium retention, vasoconstriction, and reduced exercise tolerance. Recognizing that neutral endopeptidase 24.11 degrades ANF and may be enhanced in CHF, we hypothesized that chronic neutral endopeptidase inhibition (NEP-I) would potentiate renal responses to exogenous ANF and alter the temporal evolution of sodium retention in evolving CHF by potentiation of increased endogenous ANF. We studied 13 conscious dogs with evolving CHF produced by rapid ventricular pacing at 250 beats per minute. Six of these dogs received NEP-I with candoxatril, 10 mg/kg PO BID, throughout evolving CHF. Responses to exogenous ANF, 10 micrograms/kg IV bolus, were assessed at baseline and after 6 days of CHF. Daily metabolic studies during evolving CHF with chronic NEP-I showed increased sodium excretion and renal cGMP generation consistent with enhanced renal activity of endogenous ANF compared with untreated controls. In addition, renal natriuretic and cGMP responses to exogenous ANF were intact in CHF with chronic NEP-I in contrast to markedly attenuated renal responses to exogenous ANF in untreated CHF. Despite enhanced ANF responsiveness and improved sodium balance in evolving CHF, a moderate degree of sodium retention was observed during chronic NEP-I in evolving CHF. Enzymatic degradation by neutral endopeptidase limits local renal responses to increases in endogenous and exogenous ANF in CHF independent of changes in systemic hemodynamics or augmented plasma concentrations of ANF. The moderate sodium retention observed during evolving CHF despite chronic NEP-I probably reflects the antinatriuretic effects of hemodynamic and humoral factors independent of ANF activity.

  3. Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics.

    PubMed Central

    Keck, W; Schwarz, U

    1979-01-01

    A novel endopeptidase degrading the peptide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. The enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of beta-lactam antibiotics. In addition, it is exceptional in being inhibited by single-stranded deoxyribonucleic acid and by some polynucleotides. The possible role of the enzyme in cell division is discussed. Images PMID:383691

  4. Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

    PubMed

    Nägler, D K; Storer, A C; Portaro, F C; Carmona, E; Juliano, L; Ménard, R

    1997-10-14

    The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.

  5. Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae).

    PubMed

    Jedličková, Lucie; Dvořáková, Hana; Kašný, Martin; Ilgová, Jana; Potěšil, David; Zdráhal, Zbyněk; Mikeš, Libor

    2016-04-01

    In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

  6. Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli.

    PubMed

    Ye, Wei; Wang, Haiying; Ma, Yi; Luo, Xiaochun; Zhang, Weimin; Wang, Jufang; Wang, Xiaoning

    2013-10-10

    Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is widely used in peptide recovery and synthesis because of its unique substrate specificity. However, the mechanism underlying its specificity is still not thoroughly understood. In this study, the roles of the prosegment and key amino acids involved in the proteolytic activity of GSE-BL were investigated. Loss of the GSE-BL prosegment severely restricted enzymatic activity toward Z-Phe-Leu-Glu-pNA. A homologous model of GSE-BL revealed that it contains the catalytic triad "His47, Asp96 and Ser 167", which was further confirmed by site-directed mutagenesis. In vitro mutagenesis further indicated that Val2, Arg89 and His190 are essential for enzymatic activity toward Z-Phe-Leu-Glu-pNA. Moreover, the catalytic efficiency of Phe57Ala GSE-BL toward Z-Phe-Leu-Glu-pNA was 50% higher than that of the native mature GSE-BL. This is the first study to fully elucidate the key amino acids for proteolytic activity of GSE-BL. Mature GSE-BL could be obtained through self-cleavage alone when Lys at -1 position was replaced by Glu, providing a new strategy for the preparation of mature GSE-BL. This study yielded some valuable insights into the substrate specificity of glutamate-specific endopeptidase, establishing a foundation for broadening the applications of GSE-BL.

  7. Occurrence of neutral endopeptidase activity in the cat carotid body and its significance in chemoreception.

    PubMed

    Kumar, G K; Runold, M; Ghai, R D; Cherniack, N S; Prabhakar, N R

    1990-05-28

    The carotid body contains both tachykinins and enkephalins. Neutral endopeptidase (NEP, E.C. 3.4.24.11), has been suggested to involve in the metabolism of these neuropeptides in several organs. In the present study we determined neutral endopeptidase activity of the cat carotid body and assessed its significance in chemoreception. The cytosolic and membrane fractions of the carotid body contained NEP-like activity whereas it occurred only in the membrane fractions of the superior cervical and the nodose ganglia. Phosphoramidon, thiorphan and metal ion chelators inhibited NEP-like activity of all the 3 tissues studied; other protease inhibitors, however, were ineffective. Close carotid body administration of phosphoramidon significantly potentiated the carotid body response to low PO2 but not to hypercapnia. The enhanced response to hypoxia following phosphoramidon was further augmented by naloxone, an enkephalin antagonist. These results demonstrate that the glomus tissue contains detectable amounts of NEP-like activity and its inhibition selectively affects the hypoxic response of the carotid body.

  8. Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae).

    PubMed

    Pardo, M F; López, L M; Canals, F; Avilés, F X; Natalucci, C L; Caffini, N O

    2000-09-01

    A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme. The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.

  9. Characterization of excretory/secretory endopeptidase and metallo-aminopeptidases from Taenia crassiceps metacestodes.

    PubMed

    Baig, Salman; Damian, Raymond T; Morales-Montor, Jorge; Olecki, Paula; Talhouk, Jamil; Hashmey, Rayhan; White, A Clinton

    2005-10-01

    Cysticercosis is caused by Taenia spp. metacestodes, which must survive in the host tissues to complete their life cycle. Their survival depends on their control of host immune responses. Because many parasites use proteases to modulate host responses, we examined culture media from Taenia crassiceps metacestodes for protease activity using peptide substrates. We identified prominent aminopeptidase activity at neutral pH, which was inhibited by chelating agents and partially inhibited by the aminopeptidase inhibitor, bestatin. Endopeptidase substrates were optimally cleaved at slightly acidic pH and endopeptidase activity was inhibited by cysteine protease inhibitors. Gel filtration FPLC and subsequent visualization by silver staining revealed a metallo-aminopeptidase of molecular weight 21 kDa and cysteine proteases of Mr 70 and 64 kDA. Recombinant IL-2 was digested when incubated with parasite culture supernatants, but not with control media. IL-2 degradation was completely inhibited by 1,10 phenanthroline and partially inhibited by bestatin, suggesting that a metallo-aminopeptidase was responsible. Incubation of human IgG with culture supernatants resulted in complete degradation of IgG, which was blocked by cysteine protease inhibitors. These observations demonstrate that Taenia spp. metacestodes secrete a number of proteolytic enzymes, which may target molecules from the host immune system and assist in evasion of the host immune response.

  10. Opioids, Neutral Endopeptidase, its Inhibitors and Cancer: Is There a Relationship among them?

    PubMed

    Mizerska-Dudka, Magdalena; Kandefer-Szerszeń, Martyna

    2015-06-01

    The role of endogenous animal opioids in the biology of cancer is widely recognized but poorly understood. This is, among others, because of the short half-life of these peptides, which are quickly inactivated by endopeptidases, e.g., neutral endopeptidase (NEP, CD10). It has been established that NEP is engaged in the modulation of the tumor microenvironment, among others that of colon cancer, by exerting influence on cell growth factors, the extracellular matrix and other biologically active substances. Although there are some discrepancies among the findings on the role of both opioids and NEP in cancer development, authors agree that their role seems to depend on the origin, stage and grade of tumor, and even on the method of examination. Moreover, recently, natural inhibitors of NEP, such as sialorphin, opiorphin and spinorphin have been detected. Their analgesic activity has been established. It is interesting to ask whether there is a relationship among opioid peptides, tumor-associated NEP and its inhibitors.

  11. Control of Storage Protein Metabolism in the Cotyledons of Germinating Mung Beans: Role of Endopeptidase 12

    PubMed Central

    Chrispeels, Maarten J.; Boulter, D.

    1975-01-01

    The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either α-mannosidase or N-acetyl-β-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination. PMID:16659204

  12. The Anti-Fibrotic Effect of Mycophenolic Acid–Induced Neutral Endopeptidase

    PubMed Central

    Dell'Oglio, Maria Pia; Rossini, Michele; Divella, Chiara; Pontrelli, Paola; Verrienti, Raffaella; Rutigliano, Monica; Ditonno, Pasquale; Stifanelli, Patrizia; Ancona, Nicola; Schena, Francesco Paolo; Grandaliano, Giuseppe

    2010-01-01

    Mycophenolic acid (MPA) appears to have anti-fibrotic effects, but the molecular mechanisms underlying this are unknown. We prospectively studied 35 stable kidney transplant recipients maintained on cyclosporine and azathioprine. We converted 20 patients from azathioprine to enteric-coated mycophenolate sodium (EC-MPS) and continued the remaining 15 patients on azathioprine. Exploratory mRNA expression profiling, performed on five randomly selected EC-MPS patients, revealed significant upregulation of neutral endopeptidase (NEP), which is an enzyme that degrades angiotensin II. We confirmed these microarray data by measuring levels of NEP expression in all subjects; in addition, we found that NEP gene expression correlated inversely with proteinuria. In an additional 33 patients, glomerular and tubular NEP protein levels from renal graft biopsies were significantly higher among the 13 patients receiving cyclosporine + EC-MPS than among the 12 patients receiving cyclosporine + azathioprine or 8 patients receiving cyclosporine alone. Glomerular NEP expression inversely correlated with glomerulosclerosis and proteinuria, and tubular NEP expression inversely correlated with interstitial fibrosis. Incubation of human proximal tubular cells with MPA increased NEP gene expression in a dose- and time-dependent manner. Moreover, MPA reduced angiotensin II–induced expression of the profibrotic factor plasminogen activator inhibitor-1, and a specific NEP inhibitor completely reversed this effect. Taken together, our data suggest that MPA directly induces expression of neutral endopeptidase, which may reduce proteinuria and slow the progression of renal damage in kidney transplant recipients. PMID:20864690

  13. Inhibition of endopeptidase and exopeptidase activity of cathepsin B impairs extracellular matrix degradation and tumour invasion.

    PubMed

    Mitrović, Ana; Mirković, Bojana; Sosič, Izidor; Gobec, Stanislav; Kos, Janko

    2016-01-01

    Cathepsin B is a lysosomal cysteine protease that is implicated in a number of physiological processes, including protein turnover in lysosomes. Changes in its expression are associated with a variety of pathological processes, including cancer. Due to the structural feature, termed the occluding loop, cathepsin B differs from other cysteine proteases in possessing both, endopeptidase and exopeptidase activity. Here we investigated the impact of both cathepsin B activities on intracellular and extracellular collagen IV degradation and tumour cell invasion using new selective synthetic inhibitors, 2-{[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3). All three compounds (5 μM) reduced extracellular degradation of collagen IV by MCF-10A neoT cells by 45-70% as determined by spectrofluorimetry and they (50 μM) attenuated intracellular collagen IV degradation by 40-60% as measured with flow cytometry. Furthermore, all three compounds (5 μM) impaired MCF-10A neoT cell invasion by 40-80% as assessed by measuring electrical impedance in real time. Compounds 1 and 3 (5 μM), but not compound 2, significantly reduced the growth of MMTV-PyMT multicellular tumour spheroids. Collectively, these data suggest that the efficient strategy to impair harmful cathepsin B activity in tumour progression may include simultaneous and potent inhibition of cathepsin B endopeptidase and exopeptidase activities.

  14. Identification of the Catalytic Triad of Family S46 Exopeptidases, Closely Related to Clan PA Endopeptidases

    NASA Astrophysics Data System (ADS)

    Suzuki, Yoshiyuki; Sakamoto, Yasumitsu; Tanaka, Nobutada; Okada, Hirofumi; Morikawa, Yasushi; Ogasawara, Wataru

    2014-03-01

    The exopeptidases of family S46 are exceptional, as the closest homologs of these enzymes are the endopeptidases of clan PA. The three-dimensional structure of S46 enzymes is unknown and only one of the catalytic residues, the serine, has been identified. The catalytic histidine and aspartate residues are not experimentally identified. Here we present phylogenetic and experimental data that identify all residues of the catalytic triad of S46 peptidase, dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24. Phylogenetic comparison with the protein and S46 peptidases, revealed His-86, Ser-657, and five aspartate residues as possible catalytic residues. Mutation studies identified the catalytic triad of DAP BII as His-86, Asp-224, and Ser-657, while secondary structure analysis predicted an extended alpha-helical domain in between Asp-224 and Ser-657. This domain is unique for family S46 exopeptidases and its absence from the endopeptidases of clan PA might be key to their different hydrolysis activities.

  15. A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap).

    PubMed

    Takahashi, Kenji; Suzuki, Takehiro; Nishii, Wataru; Kubota, Keiko; Shibata, Chiaki; Isobe, Toshiaki; Dohmae, Naoshi

    2011-01-01

    The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme.

  16. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

    PubMed Central

    Devault, A; Lazure, C; Nault, C; Le Moual, H; Seidah, N G; Chrétien, M; Kahn, P; Powell, J; Mallet, J; Beaumont, A

    1987-01-01

    Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase. Images Fig. 1. Fig. 3. PMID:2440677

  17. Effects of endopeptidase inhibition on the contraction-relaxation response of isolated human vaginal tissue.

    PubMed

    Rahardjo, Harrina E; Uckert, Stefan; Taher, Akmal; Sonnenberg, Joachim E; Kauffels, Wolfgang; Rahardjo, Djoko; Kuczyk, Markus A

    2013-04-01

    INTRODUCTION.: Vasoactive peptides, such as bradykinin, C-type natriuretic peptide (CNP), vasoactive intestinal polypeptide (VIP), and endothelin 1 (ET-1), are assumed to be involved in the control of female genital vascular and nonvascular smooth muscle. Tissue levels of said peptides are controlled by the activity of endopeptidase enzymes. Theoretically, in female genital tissues, inhibiting the degradation of bradykinin, CNP, and VIP, or the conversion of Big ET-1 into ET-1 should result in an enhancement in smooth muscle relaxation and, thus, an improvement in sexual response. AIM.: Elucidate the effects of the endopeptidase inhibitor KC 12615 on the contraction/relaxation response of isolated human vaginal smooth muscle to Big ET-1, bradykinin, CNP, or VIP. METHODS.: Tissue bath experiments were carried out to ascertain the responses of human vaginal tissue challenged by ET-1 (0.1 μM) to increasing concentrations of bradykinin, CNP, and VIP (0.01 μM, 0.1 μM, and 1 μM, respectively). The effects were also evaluated following preexposure to KC 12615 (10 μM, for 20 minutes). MAIN OUTCOME MEASURES.: Measure the effects of KC 12615 on the relaxation of isolated human vaginal smooth muscle brought about by bradykinin, CNP, or VIP and the contraction mediated by Big ET-1. RESULTS.: The tension induced by ET-1 was reversed by bradykinin, CNP, or VIP (-25 ± 6.6%, -13.3 ± 2.2%, and -17.6 ± 10%, respectively). Big ET-1 induced contraction of the vaginal tissue. Preexposure of the tissue to KC 12615 increased the relaxation exerted by bradykinin, CNP, or VIP (to -39.2 ± 5.8%, -40.7 ± 7.3%, and -44.6 ± 19%, respectively). The contraction induced by Big ET-1 was attenuated in the presence of KC 12615 (to approximately 25% of the initial response). CONCLUSION.: Inhibition of endopeptidase activity can antagonize the contraction of human vaginal tissue induced by Big ET-1 and increase the relaxation induced by vasoactive endogenous

  18. Lacking "Lack": A Reply to Joldersma

    ERIC Educational Resources Information Center

    Marshall, James D.

    2007-01-01

    First I would like to thank Clarence Joldersma for his review of our "Poststructuralism, Philosophy, Pedagogy" (Marshall, 2004-PPP). In particular, I would thank him for his opening sentence: "[t]his book is a response to a lack." It is the notion of a lack, noted again later in his review, which I wish to take up mainly in this response. Rather…

  19. Immobilization of Procerain B, a Cysteine Endopeptidase, on Amberlite MB-150 Beads

    PubMed Central

    Singh, Abhay Narayan; Singh, Sushant; Dubey, Vikash Kumar

    2013-01-01

    Proteases are involved in several crucial biological processes and reported to have important physiological functions. They also have multifarious applications in different industries. The immobilized form of the enzyme further improves its industrial applicability. Here, we report covalent immobilization of a novel cysteine endopeptidase (procerain B) on amberlite MB-150 beads through glutaraldehyde by Schiff base linkage. The immobilized product was examined extensively by Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron microscopy (SEM) and Energy Dispersive X-ray (EDX) analysis. The characterization of the immobilized product showed broader pH and thermal optima compared to the soluble form of the enzyme. The immobilized form of procerain B also showed lower Km (180.27±6 µM) compared to the soluble enzyme using azocasein as substrate. Further, immobilized procerain B retains 38.6% activity till the 10th use, which strongly represents its industrial candidature. PMID:23776589

  20. AmpH, a Bifunctional dd-Endopeptidase and dd-Carboxypeptidase of Escherichia coli▿

    PubMed Central

    González-Leiza, Silvia M.; de Pedro, Miguel A.; Ayala, Juan A.

    2011-01-01

    In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display dd-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related to AmpC β-lactamases. AmpH has been associated with PG recycling, although its enzymatic activity remained uncharacterized until now. Construction and purification of His-tagged AmpH from E. coli permitted a detailed study of its enzymatic properties. The N-terminal export signal of AmpH is processed, but the protein remains membrane associated. The PBP nature of AmpH was demonstrated by its ability to bind the β-lactams Bocillin FL (a fluorescent penicillin) and cefmetazole. In vitro assays with AmpH and specific muropeptides demonstrated that AmpH is a bifunctional dd–endopeptidase and dd-carboxypeptidase. Indeed, the enzyme cleaved the cross-linked dimers tetrapentapeptide (D45) and tetratetrapeptide (D44) with efficiencies (kcat/Km) of 1,200 M−1 s−1 and 670 M−1 s−1, respectively, and removed the terminal d-alanine from muropeptides with a C-terminal d-Ala-d-Ala dipeptide. Both dd-peptidase activities were inhibited by 40 μM cefmetazole. AmpH also displayed a weak β-lactamase activity for nitrocefin of 1.4 × 10−3 nmol/μg protein/min, 1/1,000 the rate obtained for AmpC under the same conditions. AmpH was also active on purified sacculi, exhibiting the bifunctional character that was seen with pure muropeptides. The wide substrate spectrum of the dd-peptidase activities associated with AmpH supports a role for this protein in PG remodeling or recycling. PMID:22001512

  1. Natriuretic peptide pharmacogenetics: membrane metallo-endopeptidase (MME): common gene sequence variation, functional characterization and degradation.

    PubMed

    Pereira, Naveen L; Aksoy, Pinar; Moon, Irene; Peng, Yi; Redfield, Margaret M; Burnett, John C; Wieben, Eric D; Yee, Vivien C; Weinshilboum, Richard M

    2010-11-01

    Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Structural basis for type VI secreted peptidoglycan dl-endopeptidase function, specificity and neutralization in Serratia marcescens

    SciTech Connect

    Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai; Trunk, Katharina; O’Rourke, Patrick E. F.; Rao, Vincenzo A.; Vollmer, Waldemar; Coulthurst, Sarah J. Hunter, William N.

    2013-12-01

    Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment. Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2

  3. Purification and characterization of human endopeptidase 3.4.24.16. Comparison with the porcine counterpart indicates a unique cleavage site on neurotensin.

    PubMed

    Vincent, B; Vincent, J P; Checler, F

    1996-02-12

    We have purified and characterized human brain endopeptidase 3.4.24.16. The enzyme behaved as a 72 kDa protein and belonged to the metalloprotease family. Human endopeptidase 3.4.24.16 cleaved neurotensin at a unique site at the Pro10-Tyr11 bond, leading to the formation of neurotensin(1-10) and neurotensin(11-13). The kinetic parameters displayed by human endopeptidase 3.4.24.16 towards a series of natural neuropeptides indicated that bradykinin was the most efficiently proteolysed. Angiotensin I, dynorphins 1-8 and 1-9 and substance P also behaved as good substrates while neuromedin N, angiotensin II, leucine and methionine enkephalin and neurokinin A resisted degradation by human endopeptidase 3.4.24.16. We have purified the porcine counterpart of endopeptidase 3.4.24.16 and compared its ability to cleave neurotensin with that of the enzyme from human origin. It appeared that, besides a major production of neurotensin(1-10), an additional formation of neurotensin(1-8) was observed with the pig enzyme, suggesting a cleavage of neurotensin not only at the Pro10-Tyr11 bond but also at the Arg8-Arg9 peptidyl bond. The latter cleavage appeared reminiscent of endopeptidase 3.4.24.15 since this peptidase was reported to cleave neurotensin at the Arg8-Arg9 bond. Our study indicated that neurotensin(1-10) formation by porcine endopeptidase 3.4.24.16 could be potently blocked with the selective endopeptidase 3.4.24.16 dipeptide inhibitor Pro-Ile without interfering with neurotensin(1-8) formation. By contrast, the formation of the latter product was highly potentiated by dithiothreitol and inhibited by the endopeptidase 3.4.24.15 inhibitor Cpp-Ala-Ala-Tyr-pAB, two effects that were not observed for neurotensin(1-10) production. Altogether, our results indicate that porcine endopeptidase 3.4.24.16 cleaves neurotensin at a unique site, leading to the formation of neurotensin(1-10) and that the production of neurotensin(1-8) is due to contaminating endopeptidase 3.4.24.15.

  4. Human endopeptidase (THOP1) is localized on chromosome 19 within the linkage region for the late-onset Alzheimer disease AD2 locus

    SciTech Connect

    Meckelein, B.; Abraham, C.R.; De Silva, H.A.R.

    1996-01-15

    A cDNA encoding the rat endopeptidase 24.15 was used to determine the chromosomal localization of the respective human gene. Hybridization to DNA from human-rodent somatic cell hybrids assigned the human gene to chromosome 19. Fluorescence in situ hybridization on human metaphase chromosomes localized the human endopeptidase 24.15 to 19q13.3. 27 refs., 1 fig., 1 tab.

  5. Purification and characterization of four new cysteine endopeptidases from fruits of Bromelia pinguin L. grown in Cuba.

    PubMed

    Payrol, Juan Abreu; Obregón, Walter D; Trejo, Sebastián A; Caffini, Néstor O

    2008-02-01

    Bromelia pinguin L. is a plant broadly distributed in Central America and Caribbean islands. The fruits have been used in traditional medicine as anthelmintic, probably owed to the presence of a mixture of cysteine endopeptidases, initially termed pinguinain. This work deals with the purification and characterization of the four main components of that mixture, two of them showing acid pI and the other two alkaline pI. Molecular masses (SDS-PAGE and MALDI-TOF), N-terminal sequence and the reactivity and kinetic parameters versus synthetic substrates (p-nitrophenyl-N-alpha-CBZ-amino acid esters, PFLNA, Z-Arg-Arg-p-NA, and Z-Phe-Arg-p-NA) of the studied peptidases are given, as well as the N-terminal sequences of the enzymes and the homology degree with other plant endopeptidases.

  6. The assignment of a Thinopyrum distichum (Thunb.) Löve-derived translocation to the long arm of wheat chromosome 7D using endopeptidase polymorphisms.

    PubMed

    Marais, G F; Marais, A S

    1990-02-01

    Endopeptidase zymograms of the translocation line 'Indis' revealed the presence of several major and minor bands that had differential expression in coleoptile and seed tissues. While 'Indis' lacks Ep-D1a, which is present in the parental cultivar 'Inia 66', it also may not express any of the Th. distichum bands. The 'Indis' zymogram was found to be identical to that of an isogenic line of 'Inia 66' possessing Lr19. Since the absence of an Ep-D1a product appears to be linked to the 7DL translocation, it is possible to use the null condition as a marker for both the Lr19 or 'Indis' translocations. The 'Indis' translocation also did not show recombination with the cn-D1 chlorophyl mutant on 7DL, confirming that a part of 7D was involved. The results of a telocentric mapping experiment involving the 7D telosomes indicated that in 'Indis' a chromosome segment from Th. distichum replaced a large section of 7DL of 'Inia 66'.

  7. Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

    PubMed

    O'Donoghue, Anthony J; Knudsen, Giselle M; Beekman, Chapman; Perry, Jenna A; Johnson, Alexander D; DeRisi, Joseph L; Craik, Charles S; Bennett, Richard J

    2015-06-16

    Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

  8. Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome

    PubMed Central

    O’Donoghue, Anthony J.; Knudsen, Giselle M.; Beekman, Chapman; Perry, Jenna A.; Johnson, Alexander D.; DeRisi, Joseph L.; Craik, Charles S.; Bennett, Richard J.

    2015-01-01

    Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host. PMID:25944934

  9. Effects of endopeptidase inhibition on the relaxation response of isolated human penile erectile tissue to vasoactive peptides.

    PubMed

    Rahardjo, H E; Reichelt, K; Sonnenberg, J E; Sohn, M; Kuczyk, M A; Ückert, S

    2016-12-01

    Peptides, such as CNP, CGRP and VIP, are involved in the function of male penile erectile tissue. Tissue levels of said peptides are controlled by the endopeptidase enzymes. Theoretically, the inhibition of the degradation of CNP, CGRP and/or VIP should result in an enhancement in penile smooth muscle relaxation. The effects were investigated of CNP or VIP (0.1 nm-1 μm), without and following pre-exposure of the tissue to a threshold concentration of the endopeptidase inhibitor KC 12615 (10 μm, for 20 min), on the reversion of tension induced by means of electrical field stimulation. Drug effects on the production of cyclic AMP/GMP were also evaluated. Neither KC 12615, CNP and VIP nor the combination of CNP plus KC 12615 or VIP plus KC 12615 increased the response of the tissue to EFS. While no effects were observed of a pre-exposure of the tissue to KC 12615 on the production of cyclic AMP in the presence of VIP, an enhancement was registered in the accumulation of cyclic AMP in the presence of CNP plus KC 12615. Further studies are indicated to investigate whether endopeptidase inhibitors might tend to be more effective in tissues affected by a decreased local production of vasoactive peptides. © 2016 Blackwell Verlag GmbH.

  10. A Comprehensive Review of the Pharmacodynamics, Pharmacokinetics, and Clinical Effects of the Neutral Endopeptidase Inhibitor Racecadotril

    PubMed Central

    Eberlin, Marion; Mück, Tobias; Michel, Martin C.

    2012-01-01

    Racecadotril, via its active metabolite thiorphan, is an inhibitor of the enzyme neutral endopeptidase (NEP, EC 3.4.24.11), thereby increasing exposure to NEP substrates including enkephalins and atrial natriuretic peptide (ANP). Upon oral administration racecadotril is rapidly and effectively converted into the active metabolite thiorphan, which does not cross the blood–brain-barrier. Racecadotril has mainly been tested in animal models and patients of three therapeutic areas. As an analgesic the effects of racecadotril across animal models were inconsistent. In cardiovascular diseases such as hypertension or congestive heart failure results from animal studies were promising, probably related to increased exposure to ANP, but clinical results have not shown substantial therapeutic benefit over existing treatment options in cardiovascular disease. In contrast, racecadotril was consistently effective in animal models and patients with various forms of acute diarrhea by inhibiting pathologic (but not basal) secretion from the gut without changing gastro-intestinal transit time or motility. This included studies in both adults and children. In direct comparative studies with loperamide in adults and children, racecadotril was at least as effective but exhibited fewer adverse events in most studies, particularly less rebound constipation. Several guidelines recommend the use of racecadotril as addition to oral rehydration treatment in children with acute diarrhea. PMID:22661949

  11. ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy.

    PubMed

    Erpenbeck, Luise; Demers, Melanie; Zsengellér, Zsuzsanna K; Gallant, Maureen; Cifuni, Stephen M; Stillman, Isaac E; Karumanchi, S Ananth; Wagner, Denisa D

    2016-01-01

    Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.

  12. Hypothalamic prolyl endopeptidase (PREP) regulates pancreatic insulin and glucagon secretion in mice

    PubMed Central

    Kim, Jung Dae; Toda, Chitoku; D’Agostino, Giuseppe; Zeiss, Caroline J.; DiLeone, Ralph J.; Elsworth, John D.; Kibbey, Richard G.; Chan, Owen; Harvey, Brandon K.; Richie, Christopher T.; Savolainen, Mari; Myöhänen, Timo; Jeong, Jin Kwon; Diano, Sabrina

    2014-01-01

    Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prepgt/gt) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prepgt/gt and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prepgt/gt and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prepgt/gt mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus–PREP reversed the glucose-intolerant phenotype of the Prepgt/gt mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function. PMID:25071172

  13. The role of neutral endopeptidase in caerulein-induced acute pancreatitis.

    PubMed

    Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

    2011-11-15

    Substance P (SP) is well known to promote inflammation in acute pancreatitis (AP) by interacting with neurokinin-1 receptor. However, mechanisms that terminate SP-mediated responses are unclear. Neutral endopeptidase (NEP) is a cell-surface enzyme that degrades SP in the extracellular fluid. In this study, we examined the expression and the role of NEP in caerulein-induced AP. Male BALB/c mice (20-25 g) subjected to 3-10 hourly injections of caerulein (50 μg/kg) exhibited reduced NEP activity and protein expression in the pancreas and lungs. Additionally, caerulein (10(-7) M) also downregulated NEP activity and mRNA expression in isolated pancreatic acinar cells. The role of NEP in AP was examined in two opposite ways: inhibition of NEP (phosphoramidon [5 mg/kg] or thiorphan [10 mg/kg]) followed by 6 hourly caerulein injections) or supplementation with exogenous NEP (10 hourly caerulein injections, treatment of recombinant mouse NEP [1 mg/kg] during second caerulein injection). Inhibition of NEP raised SP levels and exacerbated inflammatory conditions in mice. Meanwhile, the severity of AP, determined by histological examination, tissue water content, myeloperoxidase activity, and plasma amylase activity, was markedly better in mice that received exogenous NEP treatment. Our results suggest that NEP is anti-inflammatory in caerulein-induced AP. Acute inhibition of NEP contributes to increased SP levels in caerulein-induced AP, which leads to augmented inflammatory responses in the pancreas and associated lung injury.

  14. Post-proline cleaving enzyme. Purification of this endopeptidase by affinity chromatography.

    PubMed

    Koida, M; Walter, R

    1976-12-10

    The endopeptidase, post-proline cleaving enzyme, has been purified 10,500-fold in an overall yield of 18% from lamb kidney. The enzyme possesses a specific activity of 45 mumol/mg/min as tested with the substrate Z-Gly-Pro-Leu-Gly (Km = 6.0 X 10(-5)), has a molecular weight of 115,000, is comprised of two subunits with a molecular weight of 57,000, and exhibits maximal activity at pH 7.5 to 8.0. With the exception of the -Pro-Pro linkage, the -Pro-X-peptide bond (X equals L- and D-amino acid residues) located internally in the peptide sequence can be hydrolyzed (cleavage occurs faster when X = lipophilic side chain as compared to X = acidic side chain). The appropriate -Pro-X- bonds in zinc-free porcine insulin, oxytocin, arginine vasopressin, angiotensin II, bradykinin-potentiating factor were cleaved. Human gastrin, adrenocorticotropic hormone, denatured guinea pig skin collagen, and ascaris cuticle collagen were not degraded. Dipeptides with the structure Z-Pro-LD-X competitively inhibit post-proline cleaving enzyme.

  15. Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase

    PubMed Central

    Walther, Thomas; Albrecht, Doris; Becker, Matthias; Schubert, Manja; Kouznetsova, Elena; Wiesner, Burkard; Maul, Björn; Schliebs, Reinhard; Grecksch, Gisela; Furkert, Jens; Sterner-Kock, Anja; Schultheiss, Heinz-Peter; Becker, Axel; Siems, Wolf-Eberhard

    2009-01-01

    Background Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-β (Aβ) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice. Methodology/Principal Findings We found that while endogenous Aβ concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Aβ deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Aβ degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice. Conclusions/Significance Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases. PMID:19240795

  16. Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

    PubMed Central

    Skolovsky, M; Galron, R; Kloog, Y; Bdolah, A; Indig, F E; Blumberg, S; Fleminger, G

    1990-01-01

    Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu6 bond, followed by cleavage at the amino side of Ile19. The nicked peptide exhibited biochemical activities comparable to those of the intact peptide--i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was approximately 1 hr, while it was approximately 4 hr for SRTX-b and even higher for SRTX-c. These in vitro findings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs. Images PMID:2191299

  17. Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

    PubMed

    Skolovsky, M; Galron, R; Kloog, Y; Bdolah, A; Indig, F E; Blumberg, S; Fleminger, G

    1990-06-01

    Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu6 bond, followed by cleavage at the amino side of Ile19. The nicked peptide exhibited biochemical activities comparable to those of the intact peptide--i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was approximately 1 hr, while it was approximately 4 hr for SRTX-b and even higher for SRTX-c. These in vitro findings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs.

  18. Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase.

    PubMed

    Alvarez-Sieiro, Patricia; Martin, Maria Cruz; Redruello, Begoña; Del Rio, Beatriz; Ladero, Victor; Palanski, Brad A; Khosla, Chaitan; Fernandez, Maria; Alvarez, Miguel A

    2014-08-01

    Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.

  19. No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population.

    PubMed

    Diosdado, Begoña; Stepniak, Dariusz T; Monsuur, Alienke J; Franke, Lude; Wapenaar, Martin C; Mearin, Maria Luisa; Koning, Frits; Wijmenga, Cisca

    2005-09-01

    Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain approximately 40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21-22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.

  20. Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

    PubMed Central

    Alvarez-Sieiro, Patricia; Martin, Maria Cruz; Redruello, Begoña; del Rio, Beatriz; Ladero, Victor; Palanski, Brad A.; Khosla, Chaitan; Fernandez, Maria; Alvarez, Miguel A.

    2015-01-01

    Prolyl endopeptidases (PEP), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in a future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients. PMID:24752841

  1. Structural Basis of Murein Peptide Specificity of a γ-D-glutamyl-L-diamino Acid Endopeptidase

    PubMed Central

    Xu, Qingping; Sudek, Sebastian; McMullan, Daniel; Miller, Mitchell D.; Geierstanger, Bernhard; Jones, David H.; Sri Krishna, S.; Spraggon, Glen; Bursalay, Badry; Abdubek, Polat; Acosta, Claire; Ambing, Eileen; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Caruthers, Jonathan; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Elias, Ylva; Elsliger, Marc-Andre; Feuerhelm, Julie; Grzechnik, Slawomir K.; Hale, Joanna; Han, Gye Won; Haugen, Justin; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Trout, Christina V.; van den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Wolf, Guenter; Zubieta, Chloe; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    Crystal structures of two homologous peptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme at 1.05 Å and 1.60 Å resolution represent the first structures of a large class of cell-wall, cysteine peptidases that contain an N-terminal bacterial SH3-like domain (SH3b) and a C-terminal NlpC/P60 cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that that these two proteins act as γ-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site. PMID:19217401

  2. Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid.

    PubMed

    Risør, Michael W; Thomsen, Line R; Sanggaard, Kristian W; Nielsen, Tania A; Thøgersen, Ida B; Lukassen, Marie V; Rossen, Litten; Garcia-Ferrer, Irene; Guevara, Tibisay; Scavenius, Carsten; Meinjohanns, Ernst; Gomis-Rüth, F Xavier; Enghild, Jan J

    2016-01-29

    Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.

  3. Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid*

    PubMed Central

    Risør, Michael W.; Thomsen, Line R.; Sanggaard, Kristian W.; Nielsen, Tania A.; Thøgersen, Ida B.; Lukassen, Marie V.; Rossen, Litten; Garcia-Ferrer, Irene; Guevara, Tibisay; Scavenius, Carsten; Meinjohanns, Ernst; Gomis-Rüth, F. Xavier; Enghild, Jan J.

    2016-01-01

    Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms. PMID:26627834

  4. Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

    PubMed Central

    Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

    2011-01-01

    The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

  5. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

    PubMed

    Rioli, Vanessa; Gozzo, Fabio C; Heimann, Andrea S; Linardi, Alessandra; Krieger, José E; Shida, Cláudio S; Almeida, Paulo C; Hyslop, Stephen; Eberlin, Marcos N; Ferro, Emer S

    2003-03-07

    Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.

  6. Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization.

    PubMed

    Marti, Thomas; Molberg, Oyvind; Li, Qing; Gray, Gary M; Khosla, Chaitan; Sollid, Ludvig M

    2005-01-01

    Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten intake in genetically susceptible individuals. It has been suggested that prolyl endopeptidases (PEPs) may be useful catalysts for gluten detoxification. We have investigated this hypothesis using food-grade gluten as the target antigen, and a combination of mass spectrometry and patient-derived T cells as quantitative assay systems. Spectrometric characterization of physiologically proteolyzed gluten revealed a number of 10 to 50 residue peptides containing known T cell epitopes involved in Celiac Sprue pathogenesis. Several of these peptides were multivalent, suggesting they may be potent triggers of the inflammatory response to gluten in celiac patients. Treatment of proteolyzed gluten with recombinant bacterial PEP decreased the number of potentially immunostimulatory peptides. Substantially reduced immunogenicity was also quantified in 12 of 14 intestinal polyclonal T cell lines from celiac patients. Kinetic investigations using eight T cell clones showed rapid destruction of alpha-gliadin epitopes, but less complete processing of gamma-gliadin epitopes. Given the difficulty associated with a strict lifelong gluten-exclusion diet, the ability of a single enzyme to greatly reduce the antigenic burden of grocery store gluten reinforces the case for developing oral peptidase therapy against Celiac Sprue.

  7. Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

    PubMed

    Szul, Tomasz; Bratcher, Preston E; Fraser, Kyle B; Kong, Michele; Tirouvanziam, Rabindra; Ingersoll, Sarah; Sztul, Elizabeth; Rangarajan, Sunil; Blalock, J Edwin; Xu, Xin; Gaggar, Amit

    2016-03-01

    Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

  8. Funastrain c II: a cysteine endopeptidase purified from the latex of Funastrum clausum.

    PubMed

    Morcelle, Susana R; Trejo, Sebastián A; Canals, Francesc; Avilés, Francesc X; Priolo, Nora S

    2004-04-01

    A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.

  9. Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases.

    PubMed

    Oliveira, Lilian C G; Silva, Vinícius O; Okamoto, Debora N; Kondo, Marcia Y; Santos, Saara M B; Hirata, Isaura Y; Vallim, Marcelo A; Pascon, Renata C; Gouvea, Iuri E; Juliano, Maria A; Juliano, Luiz

    2012-02-01

    Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abz (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. The peptide libraries MCA-GXXX↓XXQ-EDDnp and MCA-GXXZ↓XXQ-EDDnp were cleaved as indicated (↓) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. The best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S(1) subsite. The pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. The FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis.

  10. Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

    PubMed Central

    Nykanen, M.; Saarelainen, R.; Raudaskoski, M.; Nevalainen, K.; Mikkonen, A.

    1997-01-01

    Localization of expression and secretion of a heterologous barley cysteine endopeptidase (EPB) and the homologous main cellobiohydrolase I (CBHI) in a Trichoderma reesei transformant expressing both proteins were studied. The transformant was grown on solid medium with Avicel cellulose and lactose to induce the cbh1 promoter for the synthesis of the native CBHI and the recombinant barley protein linked to a cbh1 expression cassette. Differences in localization of expression between the two proteins were clearly indicated by in situ hybridization, indirect immunofluorescence, and immunoelectron microscopy. In young hyphae, native-size recombinant epb mRNA was localized to apical compartments. In older cultures, it was also seen in subapical compartments but not in hyphae from the colony center. The recombinant EPB had a higher molecular weight than the native barley protein, probably due to glycosylation and differential processing in the fungal host. As was found with its transcripts, recombinant EPB was localized in apical and subapical compartments of hyphae. The cbh1 mRNA and CBHI were both localized to all hyphae of a colony, which suggests that the endogenous CBHI was also secreted from these. In immunoelectron microscopy, the endoplasmic reticulum and spherical vesicles assumed to contribute to secretion were labeled by both CBHI and EPB antibodies while only CBHI was localized in elongated vesicles close to the plasma membrane and in hyphal walls. The results indicate that in addition to young apical cells, more mature hyphae in a colony may secrete proteins. PMID:16535755

  11. Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin

    PubMed Central

    Burns, D M; Walker, B; Gray, J; Nelson, J

    1999-01-01

    We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP18–27, and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml−;1). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth. © 1999 Cancer Research Campaign PMID:9888460

  12. PepO, a CovRS-controlled endopeptidase, disrupts Streptococcus pyogenes quorum sensing.

    PubMed

    Wilkening, Reid V; Chang, Jennifer C; Federle, Michael J

    2016-01-01

    Group A Streptococcus (GAS, Streptococcus pyogenes) is a human-restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two-component system and the Rgg2/3 quorum-sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg(2+) and a fragment of the antimicrobial peptide LL-37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP-type quorum-sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non-virulent GAS lifestyles. © 2015 John Wiley & Sons Ltd.

  13. PepO, a CovRS-controlled endopeptidase, disrupts Streptococcus pyogenes quorum sensing

    PubMed Central

    Wilkening, Reid V.; Chang, Jennifer C.; Federle, Michael J.

    2016-01-01

    Summary Group A Streptococcus (GAS, Streptococcus pyogenes) is a human-restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two-component system and the Rgg2/3 quorum-sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg2+ and a fragment of the antimicrobial peptide LL-37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP-type quorum-sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non-virulent GAS lifestyles. PMID:26418177

  14. Role of endopeptidase 3.4.24.16 in the catabolism of neurotensin, in vivo, in the vascularly perfused dog ileum.

    PubMed

    Barelli, H; Fox-Threlkeld, J E; Dive, V; Daniel, E E; Vincent, J P; Checler, F

    1994-05-01

    1. The degradation of tritiated and unlabelled neurotensin (NT) following close intra-arterial infusion of the peptides in ileal segments of anaesthetized dogs was examined. 2. Intact NT and its catabolites recovered in the venous effluents were purified by chromatography on Sep-Pak columns followed by reverse-phase h.p.l.c. and identified by their retention times or by radioimmunoassay. 3. The half-life of neurotensin was estimated to be between 2 and 6 min. Four labelled catabolites, corresponding to free tyrosine, neurotensin (1-8), neurotensin (1-10) and neurotensin (1-11), were detected. 4. Neurotensin (1-11) was mainly generated by a phosphoramidon-sensitive cleavage, probably elicited by endopeptidase 24-11. 5. Two endopeptidase 3.4.24.16 inhibitors, phosphodiepryl 03 and the dipeptide Pro-Ile, dose-dependently potentiated the recovery of intact neurotensin. Furthermore, both agents inhibited the formation of neurotensin (1-10), the product that results from the hydrolysis of neurotensin by purified endopeptidase 3.4.24.16. In contrast, the endopeptidase 3.4.24.15 inhibitor Cpp-AAY-pAB neither protected neurotensin from degradation nor modified the production of neurotensin (1-10). 6. Our study is the first evidence to indicate that endopeptidase 3.4.24.16 contributes to the catabolism of neurotensin, in vivo, in the dog intestine.

  15. A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis.

    PubMed

    Spencer, Brian; Verma, Inder; Desplats, Paula; Morvinski, Dinorah; Rockenstein, Ed; Adame, Anthony; Masliah, Eliezer

    2014-06-20

    Alzheimer disease (AD) is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of amyloid-β peptide (Aβ) in the neuropil and around the blood vessels, and formation of neurofibrillary tangles. The endopeptidase neprilysin has been successfully used to reduce the accumulation of Aβ following intracranial viral vector delivery or ex vivo manipulated intracranial delivery. These therapies have relied on direct injections into the brain, whereas a clinically desirable therapy would involve i.v. infusion of a recombinant enzyme. We previously characterized a recombinant neprilysin that contained a 38-amino acid brain-targeting domain. Recombinant cell lines have been generated expressing this brain-targeted enzyme (ASN12). In this report, we characterize the ASN12 recombinant protein for pharmacology in a mouse as well as efficacy in two APPtg mouse models of AD. The recombinant ASN12 transited to the brain with a t½ of 24 h and accumulated to 1.7% of injected dose at 24 h following i.v. delivery. We examined pharmacodynamics in the tg2576 APPtg mouse with the prion promoter APP695 SWE mutation and in the Line41 mThy1 APP751 mutation mouse. Treatment of either APPtg mouse resulted in reduced Aβ, increased neuronal synapses, and improved learning and memory. In addition, the Line41 APPtg mice showed increased levels of C-terminal neuropeptide Y fragments and increased neurogenesis. These results suggest that the recombinant brain-targeted neprilysin, ASN12, may be an effective treatment for AD and warrant further investigation in clinical trials.

  16. The neuronal endopeptidase ECEL1 is associated with a distinct form of recessive distal arthrogryposis.

    PubMed

    Dieterich, Klaus; Quijano-Roy, Susana; Monnier, Nicole; Zhou, Jie; Fauré, Julien; Smirnow, Daniela Avila; Carlier, Robert; Laroche, Cécile; Marcorelles, Pascale; Mercier, Sandra; Mégarbané, André; Odent, Sylvie; Romero, Norma; Sternberg, Damien; Marty, Isabelle; Estournet, Brigitte; Jouk, Pierre-Simon; Melki, Judith; Lunardi, Joël

    2013-04-15

    Distal arthrogryposis (DA) is a heterogeneous subgroup of arthrogryposis multiplex congenita (AMC), a large family of disorders characterized by multiple congenital joint limitations due to reduced fetal movements. DA is mainly characterized by contractures afflicting especially the distal extremities without overt muscular or neurological signs. Although a limited number of genes mostly implicated in the contractile apparatus have been identified in DA, most patients failed to show mutations in currently known genes. Using a pangenomic approach, we demonstrated linkage of DA to chromosome 2q37 in two consanguineous families and the endothelin-converting enzyme like 1 (ECEL1) gene present in this region was associated with DA. Screening of a panel of 20 families with non-specific DA identified seven homozygous or compound heterozygous mutations of ECEL1 in a total of six families. Mutations resulted mostly in the absence of protein. ECEL1 is a neuronal endopeptidase predominantly expressed in the central nervous system and brain structures during fetal life in mice and human. ECEL1 plays a major role in intramuscular axonal branching of motor neurons in skeletal muscle during embryogenesis. A detailed review of clinical findings of DA patients with ECEL1 mutations revealed a homogeneous and recognizable phenotype characterized by limited knee flexion, flexed third to fifth fingers and severe muscle atrophy predominant on lower limbs and tongue that suggested a common pathogenic mechanism. We described a new and homogenous phenotype of DA associated with ECEL1 that resulted in symptoms involving rather the peripheral than the central nervous system and suggesting a developmental dysfunction.

  17. Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea

    PubMed Central

    Prinsen, Michael J.; Oliva, Jonathan; Campbell, Mary A.; Arnett, Stacy D.; Tajfirouz, Deena; Ruminski, Peter G.; Yu, Ying; Bond, Brian R.; Ji, Yuhua; Neckermann, Georg; Choy, Robert K. M.; de Hostos, Eugenio; Meyers, Marvin J.

    2016-01-01

    Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil–treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy. PMID:26907621

  18. Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1.

    PubMed Central

    Sugai, M; Fujiwara, T; Akiyama, T; Ohara, M; Komatsuzawa, H; Inoue, S; Suginaka, H

    1997-01-01

    A novel staphylolytic enzyme, ALE-1, acting on Staphylococcus aureus, was purified from a Staphylococcus capitis EPK1 culture supernatant. The optimal pH range for staphylolytic activity was 7 to 9. ALE-1 contains one Zn2+ atom per molecule. Analysis of peptidoglycan fragments released by ALE-1 indicated that the enzyme is a glycylglycine endopeptidase. The effects of various modulators were determined, and we found that o-phenanthroline, iodoacetic acid, diethylpyrocarbonate, and Cu2+ reduced the staphylolytic activity of ALE-1. beta-Casein, elastin, and pentaglycine were poor substrates for ALE-1. Molecular cloning data revealed that ALE-1 is composed of 362 amino acid residues and is synthesized as a precursor protein which is cleaved after Ala at position 35, thus producing a mature ALE-1 of 35.6 kDa. The primary structure of mature ALE-1 is very similar to the proenzyme form of lysostaphin. It has the modular design of an N-terminal domain of tandem repeats of a 13-amino-acid sequence fused to the active site containing C-terminal domain. Unlike lysostaphin, ALE-1 does not undergo processing of the N-terminal repeat domain in broth culture. ale-1 is encoded on the plasmid. Protein homology search suggested that ALE-1 and lysostaphin are members of the novel Zn2+ protease family with a homologous 38-amino-acid-long motif, Tyr-X-His-X(11)-Val-X(12/20)-Gly-X(5-6)-His. PMID:9023202

  19. Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity

    PubMed Central

    Rougeot, Catherine; Messaoudi, Michaël; Hermitte, Véronique; Rigault, Anne Gaëlle; Blisnick, Thierry; Dugave, Christophe; Desor, Didier; Rougeon, François

    2003-01-01

    Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4–1 μM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100–200 μg/kg doses of sialorphin required the activation of μ- and δ-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules. PMID:12835417

  20. Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein.

    PubMed

    Wong, Jaslyn E M M; Alsarraf, Husam M A B; Kaspersen, Jørn Døvling; Pedersen, Jan Skov; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2014-02-01

    Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1-4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate-binding site in LysM-containing proteins is conserved across phyla. © 2013 FEBS.

  1. Substance P and neutral endopeptidase in development of acute respiratory distress syndrome following fire smoke inhalation.

    PubMed

    Wong, Simon S; Sun, Nina N; Lantz, R Clark; Witten, Mark L

    2004-10-01

    To characterize the tachykininergic effects in fire smoke (FS)-induced acute respiratory distress syndrome (ARDS), we designed a series of studies in rats. Initially, 20 min of FS inhalation induced a significant increase of substance P (SP) in bronchoalveolar lavage fluid (BALF) at 1 h and persisted for 24 h after insult. Conversely, FS disrupted 51.4, 55.6, 46.3, and 43.0% enzymatic activity of neutral endopeptidase (NEP, a primary hydrolyzing enzyme for SP) 1, 6, 12, and 24 h after insult, respectively. Immunolabeling density of NEP in the airway epithelium largely disappeared 1 h after insult due to acute cell damage and shedding. These changes were also accompanied by extensive influx of albumin and granulocytes/lymphocytes in BALF. Furthermore, levels of BALF SP and tissue NEP activity dose dependently increased and decreased, respectively, following 0, low (10 min), and high (20 min) levels of FS inhalation. However, neither the time-course nor the dose-response study observed a significant change in the highest affinity neurokinin-1 receptor (NK-1R) for SP. Finally, treatment (10 mg/kg im) with SR-140333B, an NK-1R antagonist, significantly prevented 20-min FS-induced hypoxemia and pulmonary edema 24 h after insult. Further examination indicated that SR-140333B (1.0 or 10.0 mg/kg im) fully abolished early (1 h) plasma extravasation following FS. Collectively, these findings suggest that a combination of sustained SP and NEP inactivity induces an exaggerated neurogenic inflammation mediated by NK-1R, which may lead to an uncontrolled influx of protein-rich edema fluid and cells into the alveoli as a consequence of increased vascular permeability.

  2. The Role of Opiorphins (Endogenous Neutral Endopeptidase Inhibitors) in Urogenital Smooth Muscle Biology

    PubMed Central

    Davies, Kelvin Paul

    2010-01-01

    Introduction The opiorphins are a newly characterized class of peptides that act as potent endogenous neutral endopeptidase (NEP) inhibitors. Recent reports have suggested that they play an important role in erectile physiology. Aim This article reviews recent developments that increase our understanding of the role of the opiorphin family of peptides in erectile physiology. Methods During a microarray screen of gene changes that occur in a rat diabetic model of erectile dysfunction (ED), Vcsa1 was one of the most down-regulated genes in the rat corpora. Quantitative real-time polymerase chain reaction demonstrated that in at least three models of diseases that result in ED (diabetes, aging, and cavernous nerve [CN] transection), Vcsa1 was down-regulated in the rat corpora. The human opiorphin family of genes (hSMR3A/B and ProL1) also acts as markers of erectile function in patients with ED. Main Outcome Measures The reader will be informed of the most current research regarding the role of opiorphins in urogenital smooth muscle biology. Results These observations led to the suggestion that genes encoding opiorphins (and potentially their peptide products) can act as markers of ED. Gene transfer of plasmids overexpressing Vcsa1 in aging rats, as well as intracorporal injection of sialorphin, led to an improvement in erectile function. In organ bath studies, we demonstrated that sialorphin can cause increased rates of relaxation of corporal smooth muscle (CSM). We have also demonstrated that in vitro, Vcsa1 causes changes in the expression of G-protein-coupled receptors (GPCRs). This has led us to suggest that the action of Vcsa1 on erectile physiology may act through relaxation of CSM by its ability to act as an inhibitor of NEP, therefore prolonging the action of peptide agonists at their GPCRs. Conclusions Overall, there is a growing body of evidence that the opiorphins play a role in regulating CSM tone and thereby erectile function. PMID:19267851

  3. Rational redesign of neutral endopeptidase binding to merlin and moesin proteins.

    PubMed

    Niv, Masha Y; Iida, Katsuyuki; Zheng, Rong; Horiguchi, Akio; Shen, Ruoqian; Nanus, David M

    2009-05-01

    Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein-protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K(19)K(20)K(21) basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E(159)Q(160) in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N(159)L(160) residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N(159)L(160)Y(161)K(162)M(163) in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide-merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function.

  4. Structural basis for type VI secreted peptidoglycan dl-endopeptidase function, specificity and neutralization in Serratia marcescens

    PubMed Central

    Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai; Trunk, Katharina; O’Rourke, Patrick E. F.; Rao, Vincenzo A.; Vollmer, Waldemar; Coulthurst, Sarah J.; Hunter, William N.

    2013-01-01

    Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions. PMID

  5. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family.

    PubMed

    Bennett, D L; Bailyes, E M; Nielsen, E; Guest, P C; Rutherford, N G; Arden, S D; Hutton, J C

    1992-07-25

    Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence. The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated. Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA. One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence. This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production. Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein. It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein. Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2. The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts. N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2. This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme. It is concluded that PC2 is the type 2 endopeptidase involved

  6. Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase.

    PubMed

    Plaxton, W C

    1991-12-01

    Leucoplast pyruvate kinase (PK(p); EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4 degrees C. Purified castor seed PK(p) was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK(p)) immunoglobulin G. Proteolytic activity against PK(p) was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4 degrees C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK(p). The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nalpha-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK(p). This endopeptidase is present throughout all stages of endosperm development. Its PK(p)-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4 degrees C for up to 21 hours, no degradation of PK(p) was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK(p) remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies

  7. Effect of a novel selective and potent phosphinic peptide inhibitor of endopeptidase 3.4.24.16 on neurotensin-induced analgesia and neuronal inactivation.

    PubMed

    Vincent, B; Jiracek, J; Noble, F; Loog, M; Roques, B; Dive, V; Vincent, J P; Checler, F

    1997-06-01

    1. We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. 2. The most selective inhibitor, Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. 3. The effect of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. 4. Pro-Phe-psi(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. 5. Intracerebroventricular administration of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. 6. Altogether, our study has established Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin.

  8. The intracellular distribution and secretion of endopeptidases 24.15 (EC 3.4.24.15) and 24.16 (EC 3.4.24.16).

    PubMed

    Ferro, Emer S; Carreno, Flávia R; Goni, Camila; Garrido, Paula A G; Guimaraes, Alessander O; Castro, Leandro M; Oliveira, Vitor; Araujo, Maurício C; Rioli, Vanessa; Gomes, Marcelo D; Fontenele-Neto, José Domingues; Hyslop, Stephen

    2004-10-01

    Endopeptidase 24.15 (EC 3.4.24.15; EP24.15) and endopeptidase 24.16 (EC 3.4.24.16; EP24.16) are enzymes involved in general peptide metabolism in mammalian cells and tissues. This review will focus on morphological and biochemical aspects related to the subcellular distribution and secretion of these homologous enzymes in the central nervous system. These are important issues for a better understanding of the functions of EP24.15 and EP24.16 within neuroendocrine systems.

  9. Role of angiotensin converting enzyme in the vascular effects of an endopeptidase 24.15 inhibitor.

    PubMed Central

    Telford, S E; Smith, A I; Lew, R A; Perich, R B; Madden, A C; Evans, R G

    1995-01-01

    1. We investigated the role of angiotensin converting enzyme (ACE) in the cardiovascular effects of N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), a peptidase inhibitor selective for metalloendopeptidase (EP) E.C. 3.4.24.15. 2. In conscious rabbits, cFP (5 mg kg-1, i.v.) markedly slowed the degradation of [3H]-bradykinin, potentiated the depressor response to right atrial administration of bradykinin (10-1000 ng kg-1), and inhibited the pressor response to right atrial angiotensin I (10-100 ng kg-1). In each of these respects, the effects of cFP were indistinguishable from those of the ACE inhibitor, captopril (0.5 mg plus 10 mg kg-1h-1 i.v.). Furthermore, the effects of combined administration of cFP and captopril were indistinguishable from those of captopril alone. 3. In experimentally naive anaesthetized rats, cFP administration (9.3 mg kg-1, i.v.) was followed by a moderate but sustained fall in arterial pressure of 13 mmHg. However, in rats pretreated with bradykinin (50 micrograms kg-1) a more pronounced fall of 30 mmHg was observed. Captopril (5 mg kg-1) had similar hypotensive effects to those of cFP, and cFP had no effect when it was administered after captopril. 4. CFP displaced the binding of [125I]-351A (the p-hydroxybenzamidine derivative of lisinopril) from preparations of rat plasma ACE and solubilized lung membrane ACE (KD = 1.2 and 0.14 microM respectively), and inhibited rat plasma ACE activity (KI = 2.4 microM). Addition of phosphoramidon (10 microM), an inhibitor of a range of metalloendopeptidases, including neutral endopeptidase (E.C.3.4.24.11), markedly reduced the potency of cFP in these systems.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7620708

  10. Atrial fibrillation down-regulates renal neutral endopeptidase expression and induces profibrotic pathways in the kidney.

    PubMed

    Bukowska, Alicja; Lendeckel, Uwe; Krohn, Alexander; Keilhoff, Gerburg; ten Have, Sara; Neumann, Klaus Hinrich; Goette, Andreas

    2008-10-01

    Recent studies suggest that atrial fibrillation (AF) substantially influences microvascular flow in ventricular myocardium. This process may contribute to the occurrence of heart failure in AF. In general, development of heart failure and renal dysfunction go hand-in-hand causing systemic fluid overload and oedema. So far, it is unknown whether AF itself influences renal function. The aim of the present study was to determine the impact of AF on renal gene expression in a closed chest rapid atrial pacing model. A total of 14 pigs were studied. In five pigs, rapid atrial pacing (AT) was performed for 7 h (600 bpm); in five additional animals, rapid atrial pacing was performed in the presence of irbesartan infusion (irbesartan group). Four pigs were instrumented without interventions (sham). After the pacing period, renal expression of collagen I alpha 1 and I alpha 3, transforming growth factor-beta (TGF-beta), neutral endopeptidase (NEP; the main enzyme involved in natriuretic protein metabolism), and atrial natriuretic peptide (ANP) were determined by RT-PCR and immunoblot analysis. Functional in vitro experiments were performed using HEK-293 kidney cells. Renal mRNA expression of NEP was substantially down-regulated during AT (AT: 12.7 +/- 9.3% vs. sham: 100 +/- 43.4%; P < 0.01). Results at the mRNA level were confirmed at the protein level. Irbesartan therapy did not prevent down-regulation of NEP. In contrast, TGF-beta1 mRNA expression was up-regulated (AT: 208.5 +/- 79.3% vs. sham: 100 +/- 34.6% P< 0.05). Collagen and angiotensin II type 1 receptor (AT1R) expression were not significantly altered by AT. HEK-293 cells were used to determine the potential humoral factors involved in down-regulation of NEP. Application of aldosterone, ANP, asymmetric dimethylarginine, and angiotensin peptides failed to cause down-regulation of renal NEP expression in vitro. AT reduces NEP expression and stimulates TGF-beta1 signalling in the kidneys. Thus, even brief episodes of

  11. Dual ACE and neutral endopeptidase inhibitors: novel therapy for patients with cardiovascular disorders.

    PubMed

    Tabrizchi, Reza

    2003-01-01

    Elevated blood pressure is a risk factor for a variety of cardiovascular disorders, including coronary heart disease, peripheral vascular disease, cardiac failure and cerebrovascular disease. The prevailing view is that an elevated systolic rather than diastolic blood pressure is the major contributor in mortality and morbidity attributed to cardiovascular disorders. Isolated high systolic blood pressure, especially in the elderly, is a major risk factor and should undoubtedly be a target for drug treatment. In the general population, systolic and diastolic blood pressure are highly correlated, and thus it is difficult to dissociate the effects of these two components of the blood pressure and specifically ascribe cardiovascular risk factors to just elevated systolic blood pressure. Therefore, the goal in therapy of an individual with hypertension must be to reduce elevated systolic and diastolic blood pressure in order to reduce mortality and morbidity. ACE and neutral peptidase inhibitors are a new class of drugs that may be beneficial in the treatment of patients with hypertension and heart failure. They may also be useful in the treatment of diabetic patients with hypertension and/or heart failure. Drugs of this class are dual inhibitors of ACE and neutral endopeptidase, and are capable of affecting vascular tone and fluid balance. They are capable of producing vasodilatation by virtue of inhibiting the production of angiotensin II, degradation of natriuretic peptides and bradykinin. They also appear to promote natriuresis and diuresis by amplifying the actions of natriuretic peptidase and reducing aldosterone effects. In addition, they should also attenuate trophogenic actions of the renin angiotensin system and the sympathetic nervous system. Omapatrilat is one drug that appears to be at the advanced stages of clinical development. This drug has been shown to be quite effective in the treatment of hypertension. Evidence also seems to indicate that treatment

  12. Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.

    PubMed

    Kang, Chao; Yu, Xiao-Wei; Xu, Yan

    2015-02-01

    A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.

  13. Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Davey, J; Davis, K; Imai, Y; Yamamoto, M; Matthews, G

    1994-01-01

    The activation of pro-hormones and many precursor proteins involves cleavage by endopeptidases belonging to the subtilisin-like family of enzymes. Here we describe the isolation and characterization of the first member of this family from the fission yeast Schizosaccharomyces pombe. The enzyme, which has been named krp for KEX2-related protease, is a type I membrane-bound endopeptidase that cleaves substrates after pairs of dibasic residues. It appears to be synthesized as a pre-pro-protein that is likely to undergo processing following translocation into the endoplasmic reticulum. Processing has been characterized in a cell-free translation/translocation system prepared from Xenopus eggs. Krp is N-glycosylated on all five of its potential sites and both the pre-sequence and the pro-sequence are quickly removed following translocation, the latter probably by autocatalytic cleavage. The inhibitor profile of krp broadly reflects the known properties of the eukaryotic subtilisin proteases, while its pH and Ca2+ dependence are consistent with it being active within the secretory pathway. One of its physiological substrates is likely to be the pheromone precursor pro-P-factor, which it is shown to process in an in vitro system, but identification of other substrates is complicated because, unlike other members of this family, krp is essential for cell viability. Images PMID:7813430

  14. Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity.

    PubMed

    Cai, S; Singh, B R

    2001-12-18

    Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form. The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain. By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure. This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid. Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure. A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C.

  15. Identification in lupin seed of a serine-endopeptidase activity cleaving between twin arginine pairs and causing limited proteolysis of seed storage proteins.

    PubMed

    Magni, Chiara; Sessa, Fabio; Tedeschi, Gabriella; Negri, Armando; Scarafoni, Alessio; Consonni, Alessandro; Duranti, Marcello

    2012-09-01

    The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

  16. [Detection of AlpA and AlpB lytic endopeptidase propeptides of Lysobacter sp. XL1 by sandwich-enzyme immunoassay based on monoclonal antibodies].

    PubMed

    Rudenko, N V; Tsfasman, I M; Latypov, O R; Ledova, L A; Krasovskaia, L A; Karatovskaia, A P; Brovko, F A; Vasil'eva, N V; Stepnaia, O A

    2014-01-01

    The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

  17. Endopeptidases 24.16 and 24.15 are responsible for the degradation of somatostatin, neurotensin, and other neuropeptides by cultivated rat cortical astrocytes.

    PubMed

    Mentlein, R; Dahms, P

    1994-01-01

    Several neuropeptides, including neurotensin, somatostatin, bradykinin, angiotensin II, substance P, and luteinizing hormone-releasing hormone but not vasopressin and oxytocin, were actively metabolized through proteolytic degradation by cultivated astrocytes obtained from rat cerebral cortex. Because phenanthroline was an effective degradation inhibitor, metalloproteases were responsible for neuropeptide fragmentation. Neurotensin was cleaved by astrocytes at the Pro10-Tyr11 and Arg8-Arg9 bonds, whereas somatostatin was cleaved at the Phe6-Phe7 and Thr10-Phe11 bonds. These cleavage sites have been found previously with endopeptidases 24.16 and 24.15 purified from rat brain. Addition of specific inhibitors of these proteases, the dipeptide Pro-Ile and N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-4-aminobenzoate, significantly reduced the generation of the above neuropeptide fragments by astrocytes. The presence of endopeptidases 24.16 and 24.15 in homogenates of astrocytes could also be demonstrated by chromatographic separations of supernatant solubilized cell preparations. Proteolytic activity for neurotensin eluted after both gel and hydroxyapatite chromatography at the same positions as found for purified endopeptidase 24.16 or 24.15. In incubation experiments or in chromatographic separations no phosphoramidon-sensitive endopeptidase 24.11 (enkephalinase) or captopril-sensitive peptidyl dipeptidase A (angiotensin-converting enzyme) could be detected in cultivated astrocytes. Because astrocytes embrace the neuronal synapses where neuropeptides are released, we presume that the endopeptidases 24.16 and 24.15 on astrocytes are strategically located to contribute significantly to the inactivation of neurotensin, somatostatin, and other neuropeptides in the brain.

  18. Purification and characterization of a cysteine endopeptidase from Vasconcellea quercifolia A. St.-Hil. latex displaying high substrate specificity.

    PubMed

    Torres, M José; Trejo, Sebastián A; Martin, M Inés; Natalucci, Claudia L; Avilés, Francesc X; López, Laura M I

    2010-10-27

    A new proteolytic preparation from Vasconcellea quercifolia ("oak leaved papaya") latex containing several cysteine endopeptidases with high proteolytic activity has been obtained. The specific activity of the new enzymatic preparation (VQ) was higher than that of Carica papaya latex. VQ was able to coagulate milk and to hydrolyze caseins and then could be used to produce cheeses and/or casein hydrolysates. Ion exchange chromatography of VQ allowed the isolation of a new protease, named quercifoliain I, homogeneous when analyzed by SDS-PAGE, IEF and MALDI-TOF-MS. Molecular mass was 24195 Da, and its isoelectric point was >9.3. The N-terminal sequence was determined (YPESVDWRQ). Insulin B-chain cleavage showed higher specificity than that of papain and was restricted to glycyl and alanyl residues at P1' position. The tryptic peptide mass fingerprint of quercifoliain I analyzed with the MASCOT search tool did not find a match with papain or any other plant cysteine proteases.

  19. cDNA Cloning and Molecular Modeling of Procerain B, a Novel Cysteine Endopeptidase Isolated from Calotropis procera

    PubMed Central

    Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

    2013-01-01

    Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed. PMID:23527269

  20. A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. The HYP Consortium.

    PubMed

    1995-10-01

    X-linked hypophosphatemic rickets (HYP) is a dominant disorder characterised by impaired phosphate uptake in the kidney, which is likely to be caused by abnormal regulation of sodium phosphate cotransport in the proximal tubules. By positional cloning, we have isolated a candidate gene from the HYP region in Xp22.1. This gene exhibits homology to a family of endopeptidase genes, members of which are involved in the degradation or activation of a variety of peptide hormones. This gene (which we have called PEX) is composed of multiple exons which span at least five cosmids. Intragenic non-overlapping deletions from four different families and three mutations (two splice sites and one frameshift) have been detected in HYP patients, which suggest that the PEX gene is involved in the HYP disorder.

  1. Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

    PubMed

    Machado, M F M; Cunha, F M; Berti, D A; Heimann, A S; Klitzke, C F; Rioli, V; Oliveira, V; Ferro, E S

    2006-01-13

    Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.

  2. VAN method lacks validity

    NASA Astrophysics Data System (ADS)

    Jackson, David D.; Kagan, Yan Y.

    Varotsos and colleagues (the VAN group) claim to have successfully predicted many earthquakes in Greece. Several authors have refuted these claims, as reported in the May 27,1996, special issue of Geophysical Research Letters and a recent book, A Critical Review of VAN [Lighthill 1996]. Nevertheless, the myth persists. Here we summarize why the VAN group's claims lack validity.The VAN group observes electrical potential differences that they call “seismic electric signals” (SES) weeks before and hundreds of kilometers away from some earthquakes, claiming that SES are somehow premonitory. This would require that increases in stress or decreases in strength cause the electrical variations, or that some regional process first causes the electrical signals and then helps trigger the earthquakes. Here we adopt their notation SES to refer to the electrical variations, without accepting any link to the quakes.

  3. Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin.

    PubMed

    Eugster, Philippe J; Salamin, Karine; Grouzmann, Eric; Monod, Michel

    2015-12-01

    Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.

  4. Histidine and Aspartic Acid Residues Important for Immunoglobulin G Endopeptidase Activity of the Group A Streptococcus Opsonophagocytosis-Inhibiting Mac Protein

    PubMed Central

    Lei, Benfang; Liu, Mengyao; Meyers, Elishia G.; Manning, Heather M.; Nagiec, Michael J.; Musser, James M.

    2003-01-01

    The secreted Mac protein made by serotype M1 group A Streptococcus (GAS) (designated Mac5005) inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear neutrophils. This protein also has cysteine endopeptidase activity against human immunoglobulin G (IgG). Site-directed mutagenesis was used to identify histidine and aspartic acid residues important for Mac IgG endopeptidase activity. Replacement of His262 with Ala abolished Mac5005 IgG endopeptidase activity. Asp284Ala and Asp286Ala mutant proteins had compromised enzymatic activity, whereas 21 other Asp-to-Ala mutant proteins cleaved human IgG at the apparent wild-type level. The results suggest that His262 is an active-site residue and that Asp284 and Asp286 are important for the enzymatic activity or structure of Mac protein. These Mac mutants provide new information about structure-activity relationships in this protein and will assist study of the mechanism of inhibition of opsonophagocytosis and killing of GAS by Mac. PMID:12704162

  5. Bacillus subtilis CwlP of the SP-β Prophage Has Two Novel Peptidoglycan Hydrolase Domains, Muramidase and Cross-linkage Digesting dd-Endopeptidase*

    PubMed Central

    Sudiarta, I Putu; Fukushima, Tatsuya; Sekiguchi, Junichi

    2010-01-01

    For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-β prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn2+-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn2+ dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined. PMID:20980266

  6. Development of the first potent and selective inhibitor of the zinc endopeptidase neurolysin using a systematic approach based on combinatorial chemistry of phosphinic peptides.

    PubMed

    Jirácek, J; Yiotakis, A; Vincent, B; Checler, F; Dive, V

    1996-08-09

    A new systematic approach, based on combinatorial chemistry of phosphinic peptides, is proposed for rapid development of highly potent and selective inhibitors of zinc metalloproteases. This strategy first evaluates the effects on the inhibitory potency and selectivity of the following parameters: 1) size of the phosphinic peptides, 2) position of the phosphinic bond in the sequence, and 3) the state (free or blocked) of the peptide extremities. After this selection step, the influence of the inhibitor sequence is analyzed in order to determine the identity of the residues that optimized both the potency and the selectivity. We demonstrate the efficiency of this novel approach in rapid identification of the first potent inhibitor of the mammalian zinc endopeptidase neurolysin(24-16), able to discriminate between this enzyme and the related zinc endopeptidase thimet oligopeptidase(24-15). The most potent and selective inhibitor developed in this study, Pro-LPhePsi(PO2CH2)Gly-Pro, displays a Ki value of 4 nM for 24-16 and is 2000 times less potent on 24-15. The specific recognition of such a free phosphinic tetrapeptide by 24-16, as well as the unique specificity of the 24-16 S2 and S2' subsites for proline, unveiled by this study, are discussed in terms of their possible significance for the function of this enzyme and its related zinc endopeptidase activities.

  7. High levels of functional endopeptidase 24.11 (CD10) activity on human thymocytes: preferential expression on immature subsets.

    PubMed Central

    Mari, B; Breittmayer, J P; Guerin, S; Belhacene, N; Peyron, J F; Deckert, M; Rossi, B; Auberger, P

    1994-01-01

    Although it is now well established that cells of the immune system express most of the exopeptidases described so far, little information is available concerning the identification and the characterization of the peptidases associated with the surface of human thymocytes. In the present study we have focused on CD10 expression on thymocytes using both FACS and enzymatic analysis. Unfractionated intact human thymocytes were shown to express significant levels of CD10-specific enzymatic activity, as assessed by the hydrolysis of the neutral endopeptidase (NEP) substrate Suc-Ala-Ala-Phe-pNA and of D-Ala2-Leu-enkephalin, a typical NEP substrate. CD10 activity was abolished by specific NEP inhibitors, including thiorphan, retrothiorphan and phosphoramidon. Moreover, high performance liquid chromatography (HPLC) analysis showed that intact thymocytes and purified NEP hydrolysed thymopentin, a thymic factor known to induce the maturation of prothymocytes into thymocytes. Finally, CD 10/NEP was preferentially associated with CD3- CD3low and immature CD4- CD8- thymocytes. The data demonstrate for the first time that human thymocytes express functional NEP and suggest a role for this enzyme in the maturation of human thymocytes. PMID:7959879

  8. Alterations in plasma prolyl endopeptidase activity in depression, mania, and schizophrenia: effects of antidepressants, mood stabilizers, and antipsychotic drugs.

    PubMed

    Maes, M; Goossens, F; Scharpé, S; Calabrese, J; Desnyder, R; Meltzer, H Y

    1995-10-16

    The activity of prolyl endopeptidase (PEP), a serine proteinase, has been found to be significantly lower in the blood of patients with major depression than in normal volunteers. The present study investigates plasma PEP activity in 25 major depressed, 10 manic, and 14 schizophrenic subjects versus 30 normal volunteers. It also examines the effects of antidepressants, valproate, and neuroleptic drugs on plasma PEP activity. PEP activity was significantly lower in major depressed subjects than in normal volunteers and in patients with mania and schizophrenia. In depressed subjects, plasma PEP activity was significantly increased during treatment with antidepressant drugs, such as fluoxetine. Plasma PEP activity was significantly increased in manic and schizophrenic subjects compared with normal volunteers. In manic subjects, short-term treatment with valproate had a significant suppressive effect on PEP activity. No significant effects of neuroleptics on PEP activity could be found in the schizophrenic patients. The results support the hypothesis that lower PEP activity could play a role in the pathophysiology of major depression, while increased PEP activity may be related to psychotic conditions, such as mania and schizophrenia.

  9. Bacteriocin protein BacL1 of Enterococcus faecalis is a peptidoglycan D-isoglutamyl-L-lysine endopeptidase.

    PubMed

    Kurushima, Jun; Hayashi, Ikue; Sugai, Motoyuki; Tomita, Haruyoshi

    2013-12-27

    Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL1 and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL1 and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL1 and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL1 and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL1 nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL1 alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL1 has a peptidoglycan D-isoglutamyl-L-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL1 serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells.

  10. Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene.

    PubMed Central

    Conlin, C A; Trun, N J; Silhavy, T J; Miller, C G

    1992-01-01

    Mutations at the Escherichia coli prlC locus suppress the export defect of certain lamB signal sequence mutations. The Salmonella typhimurium opdA gene encodes an endoprotease that can participate in the catabolism of certain peptides and is required for normal development of phage P22. Plasmids carrying either the wild-type (pTC100 prlC+) or suppressor alleles of prlC complemented all phenotypes associated with an S. typhimurium opdA mutation. A plasmid carrying an amber mutation in prlC [prlC31(AM)] was unable to complement except in an amber suppressor background. Tn1000 insertions which eliminated the ability of pTC100 (prlC+) to complement opdA mapped to the region of the plasmid shown by deletion analysis and subcloning to contain prlC. The nucleotide sequence of a 2.7-kb fragment including this region was determined, revealing an open reading frame encoding a 77-kDa protein. The sequences of this open reading frame and its putative promoter region were very similar (84% nucleotide sequence identity and 95% amino acid identity) to those of S. typhimurium opdA, showing that these genes are homologs. The nucleotide sequence of the prlC1 suppressor allele was determined and predicts an in-frame duplication of seven amino acids, providing further confirmation that the prlC suppressor phenotype results from changes in the endopeptidase OpdA. PMID:1325967

  11. A Highly Unstable Transcript Makes CwlO d,l-Endopeptidase Expression Responsive to Growth Conditions in Bacillus subtilis

    PubMed Central

    Salzberg, Letal I.; Botella, Eric; Bäsell, Katrin; Becher, Dörte; Antelmann, Haike; Devine, Kevin M.

    2014-01-01

    The Bacillus subtilis cell wall is a dynamic structure, composed of peptidoglycan and teichoic acid, that is continually remodeled during growth. Remodeling is effected by the combined activities of penicillin binding proteins and autolysins that participate in the synthesis and turnover of peptidoglycan, respectively. It has been established that one or the other of the CwlO and LytE d,l-endopeptidase-type autolysins is essential for cell viability, a requirement that is fulfilled by coordinate control of their expression by WalRK and SigI RsgI. Here we report on the regulation of cwlO expression. The cwlO transcript is very unstable, with its degradation initiated by RNase Y cleavage within the 187-nucleotide leader sequence. An antisense cwlO transcript of heterogeneous length is expressed from a SigB promoter that has the potential to control cellular levels of cwlO RNA and protein under stress conditions. We discuss how a multiplicity of regulatory mechanisms makes CwlO expression and activity responsive to the prevailing growth conditions. PMID:24163346

  12. Structural and Functional Analyses Reveal That Staphylococcus aureus Antibiotic Resistance Factor HmrA Is a Zinc-dependent Endopeptidase*

    PubMed Central

    Botelho, Tiago O.; Guevara, Tibisay; Marrero, Aniebrys; Arêde, Pedro; Fluxà, Viviana S.; Reymond, Jean-Louis; Oliveira, Duarte C.; Gomis-Rüth, F. Xavier

    2011-01-01

    HmrA is an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus. Molecular analysis of this protein revealed that it is not a muramidase or β-lactamase but a nonspecific double-zinc endopeptidase consisting of a catalytic domain and an inserted oligomerization domain, which probably undergo a relative interdomain hinge rotation upon substrate binding. The active-site cleft is located at the domain interface. Four HmrA protomers assemble to a large ∼170-kDa homotetrameric complex of 125 Å. All four active sites are fully accessible and ∼50–70 Å apart, far enough apart to act on a large meshwork substrate independently but simultaneously. In vivo studies with four S. aureus strains of variable resistance levels revealed that the extracellular addition of HmrA protects against loss of viability in the presence of oxacillin and that this protection depends on proteolytic activity. All of these results indicate that HmrA is a peptidase that participates in resistance mechanisms in vivo in the presence of β-lactams. Furthermore, our results have implications for most S. aureus strains of known genomic sequences and several other cocci and bacilli, which harbor close orthologs. This suggests that HmrA may be a new widespread antibiotic resistance factor in bacteria. PMID:21622555

  13. Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism.

    PubMed

    Norman, M Ursula; Reeve, Shane B; Dive, Vincent; Smith, A Ian; Lew, Rebecca A

    2003-06-01

    The closely related metalloendopeptidases EC (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

  14. Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity.

    PubMed

    Klegeris, Andis; Li, Jane; Bammler, Theo K; Jin, Jinghua; Zhu, David; Kashima, Daniel T; Pan, Sheng; Hashioka, Sadayuki; Maguire, John; McGeer, Patrick L; Zhang, Jing

    2008-04-15

    Reactive microglial cells may exacerbate the pathology in some neurodegenerative disorders. Supernatants of stimulated human microglial cells, or their surrogate THP-1 cells, are lethal to cultured human neuroblastoma SH-SY5Y cells. To explore this neurotoxicity, we examined the spectrum of proteins generated by THP-1 cells using the technique of stable isotope labeling by amino acids in cell culture (SILAC). Unstimulated cells were grown in medium with light L-[(12)C(6)] arginine while cells stimulated by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) were grown in medium with heavy L-[(13)C(6)] arginine. Proteins isolated from the media were digested with trypsin, and relative concentrations of generated peptides determined by mass spectrometry. More than 1,500 proteins or putative proteins were identified. Of these, 174 were increased and 189 decreased by more than twofold in the stimulated cell supernatant. We selected one upregulated protein, prolyl endopeptidase (PEP), for further investigation of its potential contribution to neurotoxicity. We first confirmed its upregulation by comparing its enzymatic activity in stimulated and unstimulated cell supernatants. We then evaluated two specific PEP inhibitors, Boc-Asn-Phe-Pro-aldehyde and Z-Pro-Pro-aldehyde-dimethyl acetal, for their potential to reduce toxicity of stimulated THP-1 cell and human microglia supernatants towards SH-SY5Y cells. We found both to be partially protective in a concentration-dependent manner. Inhibition of PEP may be a therapeutic approach to neurodegenerative disorders including Alzheimer and Parkinson diseases.

  15. When Lack of Evidence Is Evidence of Lack.

    PubMed

    Pickering, Neil

    2015-12-01

    In their recent article "A Gentle Ethical Defence of Homeopathy," Levy, Gadd, Kerridge, and Komesaroff use the claim that "lack of evidence is not equivalent to evidence of lack" as a component of their ethical defence of homeopathy. In response, this article argues that they cannot use this claim to shore up their ethical arguments. This is because it is false.

  16. Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities.

    PubMed

    Imamura, Takahisa; Murakami, Yoji; Nitta, Hidetoshi

    2017-09-26

    Aeromonas sobria serine protease (ASP) is secreted from Aeromonas sobria, a pathogen causing gastroenteritis and sepsis. ASP resembles Saccharomyces cerevisiae Kex2, a member of the subtilisin family, and preferentially cleaves peptide bonds at the C-terminal side of paired basic amino acid residues; also accepting unpaired arginine at the P1 site. Unlike Kex2, however, ASP lacks an intramolecular chaperone N-terminal propeptide, instead utilizes the external chaperone ORF2 for proper folding, therefore, ASP and its homologues constitute a new subfamily in the subtilisin family. Through activation of the kallikrein/kinin system, ASP induces vascular leakage, and presumably causes edema and septic shock. ASP accelerates plasma clotting by α-thrombin generation from prothrombin, whereas it impairs plasma clottability by fibrinogen degradation, together bringing about blood coagulation disorder that occurs in disseminated intravascular coagulation, a major complication of sepsis. From complement C5 ASP liberates C5a that induces neutrophil recruitment and superoxide release, and mast cell degranulation, which are associated with pus formation, tissue injury and diarrhea, respectively. Nicked two-chain ASP also secreted from A. sobria is more resistant to inactivation by α2-macroglobulin than single-chain ASP, thereby raising virulence activities. Thus, ASP is a potent virulence factor and may participate in the pathogenesis of A. sobria infection.

  17. Potentiation of the depressor responses to atrial natriuretic peptides in conscious SHR by an inhibitor of neutral endopeptidase.

    PubMed

    Seymour, A A; Swerdel, J N; Fennell, S A; Druckman, S P; Neubeck, R; Delaney, N G

    1989-08-01

    In previous studies, neutral endopeptidase (NEP) hydrolyzed the Cys105-Phe106 bond of atrial natriuretic peptides (ANP) in vitro. Three such ring-opened peptides derived from ANP 99-126, 103-126, and 103-123 were inactive in conscious rats. In conscious spontaneously hypertensive rats (SHR) in the present study, 100 mumol/kg, intravenously (i.v.) of the NEP inhibitor, SQ 29,072 (7-[[2-(mercaptomethyl)-1-oxo-3-phenyl-propyl]amino]heptanoic acid), significantly increased the area over the curve (AOC) of the depressor response to 3 nmol/kg of ANP 103-126 from 165 +/- 36 to 792 +/- 350, 1,515 +/- 374, and 828 +/- 164 mm Hg.min at 15, 30, and 60 min after inhibitor treatment. Thirty minutes after 3, 10, 30, and 100 mumol/kg of SQ 29,072, the AOC of 3 nmol/kg of ANP 99-126 increased from 175 +/- 59 mm Hg.min in vehicle-treated rats to 296 +/- 100, 318 +/- 34, 632 +/- 194 (p less than 0.05) and 656 +/- 151 (p less than 0.05) mm Hg.min. Furthermore, 100 mumol/kg of SQ 29,072 potentiated the AOC of human ANP 99-126 and 105-126 and rat ANP 99-126, 103-126, and 103-123, suggesting that the exocyclic N-terminal residues and the C-terminal tripeptide did not influence ANP potentiation by SQ 29,072. In contrast, inhibitors of aminopeptidase, angiotensin-converting enzyme (ACE), and serine protease and an arginine vasopressin (AVP) antagonist did not substantially affect the AOC of 3 nmol/kg ANP 99-126. Finally, SQ 29,072 did not alter the activities of bradykinin, AVP, or angiotensin I or II. In conclusion, NEP may inactivate ANP in vivo by cleavage of susceptible bonds within the ANP ring.

  18. Prognostic significance of the combined expression of neutral endopeptidase and dipeptidyl peptidase IV in intrahepatic cholangiocarcinoma patients after surgery resection

    PubMed Central

    Zhu, Jianyong; Guo, XiaoDong; Qiu, Baoan; Li, Zhiyan; Xia, Nianxin; Yang, Yingxiang; Liu, Peng

    2014-01-01

    Aim The aim of this study was to investigate the relationship between the expression of neutral endopeptidase (NEP) and dipeptidyl peptidase IV (DPP IV) proteins, and the clinical significance of the two proteins in patients with intrahepatic cholangiocarcinomas (IHCC). Methods Expression patterns and subcellular localizations of NEP and DPP IV proteins in 186 primary IHCC and 60 noncancerous liver tissue specimens were detected by immunohistochemistry. Results Both the expression of NEP and DPP IV proteins in IHCC tissues were significantly higher than those in noncancerous liver tissues (both P<0.001). Of 186 patients with IHCC, 128 (68.82%) highly expressed both NEP and DPP IV proteins. In addition, the coexpression of NEP and DPP IV proteins was significantly associated with advanced tumor stage (P=0.009), positive lymph node metastasis (P=0.016) and distant metastasis (P=0.013), and the presence of recurrence (P=0.027). Moreover, Kaplan–Meier analysis showed that IHCC patients with high NEP expression, high DPP IV expression, and combined overexpression of NEP and DPP IV proteins all had poorer overall survival and early recurrence after surgery. Furthermore, Cox analysis suggested that NEP expression, DPP IV expression, and combined expression of NEP and DPP IV proteins were all independent prognostic markers for overall survival and recurrence-free survival in patients with IHCC. Conclusion Our data suggest, for the first time, that both the expression of NEP and DPP IV proteins may be upregulated in human IHCC tissues and the combined expression of NEP and DPP IV proteins may play important roles in progression and prognosis of patients with IHCC. PMID:24570591

  19. Mechanisms of toxic smoke inhalation and burn injury: role of neutral endopeptidase and vascular leakage in mice.

    PubMed

    Jacob, Sam; Deyo, Donald J; Cox, Robert A; Traber, Daniel L; Herndon, David N; Hawkins, Hal K

    2009-03-01

    The effects of neutral endopeptidase (NEP) in acute inflammation in the lung were studied using a newly developed murine model of smoke and burn (SB) injury. C57BL/6 mice were pretreated with an i.v. dose of a specific NEP antagonist CGS-24592 (10 mg/Kg) 1 h prior to SB injury (n = 5-8/group). Mice were anesthetized with i.p. ketamine/xylazine, intubated, and exposed to cooled cotton smoke (2 x 30 s). After s.c. injection of 1 ml 0.9% saline, each received a 40% total body surface area (TBSA) flame burn. Buprenorphene (2 mg/kg) was given i.p. and resuscitated by saline. Evans Blue dye (EB) was injected i.v. 15 min before sacrifice. Lung wet/dry weight ratio was measured. After vascular perfusion, lungs were analyzed for their levels of EB dye and myeloperoxidase (MPO). In mice pretreated with CGS-24592 followed by SB injury the EB levels were significantly higher (61%, p = 0.043) than those with SB injury alone. There was a significant increase (144%, p = 0.035) in EB dye in animals with SB injury alone as compared to shams. In mice pretreated with CGS-24592 prior to SB injury wet/dry weight ratios were significantly (27%, p = 0.042) higher compared to animals with SB injury alone. CGS-24592 pretreatment also caused a significant increase in MPO (29%, p = 0.026) as compared to mice with SB injury alone. In conclusion the current study indicates that specific NEP inhibitor CGS 24592 exacerbates the SB-induced lung injury and inflammation in mice.

  20. Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy.

    PubMed

    Gass, Jonathan; Ehren, Jennifer; Strohmeier, Gregg; Isaacs, Indu; Khosla, Chaitan

    2005-12-20

    Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.

  1. CD10/neutral endopeptidase inhibition augments pulmonary neuroendocrine cell hyperplasia in hamsters treated with diethylnitrosamine and hyperoxia.

    PubMed

    Willett, C G; Shahsafei, A; Graham, S A; Sunday, M E

    1999-07-01

    In previous studies, we demonstrated that pulmonary neuroendocrine cell (PNEC) hyperplasia in hamsters treated with diethylnitrosamine (DEN) plus 65% hyperoxia (DEN/O2) reflects predominantly neuroendocrine cell differentiation. Several peptides implicated in non-neoplastic PNEC hyperplasia are hydrolyzed by CD10/neutral endopeptidase 24.11 (CD10/NEP), an enzyme known to downregulate neurogenic inflammation of the lung by modulating locally effective concentrations of multiple bioactive peptides. In fetal mice, we observed that CD10/NEP inhibition by SCH32615 potentiates cell proliferation and type II cell differentiation in the lung in utero. Further, CD10/NEP messenger RNA levels parallelled relative PNEC numbers in DEN/O2-treated hamster lung, suggesting that the enzyme might mediate spontaneous regression of PNEC hyperplasia. The goals of the present study were: (1) to determine whether CD10/NEP inhibition would alter the extent of PNEC hyperplasia occurring in these hamsters, and (2) to analyze cellular mechanisms potentially involved in altering numbers of PNECs in this model. We administered SCH32615 chronically to a subset of DEN/O2-treated hamsters. Immunostaining of lungs from the CD10/ NEP-inhibited subset demonstrated significant acceleration of the development of PNEC hyperplasia, increased PNEC proliferation, and diminished PNEC apoptosis as compared with animals receiving no SCH32615. These observations indicate that PNEC hyperplasia can occur as a result of multiple cellular processes, including increased neuroendocrine cell differentiation, proliferation, and survival. CD10/NEP modulates PNEC numbers primarily by promoting cell differentiation and proliferation during lung injury, probably via increasing the half-life of bioactive peptides in the lung.

  2. The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

    PubMed

    Lai, Ghee Chuan; Cho, Hongbaek; Bernhardt, Thomas G

    2017-07-01

    Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes

  3. Cigarette smoke-induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown

    PubMed Central

    Koelink, Pim J.; Henricks, Paul A. J.; Jackson, Patricia L.; Nijkamp, Frans P.; Garssen, Johan; Kraneveld, Aletta D.; Blalock, J. Edwin; Folkerts, Gert

    2011-01-01

    There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide l-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema. PMID:21112944

  4. Autoradiographic comparison of the distribution of the neutral endopeptidase enkephalinase and of. mu. and delta opioid receptors in rat brain

    SciTech Connect

    Waksman, G.; Hamel, E.; Fournie-Zaluski, M.C.; Roques, B.P.

    1986-03-01

    The neutral endopeptidase EC 3.4.24.11, also designated enkephalinase, has been visualized by in vitro autoradiography using the tritiated inhibitor (/sup 3/H)-N-((2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)glycine, ((/sup 3/H)HACBO-Gly). Specific binding of (/sup 3/H)HACBO-Gly corresponding to 85% of the total binding to brain slices was inhibited by 1 ..mu..M thiorphan, a selective inhibitor of enkephalinase, but remained unchanged in the presence of captopril, a selective inhibitor of angiotensin-converting enzyme. Very high levels of (/sup 3/H)HACBO-Gly binding were found in the choroid plexus and the substantia nigra. High levels were present in the caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercle, and in the substantia gelatinosa of the spinal cord. The distribution of enkephalinase was compared to that of ..mu.. and delta opioid receptors, selectively labeled with (/sup 3/H)Tyr-D-Ala-Gly-MePhe-glycinol and (/sup 3/H)Try-D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate putamen, (/sup 3/H)HACBO-Gly binding overlapped the clustered ..mu.. sites but appeared more closely related to the diffusely distributed delta sites. The association of enkephalinase with delta and ..mu.. opioid receptors in these areas is consistent with the observed role of the enzyme in regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in numerous rat brain areas between the distribution of enkephalinase and that of ..mu.. and/ or delta opioid binding sites could account for most of the pharmacological effects elicited by enkephalinase inhibitors.

  5. Expression of Chironomus riparius serine-type endopeptidase gene under di-(2-ethylhexyl)-phthalate (DEHP) exposure.

    PubMed

    Park, Kiyun; Kwak, Inn-Sil

    2008-11-01

    Environmental stressors can induce changes in gene expression that can be useful as biomarkers. To identify potential biomarkers of water quality, we characterized full-length cDNA sequences of the serine-type endopeptidase (SP) gene from Chironomus riparius. Their expression was analyzed during different life-history stages and in response to treatment with various concentrations of di(2-ethylhexyl) phthalate (DEHP) for short and long periods of time. A comparative molecular and phylogenetic investigation was then conducted among different orders of insects using sequence database analysis. The sequence of the C. riparius SP gene was found to be most closely related to the sequence of SPs isolated from Aedes aegypti. In addition, the basal level of C. riparius SP mRNA was more highly expressed in larvae than in other life-history stages. However, the expression of C. riparius SP was primarily limited to the gut in larvae. When the effects of short-term exposure to DEHP were evaluated, C. riparius SP gene expression decreased within 1 h of treatment, regardless of dose. We also investigated expression of the C. riparius SP gene following long-term DEHP exposure (10 days) and found that it decreased significantly across all DEHP dosages. Finally, the response of the SP gene was more sensitive in C. riparius that were exposed to low concentrations of DEHP than in those that were exposed to high concentrations. These results show that suppression of the C. riparius SP gene by DEHP is as a potential biomarker that could be useful for monitoring aquatic quality.

  6. Transcription of G-protein coupled receptors in corporal smooth muscle is regulated by sialorphin (an endogenous neutral endopeptidase inhibitor)

    PubMed Central

    Tong, Yuehong; Tiplitsky, Scott I.; Tar, Moses; Melman, Arnold; Davies, Kelvin P.

    2009-01-01

    Purpose Several reports have suggested the rat Vcsa1 gene is down-regulated in models of erectile dysfunction (ED). Vcsa’s protein product, sialorphin, is an endogenous neutral endopeptidase (NEP), and its down-regulation could result in prolonged activation of G-protein activated signaling pathways by their peptide agonists. We investigated if down- regulation of Vcsa1 could result in adaptive change in the expression of G-protein coupled receptors (GPCR). Materials and Methods Gene expression in cultured rat corporal smooth muscle cells (CSM) following treatment with siRNA directed against Vcsa1 or the NEP gene was analyzed using microarray and quantitative RT-PCR. In rats Vcsa1 is one of the most down-regulated genes following bilateral transection of the cavernosal nerves. Using that animal model, we also investigated whether the down-regulation of Vcsa1 is accompanied by similar changes in gene expression observed in the CSM cells where Vcsa1 was knocked-down in vitro. Results Microarray analysis and quantitative RT-PCR demonstrated that CSM cells treated in vitro with siRNA against Vcsa1 resulted in up-regulation of GPCR as a functional group. In contrast, treatment of CSM cells that lowered NEP activity resulted in decreases in GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on NEP. In animals with bilaterally transected cavernous nerves the reduced expression of Vcsa1 is accompanied by increased GPCR expression in cavernosal tissue. Conclusions These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR expression. PMID:18554633

  7. Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

    PubMed

    Rhodes, C J; Lincoln, B; Shoelson, S E

    1992-11-15

    Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC

  8. Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function

    PubMed Central

    2004-01-01

    The mammalian neprilysin (NEP) family members are typically type II membrane endopeptidases responsible for the activation/inactivation of neuropeptides and peptide hormones. Differences in substrate specificity and subcellular localization of the seven mammalian NEPs contribute to their functional diversity. The sequencing of the Drosophila melanogaster genome has revealed a large expansion of this gene family, resulting in over 20 fly NEP-like genes, suggesting even greater diversity in structure and function than seen in mammals. We now report that one of these genes (Nep2) codes for a secreted endopeptidase with a highly restricted pattern of expression. D. melanogaster NEP2 is expressed in the specialized stellate cells of the renal tubules and in the cyst cells that surround the elongating spermatid bundles in adult testis, suggesting roles for the peptidase in renal function and in spermatogenesis. D. melanogaster NEP2 was found in vesicle-like structures in the syncytial cytoplasm of the spermatid bundles, suggesting that the protein was acquired by endocytosis of protein secreted from the cyst cells. Expression of NEP2 cDNA in D. melanogaster S2 cells confirmed that the peptidase is secreted and is only weakly inhibited by thiorphan, a potent inhibitor of human NEP. D. melanogaster NEP2 also differs from human NEP in the manner in which the peptidase cleaves the tachykinin, GPSGFYGVR-amide. Molecular modelling suggests that there are important structural differences between D. melanogaster NEP2 and human NEP in the S1′ and S2′ ligand-binding subsites, which might explain the observed differences in inhibitor and substrate specificities. A soluble isoform of a mouse NEP-like peptidase is strongly expressed in spermatids, suggesting an evolutionarily conserved role for a soluble endopeptidase in spermatogenesis. PMID:15554877

  9. Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.

    PubMed

    Becker, C; Senyuk, V I; Shutov, A D; Nong, V H; Fischer, J; Horstmann, C; Müntz, K

    1997-09-01

    Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

  10. A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase.

    PubMed Central

    Bailyes, E M; Shennan, K I; Seal, A J; Smeekens, S P; Steiner, D F; Hutton, J C; Docherty, K

    1992-01-01

    PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones. Images Fig. 1. Fig. 3. PMID:1637332

  11. Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake.

    PubMed

    Pollard, Ricquita D; Blesso, Christopher N; Zabalawi, Manal; Fulp, Brian; Gerelus, Mark; Zhu, Xuewei; Lyons, Erica W; Nuradin, Nebil; Francone, Omar L; Li, Xiang-An; Sahoo, Daisy; Thomas, Michael J; Sorci-Thomas, Mary G

    2015-06-19

    Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.

  12. Endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 of Arabidopsis (AtCEP1) is involved in pathogen defense.

    PubMed

    Höwing, Timo; Huesmann, Christina; Hoefle, Caroline; Nagel, Marie-Kristin; Isono, Erika; Hückelhoven, Ralph; Gietl, Christine

    2014-01-01

    Programmed cell death (PCD) is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type cysteine endopeptidases (CysEP) with a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEP). KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodeling. We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.

  13. Effect of an inhaled neutral endopeptidase inhibitor, phosphoramidon, on baseline airway calibre and bronchial responsiveness to bradykinin in asthma.

    PubMed Central

    Crimi, N.; Polosa, R.; Pulvirenti, G.; Magrì, S.; Santonocito, G.; Prosperini, G.; Mastruzzo, C.; Mistretta, A.

    1995-01-01

    BACKGROUND--Bradykinin is a potent vasoactive peptide which has been proposed as an important inflammatory mediator in asthma since it provokes potent bronchoconstriction in asthmatic subjects. Little is known at present about the potential role of lung peptidases in modulating bradykinin-induced airway dysfunction in vivo in man. The change in bronchial reactivity to bradykinin was therefore investigated after treatment with inhaled phosphoramidon, a potent neutral endopeptidase (NEP) inhibitor, in a double blind, placebo controlled, randomised study of 10 asthmatic subjects. METHODS--Subjects attended on six separate occasions at the same time of day during which concentration-response studies with inhaled bradykinin and histamine were carried out, without treatment and after each test drug. Subjects received nebulised phosphoramidon sodium salt (10(-5) M, 3 ml) or matched placebo for 5-7 minutes using an Inspiron Mini-neb nebuliser 5 minutes before the bronchoprovocation test with bradykinin or histamine. Agonists were administered in increasing concentrations as an aerosol generated from a starting volume of 3 ml in a nebuliser driven by compressed air at 8 1/min. Changes in airway calibre were measured as forced expiratory volume in one second (FEV1) and responsiveness as the provocative concentration causing a 20% fall in FEV1 (PC20). RESULTS--Phosphoramidon administration caused a transient fall in FEV1 from baseline, FEV1 values decreasing 6.3% and 5.3% on the bradykinin and histamine study days, respectively. When compared with placebo, phosphoramidon elicited a small enhancement of the airways response to bradykinin, the geometric mean PC20 value (range) decreasing from 0.281 (0.015-5.575) to 0.136 (0.006-2.061) mg/ml. In contrast, NEP blockade failed to alter the airways response to a subsequent inhalation with histamine, the geometric mean (range) PC20 histamine value of 1.65 (0.17-10.52) mg/ml after placebo being no different from that of 1.58 (0

  14. epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

    PubMed Central

    Sugai, M; Fujiwara, T; Ohta, K; Komatsuzawa, H; Ohara, M; Suginaka, H

    1997-01-01

    Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide. PMID:9209049

  15. Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

    PubMed Central

    Sridhar, Vidya R.; Hughes, Joanne E.; Welker, Dennis L.; Broadbent, Jeffery R.; Steele, James L.

    2005-01-01

    Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and αS1-casein (αS1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards αS1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and αS1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides αS1-CN (f1-9), αS1-CN (f1-13), and αS1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and αS1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese. PMID:15932998

  16. Recovery, viscoelastic and functional properties of Barbel skin gelatine: investigation of anti-DPP-IV and anti-prolyl endopeptidase activities of generated gelatine polypeptides.

    PubMed

    Sila, Assaâd; Martinez-Alvarez, Oscar; Haddar, Anissa; Gómez-Guillén, M Carmen; Nasri, Moncef; Montero, M Pilar; Bougatef, Ali

    2015-02-01

    The characteristics and functional properties of gelatine from freshwater fish skin (Barbus callensis) were investigated. The gelatine extraction efficiency was improved by an acid-swelling process in the presence of barbel crude acid protease extract. Barbel skin gelatine (BSG) contained 92.15% protein, 0.31% lipid and 0.72% ash. The amino acid profile of BSG showed a high percentage of imino acids. The electrophoretic profile showed that BSG is mainly composed of α- and β-components. BSG showed an excellent solubility and possessed interfacial properties, which were governed by the protein concentration. Biological activities of the hydrolysates obtained after digestion of BSG with several commercial proteases were evaluated. The results suggested that these hydrolysates are a good source of natural inhibitors of dipeptidyl peptidase-IV and prolyl endopeptidase and could potentially be used as dietary ingredients in the management of type 2-diabetes and/or neuropathological disorders. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Peptides in Seminal Fluid and Their Role in Infertility: A Potential Role for Opiorphin Inhibition of Neutral Endopeptidase Activity as a Clinically Relevant Modulator of Sperm Motility

    PubMed Central

    Davies, Kelvin P.; Neal-Perry, Genevieve S.

    2014-01-01

    Infertility is a devastating medical condition that adversely affects emotional health and well-being of couples who desire pregnancy and parenthood. The overall demographic data suggest that the indication for more than one-third of assisted reproductive technology cycles performed in the United States includes male factor infertility. There is increasing recognition of the role that peptides present in seminal plasma have in determining sperm motility. Several recent studies suggest that peptidases, such as neutral endopeptidase (NEP) and aminopeptidase N (APN), impose significant adverse effects on sperm motility. Interestingly, several recent studies demonstrate that there is an endogenous NEP/APN inhibitor peptide called opiorphin in human seminal plasma. Our pilot studies suggest opiorphin promotes sperm motility and may positively influence sperm motility parameters in some cases of males infertility characterized by asthenozoospermia. PMID:24855109

  18. Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

    PubMed

    Panda, Rakhi; Fiedler, Katherine L; Cho, Chung Y; Cheng, Raymond; Stutts, Whitney L; Jackson, Lauren S; Garber, Eric A E

    2015-12-09

    The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

  19. Variation in bull beef quality due to ultimate muscle pH is correlated to endopeptidase and small heat shock protein levels.

    PubMed

    Pulford, D J; Dobbie, P; Fraga Vazquez, S; Fraser-Smith, E; Frost, D A; Morris, C A

    2009-09-01

    This study set out to determine if ultimate pH (pH(u)) affected the performance of intracellular small heat shock protein and endopeptidase dynamics in muscle during beef ageing. Longissimus dorsi muscles from 39 Angus or Limousin×Angus bulls were examined to see if pH(u) achieved at 22h post mortem (rigor) affected tenderness and water holding capacity of beef. Samples were segregated into three pH(u) groups termed high (pH>6.3), intermediate (5.73 days post mortem for intermediate pH(u) beef. High levels of alpha β-crystallin (aβC) at 22h post mortem coincided with delayed muscle protein degradation for low pH(u) beef. Our results support the hypothesis that aβC shields myofibrils and buffers against endopeptidase degradation of beef structure during ageing.

  20. Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM.

    PubMed

    Kohler, Verena; Probst, Ines; Aufschnaiter, Andreas; Büttner, Sabrina; Schaden, Lisa; Rechberger, Gerald N; Koraimann, Günther; Grohmann, Elisabeth; Keller, Walter

    2017-02-17

    Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.

  1. Purification of the main somatostatin-degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16.

    PubMed

    Dahms, P; Mentlein, R

    1992-08-15

    The main somatostatin-degrading proteases were purified from rat and pig brain homogenates and characterized as thiol- and metal-dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68-kDa protease cleaved somatostatin at three bonds (Asn5-Phe6, Phe6-Phe7 and Thr10-Phe11) and neurotensin only at the Arg8-Arg9 bond, whereas the 70-kDa protease digested somatostatin at only two bonds (Phe6-Phe7 and Thr10-Phe11) and neurotensin as well as acetylneurotensin-(8-13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10-Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68-kDa protease was found to be mainly, but not exclusively, soluble and not membrane-associated, whereas the inverse was detected for the 70-kDa protease. Based on distinct molecular and catalytic properties, the 68-kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70-kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensin-degrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species-dependent activity. Species-independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin-(8-13) and liberation of free Phe from somatostatin only by

  2. Neutral endopeptidase-resistant C-type natriuretic peptide variant represents a new therapeutic approach for treatment of fibroblast growth factor receptor 3-related dwarfism.

    PubMed

    Wendt, Daniel J; Dvorak-Ewell, Melita; Bullens, Sherry; Lorget, Florence; Bell, Sean M; Peng, Jeff; Castillo, Sianna; Aoyagi-Scharber, Mika; O'Neill, Charles A; Krejci, Pavel; Wilcox, William R; Rimoin, David L; Bunting, Stuart

    2015-04-01

    Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase, that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B. The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants (BMN 111) resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.

  3. Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

    PubMed

    Valdés-Rodríguez, Silvia; Galván-Ramírez, Juan Pablo; Guerrero-Rangel, Armando; Cedro-Tanda, Alberto

    2015-01-01

    In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity.

  4. Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli.

    PubMed

    Sharma, Rahul; Sharma, Poonam R; Choudhary, Manohar L; Pande, Amit; Khatri, Ghan Shyam

    2006-01-01

    Methicillin-resistant Staphylococcus aureus is a major problem in the world, causing hospital acquired infections and the infections/pathogenesis in community. Lysostaphin is a novel therapeutic molecule to kill the multidrug-resistant S. aureus. Mature lysostaphin is a single polypeptide (approximately 27 kDa) chain metalloprotease glycylglycine endopeptidase, capable of specifically hydrolyzing penta-glycine crosslinks present in the peptidoglycan of the S. aureus cell wall. The mature lysostaphin gene of Staphylococcus simulans has been cloned and overexpressed in the cytoplasm of E. coli with amino terminal hexa-histidine as a fusion partner under the transcriptional control of bacteriophage T7 phi 10 promoter/lac operator and ribosome binding site. The transformed E. coli BL21 (lambdaDE3) cells produced catalytically active soluble (His)6-lysostaphin fusion protein in the cytoplasm representing approximately 20% of the total cellular proteins. The fusion protein was purified to homogeneity using a single chromatographic step of IMAC on Ni-NTA agarose. The present cloning, expression, and purification procedure of recombinant lysostaphin from a non-pathogenic organism E. coli enables preparation of large quantity of r-lysostaphin for structure function studies and evaluation of its clinical potential in therapy and prophylaxis of staphylococcal infections.

  5. Functional up-regulation of endopeptidase neurolysin during post-acute and early recovery phases of experimental stroke in mouse brain.

    PubMed

    Rashid, Mamoon; Wangler, Naomi J; Yang, Li; Shah, Kaushik; Arumugam, Thiruma V; Abbruscato, Thomas J; Karamyan, Vardan T

    2014-04-01

    In this study, we provide evidence for the first time that membrane-bound endopeptidase neurolysin is up-regulated in different parts of mouse brain affected by focal ischemia-reperfusion in a middle cerebral artery occlusion model of stroke. Radioligand binding, enzymatic and immunoblotting experiments in membrane preparations of frontoparietal cortex, striatum, and hippocampus isolated from the ischemic hemisphere of mouse brain 24 h after reperfusion revealed statistically significant increase (≥ twofold) in quantity and activity of neurolysin compared with sham-operated controls. Cerebellar membranes isolated from the ischemic hemisphere served as negative control supporting the observations that up-regulation of neurolysin occurs in post-ischemic brain regions. This study also documents sustained functional up-regulation of neurolysin in frontoparietal cortical membranes for at least 7 days after stroke, which appears not to be transcriptionally or translationally regulated, but rather depends on translocation of cytosolic neurolysin to the membranes and mitochondria. Considering diversity of endogenous neurolysin substrates (neurotensin, bradykinin, angiotensins I/II, substance P, hemopressin, dynorphin A(1-8), metorphamide, somatostatin) and the well-documented role of these peptidergic systems in pathogenesis of stroke, resistance to ischemic injury and/or post-stroke brain recovery, our findings suggest that neurolysin may play a role in processes modulating the brain's response to stroke and its recovery after stroke.

  6. Substrate specificity of an elongation-specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae

    PubMed Central

    Dörr, Tobias; Cava, Felipe; Lam, Hubert; Davis, Brigid M.; Waldor, Matthew K.

    2013-01-01

    The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross-linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide crosslinks that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth – particularly elongation - of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG-binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,D-endopeptidase able to cleave most peptide chain crosslinks in V. cholerae’s PG. PG from a shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA’s substrate-dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side-wall growth are discussed. PMID:23834664

  7. Lack of Exercise Might Invite Dementia

    MedlinePlus

    ... medlineplus.gov/news/fullstory_163264.html Lack of Exercise Might Invite Dementia Study found being sedentary may ... the study didn't prove that lack of exercise caused dementia risk to increase. It only found ...

  8. UK-414,495, a selective inhibitor of neutral endopeptidase, potentiates pelvic nerve-stimulated increases in female genital blood flow in the anaesthetized rabbit

    PubMed Central

    Wayman, CP; Baxter, D; Turner, L; Van Der Graaf, PH; Naylor, AM

    2010-01-01

    Background and purpose: Female sexual arousal consists of a number of physiological responses resulting from increased genital blood. Vasoactive intestinal peptide (VIP), neuropeptide Y and to a lesser extent nitric oxide are neurotransmitters found in the vasculature of the genitalia. Neutral endopeptidase (NEP) modulates the activity of neuropeptides including VIP. The aim of this study was to investigate the control of genital blood flow by VIP and endogenous neuropeptides using a selective NEP inhibitor [UK-414,495, ((R)-2-({1-[(5-ethyl-1,3,4-thiadiazol-2-yl) carbamoyl]cyclopentyl}methyl) valeric acid)]. Experimental approach: Vaginal and clitoral blood flow (VBF and CBF) were monitored using laser Doppler in terminally anaesthetized New Zealand rabbits. Increases in VBF and CBF were induced by either electrical stimulation of the pelvic nerve or by i.v. infusion of VIP. Key results: Stimulation of the pelvic nerve increased VBF and CBF, compared with basal flow. Increases were mimicked by infusion of exogenous VIP. UK-414,495 dose-dependently potentiated pelvic nerve-stimulated increases in VBF (EC50= 37 ± 9 nM; 3.6 × IC50 rabbit NEP). Nerve-stimulated increases in VBF and CBF were both enhanced after UK-414,495. UK-414,495 increased the amplitude and duration of VIP-induced increases in VBF. UK-414,495 had no effect on basal VBF or cardiovascular parameters. Conclusions and implications: Inhibition of NEP potentiates pelvic nerve-stimulated increases in genital blood flow. This suggests that the endogenous neurotransmitter mediating genital blood flow is a substrate for NEP (most likely VIP). NEP inhibitors may restore sexual arousal in women adversely affected by female sexual arousal disorder. This article is commented on by Angulo, pp. 48–50 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00693.x PMID:20412068

  9. Effects of chronic neutral endopeptidase inhibition on the progression of left ventricular dysfunction and remodeling in dogs with moderate heart failure.

    PubMed

    Mishima, Takayuki; Tanimura, Mitsuhiro; Suzuki, George; Todor, Anastassia; Sharov, Victor G; Tanhehco, Elaine J; Goldstein, Sidney; Sabbah, Hani N

    2002-05-01

    The diuretic actions of endogenously produced atrial natriuretic factor (ANF) may be beneficial in the treatment of chronic heart failure (CHF). Neutral endopeptidase (NEP) is the primary enzyme responsible for the degradation of ANF. The present study investigates the effects of long-term NEP inhibition on the progression of left ventricular (LV) dysfunction and remodeling in dogs with moderate heart failure. LV dysfunction was produced in 12 dogs by multiple sequential intracoronary microembolizations. Embolizations were discontinued when LV ejection fraction (EF) was between 30-40%. Two weeks after the last embolization, dogs were randomized to 3 months of oral therapy with the NEP inhibitor ecadotril (100 mg, once daily, n = 6) or to no therapy at all (control, n = 6). During the 3 months of follow-up, LV EF in control dogs decreased from 37 +/- 1% to 28 +/- 1% (P < 0.01) and LV end-diastolic volume (EDV) and end-systolic volume (ESV) increased (EDV: 72 +/- 3 vs. 84 +/- 5 ml, P < 0.01); ESV: 45 +/- 1 vs. 60 +/- 4 ml, P < 0.01). In dogs treated with ecadotril, LV EF (34 +/- 1% vs. 37 +/- 2%), EDV (79+/- 5 vs. 78+/- 6 ml) and ESV (52 +/- 3 vs. 49 +/- 4) remained essentially unchanged after 3 months of therapy. Histomorphometric measurements at the termination of the study showed that ecadotril was associated with significantly reduced cardiomyocyte hypertrophy compared to control. Early, long-term NEP inhibition with ecadotril prevents the progression of LV dysfunction and attenuates progressive LV remodeling in dogs with moderate heart failure.

  10. The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts

    PubMed Central

    Nakajima, Hiroaki; Terazawa, Shuko; Niwano, Takao; Yamamoto, Yorihiro; Imokawa, Genji

    2016-01-01

    We recently reported that the over-expression of skin fibroblast-derived neutral endopeptidase (NEP) plays a pivotal role in impairing the three-dimensional architecture of dermal elastic fibers during the biological mechanism of ultraviolet (UV)-induced skin wrinkling. In that process, a UVB-associated epithelial-mesenchymal cytokine interaction as well as a direct UVA-induced cellular stimulation are associated with the up-regulation of NEP in human fibroblasts. In this study, we characterized the mode of action of ubiquinol10 which may abrogate the up-regulation of NEP by dermal fibroblasts, resulting in a reported in vivo anti-wrinkling action, and compared that with 3 other anti-oxidants, astaxanthin (AX), riboflavin (RF) and flavin mononucleotide (FMN). Post-irradiation treatment with all 4 of those anti-oxidants elicited an interrupting effect on the UVB-associated epithelial-mesenchymal cytokine interaction leading to the up-regulation of NEP in human fibroblasts but with different modes of action. While AX mainly served as an inhibitor of the secretion of wrinkle-inducing cytokines, such as interleukin-1α (IL-1α) and granulocyte macrophage colony stimulatory factor (GM-CSF) in UVB-exposed epidermal keratinocytes, ubiquinol10, RF and FMN predominantly interrupted the IL-1α and GM-CSF-stimulated expression of NEP in dermal fibroblasts. On the other hand, as for the UVA-associated mechanism, similar to the abrogating effects reported for AX and FMN, ubiquinol10 but not RF had the potential to abrogate the increased expression of NEP and matrix-metalloproteinase-1 in UVA-exposed human fibroblasts. Our findings strongly support the in vivo anti-wrinkling effects of ubiquinol10 and AX on human and animal skin and provide convincing proof of the UV-induced wrinkling mechanism that essentially focuses on the over-expression of NEP by dermal fibroblasts as an intrinsic causative factor. PMID:27648570

  11. A Systematic Approach to the Comparison of Cost Efficiency of Endopeptidases for the Hydrolysis of Atlantic Salmon (Salmo salar) By-Products

    PubMed Central

    Egede-Nissen, Henning; Oterhals, Ĺge

    2016-01-01

    Summary The hydrolytic and cost efficiencies of five endopeptidases (Alcalase 2.4L, Corolase 7089, Neutrase 0.8L, Promod 671L and Protex 7L) to hydrolyze Atlantic salmon by-products were compared at standardized activity levels based on a casein assay. The substrate was characterized prior to the hydrolytic experiments (pH=6.5, t=50 °C) to obtain substrate--specific constants for nitrogen to protein mass (in g) ratio, i.e. conversion factor fN=5.23 and total amount of peptide bonds (htot)=9.3 mmol per g of protein. At low enzyme activity to substrate ratio, all enzymes were equally efficient in hydrolyzing the substrate. At highest enzyme activity to substrate ratio, Protex 7L, Alcalase 2.4L and Promod 671L gave higher degree of hydrolysis (DH=14.2–14.6%) than Corolase 7089 (13.2%) and Neutrase 0.8L (11.6%) after 120 min of hydrolysis. No differences were observed in protein recovery (yield of solubilized protein) relative to DH. Determination of DH was followed by the pH-STAT and o-phthaldialdehyde methods. Based on pH-STAT data, response surface regression models were established based on the combined effects of hydrolysis time and enzyme activity to substrate ratio on DH and protein recovery. The modelling approach was combined with enzyme cost to identify the most cost-efficient enzyme (Protex 7L). PMID:28115899

  12. A Highly Active and Negatively Charged Streptococcus pyogenes Lysin with a Rare d-Alanyl-l-Alanine Endopeptidase Activity Protects Mice against Streptococcal Bacteremia

    PubMed Central

    Lood, Rolf; Raz, Assaf; Molina, Henrik; Euler, Chad W.

    2014-01-01

    Bacteriophage endolysins have shown great efficacy in killing Gram-positive bacteria. PlyC, a group C streptococcal phage lysin, represents the most efficient lysin characterized to date, with a remarkably high specificity against different streptococcal species, including the important pathogen Streptococcus pyogenes. However, PlyC is a unique lysin, in terms of both its high activity and structure (two distinct subunits). We sought to discover and characterize a phage lysin active against S. pyogenes with an endolysin architecture distinct from that of PlyC to determine if it relies on the same mechanism of action as PlyC. In this study, we identified and characterized an endolysin, termed PlyPy (phage lysin from S. pyogenes), from a prophage infecting S. pyogenes. By in silico analysis, PlyPy was found to have a molecular mass of 27.8 kDa and a pI of 4.16. It was active against a majority of group A streptococci and displayed high levels of activity as well as binding specificity against group B and C streptococci, while it was less efficient against other streptococcal species. PlyPy showed the highest activity at neutral pH in the presence of calcium and NaCl. Surprisingly, its activity was not affected by the presence of the group A-specific carbohydrate, while the activity of PlyC was partly inhibited. Additionally, PlyPy was active in vivo and could rescue mice from systemic bacteremia. Finally, we developed a novel method to determine the peptidoglycan bond cleaved by lysins and concluded that PlyPy exhibits a rare d-alanyl-l-alanine endopeptidase activity. PlyPy thus represents the first lysin characterized from Streptococcus pyogenes and has a mechanism of action distinct from that of PlyC. PMID:24637688

  13. Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q.

    PubMed

    Honda-Ogawa, Mariko; Sumitomo, Tomoko; Mori, Yasushi; Hamd, Dalia Talat; Ogawa, Taiji; Yamaguchi, Masaya; Nakata, Masanobu; Kawabata, Shigetada

    2017-03-10

    Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis. However, full understanding of the molecules possessed by this organism that interact with complement C1q, an initiator of the classical complement pathway, remains elusive. In this study, we identified an endopeptidase of S. pyogenes, PepO, as an interacting molecule, and investigated its effects on complement immunity and pathogenesis. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis findings revealed that S. pyogenes recombinant PepO bound to human C1q in a concentration-dependent manner under physiological conditions. Sites of inflammation are known to have decreased pH levels, thus the effects of PepO on bacterial evasion from complement immunity was analyzed in a low pH condition. Notably, under low pH conditions, PepO exhibited a higher affinity for C1q as compared with IgG, and PepO inhibited the binding of IgG to C1q. In addition, pepO deletion rendered S. pyogenes more susceptible to the bacteriocidal activity of human serum. Also, observations of the morphological features of the pepO mutant strain (ΔpepO) showed damaged irregular surfaces as compared with the wild-type strain (WT). WT-infected tissues exhibited greater severity and lower complement activity as compared with those infected by ΔpepO in a mouse skin infection model. Furthermore, WT infection resulted in a larger accumulation of C1q than that with ΔpepO. Our results suggest that interaction of S. pyogenes PepO with C1q interferes with the complement pathway, which enables S. pyogenes to evade complement-mediated bacteriolysis under acidic conditions, such as seen in inflammatory sites. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Structures of a bifunctional cell-wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 dl-endopeptidase

    PubMed Central

    Xu, Qingping; Chiu, Hsiu-Ju; Farr, Carol L.; Jaroszewski, Lukasz; Knuth, Mark W.; Miller, Mitchell D.; Lesley, Scott A.; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2013-01-01

    Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell-wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of two CwlT from pathogens Staphylococcus aureus mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific for tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release, or closing during catalysis. PMID:24051416

  15. Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.

    PubMed

    Xu, Qingping; Chiu, Hsiu-Ju; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Lesley, Scott A; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

    2014-01-09

    Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

  16. Abundance of cysteine endopeptidase dionain in digestive fluid of Venus flytrap (Dionaea muscipula Ellis) is regulated by different stimuli from prey through jasmonates.

    PubMed

    Libiaková, Michaela; Floková, Kristýna; Novák, Ondřej; Slováková, L'udmila; Pavlovič, Andrej

    2014-01-01

    The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey's movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH4Cl, KH2PO4, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed.

  17. Abundance of Cysteine Endopeptidase Dionain in Digestive Fluid of Venus Flytrap (Dionaea muscipula Ellis) Is Regulated by Different Stimuli from Prey through Jasmonates

    PubMed Central

    Libiaková, Michaela; Floková, Kristýna; Novák, Ondřej; Slováková, L'udmila; Pavlovič, Andrej

    2014-01-01

    The trap of the carnivorous plant Venus flytrap (Dionaea muscipula) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey's movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH4Cl, KH2PO4, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of cis-12-oxophytodienoic acid (cis-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed. PMID:25153528

  18. Comparative fine structural distribution of endopeptidase 24.15 (EC3.4.24.15) and 24.16 (EC3.4.24.16) in rat brain.

    PubMed

    Fontenele-Neto, J D; Massarelli, E E; Gurgel Garrido, P A; Beaudet, A; Ferro, E S

    2001-10-01

    Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.

  19. Neurotensin high affinity binding sites and endopeptidase 24. 11 are present respectively in the meningothelial and in the fibroblastic components of human meningiomas

    SciTech Connect

    Mailleux, P.; Przedborski, S.; Beaumont, A.; Verslijpe, M.; Depierreux, M.; Levivier, M.; Kitabgi, P.; Roques, B.P.; Vanderhaeghen, J.J. )

    1990-11-01

    The presence of neurotensin receptors and endopeptidase 24.11 (E-24.11) in 16 human meningioma specimens, obtained at surgery, was assessed by measuring the binding of {sup 125}I-(tyrosyl3)neurotensin(1-13) ({sup 125}I-NT) and the inhibitor {sup 3}H-N(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-(oxopropyl)glycine ({sup 3}H-HACBO-Gly), for the receptor and enzyme, respectively. E-24.11 activity was also measured. Autoradiography, on the 16 meningiomas, showed that specific {sup 125}I-NT labeling (nonspecific labeling was assessed in the presence of excess NT) was exclusively located in the meningothelial regions. In contrast, specific {sup 3}H-HACBO-Gly labeling (nonspecific labeling was assessed in the presence of an excess of the E-24.11 inhibitor thiorphan) was exclusively found in fibroblastic regions. No specific labeling of either ligand was found on collagen or blood vessels. In vitro binding assays were performed on membranes of 10 of the 16 meningiomas. In the 4 meningiomas rich in meningothelial cells, {sup 125}I-NT specifically bound to one population of sites with Bmax ranging from 57 to 405 fmol/mg protein and Kd around 0.3 nM. These sites share common properties with the brain NT receptor, since the carboxy terminal acetyl NT(8-13) fragment bound to the same sites but with a higher affinity. The carboxy terminal analogue of NT, neuromedin N, also bound to the same sites with a 10-fold lower affinity and the sites were bradykinin and levocabastine insensitive. In the 4 meningiomas rich in fibroblastic cells, {sup 3}H-HACBO-Gly specifically bound to one population of sites with Bmax ranging from 251 to 739 fmol/mg protein and Kd around 2.8 nM.

  20. Genetic homogeneity but IgG subclass-dependent clinical variability of alloimmune membranous nephropathy with anti-neutral endopeptidase antibodies.

    PubMed

    Vivarelli, Marina; Emma, Francesco; Pellé, Thimothée; Gerken, Christopher; Pedicelli, Stefania; Diomedi-Camassei, Francesca; Klaus, Günter; Waldegger, Siegfried; Ronco, Pierre; Debiec, Hanna

    2015-03-01

    Alloimmune antenatal membranous nephropathy (MN) during pregnancy results from antibodies produced by a neutral endopeptidase (NEP)-deficient mother. Here we report two recent cases that provide clues to the severity of renal disease. Mothers of the two children had circulating antibodies against NEP showing the characteristic species-dependent pattern by immunofluorescence on kidney slices. A German mother produced predominantly anti-NEP IgG4 accompanied by a low amount of IgG1. Her child recovered renal function within a few weeks. In sharp contrast, an Italian mother mainly produced complement-fixing anti-NEP IgG1, which also inhibits NEP enzymatic activity, whereas anti-NEP IgG4 has a weak inhibitory potency. Her child was dialyzed for several weeks. A kidney biopsy performed at 12 days of age showed MN, ischemic glomeruli, and arteriolar and tubular lesions. A second biopsy performed at 12 weeks of age showed aggravation with an increased number of collapsed capillary tufts. Both mothers were homozygous for the truncating deletion mutation 466delC and were thus NEP deficient. The 466delC mutation, identified in three previously described families, suggests a founder effect. Because of the potential severity of alloimmune antenatal MN, it is essential to identify families at risk by the detection of anti-NEP antibodies and NEP antigen in urine. On the basis of the five families identified to date, we propose an algorithm for the diagnosis of the disease and the prevention of complications.

  1. Lack of Set Theory Relevant Prerequisite Knowledge

    ERIC Educational Resources Information Center

    Dogan-Dunlap, Hamide

    2006-01-01

    Many students struggle with college mathematics topics due to a lack of mastery of prerequisite knowledge. Set theory language is one such prerequisite for linear algebra courses. Many students' mistakes on linear algebra questions reveal a lack of mastery of set theory knowledge. This paper reports the findings of a qualitative analysis of a…

  2. Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin

    PubMed Central

    2011-01-01

    Background Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use. Results Diversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screening with several rounds of increasing stringency, as is usual with phage technology, the library got overwhelmed by phagemids encoding incomplete scFvs. The screening was successfully re-performed with a single round of high stringency. In particular, one of the isolated scFvs, 2H8, bound BoNT/A1 with a 3.3 nM affinity and effectively inhibited BoNT/A1 endopeptidase activity. The sequence encoding 2H8 was 88% identical to human germline genes and its average G-score was -0.72, quantifying the high human-like quality of 2H8. Conclusions The presence of restrictions sites within many of the sequences that were to be part of the library did not prevent the isolation of an scFv, 2H8, by an adapted panning strategy. ScFv 2H8 inhibited toxin endopeptidase activity in vitro and possessed human-like quality required for clinical development. More generally, the construction and screening of phage-displayed libraries built from hyper-immunized non-human primates is an efficient solution to isolate

  3. Report: EPA Travel Program Lacks Necessary Controls

    EPA Pesticide Factsheets

    Report #10-P-0078, March 9, 2010. The EPA travel program, which comprises EPA policies and GovTrip, lacks necessary control procedures to assure all travel authorizations were necessary and in the best interest of the government.

  4. Pharmacokinetics of M100240 and MDL 100,173, a dual angiotensin-converting enzyme/neutral endopeptidase inhibitor, in healthy young and elderly volunteers.

    PubMed

    Emmons, Gary T; Argenti, Rick; Martin, Louis L; Martin, Nancy E; Jensen, Bradford K

    2004-08-01

    M100240 is an acetate thioester of MDL 100,173-a dual angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitor-in phase II development. The pharmacokinetics of M100240 and MDL 100,173 were compared in young and elderly subjects. Pharmacokinetic data were obtained from 12 young (ages 18-45 years, 10 male, 2 female) and 12 elderly (ages 65-85 years, 7 male, 5 female) healthy subjects in a parallel-group, open-label study. Following an overnight fast, subjects received a single 25-mg oral dose of M100240. Serial plasma concentrations of M100240 and MDL 100,173 were determined using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, and pharmacokinetic parameters were calculated with noncompartmental methods. Single-dose treatment with M100240 was well tolerated in both groups of subjects, with no clinically significant changes in vital signs, ECG recordings, or laboratory safety parameters. M100240 was rapidly absorbed and converted to MDL 100,173, with M100240 concentrations no longer detectable at 3 to 4 hours postdose in both groups. The pharmacokinetics of the pharmacologically active MDL 100,173 were similar for both groups. Although maximum concentrations of M100240 were generally higher in elderly versus young subjects (C(max) 0.48 ng/mL vs. 0.17 ng/mL), systemic availability of M100240 was quite low and variable with plasma, and this apparent difference in parent drug exposure is unlikely to have important clinical implications. No age-related differences in the pharmacokinetic parameters of MDL 100,173 (C(max) 8.16 vs. 9.62 ng/mL, t(max) 1.25 vs. 1.5 h, AUC((0-last)) 81.6 vs. 72.2 ng x h/mL) were observed between young and elderly subjects, respectively. In conclusion, there are no age-related differences in the pharmacokinetics of MDL 100,173 between young and elderly subjects.

  5. Teichoic Acid Polymers Affect Expression and Localization of dl-Endopeptidase LytE Required for Lateral Cell Wall Hydrolysis in Bacillus subtilis

    PubMed Central

    Kasahara, Jun; Kiriyama, Yuuka; Miyashita, Mari; Kondo, Takuma; Yamada, Takeshi; Yazawa, Kazuya; Yoshikawa, Ritsuko

    2016-01-01

    ABSTRACT In Bacillus subtilis, the dl-endopeptidase LytE is responsible for lateral peptidoglycan hydrolysis during cell elongation. We found that σI-dependent transcription of lytE is considerably enhanced in a strain with a mutation in ltaS, which encodes a major lipoteichoic acid (LTA) synthase. Similar enhancements were observed in mutants that affect the glycolipid anchor and wall teichoic acid (WTA) synthetic pathways. Immunofluorescence microscopy revealed that the LytE foci were considerably increased in these mutants. The localization patterns of LytE on the sidewalls appeared to be helix-like in LTA-defective or WTA-reduced cells and evenly distributed on WTA-depleted or -defective cell surfaces. These results strongly suggested that LTA and WTA affect both σI-dependent expression and localization of LytE. Interestingly, increased LytE localization along the sidewall in the ltaS mutant largely occurred in an MreBH-independent manner. Moreover, we found that cell surface decorations with LTA and WTA are gradually reduced at increased culture temperatures and that LTA rather than WTA on the cell surface is reduced at high temperatures. In contrast, the amount of LytE on the cell surface gradually increased under heat stress conditions. Taken together, these results indicated that reductions in these anionic polymers at high temperatures might give rise to increases in SigI-dependent expression and cell surface localization of LytE at high temperatures. IMPORTANCE The bacterial cell wall is required for maintaining cell shape and bearing environmental stresses. The Gram-positive cell wall consists of mesh-like peptidoglycan and covalently linked wall teichoic acid and lipoteichoic acid polymers. It is important to determine if these anionic polymers are required for proliferation and environmental adaptation. Here, we demonstrated that these polymers affect the expression and localization of a peptidoglycan hydrolase LytE required for lateral cell wall

  6. Effects of SA7060, a novel dual inhibitor of neutral endopeptidase and angiotensin-converting enzyme, on deoxycorticosterone acetate-salt-induced hypertension in rats.

    PubMed

    Kuro, T; Okahara, A; Nose, M; Ikuse, T; Matsumura, Y

    2000-07-01

    We evaluated whether a novel dual inhibitor of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), SA7060, (S)-2-[3-[(S)-2-(butoxycarbonyl)-2-hydroxyethyl]-3-isobutylureido] -3-(2-naphtyl) propionic acid, prevents deoxycorticosterone acetate (DOCA)-salt-induced hypertension and related organ damage, such as cardiovascular hypertrophy, renal dysfunction and renal tissue injury in rats. The effectiveness was compared with candoxatril and enalapril, which are a selective NEP and ACE inhibitor, respectively. During DOCA-salt treatment for 4 weeks, the rats were given SA7060, candoxatril, enalapril or vehicle, once daily by gavage. The 4-weeks treatment with DOCA and salt produced progressive increases in systolic blood pressure. Daily administration of SA7060, candoxatril or enalapril significantly suppressed the development of hypertension induced by DOCA and salt, although the effect of enalapril was less potent at 4-weeks of the treatment period. In vehicle-treated DOCA-salt rats, decreases in creatinine clearance and increases in urinary excretion of protein and blood urea nitrogen were observed. This functional damage was improved most efficiently by the treatment with SA7060. There were significant increases in urinary excretions of atrial natriuretic peptide and cyclic GMP in SA7060- or candoxatril-treated animals. Histopathological examination of the kidney in DOCA-salt rats revealed tubular, glomerular and vascular lesions, all of which were improved in animals given SA7060 or candoxatril. When the vascular hypertrophy of the aorta was evaluated, there were significant increases in wall thickness, wall area and the wall-to-lumen ratio in vehicle-treated DOCA-salt rats compared with the sham rats. The development of vascular hypertrophy was suppressed by the treatment with SA7060, candoxatril or enalapril. Our findings indicate that SA7060 efficiently prevents DOCA-salt-induced hypertension and related tissue injury, mainly by inhibiting NEP

  7. Lacking control increases illusory pattern perception.

    PubMed

    Whitson, Jennifer A; Galinsky, Adam D

    2008-10-03

    We present six experiments that tested whether lacking control increases illusory pattern perception, which we define as the identification of a coherent and meaningful interrelationship among a set of random or unrelated stimuli. Participants who lacked control were more likely to perceive a variety of illusory patterns, including seeing images in noise, forming illusory correlations in stock market information, perceiving conspiracies, and developing superstitions. Additionally, we demonstrated that increased pattern perception has a motivational basis by measuring the need for structure directly and showing that the causal link between lack of control and illusory pattern perception is reduced by affirming the self. Although these many disparate forms of pattern perception are typically discussed as separate phenomena, the current results suggest that there is a common motive underlying them.

  8. Lack of Communications: The Most Common Deficiency.

    ERIC Educational Resources Information Center

    Allen, Thomas R., Jr.

    1979-01-01

    A survey with employers and teacher-coordinators of cooperative education programs showed that young employees' most common deficiencies are in communication skills, both written and oral. Poor handwriting was the leading complaint, followed by misspelling, ignorance of grammar and rhetoric, poor customer relations, and lack of comprehension and…

  9. Kid's Green Movement Lacks Basis in Reality.

    ERIC Educational Resources Information Center

    Chemecology, 1992

    1992-01-01

    Discussed is the idea that kids are highly politically correct environmentalists on the surface, but underneath they seem to be missing the connection with nature. The author attributes this ignorance of all things natural to things such as kid's preference for video games, television, and lack of access and time. The importance of parental…

  10. Kid's Green Movement Lacks Basis in Reality.

    ERIC Educational Resources Information Center

    Chemecology, 1992

    1992-01-01

    Discussed is the idea that kids are highly politically correct environmentalists on the surface, but underneath they seem to be missing the connection with nature. The author attributes this ignorance of all things natural to things such as kid's preference for video games, television, and lack of access and time. The importance of parental…

  11. Apathy in aging: are lack of interest and lack of initiative dissociable?

    PubMed

    Esposito, Fabienne; Rochat, Lucien; Juillerat Van der Linden, Anne-Claude; Lekeu, Françoise; Charnallet, Annik; Van der Linden, Martial

    2014-01-01

    Apathy is common in aging and generally defined on the basis of three dimensions: lack of initiative, lack of interest and emotional blunting. Curiously, no study until now has examined the associations and dissociations between these dimensions in elderly people (with or without dementia). These questions were addressed in two studies. In the first study, we explored the distribution of scores and the relationships between the three dimensions of apathy in 56 patients with dementia, focusing mainly on lack of initiative and lack of interest. Apathy was hetero-evaluated with the Apathy Inventory (AI), a scale widely used to assess the apathy dimensions in aging. In the second study, given the AI's limitations, we investigated in more detail the relationship between lack of initiative and interest in 115 elderly people using a new questionnaire specifically designed to assess these two dimensions. Results showed that lack of initiative was closely related to lack of interest (Study 1). Although we used a more specific questionnaire, these facets of apathy did not constitute two separable dimensions, but reflected a common main factor of apathy in aging (Study 2). Thus, the distinction between lack of initiative and lack of interest seems questionable. Only a multifactorial approach that includes the various psychological factors involved in apathy would enable one to gain a better understanding of the different manifestations of apathy and to highlight possible dissociations between them.

  12. Conceptualising the lack of health insurance coverage.

    PubMed

    Davis, J B

    2000-01-01

    This paper examines the lack of health insurance coverage in the US as a public policy issue. It first compares the problem of health insurance coverage to the problem of unemployment to show that in terms of the numbers of individuals affected lack of health insurance is a problem comparable in importance to the problem of unemployment. Secondly, the paper discusses the methodology involved in measuring health insurance coverage, and argues that the current method of estimation of the uninsured underestimates the extent that individuals go without health insurance. Third, the paper briefly introduces Amartya Sen's functioning and capabilities framework to suggest a way of representing the extent to which individuals are uninsured. Fourth, the paper sketches a means of operationalizing the Sen representation of the uninsured in terms of the disability-adjusted life year (DALY) measure.

  13. Peptides in seminal fluid and their role in infertility: a potential role for opiorphin inhibition of neutral endopeptidase activity as a clinically relevant modulator of sperm motility: a review.

    PubMed

    Bosler, Jayme S; Davies, Kelvin P; Neal-Perry, Genevieve S

    2014-11-01

    Infertility is a devastating medical condition that adversely affects emotional health and well-being of couples who desire pregnancy and parenthood. The overall demographic data suggest that the indication for more than one-third of assisted reproductive technology cycles performed in the United States includes male factor infertility. There is increasing recognition of the role that peptides present in seminal plasma have in determining sperm motility. Several recent studies suggest that peptidases, such as neutral endopeptidase (NEP) and aminopeptidase N (APN), impose significant adverse effects on sperm motility. Interestingly, several recent studies demonstrate that there is an endogenous NEP/APN inhibitor peptide called opiorphin in human seminal plasma. Our pilot studies suggest opiorphin promotes sperm motility and may positively influence sperm motility parameters in some cases of males infertility characterized by asthenozoospermia.

  14. Structure of the γ-d-glutamyl-l-diamino acid endopeptidase YkfC from Bacillus cereus in complex with l-Ala-γ-d-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases

    PubMed Central

    Xu, Qingping; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Lam, Winnie W.; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Yeh, Andrew; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-d-glutamyl-l-­diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (l-Ala-γ-d-Glu) enabled the identification of conserved sequence and structural signatures for recognition of l-Ala and γ-d-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial l-­alanine-γ-d-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site. PMID:20944232

  15. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  16. Does the Autistic Brain Lack Core Modules?

    PubMed Central

    Gernsbacher, Morton Ann; Frymiare, Jennifer L.

    2014-01-01

    Researchers have hypothesized that autistics are missing core modules of the brain, critical neural tissue necessary for accomplishing various processes. In this article, we critically review the evidence supporting two such hypothesized deficits. We ask whether autistic brains lack a module for understanding the behavior of others (i.e., theory of mind) and whether they lack a module for processing faces. We illustrate that successful performance on theory of mind tasks depends on linguistic ability; therefore, it is not surprising that autistics are more likely to fail theory of mind tasks because a qualitative impairment in communication is one of the primary diagnostic criteria for autism. Similarly, we illustrate that autistics are less likely to fixate the eye region of facial photographs and that the amount of time spent fixating the eye region correlates with activation in the face processing “module”; therefore, it is not surprising that autistics are less likely to activate the putative face processing area. These illustrations cast doubt on the arguments that the autistic brain is missing the core modules responsible for understanding theory of mind and for processing faces. PMID:25520587

  17. Analysing the lack of Demand Organisation

    NASA Astrophysics Data System (ADS)

    Boxer, Philip; Cohen, Bernard

    1998-07-01

    We seek to develop means of intervention in Enterprises that will enable them to react in an effective, sustainable and timely fashion to changes in the ways that markets and demand are organized; that is, to act strategically. We take an enterprise to be some entity that seeks to provide its clients with services that they value while maintaining its ability to do so in the face of changes in the demands of its clients and in the resources at its disposal. The services that clients value form around what the organization of their demands lack. The concept of strategy therefore rests on critically evaluating the ontology and semantics of the Enterprise in relation to these holes in demand organization. We access ontology and semantics by constructing and manipulating hypothetical, first-order, mathematical models of the Enterprise's services and of its value-adding processes. Because an enterprise is an anticipatory system, its semantic domain must include representations of the enterprise's model of itself and of the market and demand organizations within which it competes. First-order (set) theory provides adequate expressive power here, but alternative, higher order, mathematical frameworks, such as Dubois' hyperincursion, provide inadequate power, particularly in relation to the analysis of the properties of emergence. Knowing exactly why and where this mathematical lack manifests in the analysis process enables effective collaboration between systems analysts and psychoanalysts, and suggest directions for mathematical research.

  18. Quantum preparation uncertainty and lack of information

    NASA Astrophysics Data System (ADS)

    Rozpędek, Filip; Kaniewski, Jędrzej; Coles, Patrick J.; Wehner, Stephanie

    2017-02-01

    The quantum uncertainty principle famously predicts that there exist measurements that are inherently incompatible, in the sense that their outcomes cannot be predicted simultaneously. In contrast, no such uncertainty exists in the classical domain, where all uncertainty results from ignorance about the exact state of the physical system. Here, we critically examine the concept of preparation uncertainty and ask whether similarly in the quantum regime, some of the uncertainty that we observe can actually also be understood as a lack of information (LOI), albeit a lack of quantum information. We answer this question affirmatively by showing that for the well known measurements employed in BB84 quantum key distribution (Bennett and Brassard 1984 Int. Conf. on Computer System and Signal Processing), the amount of uncertainty can indeed be related to the amount of available information about additional registers determining the choice of the measurement. We proceed to show that also for other measurements the amount of uncertainty is in part connected to a LOI. Finally, we discuss the conceptual implications of our observation to the security of cryptographic protocols that make use of BB84 states.

  19. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    DTIC Science & Technology

    2011-07-01

    Domann, FE Page 11 Appendices – Figure 2 Figure 2. PHD3 mRNA expression is down-regulated in multiple ...containing proteins (PHD/EGLN/HPH) which utilize iron , oxygen and 2-oxoglutarate as co-factors to enzymatically catalyze hydroxylation on the oxygen-dependent...following hypoxia has been observed in multiple human cell lines from tumors of the breast, prostate and brain [10,16]. A recent clinical study of

  20. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    DTIC Science & Technology

    2009-07-01

    Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14 . ABSTRACT In many solid tumors, including prostate cancer, hypoxia...7 The HIF-1α promoter construct will allow for identification of proper HIF transactivation within a cell population to aid in ruling out HIF...Figure 6. All low PHD3 expressing cell lines show reactivation with 5-aza-dC. Cells were treated with 5µM 5-aza-dC every other day for 7 days

  1. Epigenetic Control of Prolyl and Asparaginyl Hydroxylases in Prostate Cancer

    DTIC Science & Technology

    2010-07-01

    in the adaptive immune system. (In preparation for Free Radical Biology and Medicine ). Case, A.J. et al (2010). The conditional loss of SOD2...et al. (2009). Heightened Susceptibility to Influenza Mortality in Immunodeficient Mice Caused by a T-cell Specific Defect in SOD2. Free Radical Biology and Medicine . 47

  2. Lack of transplacental transmission of Bartonella bovis.

    PubMed

    Chastant-Maillard, S; Boulouis, H-J; Reynaud, K; Thoumire, S; Gandoin, C; Bouillin, C; Cordonnier, N; Maillard, R

    2015-02-01

    Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.

  3. Lack of belowground mutualisms hinders Pinaceae invasions.

    PubMed

    Nuñez, Martin A; Horton, Thomas R; Simberloff, Daniel

    2009-09-01

    Why particular invasions succeed and others fail is not well understood. The role of soil biota has been proposed as important. However, the role of mutualists has received much less attention than that of pathogens. Here we report that lack of adequate ectomycorrhizal fungi hinders invasion by exotic Pinaceae on Isla Victoria, Argentina, by reducing both the probability of establishment and growth of invading individuals. More than one hundred exotic tree species were introduced to this island ca. 80 years ago, but invasive trees are found in high densities only in areas adjacent to plantations. With a series of greenhouse and field experiments we found lower mycorrhizal colonization levels and few fungal species far from original plantings, and key fungal mutualists are confined to areas near plantations, probably owing to dispersal limitations. Low inoculum levels far from the plantations are retarding the invasion. Our experiments indicate that positive interactions belowground can play a key but underappreciated role in invasion dynamics.

  4. The lack of large compact symmetric objects

    NASA Astrophysics Data System (ADS)

    Augusto, P.

    2009-02-01

    In recent years, `baby' (< 103 yr) and `young' (103-105 yr) radio galaxies have been found and classified, although their numbers are still small (tens). Also, they have many different names, depending on the type of survey and scientific context in which they were found: compact steep spectrum sources (CSS), giga-Hertz peaked spectrum sources (GPS) and compact-medium symmetric objects (C-MSO). The latter have the radio galaxy structure more obvious and correspond to the `babies' (CSOs; < 1 kpc) and `young' (MSOs; 1-15 kpc) radio galaxies. The log-size distribution of CSOs shows a sharp drop at 0.3 kpc. This trend continues through flat-spectrum MSOs (over the full 1-15 kpc size range). In order to find out if this lack of large CSOs and flat-spectrum MSOs is due to poor sampling (lack of surveys that probe efficiently the 0.3-15 kpc size range) and/or has physical meaning (e.g. if the lobes of CSOs expand as they grow and age, they might become CSSs, `disappearing' from the flat-spectrum MSO statistics), we have built a sample of 157 flat-spectrum radio sources with structure on ˜0.3-15 kpc scales. We are using new, archived and published data to produce and inspect hundreds of multi-frequency multi-instrument maps and models. We have already found 13 new secure CSO/MSOs. We expect to uncover ˜30-40 new CSOs and MSOs, most on the 0.3-15 kpc size range, when our project is complete.

  5. A chenopod extensin lacks repetitive tetrahydroxyproline blocks

    SciTech Connect

    Li, Xiongbiao; Kieliszewski, M.; Lamport, D.T.A. )

    1990-02-01

    An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criteria for membership of the extensin family save one, notably, lack of the diagnostic pentamer Ser-Hyp-Hyp-Hyp-Hyp. However, sequence analysis of the major tryptic peptides shows that sugar beet extensin shares a motif in common with tomato extensin P1 but differs by the position of an insertion sequence (X) or (Y) which, in sugar beet, splits the tetrahydroxyproline block: Ser-Hyp-Hyp-(X)-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, where (X) is (Val-His-Glu/Lys-Tyr-Pro), while in tomato the insertion sequence (Y) = (Val-Lys-Pro-Tyr-His-Pro) and, when it occurs, immediately follows the tetrahydroxyproline block: Ser-Hyp-Hyp-Hyp-Hyp-(Y)-Thr-Hyp-Val-Tyr-Lys. Based on these data were reinterpret three highly repetitive cDNA sequences, including nodulin N75 from soybean and wound-induced P33 of carrot, as extensins with split tetra(hydroxy)proline blocks.

  6. Perturbed hematopoiesis in mice lacking ATMIN

    PubMed Central

    Anjos-Afonso, Fernando; Loizou, Joanna I.; Bradburn, Amy; Kanu, Nnennaya; Purewal, Sukhveer; Da Costa, Clive; Behrens, Axel

    2016-01-01

    The ataxia telangiectasia mutated (ATM)-interacting protein ATMIN mediates noncanonical ATM signaling in response to oxidative and replicative stress conditions. Like ATM, ATMIN can function as a tumor suppressor in the hematopoietic system: deletion of Atmin under the control of CD19-Cre results in B-cell lymphomas in aging mice. ATM signaling is essential for lymphopoiesis and hematopoietic stem cell (HSC) function; however, little is known about the role of ATMIN in hematopoiesis. We thus sought to investigate whether the absence of ATMIN would affect primitive hematopoietic cells in an ATM-dependent or -independent manner. Apart from its role in B-cell development, we show that ATMIN has an ATM-independent function in the common myeloid progenitors (CMPs) by deletion of Atmin in the entire hematopoietic system using Vav-Cre. Despite the lack of lymphoma formation, ATMIN-deficient mice developed chronic leukopenia as a result of high levels of apoptosis in B cells and CMPs and induced a compensatory mechanism in which HSCs displayed enhanced cycling. Consequently, ATMIN-deficient HSCs showed impaired regeneration ability with the induction of the DNA oxidative stress response, especially when aged. ATMIN, therefore, has multiple roles in different cell types, and its absence results in perturbed hematopoiesis, especially during stress conditions and aging. PMID:27581360

  7. Lack of RNase L Attenuates Macrophage Functions

    PubMed Central

    Yi, Xin; Zeng, Chun; Liu, Hongli; Chen, Xiaoli; Zhang, Ping; Yun, Boo Seok; Jin, Ge; Zhou, Aimin

    2013-01-01

    Background Macrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL–6, and cyclooxygenase-2 (Cox-2) by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN) inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown. Methodology Bone marrow-derived macrophages (BMMs) were generated from RNase L+/+and −/− mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC)-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays. Conclusions/Findings Lack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2). Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions. PMID:24324683

  8. Lack of convergence in aquatic Anolis lizards.

    PubMed

    Leal, Manuel; Knox, Alison K; Losos, Jonathan B

    2002-04-01

    Why convergent evolution occurs among some species occupying similar habitats but not among others is a question that has received surprisingly little attention. Caribbean Anolis lizards, known for their extensive convergent evolution among islands in the Greater Antilles, are an appropriate group with which to address this question. Despite the well-documented pattern of between-island convergence, some Greater Antillean anoles are not obviously part of the convergence syndrome. One example involves aquatic anoles--species that are found near to and readily enter streams-which have evolved independently twice in the Caribbean and also twice on mainland Central America. Despite being found in similar habitats, no previous study has investigated whether aquatic anoles represent yet another case of morphological convergence. We tested this hypothesis by collecting morphological data for seven aquatic anole species and 29 species from the six convergent types of Greater Antillean habitat specialists. We failed to find evidence for morphological convergence: the two Caribbean aquatic species are greatly dissimilar to each other and to the Central American species, which, however, may be convergent upon each other. We suggest two possible reasons for this lack of convergence in an otherwise highly convergent system: either there is more than one habitat type occupied by anoles in the proximity of water, or there is more than one way to adapt to a single aquatic habitat. We estimate that almost all of the 113 species of Greater Antillean anoles occupy habitats that are also used by distantly related species, but only 15% of these species are not morphologically similar to their distantly related ecological counterparts. Comparative data from other taxa would help enlighten the question of why the extent of convergence is so great in some lineages and not in others.

  9. Caterpillars lack a resident gut microbiome.

    PubMed

    Hammer, Tobin J; Janzen, Daniel H; Hallwachs, Winnie; Jaffe, Samuel P; Fierer, Noah

    2017-09-05

    Many animals are inhabited by microbial symbionts that influence their hosts' development, physiology, ecological interactions, and evolutionary diversification. However, firm evidence for the existence and functional importance of resident microbiomes in larval Lepidoptera (caterpillars) is lacking, despite the fact that these insects are enormously diverse, major agricultural pests, and dominant herbivores in many ecosystems. Using 16S rRNA gene sequencing and quantitative PCR, we characterized the gut microbiomes of wild leaf-feeding caterpillars in the United States and Costa Rica, representing 124 species from 15 families. Compared with other insects and vertebrates assayed using the same methods, the microbes that we detected in caterpillar guts were unusually low-density and variable among individuals. Furthermore, the abundance and composition of leaf-associated microbes were reflected in the feces of caterpillars consuming the same plants. Thus, microbes ingested with food are present (although possibly dead or dormant) in the caterpillar gut, but host-specific, resident symbionts are largely absent. To test whether transient microbes might still contribute to feeding and development, we conducted an experiment on field-collected caterpillars of the model species Manduca sexta Antibiotic suppression of gut bacterial activity did not significantly affect caterpillar weight gain, development, or survival. The high pH, simple gut structure, and fast transit times that typify caterpillar digestive physiology may prevent microbial colonization. Moreover, host-encoded digestive and detoxification mechanisms likely render microbes unnecessary for caterpillar herbivory. Caterpillars illustrate the potential ecological and evolutionary benefits of independence from symbionts, a lifestyle that may be widespread among animals.

  10. Interplanetary Shocks Lacking Type 2 Radio Bursts

    NASA Technical Reports Server (NTRS)

    Gopalswamy, N.; Xie, H.; Maekela, P.; Akiyama, S.; Yashiro, S.; Kaiser, M. L.; Howard, R. A.; Bougeret, J.-L.

    2010-01-01

    We report on the radio-emission characteristics of 222 interplanetary (IP) shocks detected by spacecraft at Sun-Earth L1 during solar cycle 23 (1996 to 2006, inclusive). A surprisingly large fraction of the IP shocks (approximately 34%) was radio quiet (RQ; i.e., the shocks lacked type II radio bursts). We examined the properties of coronal mass ejections (CMEs) and soft X-ray flares associated with such RQ shocks and compared them with those of the radio-loud (RL) shocks. The CMEs associated with the RQ shocks were generally slow (average speed approximately 535 km/s) and only approximately 40% of the CMEs were halos. The corresponding numbers for CMEs associated with RL shocks were 1237 km/s and 72%, respectively. Thus, the CME kinetic energy seems to be the deciding factor in the radio-emission properties of shocks. The lower kinetic energy of CMEs associated with RQ shocks is also suggested by the lower peak soft X-ray flux of the associated flares (C3.4 versus M4.7 for RL shocks). CMEs associated with RQ CMEs were generally accelerating within the coronagraph field of view (average acceleration approximately +6.8 m/s (exp 2)), while those associated with RL shocks were decelerating (average acceleration approximately 3.5 m/s (exp 2)). This suggests that many of the RQ shocks formed at large distances from the Sun, typically beyond 10 Rs, consistent with the absence of metric and decameter-hectometric (DH) type II radio bursts. A small fraction of RL shocks had type II radio emission solely in the kilometric (km) wavelength domain. Interestingly, the kinematics of the CMEs associated with the km type II bursts is similar to those of RQ shocks, except that the former are slightly more energetic. Comparison of the shock Mach numbers at 1 AU shows that the RQ shocks are mostly subcritical, suggesting that they were not efficient in accelerating electrons. The Mach number values also indicate that most of these are quasi-perpendicular shocks. The radio-quietness is

  11. INTERPLANETARY SHOCKS LACKING TYPE II RADIO BURSTS

    SciTech Connect

    Gopalswamy, N.; Kaiser, M. L.; Xie, H.; Maekelae, P.; Akiyama, S.; Yashiro, S.; Howard, R. A.; Bougeret, J.-L.

    2010-02-20

    We report on the radio-emission characteristics of 222 interplanetary (IP) shocks detected by spacecraft at Sun-Earth L1 during solar cycle 23 (1996 to 2006, inclusive). A surprisingly large fraction of the IP shocks ({approx}34%) was radio quiet (RQ; i.e., the shocks lacked type II radio bursts). We examined the properties of coronal mass ejections (CMEs) and soft X-ray flares associated with such RQ shocks and compared them with those of the radio-loud (RL) shocks. The CMEs associated with the RQ shocks were generally slow (average speed {approx}535 km s{sup -1}) and only {approx}40% of the CMEs were halos. The corresponding numbers for CMEs associated with RL shocks were 1237 km s{sup -1} and 72%, respectively. Thus, the CME kinetic energy seems to be the deciding factor in the radio-emission properties of shocks. The lower kinetic energy of CMEs associated with RQ shocks is also suggested by the lower peak soft X-ray flux of the associated flares (C3.4 versus M4.7 for RL shocks). CMEs associated with RQ CMEs were generally accelerating within the coronagraph field of view (average acceleration {approx}+6.8 m s{sup -2}), while those associated with RL shocks were decelerating (average acceleration {approx}-3.5 m s{sup -2}). This suggests that many of the RQ shocks formed at large distances from the Sun, typically beyond 10 Rs, consistent with the absence of metric and decameter-hectometric (DH) type II radio bursts. A small fraction of RL shocks had type II radio emission solely in the kilometric (km) wavelength domain. Interestingly, the kinematics of the CMEs associated with the km type II bursts is similar to those of RQ shocks, except that the former are slightly more energetic. Comparison of the shock Mach numbers at 1 AU shows that the RQ shocks are mostly subcritical, suggesting that they were not efficient in accelerating electrons. The Mach number values also indicate that most of these are quasi-perpendicular shocks. The radio-quietness is predominant

  12. The endopeptidase activity and the activation by Cl- of angiotensin-converting enzyme is evolutionarily conserved: purification and properties of an an angiotensin-converting enzyme from the housefly, Musca domestica.

    PubMed Central

    Lamango, N S; Sajid, M; Isaac, R E

    1996-01-01

    A soluble 67 kDa angiotensin-converting enzyme (ACE) has been purified by lisinopril-Sepharose affinity column chromatography from adult houseflies, Musca domestica. The dipeptidyl carboxypeptidase activity towards benzoyl-Gly-His-Leu was inhibited by captopril (IC50 50 nM) and fosinoprilat (IC50 251 nM), two inhibitors of mammalian ACE, and was activated by Cl- (optimal Cl- concentration 600 mM). Musca ACE removed C-terminal dipeptides from angiotensin I, bradykinin [Leu5]enkephalin and [Met5]enkephalin and also functioned as an endopeptidase by hydrolysing dipeptideamides from [Leu5]enkephalinamide and [Met5]enkephalinamide, and a dipeptideamide and a tripeptideamide from substance P. Musca ACE was also able to cleave a tripeptide from both the N-terminus and C-terminus of luteinizing hormone-releasing hormone, with C-terminal hydrolysis predominating. Maximal N-terminal tripeptidase activity occurred at 150 mM NaCl, whereas the C-terminal tripeptidase activity continued to rise with increasing concentration of Cl- (0-0.5 M). Musca ACE displays properties of both the N- and C-domains of human ACE, indicating a high degree of conservation during evolution of the substrate specificity of ACE and its response to Cl-. PMID:8670080

  13. Treatment of both native and deamidated gluten peptides with an endo-peptidase from Aspergillus niger prevents stimulation of gut-derived gluten-reactive T cells from either children or adults with celiac disease.

    PubMed

    Toft-Hansen, Henrik; Rasmussen, Karina S; Staal, Anne; Roggen, Erwin L; Sollid, Ludvig M; Lillevang, Søren T; Barington, Torben; Husby, Steffen

    2014-08-01

    Celiac disease (CD) is characterized by an inappropriate immunological reaction against gluten driven by gluten-specific CD4+ T cells. We screened 25 proteases and tested 10 for their potential to degrade gluten in vitro. Five proteases were further tested for their ability to prevent the proliferative response by a gluten-specific CD4+ T cell clone and seven gluten-reactive T cell lines to protease-digested gluten peptides. A proline-specific endo-peptidase from Aspergillus niger (AnP2) was particularly efficient at diminishing proliferation after stimulation with cleaved antigen, and could completely block the response against both native and deamidated gluten peptides. We found that AnP2 was efficient down to a 1:64 protease:substrate ratio (w:w). When AnP2 was tested in assays using seven gluten-reactive T cell lines from individual CD patients (three adults and four children), the response to gluten was diminished in all cases. Our study indicates a therapeutic benefit of AnP2 to CD patients.

  14. Gaps in employer coverage: lack of supply or lack of demand?

    PubMed

    Long, S H; Marquis, M S

    1993-01-01

    According to data from the May 1988 Current Population Survey, 18 percent of workers are in firms that do not offer health insurance. The question explored here is whether the absence of insurance in these firms is related to lack of supply (that is, a failure of the firm to offer the benefit because the price it faces is too high or the benefit too low) or lack of demand (that is, employees in these firms would not purchase the insurance even if it were offered). Characteristics hypothesized to affect the supply of insurance by firms (size, rate of turnover, and union status) are found to distinguish whether or not firms offer insurance. The data show near-universal acceptance of group insurance among employees offered the opportunity to participate. Both of these factors suggest a failure of supply. However, employees in firms that do not offer insurance are young, low-wage earners who work part time. These are also characteristics of workers who do not purchase group insurance even when it is offered, suggesting that many of the workers who are not offered group insurance would not participate in a plan even if the supply failure were corrected. These findings have implications for the effectiveness of voluntary strategies to improve access, but they also raise concern over the fairness to workers of mandates requiring that they purchase coverage.

  15. Lack of Sleep Takes Big Bite Out of World Economies

    MedlinePlus

    ... medlineplus.gov/news/fullstory_162298.html Lack of Sleep Takes Big Bite Out of World Economies More ... increased risk of death linked to lack of sleep among U.S. workers cost the nation's economy as ...

  16. Effect on blood pressure of combined inhibition of endothelin-converting enzyme and neutral endopeptidase with daglutril in patients with type 2 diabetes who have albuminuria: a randomised, crossover, double-blind, placebo-controlled trial.

    PubMed

    Parvanova, Aneliya; van der Meer, Irene M; Iliev, Ilian; Perna, Annalisa; Gaspari, Flavio; Trevisan, Roberto; Bossi, Antonio; Remuzzi, Giuseppe; Benigni, Ariela; Ruggenenti, Piero

    2013-09-01

    Effective reduction of albuminuria and blood pressure in patients with type 2 diabetes who have nephropathy is seldom achieved with available treatments. We tested the effects of treatment of such patients with daglutril, a combined endothelin-converting enzyme and neutral endopeptidase inhibitor. We did this randomised, crossover trial in two hospitals in Italy. Eligibility criteria were: age 18 years or older, urinary albumin excretion 20-999 μg/min, systolic blood pressure (BP) less than 140 mm Hg, and diastolic BP less than 90 mm Hg. Patients were randomly assigned (1:1) with a computer-generated randomised sequence to receive either daglutril (300 mg/day) then placebo for 8 weeks each or vice versa, with a 4-week washout period. Patients also took losartan throughout. Participants and investigators were masked to treatment allocation. The primary endpoint was 24-h urinary albumin excretion in the intention-to-treat population. Secondary endpoints were median office and ambulatory (24 h, daytime, and night-time) BP, renal haemodynamics and sieving function, and metabolic and laboratory test results. This study is registered with ClinicalTrials.gov, number NCT00160225. We screened 58 patients, of whom 45 were enrolled (22 assigned to daglutril then placebo, 23 to placebo then daglutril; enrolment from May, 2005, to December, 2006) and 42 (20 vs 22) were included in the primary analysis. Daglutril did not significantly affect 24-h urinary albumin excretion compared with placebo (difference in change -7·6 μg/min, IQR -78·7 to 19·0; p=0·559). 34 patients had complete 24-h BP readings; compared with placebo, daglutril significantly reduced 24-h systolic (difference -5·2 mm Hg, SD 9·4; p=0·0013), diastolic (-2·5, 6·2; p=0·015), pulse (-3·0, 6·3; p=0·019), and mean (-3·1, 6·2; p=0·003) BP, as well as all night-time BP readings and daytime systolic, pulse, and mean BP, but not diastolic BP. Compared with placebo, daglutril also significantly reduced

  17. B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8− dendritic cells require the intramembrane endopeptidase SPPL2A

    PubMed Central

    Bergmann, Hannes; Yabas, Mehmet; Short, Alanna; Miosge, Lisa; Barthel, Nadine; Teh, Charis E.; Roots, Carla M.; Bull, Katherine R.; Jeelall, Yogesh; Horikawa, Keisuke; Whittle, Belinda; Balakishnan, Bhavani; Sjollema, Geoff; Bertram, Edward M.; Mackay, Fabienne; Rimmer, Andrew J.; Cornall, Richard J.; Field, Matthew A.; Andrews, T. Daniel

    2013-01-01

    Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase–like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell–activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74–MHC II pathway and a new target for protease inhibitor treatment of B cell diseases. PMID:23267016

  18. B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

    PubMed

    Bergmann, Hannes; Yabas, Mehmet; Short, Alanna; Miosge, Lisa; Barthel, Nadine; Teh, Charis E; Roots, Carla M; Bull, Katherine R; Jeelall, Yogesh; Horikawa, Keisuke; Whittle, Belinda; Balakishnan, Bhavani; Sjollema, Geoff; Bertram, Edward M; Mackay, Fabienne; Rimmer, Andrew J; Cornall, Richard J; Field, Matthew A; Andrews, T Daniel; Goodnow, Christopher C; Enders, Anselm

    2013-01-14

    Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.

  19. Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM

    PubMed Central

    Lemieux, Maxime; D. Laflamme, Olivier; Thiry, Louise; Boulanger-Piette, Antoine; Frenette, Jérôme

    2015-01-01

    Down syndrome cell adherence molecule (DSCAM) contributes to the normal establishment and maintenance of neural circuits. Whereas there is abundant literature regarding the role of DSCAM in the neural patterning of the mammalian retina, less is known about motor circuits. Recently, DSCAM mutation has been shown to impair bilateral motor coordination during respiration, thus causing death at birth. DSCAM mutants that survive through adulthood display a lack of locomotor endurance and coordination in the rotarod test, thus suggesting that the DSCAM mutation impairs motor control. We investigated the motor and locomotor functions of DSCAM2J mutant mice through a combination of anatomical, kinematic, force, and electromyographic recordings. With respect to wild-type mice, DSCAM2J mice displayed a longer swing phase with a limb hyperflexion at the expense of a shorter stance phase during locomotion. Furthermore, electromyographic activity in the flexor and extensor muscles was increased and coactivated over 20% of the step cycle over a wide range of walking speeds. In contrast to wild-type mice, which used lateral walk and trot at walking speed, DSCAM2J mice used preferentially less coordinated gaits, such as out-of-phase walk and pace. The neuromuscular junction and the contractile properties of muscles, as well as their muscle spindles, were normal, and no signs of motor rigidity or spasticity were observed during passive limb movements. Our study demonstrates that the DSCAM mutation induces dystonic hypertonia and a disruption of locomotor gaits. PMID:26683069

  20. Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM.

    PubMed

    Lemieux, Maxime; Laflamme, Olivier D; Thiry, Louise; Boulanger-Piette, Antoine; Frenette, Jérôme; Bretzner, Frédéric

    2016-03-01

    Down syndrome cell adherence molecule (DSCAM) contributes to the normal establishment and maintenance of neural circuits. Whereas there is abundant literature regarding the role of DSCAM in the neural patterning of the mammalian retina, less is known about motor circuits. Recently, DSCAM mutation has been shown to impair bilateral motor coordination during respiration, thus causing death at birth. DSCAM mutants that survive through adulthood display a lack of locomotor endurance and coordination in the rotarod test, thus suggesting that the DSCAM mutation impairs motor control. We investigated the motor and locomotor functions of DSCAM(2J) mutant mice through a combination of anatomical, kinematic, force, and electromyographic recordings. With respect to wild-type mice, DSCAM(2J) mice displayed a longer swing phase with a limb hyperflexion at the expense of a shorter stance phase during locomotion. Furthermore, electromyographic activity in the flexor and extensor muscles was increased and coactivated over 20% of the step cycle over a wide range of walking speeds. In contrast to wild-type mice, which used lateral walk and trot at walking speed, DSCAM(2J) mice used preferentially less coordinated gaits, such as out-of-phase walk and pace. The neuromuscular junction and the contractile properties of muscles, as well as their muscle spindles, were normal, and no signs of motor rigidity or spasticity were observed during passive limb movements. Our study demonstrates that the DSCAM mutation induces dystonic hypertonia and a disruption of locomotor gaits. Copyright © 2016 the American Physiological Society.

  1. Endopeptidase Cleavage Generates a Functionally Distinct Isoform of C1q/Tumor Necrosis Factor-related Protein-12 (CTRP12) with an Altered Oligomeric State and Signaling Specificity*

    PubMed Central

    Wei, Zhikui; Lei, Xia; Seldin, Marcus M.; Wong, G. William

    2012-01-01

    Adipose tissue-derived adipokines are an important class of secreted metabolic regulators that mediate tissue cross-talk to control systemic energy balance. We recently described C1q/TNF-related protein-12 (CTRP12), a novel insulin-sensitizing adipokine that regulates glucose metabolism in liver and adipose tissue. However, the biochemical properties of CTRP12 and its naturally occurring cleaved isoform have not been characterized. Here, we show that CTRP12 is a secreted hormone subjected to multiple functionally relevant posttranslational modifications at highly conserved residues. For example, Asn39 is glycosylated, whereas Cys85 mediates the assembly of higher order oligomeric structure. Endopeptidase cleavage at Lys91 generates a cleaved globular gCTRP12 isoform, the expression of which is increased by insulin. PCSK3/furin was identified as the major proprotein convertase expressed by adipocytes that mediates the endogenous cleavage of CTRP12. Cleavage at Lys91 is context-dependent: mutation of the charged Arg93 to Ala on the P2′ position enhanced cleavage, and triple mutations (K90A/K91A/R93A) abolished cleavage. Importantly, the two isoforms of CTRP12 differ in oligomeric structures and are functionally distinct. The full-length protein forms trimers and larger complexes, and the cleaved isoform consisted of predominantly dimers. Whereas full-length fCTRP12 strongly activated Akt signaling in H4IIE hepatocytes and 3T3-L1 adipocytes, gCTRP12 preferentially activated MAP kinase (ERK1/2 and p38 MAPK) signaling. Further, only fCTRP12 improved insulin-stimulated glucose uptake in adipocytes. These results reveal a novel mechanism controlling signaling specificity and function of a hormone via cleavage-dependent alteration in oligomeric state. PMID:22942287

  2. 29 CFR 18.602 - Lack of personal knowledge.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Lack of personal knowledge. 18.602 Section 18.602 Labor... OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A... witness has personal knowledge of the matter. Evidence to prove personal knowledge may, but need...

  3. 10 CFR 503.21 - Lack of alternate fuel supply.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 4 2013-01-01 2013-01-01 false Lack of alternate fuel supply. 503.21 Section 503.21 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS NEW FACILITIES Temporary Exemptions for New Facilities § 503.21 Lack of alternate fuel supply. (a) Eligibility. Section 211(a)(1) of the Act provides for...

  4. 10 CFR 503.21 - Lack of alternate fuel supply.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Lack of alternate fuel supply. 503.21 Section 503.21 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS NEW FACILITIES Temporary Exemptions for New Facilities § 503.21 Lack of alternate fuel supply. (a) Eligibility. Section 211(a)(1) of the Act provides for...

  5. 10 CFR 503.21 - Lack of alternate fuel supply.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Lack of alternate fuel supply. 503.21 Section 503.21 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS NEW FACILITIES Temporary Exemptions for New Facilities § 503.21 Lack of alternate fuel supply. (a) Eligibility. Section 211(a)(1) of the Act provides for...

  6. 10 CFR 503.21 - Lack of alternate fuel supply.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 4 2014-01-01 2014-01-01 false Lack of alternate fuel supply. 503.21 Section 503.21 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS NEW FACILITIES Temporary Exemptions for New Facilities § 503.21 Lack of alternate fuel supply. (a) Eligibility. Section 211(a)(1) of the Act provides for...

  7. 29 CFR 18.602 - Lack of personal knowledge.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 1 2014-07-01 2013-07-01 true Lack of personal knowledge. 18.602 Section 18.602 Labor Office of the Secretary of Labor RULES OF PRACTICE AND PROCEDURE FOR ADMINISTRATIVE HEARINGS BEFORE THE OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A...

  8. 29 CFR 18.602 - Lack of personal knowledge.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 1 2011-07-01 2011-07-01 false Lack of personal knowledge. 18.602 Section 18.602 Labor Office of the Secretary of Labor RULES OF PRACTICE AND PROCEDURE FOR ADMINISTRATIVE HEARINGS BEFORE THE OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A...

  9. 29 CFR 18.602 - Lack of personal knowledge.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 1 2013-07-01 2013-07-01 false Lack of personal knowledge. 18.602 Section 18.602 Labor Office of the Secretary of Labor RULES OF PRACTICE AND PROCEDURE FOR ADMINISTRATIVE HEARINGS BEFORE THE OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A...

  10. 29 CFR 18.602 - Lack of personal knowledge.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 1 2012-07-01 2012-07-01 false Lack of personal knowledge. 18.602 Section 18.602 Labor Office of the Secretary of Labor RULES OF PRACTICE AND PROCEDURE FOR ADMINISTRATIVE HEARINGS BEFORE THE OFFICE OF ADMINISTRATIVE LAW JUDGES Rules of Evidence Witnesses § 18.602 Lack of personal knowledge. A...

  11. Lack of progress in labor as a reason for cesarean.

    PubMed

    Gifford, D S; Morton, S C; Fiske, M; Keesey, J; Keeler, E; Kahn, K L

    2000-04-01

    To estimate the prevalence of lack of progress in labor as a reason for cesarean delivery and to compare published diagnostic criteria with the labor characteristics of women with this diagnosis. We reviewed medical records and did a postpartum telephone survey to collect data from 733 women who delivered full-term, nonbreech infants by unplanned cesarean between March 1993 and February 1994. These were a subset of 2447 births sampled at delivery from 30 hospitals in Los Angeles County and Iowa. We measured the proportion of unplanned cesareans done for lack of progress in labor, the cervical dilatation at the time of cesarean, length of the second stage, and slope of the active phase among the women. We estimated the proportion of these cesareans that conformed to the ACOG criteria for the diagnosis of lack of progress. Lack of progress was a reason for 68% of unplanned, vertex cesareans. At least 16% of the subjects who had cesareans for lack of progress were in the latent phase of labor according to ACOG criteria. The second stage was not prolonged in 36% of the women who delivered at 10 cm. Lack of progress in labor is a dominant reason for cesarean delivery. Many cesareans are done during the latent phase of labor, and in the second stage of labor when it is not prolonged. These practices do not conform to published diagnostic criteria for lack of progress.

  12. Many College Football Players Lack Vitamin D: Study

    MedlinePlus

    ... fullstory_164139.html Many College Football Players Lack Vitamin D: Study Deficiency could put them at risk ... 2017 THURSDAY, March 16, 2017 (HealthDay News) -- Low vitamin D levels are common among college football players ...

  13. 7. VIEW OF ARIZONA CANAL ABOVE EVERGREEN, SHOWING LACK OF ...

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

    7. VIEW OF ARIZONA CANAL ABOVE EVERGREEN, SHOWING LACK OF SILT. OLD TOOTH MARKS OF DRAGLINE BUCKET MADE IN 1909 CALICHE BOTTOM WERE STILL VISIBLE Photographer: unknown. February 1938 - Arizona Canal, North of Salt River, Phoenix, Maricopa County, AZ

  14. Anti-Angiogenic Action of Neutral Endopeptidase

    DTIC Science & Technology

    2006-11-01

    antagonist of angiogenesis. We report that NEP is indeed antiangiogenic in vivo, significantly inhibiting angiogenesis. Surprisingly, we...Figure 6. Quantitative real time PCR analysis NEP transcripts as a function of oxygen tension in C42 and LNCaP prostate cancer cell lines cultured

  15. Self Purification of Two Serine Endopeptidases*

    PubMed Central

    Awad, W. M.; Ochoa, Maria S.; Toomey, T. P.

    1972-01-01

    We have reported that a serine protease from Pronase, homologous with bovine chymotrypsin, is both active and stable in 6 M guanidinium chloride. The present investigation examined the possibility that this unique property might be used to permit the enzyme to engage in its own purification by cleaving companion proteins to low-molecular-weight products. Analysis with model substrates of the several specific activities that were originally present revealed that only the activity against Nα-acetyl-L-tyrosine ethyl ester was demonstrable after incubation for 100 hr in the denaturant. After a moderate loss within the first 24 hr, the remaining activity against this ester was conserved for many days thereafter. Pronase was routinely incubated for 1 week at 22° in 6 M guanidinium chloride at pH 8.0 where the esterases showed maximal activity. Analysis of the products of incubation revealed unexpectedly the presence of two serine proteases that were easily separated. After purification to homogeneity these components proved themselves to be the previously demonstrated subtilisin-like and stable chymotrypsin-like enzymes. The only amino-terminal residue of the chymotrypsin-like enzyme is isoleucine, as it is in the earlier, conventionally purified product. The migration of the single band of this enzyme during acrylamide gel electrophoresis was the same whether purified by the past or present technique. No free amino-terminal group was demonstrable in the subtilisin-like enzyme. This study presents a unique and rapid technique for isolation of these proteases, with the first reported purification to homogeneity of the subtilisin-like component. These enzymes may be useful as probes for local relaxations of conformation in substrate proteins. Furthermore, they may contribute to the preparation of enzyme-free non-protein macromolecules. Images PMID:4506775

  16. Anti-Angiogenic Action of Neutral Endopeptidase

    DTIC Science & Technology

    2007-11-01

    FGF-2 and Maltose -binding Protein-FGF-2 Fusion Proteins—Full-length human FGF-2 cDNA (kindly provided by Dr. Daniel Rifkin, New York University Medical...GAGCATC-3 (sense) and 5-CCCCAAGCTTTTAGCTCT- TAGCAGACAT-3 (antisense) for maltose -binding protein fusion proteins, as previously described (13...terminal neprilysin cleavage product), and DNA sequencing was performed to confirm their accuracy. For maltose -binding protein constructs, the PCR product

  17. Evidence for Golgi bodies in proposed 'Golgi-lacking' lineages.

    PubMed Central

    Dacks, Joel B; Davis, Lesley A M; Sjögren, Asa M; Andersson, Jan O; Roger, Andrew J; Doolittle, W Ford

    2003-01-01

    Golgi bodies are nearly ubiquitous in eukaryotic cells. The apparent lack of such structures in certain eukaryotic lineages might be taken to mean that these protists evolved prior to the acquisition of the Golgi, and it raises questions of how these organisms function in the absence of this crucial organelle. Here, we report gene sequences from five proposed 'Golgi-lacking' organisms (Giardia intestinalis, Spironucleus barkhanus, Entamoeba histolytica, Naegleria gruberi and Mastigamoeba balamuthi). BLAST and phylogenetic analyses show these genes to be homologous to those encoding components of the retromer, coatomer and adaptin complexes, all of which have Golgi-related functions in mammals and yeast. This is, to our knowledge, the first molecular evidence for Golgi bodies in two major eukaryotic lineages (the pelobionts and heteroloboseids). This substantiates the suggestion that there are no extant primitively 'Golgi-lacking' lineages, and that this apparatus was present in the last common eukaryotic ancestor, but has been altered beyond recognition several times. PMID:14667372

  18. Garlic exhibits lack of control over gastrointestinal nematodes in goats

    USDA-ARS?s Scientific Manuscript database

    Gastrointestinal nematodes (GIN) continue to hinder small ruminant production because of anthelmintic resistance and lack of effective products for GIN control in organic production. The objective of this study was to examine the effectiveness of a commercially available certified organic garlic pr...

  19. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

    PubMed

    Williams, Margaret M; Sen, Kathryn; Weigand, Michael R; Skoff, Tami H; Cunningham, Victoria A; Halse, Tanya A; Tondella, M Lucia

    2016-02-01

    A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.

  20. Barriers to College: Lack of Preparation vs. Financial Need

    ERIC Educational Resources Information Center

    Cavanagh, Sean

    2004-01-01

    As politicians, academic leaders, and researchers decry the impact of college tuition fee increases for needy students, others say such concerns mask a more serious barrier for college aspirants: lack of academic preparation. The debate was renewed last week with the publication of a book from the Century Foundation analyzing the reasons…

  1. Understanding the Lack of Female Leadership in Collegiate Athletics

    ERIC Educational Resources Information Center

    Camarco, Lisa

    2016-01-01

    This study sought an understanding of the current trends in the lack of females in leadership positions within collegiate athletic departments amongst California Community Colleges. The passage of Title IX created a new funding stream for women's athletics, resulting in male coaches and administrators entering into the female realm, therefore…

  2. 10 CFR 503.21 - Lack of alternate fuel supply.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... substantially exceed the cost of using imported petroleum as a primary energy source as defined in § 503.6 (Cost... 10 Energy 4 2011-01-01 2011-01-01 false Lack of alternate fuel supply. 503.21 Section 503.21 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS NEW FACILITIES Temporary Exemptions for...

  3. Children's Lack of Playtime Seen as Troubling Health, School Issue

    ERIC Educational Resources Information Center

    Jacobson, Linda

    2008-01-01

    Teachers and parents are frequently warned that students in the United States are lacking the academic skills they need for the 21st century. But a growing contingent of educators, psychologists, and other professionals are voicing worries that today's children are also growing up without the chance to play. Test preparation in kindergarten,…

  4. Domestic properties in the UK and a lack of sustainability

    NASA Astrophysics Data System (ADS)

    Ugochukwu, Nnadozie

    This research aims to provide sufficient insight into the lack of sustainable domestic properties in the UK. The paper reviewed relevant theories of sustainability, with respect to energy performance and environmental friendliness of the built environment. The research also studied the efforts made by the UK Government and other Stakeholders to ensure availability of sustainable domestic properties in the UK, by introducing the Code for Sustainable Homes. The research identified constraints that cause the lack of sustainable domestic properties in the UK, they are: The extra costs associated with building homes to sustainable standards, flexible government legislation, lack of information of the benefits of owning a home built to sustainable standards, and lack of community participation in the formulation of sustainable policies. Recommendations for the availability of more homes built to sustainable standards include the need for mandatory government legislation, making the formulation of policies more participatory amongst the communities where they will be implemented, creating public awareness about the benefits of owning a home built to sustainable standards and the fact that the costs associated with owning such a home is recoverable through savings made in energy costs.

  5. Lack of Emphasis on Nutrition in Medical School Curriculum.

    ERIC Educational Resources Information Center

    Friedman, Suanne

    The need and concern for the apparent lack of nutrition education provided in training programs for physicians was the impetus for begining a 10-session nutrition lecture series program. The program was developed and implemented in a large teaching medical center hospital and given to 16 third-year medical students. The program's purpose was to…

  6. Special Relativity in Week One: 4) Lack of Simultaneity

    ERIC Educational Resources Information Center

    Huggins, Elisha

    2011-01-01

    This is our final article on teaching special relativity in the first week of an introductory physics course. One of the profound changes in our view of the world was Einstein's discovery of the lack of simultaneity. He illustrated this result with a thought experiment in which we observe a railroad car passing by us. We see the two ends of the…

  7. Loneliness and Lack of Social Support: Same or Different Phenomena?

    ERIC Educational Resources Information Center

    Rook, Karen

    Research on loneliness and research on social support offer complementary perspectives on how social relationships affect health and well being. However, despite considerable overlap, loneliness and lack of social support reflect deficits of different kinds of social exchanges and these deficits have distinct consequences for well being. Social…

  8. Understanding the Lack of Female Leadership in Collegiate Athletics

    ERIC Educational Resources Information Center

    Camarco, Lisa

    2016-01-01

    This study sought an understanding of the current trends in the lack of females in leadership positions within collegiate athletic departments amongst California Community Colleges. The passage of Title IX created a new funding stream for women's athletics, resulting in male coaches and administrators entering into the female realm, therefore…

  9. Barriers to College: Lack of Preparation vs. Financial Need

    ERIC Educational Resources Information Center

    Cavanagh, Sean

    2004-01-01

    As politicians, academic leaders, and researchers decry the impact of college tuition fee increases for needy students, others say such concerns mask a more serious barrier for college aspirants: lack of academic preparation. The debate was renewed last week with the publication of a book from the Century Foundation analyzing the reasons…

  10. Lack of Emphasis on Nutrition in Medical School Curriculum.

    ERIC Educational Resources Information Center

    Friedman, Suanne

    The need and concern for the apparent lack of nutrition education provided in training programs for physicians was the impetus for begining a 10-session nutrition lecture series program. The program was developed and implemented in a large teaching medical center hospital and given to 16 third-year medical students. The program's purpose was to…

  11. Lack of Clarity in University Teaching: A Case Study.

    ERIC Educational Resources Information Center

    Hativa, Nira

    1998-01-01

    A study used qualitative methods to examine lack of clarity and need for inference in teaching of an undergraduate physics course for nonscience majors. Evidence from several data sources converge, revealing very low levels of student understanding of material presented and strong dissatisfaction with instruction, and also insights into teacher…

  12. Special Relativity in Week One: 4) Lack of Simultaneity

    ERIC Educational Resources Information Center

    Huggins, Elisha

    2011-01-01

    This is our final article on teaching special relativity in the first week of an introductory physics course. One of the profound changes in our view of the world was Einstein's discovery of the lack of simultaneity. He illustrated this result with a thought experiment in which we observe a railroad car passing by us. We see the two ends of the…

  13. Lack of Resources Hampers Graduation Rates in Division II.

    ERIC Educational Resources Information Center

    Suggs, Welch

    2001-01-01

    A recent report by the National Collegiate Athletic Association on graduation rates indicates that athletes in Division II schools face as many challenges completing college as do their peers in Division I. Division II institutions are further hampered by lack of resources causing Division II students less likely to graduate in general. (SLD)

  14. DETECTION OF LACK OF FUSION WELD DEFECTS BY RADIOGRAPHY

    SciTech Connect

    Souza, M. P.; Almeida, R. M.; Rebello, J. M. A.

    2009-03-03

    In this work, radiography was employed as the NDT technique for detection of flaws in circumferential girth welds of steel pipelines used in offshore installations in the petroleum industry. The kind of defect specifically focused was lack of fusion. It is currently accepted in the literature that radiography is not as sensitive as ultrasonics to detect lack of fusion defects. Unfortunately, the radiographic inspection can barely detect lack of fusion and only when it is associated to inclusions and voids of considerable size. However, in a previous article ('Reliability of radiographic inspection of steel pipeline girth welds', QNDE Conference, 2007), the authors showed that it is possible to detect lack of fusion defects if, in the radiographic tests, the angle of incidence is the same angle that the weld bevel makes with the test piece surface, which means lowering the angle of disorientation between the flaw and the radiographic beam. However, no concerns were made to sizing the defects. Computational simulation was used with XRSIM software to establish the optimal radiographic parameters to reach the lower limit for detection for this kind of defect.

  15. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin

    PubMed Central

    Sen, Kathryn; Weigand, Michael R.; Skoff, Tami H.; Cunningham, Victoria A.; Halse, Tanya A.; Tondella, M. Lucia

    2016-01-01

    A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin–deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes. PMID:26812174

  16. Early Neurobehavioral Development of Mice Lacking Endogenous PACAP.

    PubMed

    Farkas, Jozsef; Sandor, Balazs; Tamas, Andrea; Kiss, Peter; Hashimoto, Hitoshi; Nagy, Andras D; Fulop, Balazs D; Juhasz, Tamas; Manavalan, Sridharan; Reglodi, Dora

    2017-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.

  17. Mice completely lacking immunoproteasomes display major alterations in antigen presentation

    PubMed Central

    Kincaid, Eleanor Z; Che, Jenny W; York, Ian; Escobar, Hernando; Reyes-Vargas, Eduardo; Delgado, Julio C.; Welsh, Raymond M; Karow, Margaret L.; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Rock, Kenneth L

    2011-01-01

    The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought. PMID:22197977

  18. Female Migraineurs Show Lack of Insular Thinning with Age

    PubMed Central

    Maleki, Nasim; Barmettler, Gabi; Moulton, Eric A.; Scrivani, Steven; Veggeberg, Rosanna; Spierings, Egilius L.H.; Burstein, Rami; Becerra, Lino; Borsook, David

    2015-01-01

    Gray matter loss in cortical regions is a normal ageing process for the healthy brain. There have been few studies on the process of ageing of the brain in chronic neurological disorders. In this study, we evaluated changes in the cortical thickness by age in 92 female subjects (46 migraine patients, and 46 healthy controls) using high field MRI. The results indicate that in contrast to healthy subjects migraineurs show lack of thinning in the insula by age. The functional significance of the lack of thinning is unknown, but may contribute to the overall cortical hyperexcitability of the migraine brain since the region is tightly involved in a number of majo brain networks involved in interoception, salience, nociception, and autonomic function, including the default mode network. PMID:25775358

  19. Lack of pharmacokinetic interaction as an equivalence problem.

    PubMed

    Steinijans, V W; Hartmann, M; Huber, R; Radtke, H W

    1991-08-01

    The demonstration that concomitant administration of drug B does not affect the pharmacokinetics of drug A can be adequately handled as an equivalence problem. Administration of drug A alone serves as reference and simultaneous administration of drugs A and B as test situation. The range of clinically acceptable variation in the pharmacokinetic characteristics of drug A defines the equivalence range. This will usually correspond to the bioequivalence range accepted for the comparison of different formulations of drug A. Equivalence, i.e. lack of pharmacokinetic interaction, is concluded if the 90%-confidence interval for the ratio (difference) of the expected medians for test and reference is entirely within the equivalence range. This decision procedure ensures that the consumer risk of incorrectly concluding "lack of interaction" is limited to 5%. Moreover, the producer risk of incorrectly concluding "interaction" can be controlled by appropriate sample sizes.

  20. Internal epitope tagging informed by relative lack of sequence conservation

    PubMed Central

    Burg, Leonard; Zhang, Karen; Bonawitz, Tristan; Grajevskaja, Viktorija; Bellipanni, Gianfranco; Waring, Richard; Balciunas, Darius

    2016-01-01

    Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function. PMID:27892520

  1. Rural ED transfers due to lack of radiology services.

    PubMed

    Lyon, Matthew; Sturgis, LaShon; Lendermon, Darren; Kuchinski, Ann Marie; Mueller, Taylor; Loeffler, Patrick; Xu, Hongyan; Gibson, Robert

    2015-11-01

    Our objectives were to determine the frequency of patient transfers to a tertiary care emergency department (Tertiary ED) due to a lack of radiology services in rural hospital EDs (Rural EDs), and examine the community and patient attributes that are associated with these transfers. This was a retrospective chart review of patients transferred to a Tertiary ED from Rural EDs. Transfers excluded from the study included pediatric patients (age <18 years old) and patients transferred for trauma surgeon evaluation. Only those patients who were transferred for radiology services were included in the final analysis. Over a 12-month period, 1445 patients were transferred to the Tertiary ED with 73.8% (n = 1066) of this population being transferred from a Rural ED. Excluding 381 trauma and pediatric patients, 64.3% (n = 685) of patients were transferred from a Rural ED and were included in the study. Of these 685 transfers, 24.5% (n = 168) were determined to be due primarily to a lack of a radiology service. Lack of radiology services in Rural EDs leads to numerous patient transfers to the Tertiary ED each year. A disproportionate number of these transfer patients are African American. These transfers place additional financial and social burdens on patients and their families. This study discusses these findings and alternative diagnostic options (ie, telemedicine and ultrasound video transfer) to address the lack of radiology services available in Rural EDs. The use of these alternate diagnostic options will likely reduce the number of patient transfers to Tertiary EDs. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Sewer deterioration modeling with condition data lacking historical records.

    PubMed

    Egger, C; Scheidegger, A; Reichert, P; Maurer, M

    2013-11-01

    Accurate predictions of future conditions of sewer systems are needed for efficient rehabilitation planning. For this purpose, a range of sewer deterioration models has been proposed which can be improved by calibration with observed sewer condition data. However, if datasets lack historical records, calibration requires a combination of deterioration and sewer rehabilitation models, as the current state of the sewer network reflects the combined effect of both processes. Otherwise, physical sewer lifespans are overestimated as pipes in poor condition that were rehabilitated are no longer represented in the dataset. We therefore propose the combination of a sewer deterioration model with a simple rehabilitation model which can be calibrated with datasets lacking historical information. We use Bayesian inference for parameter estimation due to the limited information content of the data and limited identifiability of the model parameters. A sensitivity analysis gives an insight into the model's robustness against the uncertainty of the prior. The analysis reveals that the model results are principally sensitive to the means of the priors of specific model parameters, which should therefore be elicited with care. The importance sampling technique applied for the sensitivity analysis permitted efficient implementation for regional sensitivity analysis with reasonable computational outlay. Application of the combined model with both simulated and real data shows that it effectively compensates for the bias induced by a lack of historical data. Thus, the novel approach makes it possible to calibrate sewer pipe deterioration models even when historical condition records are lacking. Since at least some prior knowledge of the model parameters is available, the strength of Bayesian inference is particularly evident in the case of small datasets.

  3. Pig lacks functional NLRC4 and NAIP genes.

    PubMed

    Sakuma, Chisato; Toki, Daisuke; Shinkai, Hiroki; Takenouchi, Takato; Sato, Mitsuru; Kitani, Hiroshi; Uenishi, Hirohide

    2017-02-01

    The NLRC4 inflammasome, which recognizes flagellin and components of the type III secretion system, plays an important role in the clearance of intracellular bacteria. Here, we examined the genomic sequences carrying two genes encoding key components of the NLRC4 inflammasome-NLR family, CARD-containing 4 (NLRC4), and NLR apoptosis inhibitory protein (NAIP)-in pigs. Pigs have a single locus encoding NLRC4 and NAIP. Comparison of the sequences thus obtained with the corresponding regions in humans revealed the deletion of intermediate exons in both pig genes. In addition, the genomic sequences of both pig genes lacked valid open reading frames encoding functional NLRC4 or NAIP protein. Additional pigs representing multiple breeds and wild boars also lacked the exons that we failed to find through genome sequencing. Furthermore, neither the NLRC4 nor the NAIP gene was expressed in pigs. These findings indicate that pigs lack the NLRC4 inflammasome, an important factor involved in monitoring bacterial proteins and contributing to the clearance of intracellular pathogens. These results also suggest that genetic polymorphisms affecting the molecular functions of TLR2, TLR4, TLR5, and other pattern recognition receptors associated with the recognition of bacteria have a more profound influence on disease resistance in pigs than in other species.

  4. Microstructure of iridescence-lacking pearl formed in Pinctada fucata

    NASA Astrophysics Data System (ADS)

    Suzuki, Michio; Mukai, Hiroki; Aoki, Hideo; Yoshimura, Etsuro; Sakuda, Shohei; Nagasawa, Hiromichi; Kogure, Toshihiro

    2016-01-01

    The iridescence-lacking pearl is regarded as a low-quality product because it shows no iridescent color which is generated by the interference of the light reflected at the organic-inorganic boundaries in the regulated interstratification of organic sheets and thin aragonite tablets. In this study, we investigated the microstructural difference between normal and iridescence-lacking pearls, as well as original nacreous layers in the shell of the pearl oyster, Pinctada fucata. Cross-sectional observation by scanning electron microscopy revealed abundant organic spherules of a few hundred nanometers in diameter attached to the inter-crystalline organic sheets in the iridescence-lacking pearl. The incoherent light scattered by the spherules inhibit the formation or emission of the iridescent color. The same spherules were also observed in hazy nacreous layers of the shell. The organic spherules often connected to the gap of inter-crystalline organic sheets implying that the spherules consist of same components of the organic sheets. Their abundance varies along the thickness of nacre, suggesting that their formation is determined by environmental factors, as well as genetic ones.

  5. Reduced passive force in skeletal muscles lacking protein arginylation

    PubMed Central

    Minozzo, Fábio C.; Kalganov, Albert; Cornachione, Anabelle S.; Cheng, Yu-Shu; Leu, Nicolae A.; Han, Xuemei; Saripalli, Chandra; Yates, John R.; Granzier, Henk; Kashina, Anna S.

    2015-01-01

    Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles. PMID:26511365

  6. Transcriptional changes associated with lack of lipid synthesis in parasitoids.

    PubMed

    Visser, Bertanne; Roelofs, Dick; Hahn, Daniel A; Teal, Peter E A; Mariën, Janine; Ellers, Jacintha

    2012-01-01

    Phenotypic regression of morphological, behavioral, or physiological traits can evolve when reduced trait expression has neutral or beneficial effects on overall performance. Studies on the evolution of phenotypic degradation in animals have concentrated mostly on the evaluation of resulting phenotypes, whereas much less research has been dedicated to uncovering the molecular mechanisms that underlie phenotypic regression. The majority of parasitoids (i.e., insects that develop on or inside other arthropods), do not accumulate lipid reserves during their free-living adult life-stage and represent an excellent system to study phenotypic regression in animals. Here, we study transcriptional patterns associated with lack of lipogenesis in the parasitic wasp Nasonia vitripennis. We first confirmed that N. vitripennis does not synthesize lipids by showing a reduction in lipid reserves despite ingestion of dietary sugar, and a lack of incorporation of isotopic labels into lipid reserves when fed deuterated sugar solution. Second, we investigated transcriptional responses of 28 genes involved in lipid and sugar metabolism in short- and long-term sugar-fed females relative to starved females of N. vitripennis. Sugar feeding did not induce transcription of fatty acid synthase (fas) or other key genes involved in the lipid biosynthesis pathway. Furthermore, several genes involved in carbohydrate metabolism had a lower transcription in fed than in starved females. Our results reveal that N. vitripennis gene transcription in response to dietary sugar deviates markedly from patterns typically observed in other organisms. This study is the first to identify differential gene transcription associated with lack of lipogenesis in parasitoids and provides new insights into the molecular mechanism that underlies phenotypic regression of this trait.

  7. Transcriptional Changes Associated with Lack of Lipid Synthesis in Parasitoids

    PubMed Central

    Visser, Bertanne; Roelofs, Dick; Hahn, Daniel A.; Teal, Peter E. A.; Mariën, Janine; Ellers, Jacintha

    2012-01-01

    Phenotypic regression of morphological, behavioral, or physiological traits can evolve when reduced trait expression has neutral or beneficial effects on overall performance. Studies on the evolution of phenotypic degradation in animals have concentrated mostly on the evaluation of resulting phenotypes, whereas much less research has been dedicated to uncovering the molecular mechanisms that underlie phenotypic regression. The majority of parasitoids (i.e., insects that develop on or inside other arthropods), do not accumulate lipid reserves during their free-living adult life-stage and represent an excellent system to study phenotypic regression in animals. Here, we study transcriptional patterns associated with lack of lipogenesis in the parasitic wasp Nasonia vitripennis. We first confirmed that N. vitripennis does not synthesize lipids by showing a reduction in lipid reserves despite ingestion of dietary sugar, and a lack of incorporation of isotopic labels into lipid reserves when fed deuterated sugar solution. Second, we investigated transcriptional responses of 28 genes involved in lipid and sugar metabolism in short- and long-term sugar-fed females relative to starved females of N. vitripennis. Sugar feeding did not induce transcription of fatty acid synthase (fas) or other key genes involved in the lipid biosynthesis pathway. Furthermore, several genes involved in carbohydrate metabolism had a lower transcription in fed than in starved females. Our results reveal that N. vitripennis gene transcription in response to dietary sugar deviates markedly from patterns typically observed in other organisms. This study is the first to identify differential gene transcription associated with lack of lipogenesis in parasitoids and provides new insights into the molecular mechanism that underlies phenotypic regression of this trait. PMID:22820524

  8. No pain, no gain: lack of exercise obstructs neurogenesis.

    PubMed

    Watson, Nate; Ji, Xunming; Yasuhara, Takao; Date, Isao; Kaneko, Yuji; Tajiri, Naoki; Borlongan, Cesar V

    2015-01-01

    Bedridden patients develop atrophied muscles, their daily activities greatly reduced, and some display a depressive mood. Patients who are able to receive physical rehabilitation sometimes show surprising clinical improvements, including reduced depression and attenuation of other stress-related behaviors. Regenerative medicine has advanced two major stem cell-based therapies for CNS disorders, namely, transplantation of exogenous stem cells and amplification of endogenous neurogenesis. The latter strategy embraces a natural way of reinnervating the damaged brain and correcting the neurological impairments. In this study, we discussed how immobilization-induced disuse atrophy, using the hindlimb suspension model, affects neurogenesis in rats. The overarching hypothesis is that immobilization suppresses neurogenesis by reducing the circulating growth or trophic factors, such as vascular endothelial growth factor or brain-derived neurotrophic factor. That immobilization alters neurogenesis and stem cell differentiation in the CNS requires characterization of the stem cell microenvironment by examining the trophic and growth factors, as well as stress-related proteins that have been implicated in exercise-induced neurogenesis. Although accumulating evidence has revealed the contribution of "increased" exercise on neurogenesis, the reverse paradigm involving "lack of exercise," which mimics pathological states (e.g., stroke patients are often immobile), remains underexplored. This novel paradigm will enable us to examine the effects on neurogenesis by a nonpermissive stem cell microenvironment likely produced by lack of exercise. BrdU labeling of proliferative cells, biochemical assays of serum, cerebrospinal fluid and brain levels of trophic factors, growth factors, and stress-related proteins are proposed as indices of neurogenesis, while quantitative measurements of spontaneous movements will reveal psychomotor components of immobilization. Studies designed to

  9. Lack of oblique astigmatism in the chicken eye.

    PubMed

    Maier, Felix M; Howland, Howard C; Ohlendorf, Arne; Wahl, Siegfried; Schaeffel, Frank

    2015-04-01

    Primate eyes display considerable oblique off-axis astigmatism which could provide information on the sign of defocus that is needed for emmetropization. The pattern of peripheral astigmatism is not known in the chicken eye, a common model of myopia. Peripheral astigmatism was mapped out over the horizontal visual field in three chickens, 43 days old, and in three near emmetropic human subjects, average age 34.7years, using infrared photoretinoscopy. There were no differences in astigmatism between humans and chickens in the central visual field (chicks -0.35D, humans -0.65D, n.s.) but large differences in the periphery (i.e. astigmatism at 40° in the temporal visual field: humans -4.21D, chicks -0.63D, p<0.001, unpaired t-test). The lack of peripheral astigmatism in chicks was not due to differences in corneal shape. Perhaps related to their superior peripheral optics, we found that chickens had excellent visual performance also in the far periphery. Using an automated optokinetic nystagmus paradigm, no difference was observed in spatial visual performance with vision restricted to either the central 67° of the visual field or to the periphery beyond 67°. Accommodation was elicited by stimuli presented far out in the visual field. Transscleral images of single infrared LEDs showed no sign of peripheral astigmatism. The chick may be the first terrestrial vertebrate described to lack oblique astigmatism. Since corneal shape cannot account for the difference in astigmatism in humans and chicks, it must trace back to the design of the crystalline lens. The lack of peripheral astigmatism in chicks also excludes a role in emmetropization. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The Baryon Cycle and its (Lack of) Environmental Dependencies

    NASA Astrophysics Data System (ADS)

    Grootes, Meiert W.

    2017-07-01

    The cycle of gas into and out of galaxies, fueling the conversion of gas into stars, is fundamental to the evolution of galaxies. Indeed, the paradigm that this baryon cycle is self-regulated has proven to be successful at explaining a number of trends, foremost amongst which is the main sequence of star forming galaxies (MS). Combined with a halo mass dependence of the inflow rate and environmental quenching of satellite galaxies, this paradigm represents a compellingly simple picture of galaxy evolution. We use the current SFRs of morphologically selected samples of disk galaxies drawn from the GAMA survey to test this paradigm in the local universe (z<0.13). We find support for the paradigm of gradually evolving self-regulated balance in the baryon cycle of galaxies in the SFR - M* relation of non-group disk galaxies, which we demonstrate evolves smoothly even over a redshift baseline of dz=0.1. Surprisingly, however, the SFR - M* of non-grouped and group central disk galaxies coincide, evidencing a lack of halo mass dependence even on the mass scale of group halos, and leading to a degree of tension with the picture of a simple self-regulated balance for group central galaxies. This lack of environmental dependence is only exacerbated by the fact that the gas-fueling of the majority of disk dominated satellite group galaxies is nigh identical to that of their group central and non-grouped counterparts, high-lighting a pronounced lack of dependence of the baryon-cycle on the environment and on halo mass in particular. Finally, we empirically quantify the group-wide impact of an AGN in the group central galaxy on the star-formation of galaxies in galaxy groups as a new and potentially significant environmental influence.

  11. Disclosing personal health information relating to adults who lack capacity.

    PubMed

    Griffith, Richard

    2014-03-01

    The need to share information about patients is vital to effective care and protection, especially where it relates to adults who lack decision-making capacity but it has to be balanced against the right to confidentiality. Like other health professionals, district nurses have a duty to maintain the confidentiality of patient information, and incapable adults have the right to expect their personal health information to be kept private. This right is guaranteed by the common-law duty of confidence, the Data Protection Act 1998 and the NHS Care Record Guarantee and confidentiality policy. This article discusses the district nurse's legal obligations when considering sharing information in relation to an incapable adult

  12. Inhibition of myoblast differentiation by lack of zinc.

    PubMed Central

    Petrie, L; Chesters, J K; Franklin, M

    1991-01-01

    The impact of restricted zinc availability on myoblast differentiation was investigated. Lack of zinc prevented myoblast fusion and the increase in muscle-specific creatine kinase activity. The depression of activity of creatine kinase in the zinc-deficient cultures was accompanied by a similar decrease in the concentration of creatine kinase mRNA and was apparent even when fusion of the myoblasts was inhibited by cytochalasin B. Thus zinc appears to be necessary for the expression of creatine kinase during myoblast differentiation. Images Fig. 1. Fig. 2. PMID:2039464

  13. Lack of semantic parafoveal preview benefit in reading revisited

    PubMed Central

    Rayner, Keith; Schotter, Elizabeth R.; Drieghe, Denis

    2014-01-01

    In contrast to earlier research, evidence for semantic preview benefit in reading has been reported by Hohenstein and Kliegl (2013) in an alphabetic writing system; they also implied that prior demonstrations of a lack of semantic preview benefit needed to be re-examined. In the present article we report a rather direct replication of an experiment reported by Rayner, Balota, and Pollatsek (1986). Using the gaze-contingent boundary paradigm, subjects read sentences that contained a target word (razor), but different preview words were initially presented in the sentence. The preview was either identical to the target word (i.e., razor), semantically related to the target word (i.e., blade), semantically unrelated to the target word (i.e., sweet), or a visually similar non-word (i.e., razar). When the reader's eyes crossed an invisible boundary location just to the left of the target word location, the preview changed to the target word. Like Rayner et al. (1986), we found that fixations on the target word were significantly shorter in the identical condition than in the unrelated condition, which did not differ from the semantically related condition; when an orthographically similar preview had been initially present in the sentence fixations were shorter than when a semantically unrelated preview had been present. Thus, the present experiment replicates the earlier data reported by Rayner et al. (1986) indicating evidence for orthographic preview benefit, but a lack of semantic preview benefit in reading English. PMID:24496738

  14. Asynchronous hatching and food limitation: A test of Lack's hypothesis

    USGS Publications Warehouse

    Skagen, Susan Knight

    1988-01-01

    Lack's (1954, 1968) hypothesis that asynchronous hatching of altricial birds is an adaptive response to unpredictable food shortages during the breeding season was examined in the highly granivorous Zebra Finch (Poephila guttata). I compared growth and survival of nestlings in asynchronous and artificially created synchronous broods reared under food-limited and food-abundant conditions in an aviary. I also examined the role of parental experience on survival and growth of nestlings.There was no differential mortality of Zebra Finch nestlings due to either asynchrony or food abundance. Young in abundant food treatments grew more rapidly, however, than those in food-restricted treatments. Heaviest Zebra Finch nestlings in a brood grew more quickly than their lightest siblings when food was limited, supporting Lack's hypothesis. Further, differential survival of light and heavy siblings occurred when food was abundant, suggesting that asynchronous hatching can be maladaptive under some ecological conditions. Nestlings reared by inexperienced parents suffered greater mortality and slower growth when food was abundant than nestlings raised by experienced parents. Prefledging mass was correlated with size at adulthood

  15. Sensory quality of soymilk and tofu from soybeans lacking lipoxygenases.

    PubMed

    Yang, Aijun; Smyth, Heather; Chaliha, Mridusmita; James, Andrew

    2016-03-01

    The oxidation of unsaturated lipids by lipoxygenases in soybeans causes undesirable flavors in soy foods. Using a traditional and a nontraditional soy food user group, we examined the cultural difference in perceiving the sensory characteristics of soymilk and tofu produced from soybeans with or without lipoxygenases (Lx123). The two groups described the samples using similar terms. The traditional users preferred the control soy milk and lipoxygenase-free tofu while the nontraditional users preferred the lipoxygenase-free soymilk with no preference for tofu. In a separate study, a trained descriptive taste panel compared the odor of soymilk and tofu from control soybeans or those lacking lipoxygenase-1 and lipoxygenase-2 (Lx12) or all three isomers (Lx123). The rancid/grassy odor was rated the lowest in Lx123 products, followed by Lx12 products with the control products given the highest rating. The Lx12 and Lx123 products were also sweeter and less bitter than the controls. Taken together, our results demonstrated that soybeans lacking lipoxygenases can produce soy foods with less undesirable aromas and are therefore likely more acceptable to the consumers.

  16. [Lack of assertiveness in patients with eating disorders].

    PubMed

    Behar A, Rosa; Manzo G, Rodrigo; Casanova Z, Dunny

    2006-03-01

    Low self-assertion has been noted as an important feature among patients with eating disorders. To verify, in a female population, if assertiveness is related or has a predictive capacity for the development of eating disorders. An structured clinical interview, the Eating Attitudes Test (EAT-40) and the Rathus Assertiveness Scale (RAS) were administered to 62 patients that fulfilled the DSM-IV diagnostic criteria for eating disorders and to 120 female students without eating problems. Patients with eating disorders ranked significantly higher on the EAT-40 and its factors (p <0.001) and showed a lower level of assertiveness on the RAS (p <0.001). Assertiveness measured by RAS and its factors was inversely related to EAT-40 and its items (r= -0.21). The predictive capability of the lack of self-assertion in the development of an eating disorder reached 53%, when patients with eating disorders and subjects at risk were considered together and compared to students without such disorder. Lack of assertiveness is a significant trait in patients with eating disorders; it may worsen its outcome and even perpetuate symptoms. Low self-assertion may be considered a predictive factor in the development of an eating disorder and must be managed from a preventive or therapeutic point of view.

  17. Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

    PubMed Central

    Kim, Tom S.; Heinlein, Cynthia; Hackman, Robert C.; Nelson, Peter S.

    2006-01-01

    Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2−/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2−/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2−/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2−/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation. PMID:16428450

  18. Replication of Mu prophages lacking the central strong gyrase site.

    PubMed

    Pato, Martin L

    2004-09-01

    Replication of Mu prophages lacking the central strong gyrase site (SGS) is severely slowed. To study details of the replication of these prophages, an assay was developed for determining the rate and extent of introduction of nicks at the 3'-ends of a Mu prophage, an early step in Mu replicative transposition. The maximal level of end-nicking of a prophage with the SGS, about 70-90% depending upon the host strain, was achieved within about 15 min after induction, whereas at that time less than 5% nicking was observed with a prophage lacking the SGS. The amount of nicking at the end of the SGS(-) prophage increased with time, and approx. 30% nicking of the SGS(-) prophage was achieved by 60 min post-induction. Nicking kinetics were identical at each end of the prophages, and no nicking was observed at the 5'-ends of the prophages, verifying in vivo the earlier results with in vitro systems. To determine if prophage location affects the kinetics of replication, we examined prophages at numerous chromosomal locations. SGS(+) prophages at different chromosomal locations showed essentially identical replication kinetics. SGS(-) prophages showed a range of delays in replication and host lysis. A gradient of delays was apparent, with prophages further from the chromosomal origin of replication, oriC, showing longer delays than ones nearer to oriC. However, there were also exceptions to this overall gradient. Possible explanations for the differences in the delays observed with SGS(-) prophages are discussed.

  19. Sleep Apneas are Increased in Mice Lacking Monoamine Oxidase A

    PubMed Central

    Real, Caroline; Popa, Daniela; Seif, Isabelle; Callebert, Jacques; Launay, Jean-Marie; Adrien, Joëlle; Escourrou, Pierre

    2007-01-01

    Study Objectives: Alterations in the serotonin (5-HT) system have been suggested as a mechanism of sleep apnea in humans and rodents. The objective is to evaluate the contribution of 5-HT to this disorder. Design: We studied sleep and breathing (whole-body plethysmography) in mutant mice that lack monoamine oxidase A (MAOA) and have increased concentrations of monoamines, including 5-HT. Measurements and Results: Compared to wild-type mice, the mutants showed similar amounts of slow wave sleep (SWS) and rapid eye movement sleep (REMS), but exhibited a 3-fold increase in SWS and REMS apnea indices. Acute administration of the MAOA inhibitor clorgyline decreased REMS amounts and increased the apnea index in wild-type but not mutant mice. Parachlorophenylalanine, a 5-HT synthesis inhibitor, reduced whole brain concentrations of 5-HT in both strains, and induced a decrease in apnea index in mutant but not wild-type mice. Conclusion: Our results show that MAOA deficiency is associated with increased sleep apnea in mice and suggest that an acute or chronic excess of 5-HT contributes to this phenotype. Citation: Real C; Popa D; Seif I; Callebert J; Launay JM; Adrien J; Escourrou P. Sleep apneas are increased in mice lacking monoamine oxidase A. PMID:17969463

  20. Individuals With OCD Lack Unrealistic Optimism Bias in Threat Estimation.

    PubMed

    Zetsche, Ulrike; Rief, Winfried; Exner, Cornelia

    2015-07-01

    Overestimating the occurrence of threatening events has been highlighted as a central cognitive factor in the maintenance of obsessive-compulsive disorder (OCD). The present study examined the different facets of this cognitive bias, its underlying mechanisms, and its specificity to OCD. For this purpose, threat estimation, probabilistic classification learning (PCL) and psychopathological measures were assessed in 23 participants with OCD, 30 participants with social phobia, and 31 healthy controls. Whereas healthy participants showed an optimistic expectation bias regarding positive and negative future events, OCD participants lacked such a bias. This lack of an optimistic expectation bias was not specific to OCD. Compared to healthy controls, OCD participants overestimated their personal risk for experiencing negative events, but did not differ from controls in their risk estimation regarding other people. Finally, OCD participants' biases in the prediction of checking-related events were associated with their impairments in learning probabilistic cue-outcome associations in a disorder-relevant context. In sum, the present results add to a growing body of research demonstrating that cognitive biases in OCD are context-dependent.

  1. Metabolic alterations in yeast lacking copper-zinc superoxide dismutase.

    PubMed

    Sehati, Sadaf; Clement, Matthew H S; Martins, Jake; Xu, Lei; Longo, Valter D; Valentine, Joan S; Gralla, Edith B

    2011-06-01

    Yeast lacking copper-zinc superoxide dismutase (sod1∆) have a number of oxygen-dependent defects, including auxotrophies for lysine and methionine and sensitivity to oxygen. Here we report additional defects in metabolic regulation. Under standard growth conditions with glucose as the carbon source, yeast undergo glucose repression in which mitochondrial respiration is deemphasized, energy is mainly derived from glycolysis, and ethanol is produced. When glucose is depleted, the diauxic shift is activated, in which mitochondrial respiration is reemphasized and stress resistance increases. We find that both of these programs are adversely affected by the lack of Sod1p. Key events in the diauxic shift do not occur and sod1∆ cells do not utilize ethanol and stop growing. The ability to shift to growth on ethanol is gradually lost as time in culture increases. In early stages of culture, sod1∆ cells consume more oxygen and have more mitochondrial mass than wild-type cells, indicating that glucose repression is not fully activated. These changes are at least partially dependent on the activity of the Hap2,3,4,5 complex, as indicated by CYC1-lacZ reporter assays. These changes may indicate a role for superoxide in metabolic signaling and regulation and/or a role for glucose derepression in defense against oxidative stress. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Optical stimulation in mice lacking the TRPV1 channel

    NASA Astrophysics Data System (ADS)

    Suh, Eul; Izzo Matic, Agnella; Otting, Margarete; Walsh, Joseph T., Jr.; Richter, Claus-Peter

    2009-02-01

    Lasers can be used to stimulate neural tissue, including the sciatic nerve or auditory neurons. Wells and coworkers suggested that neural tissue is likely stimulated by heat.[1,2] Ion channels that can be activated by heat are the TRPV channels, a subfamily of the Transient Receptor Potential (TRP) ion channels. TRPV channels are nonselective cation channels found in sensory neurons involved in nociception. In addition to various chemicals, TRPV channels can also be thermally stimulated. The activation temperature for the different TRPV channels varies and is 43°C for TRPV1 and 39°C for TRPV3. By performing an immunohistochemical staining procedure on frozen 20 μm cochlear slices using a primary TRPV1 antibody, we observed specific immunostaining of the spiral ganglion cells. Here we show that in mice that lack the gene for the TRPV1 channel optical radiation cannot evoke action potentials on the auditory nerve.

  3. Lack of Evidence for Zoonotic Transmission of Schmallenberg Virus

    PubMed Central

    van den Wijngaard, Cees; van Beek, Paul; Beer, Martin; Bouwstra, Ruth; Godeke, Gert-Jan; Isken, Leslie; van den Kerkhof, Hans; van Pelt, Wilfrid; van der Poel, Wim; Reimerink, Johan; Schielen, Peter; Schmidt-Chanasit, Jonas; Vellema, Piet; de Vries, Ankje; Wouters, Inge; Koopmans, Marion

    2012-01-01

    The emergence of Schmallenberg virus (SBV), a novel orthobunyavirus, in ruminants in Europe triggered a joint veterinary and public health response to address the possible consequences to human health. Use of a risk profiling algorithm enabled the conclusion that the risk for zoonotic transmission of SBV could not be excluded completely. Self-reported health problems were monitored, and a serologic study was initiated among persons living and/or working on SBV-affected farms. In the study set-up, we addressed the vector and direct transmission routes for putative zoonotic transfer. In total, 69 sheep farms, 4 goat farms, and 50 cattle farms were included. No evidence for SBV-neutralizing antibodies was found in serum of 301 participants. The lack of evidence for zoonotic transmission from either syndromic illness monitoring or serologic testing of presumably highly exposed persons suggests that the public health risk for SBV, given the current situation, is absent or extremely low. PMID:23092696

  4. Vaccinia virions deficient in transcription enzymes lack a nucleocapsid

    SciTech Connect

    McFadden, Baron D.H.; Moussatche, Nissin; Kelley, Karen; Kang, Byung-Ho; Condit, Richard C.

    2012-12-05

    The poxvirus virion contains an inner tubular nucleocapsid structure. The nucleocapsid is apparently labile to conventional electron microscopy fixation procedures and has therefore been largely ignored for decades. Advancements in electron microscopy sample preparation, notably high pressure freezing, better preserve the nucleocapsid structure. Using high pressure freezing and electron microscopy, we have compared the virion structures of wt virus and mutant viruses known to be deficient in packaging of viral transcription enzymes. We show that the mutant viruses lack a defined nucleocapsid. These results support the hypothesis that the nucleocapsid contains the viral DNA genome complexed with viral transcription enzymes and structural proteins. The studies open the door to further investigation of the composition and ultrastructure of the poxvirus nucleocapsid.

  5. Mice lacking a transcriptional corepressor Tob are predisposed to cancer

    PubMed Central

    Yoshida, Yutaka; Nakamura, Takahisa; Komoda, Masato; Satoh, Hitoshi; Suzuki, Toru; Tsuzuku, Junko K.; Miyasaka, Takashi; Yoshida, Eri H.; Umemori, Hisashi; Kunisaki, Reiko K.; Tani, Kenzaburo; Ishii, Shunsuke; Mori, Shigeo; Suganuma, Masami; Noda, Tetsuo; Yamamoto, Tadashi

    2003-01-01

    tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob−/− mice than in wild-type mice. tob−/−p53−/− mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development. PMID:12756225

  6. Lack of production sharing laws slows joint ventures in Russia

    SciTech Connect

    Knott, D.

    1995-10-30

    When Russia opened its doors to foreign oil companies in 1990, there was a rush to secure a piece of the country`s potentially vast oil wealth. Since then, many of the ventures between Russian and non-Russian partners have become bogged down with operational problems and an ever changing tax and legal regime. There is a stockpile of massive developments building, while government grinds with seeming reluctance toward passing laws that will allow outside firms to do big business. For major development projects the main stumbling block is the lack of production sharing contract legislation. The paper describes the problems, the current legislation, and operating problems, then highlights several joint ventures that have been successful and several that have ended in pullouts of the foreign investor.

  7. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart.

    PubMed

    Xu, Wenjing; Barrientos, Tomasa; Mao, Lan; Rockman, Howard A; Sauve, Anthony A; Andrews, Nancy C

    2015-10-20

    Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

  8. Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom.

    PubMed

    Wooldridge, B J; Pineda, G; Banuelas-Ornelas, J J; Dagda, R K; Gasanov, S E; Rael, E D; Lieb, C S

    2001-09-01

    The venom composition of Mojave rattlesnakes (Crotalus scutulatus scutulatus) differs in that some individuals have Mojave toxin and others do not. In order to understand the genetic basis for this difference, genomic DNA samples from Mojave rattlesnakes collected in Arizona, New Mexico, and Texas were analyzed for the presence of DNA sequences that relate to the acidic (Mta) and basic (Mtb) subunits of this toxin. DNA samples were subjected to PCR to amplify nucleotide sequences from second to fourth exons of the acidic and basic subunits. These nucleotide sequences were cloned and sequenced. The nucleotide sequences generated aligned exactly to previously published nucleotide sequences of Mojave toxin. All DNA samples analyzed generated product using the basic subunit primers, and aligned identically to the Mtb nucleotide sequence. However, only 11 out of the 14 samples generated a product with the acidic subunit primers. These 11 sequences aligned identically to the Mta nucleotide sequence. The venom from the three snakes whose DNA did not amplify with the acidic subunit primers were not recognized by antibodies to Mojave toxin. This suggests that snakes with venom lacking Mojave toxin also lack the productive nucleotide sequence for the acidic subunit in their DNA.

  9. Albumins and their processing machinery are hijacked for cyclic peptides in sunflower.

    PubMed

    Mylne, Joshua S; Colgrave, Michelle L; Daly, Norelle L; Chanson, Aurelie H; Elliott, Alysha G; McCallum, Emily J; Jones, Alun; Craik, David J

    2011-05-01

    The cyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) blocks trypsin and is a promising drug lead and protein engineering scaffold. We show that SFTI-1 and the newfound SFT-L1 are buried within PawS1 and PawS2, precursors for seed storage protein albumins. Proalbumins are matured by asparaginyl endopeptidase, which we show is required to liberate both ends of SFTI-1 as well as to mature PawS1 albumin. Thus, these peptides emerge from within an albumin precursor by the action of albumin's own processing enzyme.

  10. Lack of size selectivity for paddlefish captured in hobbled gillnets

    USGS Publications Warehouse

    Scholten, G.D.; Bettoli, P.W.

    2007-01-01

    A commercial fishery for paddlefish Polyodon spathula caviar exists in Kentucky Lake, a reservoir on the lower Tennessee River. A 152-mm (bar-measure) minimum mesh size restriction on entanglement gear was enacted in 2002 and the minimum size limit was increased to 864 mm eye-fork length to reduce the possibility of recruitment overfishing. Paddlefish were sampled in 2003-2004 using experimental monofilament gillnets with panels of 89, 102, 127, 152, 178, and 203-mm meshes and the efficacy of the mesh size restriction was evaluated. Following the standards of commercial gear used in that fishery, nets were "hobbled" (i.e., 128 m ?? 3.6 m nets were tied down to 2.4 m; 91 m ?? 9.1 m nets were tied down to 7.6 m). The mean lengths of paddlefish (Ntotal = 576 fish) captured in each mesh were similar among most meshes and bycatch rates of sublegal fish did not vary with mesh size. Selectivity curves could not be modeled because the mean and modal lengths of fish captured in each mesh did not increase with mesh size. Ratios of fish girth to mesh perimeter (G:P) for individual fish were often less than 1.0 as a result of the largest meshes capturing small paddlefish. It is unclear whether lack of size selectivity for paddlefish was because the gillnets were hobbled, the unique morphology of paddlefish, or the fact that they swim with their mouths agape when filter feeding. The lack of size selectivity by hobbled gillnets fished in Kentucky Lake means that managers cannot influence the size of paddlefish captured by commercial gillnet gear by changing minimum mesh size regulations. ?? 2006 Elsevier B.V. All rights reserved.

  11. Phenotypic analysis of mice completely lacking netrin 1.

    PubMed

    Yung, Andrea R; Nishitani, Allison M; Goodrich, Lisa V

    2015-11-01

    Netrin 1 (Ntn1) is a multifunctional guidance cue expressed in the ventricular zone and floor plate of the embryonic neural tube. Although Ntn1 is best known for acting as an axon guidance cue through Dcc and neogenin receptors, it is also thought to regulate neuronal survival and blood vessel development through Unc5 family receptors. However, the Ntn1 gene trap mutant mouse does not display all the phenotypes predicted from in vitro assays or analyses of mice lacking predicted receptors. Since the gene trap strain still produces wild-type Ntn1 protein, it is unclear whether the absence of phenotypes reflects the activity of alternative cues or of residual Ntn1. To resolve the full contribution of Ntn1 to development, we generated a null allele of Ntn1 and re-examined tissues exhibiting phenotypic discrepancies between receptor mutants and Ntn1 hypomorphs. We found that in Ntn1 null animals commissural axons rarely cross the midline, resulting in a strongly enhanced phenotype relative to Ntn1 hypomorphs, which retain many axons with normal trajectories. Thus, low levels of Ntn1 can account for persistent attraction to the midline in hypomorphs. By contrast, Ntn1 null mice do not show all of the phenotypes reported for Unc5 receptor mutants, indicating that Ntn1 is not necessarily the dominant ligand for Unc5 family members in vivo and ruling out primary roles in survival or angiogenesis. © 2015. Published by The Company of Biologists Ltd.

  12. Phenotypic analysis of mice completely lacking netrin 1

    PubMed Central

    Yung, Andrea R.; Nishitani, Allison M.; Goodrich, Lisa V.

    2015-01-01

    Netrin 1 (Ntn1) is a multifunctional guidance cue expressed in the ventricular zone and floor plate of the embryonic neural tube. Although Ntn1 is best known for acting as an axon guidance cue through Dcc and neogenin receptors, it is also thought to regulate neuronal survival and blood vessel development through Unc5 family receptors. However, the Ntn1 gene trap mutant mouse does not display all the phenotypes predicted from in vitro assays or analyses of mice lacking predicted receptors. Since the gene trap strain still produces wild-type Ntn1 protein, it is unclear whether the absence of phenotypes reflects the activity of alternative cues or of residual Ntn1. To resolve the full contribution of Ntn1 to development, we generated a null allele of Ntn1 and re-examined tissues exhibiting phenotypic discrepancies between receptor mutants and Ntn1 hypomorphs. We found that in Ntn1 null animals commissural axons rarely cross the midline, resulting in a strongly enhanced phenotype relative to Ntn1 hypomorphs, which retain many axons with normal trajectories. Thus, low levels of Ntn1 can account for persistent attraction to the midline in hypomorphs. By contrast, Ntn1 null mice do not show all of the phenotypes reported for Unc5 receptor mutants, indicating that Ntn1 is not necessarily the dominant ligand for Unc5 family members in vivo and ruling out primary roles in survival or angiogenesis. PMID:26395479

  13. Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

    PubMed

    Dusaulcy, R; Daviaud, D; Pradère, J P; Grès, S; Valet, Ph; Saulnier-Blache, J S

    2009-12-01

    The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

  14. Lack of Galanin 3 Receptor Aggravates Murine Autoimmune Arthritis.

    PubMed

    Botz, Bálint; Kemény, Ágnes; Brunner, Susanne M; Locker, Felix; Csepregi, Janka; Mócsai, Attila; Pintér, Erika; McDougall, Jason J; Kofler, Barbara; Helyes, Zsuzsanna

    2016-06-01

    Neurogenic inflammation mediated by peptidergic sensory nerves has a crucial impact on the pathogenesis of various joint diseases. Galanin is a regulatory sensory neuropeptide, which has been shown to attenuate neurogenic inflammation, modulate neutrophil activation, and be involved in the development of adjuvant arthritis, but our current understanding about its targets and physiological importance is incomplete. Among the receptors of galanin (GAL1-3), GAL3 has been found to be the most abundantly expressed in the vasculature and on the surface of some immune cells. However, since there are minimal in vivo data on the role of GAL3 in joint diseases, we analyzed its involvement in different inflammatory mechanisms of the K/BxN serum transfer-model of autoimmune arthritis employing GAL 3 gene-deficient mice. After arthritis induction, GAL3 knockouts demonstrated increased clinical disease severity and earlier hindlimb edema than wild types. Vascular hyperpermeability determined by in vivo fluorescence imaging was also elevated compared to the wild-type controls. However, neutrophil accumulation detected by in vivo luminescence imaging or arthritic mechanical hyperalgesia was not altered by the lack of the GAL3 receptor. Our findings suggest that GAL3 has anti-inflammatory properties in joints by inhibiting vascular hyperpermeability and consequent edema formation.

  15. Cells lacking DNA topoisomerase II beta are resistant to genistein.

    PubMed

    López-Lazaro, Miguel; Willmore, Elaine; Austin, Caroline A

    2007-05-01

    Evidence suggests that DNA topoisomerases (topos) may be involved in the anticancer and carcinogenic properties attributed to flavonoids. Using the cell-based assay TARDIS, the dietary flavonoids genistein (1) and luteolin (2) have been evaluated as topo I and topo II poisons and catalytic inhibitors in K562 leukemia cells. Both flavonoids induced topo II-DNA complexes, but they did not induce significant levels of topo I-DNA complexes. Genistein decreased the topo II-DNA complexes induced by the topo II poison etoposide, suggestive of a catalytic inhibition of topo II, and luteolin decreased the topo I-DNA complexes induced by the topo I poison camptothecin, indicative of a catalytic inhibition of topo I. Murine transgenic cells lacking topo II beta were resistant to genistein-induced cell growth inhibition (XTT assays) and cytotoxicity (clonogenic assay). High levels of topo II beta-DNA complexes were also observed in K562 cells exposed to genistein. These data suggest that topo II beta has an important function in genistein-induced cell growth inhibition and cell death. The possible role of topoisomerases in the putative anticancer and carcinogenic properties of genistein and luteolin is discussed.

  16. Lack of eyeblink entrainments in autism spectrum disorders.

    PubMed

    Nakano, Tamami; Kato, Nobumasa; Kitazawa, Shigeru

    2011-07-01

    Interpersonal synchrony is the temporal coordination of movements between individuals during social interactions. For example, it has been shown that listeners synchronize their eyeblinks to a speaker's eyeblinks, especially at breakpoints of speech, when viewing a close-up video clip of the speaker's face. We hypothesized that this interpersonal synchronous behavior would not be observed in individuals with autism spectrum disorders (ASD), which are characterized by impaired social communication. To test this hypothesis, we examined eyeblink entrainments in adults with ASD. As we reported previously, the eyeblinks of adults without ASD were significantly synchronized with the speaker's eyeblinks at pauses in his speech when they viewed the speaker's entire face. However, the significant eyeblink synchronization disappeared when adults without ASD viewed only the speaker's eyes or mouth, suggesting that information from the whole face, including information from both the eyes and the mouth, was necessary for eyeblink entrainment. By contrast, the ASD participants did not show any eyeblink synchronization with the speaker, even when viewing the speaker's eyes and mouth simultaneously. The lack of eyeblink entrainment to the speaker in individuals with ASD suggests that they are not able to temporally attune themselves to others' behaviors. The deficits in temporal coordination may impair effective social communication with others.

  17. Prefrontal glucose deficits in murderers lacking psychosocial deprivation.

    PubMed

    Raine, A; Phil, D; Stoddard, J; Bihrle, S; Buchsbaum, M

    1998-01-01

    Previous research has suggested that links between autonomic nervous system functioning and violence are strongest in those who come from benign home backgrounds, but there appears to be no similar research using brain-imaging measures of central nervous system functioning. It was hypothesized that murderers who had no early psychosocial deprivation (e.g., no childhood abuse, family neglect) would demonstrate lower prefrontal glucose metabolism than murderers with early psychosocial deprivation and a group of normal controls. Murderers from a previous study, which showed prefrontal deficits in murderers, were assessed for psychosocial deprivation and divided into those with and without deprivation. Murderers without any clear psychosocial deficits were significantly lower on prefrontal glucose metabolism than murderers with psychosocial deficits and controls. These results suggest that murderers lacking psychosocial deficits are characterized by prefrontal deficits. It is argued that among violent offenders without deprived home backgrounds, the "social push" to violence is minimized, and consequently, brain abnormalities provide a relatively stronger predisposition to violence in this group.

  18. Association between friction and wear in diarthrodial joints lacking lubricin.

    PubMed

    Jay, Gregory D; Torres, Jahn R; Rhee, David K; Helminen, Heikki J; Hytinnen, Mika M; Cha, Chung-Ja; Elsaid, Khaled; Kim, Kyung-Suk; Cui, Yajun; Warman, Matthew L

    2007-11-01

    The glycoprotein lubricin (encoded by the gene Prg4) is secreted by surface chondrocytes and synovial cells, and has been shown to reduce friction in vitro. In contrast to man-made bearings, mammalian diarthrodial joints must endogenously produce friction-reducing agents. This study was undertaken to investigate whether friction is associated with wear. The lubricating ability of synovial fluid (SF) samples from humans with genetic lubricin deficiency was tested in vitro. The coefficient of friction in the knee joints of normal and lubricin-null mice was measured ex vivo; these joints were also studied by light and electron microscopy. Atomic force microscopy was used to image and measure how lubricin reduces friction in vitro. SF lacking lubricin failed to reduce friction in the boundary mode. Joints of lubricin-null mice showed early wear and higher friction than joints from their wild-type counterparts. Lubricin self-organized and reduced the work of adhesion between apposing asperities. These data show that friction is coupled with wear at the cartilage surface in vivo. They imply that acquired lubricin degradation occurring in inflammatory joint diseases predisposes the cartilage to damage. Lastly, they suggest that lubricin, or similar biomolecules, will have applications in man-made devices in which reducing friction is essential.

  19. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

  20. A Chenopod Extensin Lacks Repetitive Tetrahydroxyproline Blocks 1

    PubMed Central

    Li, Xiong-biao; Kieliszewski, Marcia; Lamport, Derek T. A.

    1990-01-01

    An extensin isolated from sugar beet (Beta vulgaris) cell suspension cultures fulfills all criteria for membership of the extensin family save one, notably, lack of the `diagnostic' pentamer Ser-Hyp-Hyp-Hyp-Hyp. However, sequence analysis of the major tryptic peptides shows that sugar beet extensin shares a motif in common with tomato extensin P1 but differs by the position of an insertion sequence [X] or [Y] which, in sugar beet, splits the tetrahydroxyproline block: Ser-Hyp-Hyp-[X]-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, where [X] is [Val-His-Glu/Lys-Tyr-Pro], while in tomato the insertion sequence [Y] = [Val-Lys-Pro-Tyr-His-Pro] and, when it occurs, immediately follows the tetrahydroxyproline block: Ser-Hyp-Hyp-Hyp-Hyp-[Y]-Thr-Hyp-Val-Tyr-Lys. Based on these data we reinterpret three highly repetitive cDNA sequences, including nodulin N75 from soybean and wound-induced P33 of carrot, as extensins with split tetra(hydroxy)proline blocks. Images Figure 4 PMID:16667277

  1. Rhesus Monkeys (Macaca mulatta) Lack Expertise in Face Processing

    PubMed Central

    Parr, Lisa A.; Heintz, Matthew; Pradhan, Gauri

    2010-01-01

    Faces are salient stimuli for primates that rely predominantly on visual cues for recognizing conspecifics and maintaining social relationships. While previous studies have shown similar face discrimination processes in chimpanzees and humans, data from monkeys are unclear. Therefore, three studies examined face processing in rhesus monkeys using the face inversion effect, a fractured face task, and an individual recognition task. Unlike chimpanzees and humans, the monkeys showed a general face inversion effect reflected by significantly better performance on upright compared to inverted faces (conspecifics, human and chimpanzees faces) regardless of the subjects’ expertise with those categories. Fracturing faces alters first- and second-order configural manipulations whereas previous studies in chimpanzees showed selective deficits for second-order configural manipulations. Finally, when required to individuate conspecific’s faces, i.e., matching two different photographs of the same conspecific, monkeys showed poor discrimination and repeated training. These results support evolutionary differences between rhesus monkeys and Hominoids in the importance of configural cues and their ability to individuate conspecifics’ faces, suggesting a lack of face expertise in rhesus monkeys. PMID:19014263

  2. Increased Bone Mass in Female Mice Lacking Mast Cell Chymase

    PubMed Central

    Lind, Thomas; Gustafson, Ann-Marie; Calounova, Gabriela; Hu, Lijuan; Rasmusson, Annica; Jonsson, Kenneth B.; Wernersson, Sara; Åbrink, Magnus; Andersson, Göran; Larsson, Sune; Melhus, Håkan; Pejler, Gunnar

    2016-01-01

    Here we addressed the potential impact of chymase, a mast-cell restricted protease, on mouse bone phenotype. We show that female mice lacking the chymase Mcpt4 acquired a persistent expansion of diaphyseal bone in comparison with wild type controls, reaching a 15% larger diaphyseal cross sectional area at 12 months of age. Mcpt4-/- mice also showed increased levels of a bone anabolic serum marker and higher periosteal bone formation rate. However, they were not protected from experimental osteoporosis, suggesting that chymase regulates normal bone homeostasis rather than the course of osteoporosis. Further, the absence of Mcpt4 resulted in age-dependent upregulation of numerous genes important for bone formation but no effects on osteoclast activity. In spite of the latter, Mcpt4-/- bones had increased cortical porosity and reduced endocortical mineralization. Mast cells were found periosteally and, notably, bone-proximal mast cells in Mcpt4-/- mice were degranulated to a larger extent than in wild type mice. Hence, chymase regulates degranulation of bone mast cells, which could affect the release of mast cell-derived factors influencing bone remodelling. Together, these findings reveal a functional impact of mast cell chymase on bone. Further studies exploring the possibility of using chymase inhibitors as a strategy to increase bone volume may be warranted. PMID:27936149

  3. Lack of activity of cadmium in in vitro estrogenicity assays

    SciTech Connect

    Silva, Elisabete . E-mail: elisabete.silva@pharmacy.ac.uk; Lopez-Espinosa, Maria Jose; Molina-Molina, Jose-Manuel; Fernandez, Marieta; Olea, Nicolas; Kortenkamp, Andreas

    2006-10-01

    Prompted by reports about strong estrogenic effects of cadmium, attempts were made to reproduce these observations using the yeast estrogen screen (YES) and the E-Screen assays. For the first time, possible activation of the Src/MAPK pathway was also investigated. In the YES, only a slight activation (10% of a maximal effect) of the estrogen receptor alpha (ER{alpha}) was observed at cadmium concentrations between 5 x 10{sup -7} M and 5 x 10{sup -6} M. In the E-Screen assay, carried out by two laboratories, the heavy metal was without observable cell proliferative effects when tested in the range between 6 x 10{sup -11} M and 1 x 10{sup -5} M. However, in both assays, cadmium led to a reduction of the effects of 17{beta}-estradiol (E2). Treatment of MCF-7 human breast cancer cells with 1 x 10{sup -7} M cadmium failed to induce phosphorylation of Src and the MAP kinases Erk1 and Erk2-effects shown to occur with E2 and epidermal growth factor (EGF). In summary, we were unable to confirm the strong estrogenicity of cadmium reported recently by a number of laboratories. This apparent absence of effects in our hands is not due to a lack of uptake of the metal or to effective protection against cadmium by high levels of glutathione or metallothionein, since toxicity and an antagonism of E2 responses were observed both in the YES and the E-Screen.

  4. Enhanced insulin clearance in mice lacking TRPM8 channels

    PubMed Central

    McCoy, Daniel D.; Zhou, Ligang; Nguyen, Anh-Khoi; Watts, Alan G.; Donovan, Casey M.

    2013-01-01

    Blood glucose concentration is tightly regulated by the rate of insulin secretion and clearance, a process partially controlled by sensory neurons serving as metabolic sensors in relevant tissues. The activity of these neurons is regulated by the products of metabolism which regulate transmitter release, and recent evidence suggests that neuronally expressed ion channels of the transient receptor potential (TRP) family function in this critical process. Here, we report the novel finding that the cold and menthol-gated channel TRPM8 is necessary for proper insulin homeostasis. Mice lacking TRPM8 respond normally to a glucose challenge while exhibiting prolonged hypoglycemia in response to insulin. Additionally, Trpm8-/- mice have increased rates of insulin clearance compared with wild-type animals and increased expression of insulin-degrading enzyme in the liver. TRPM8 channels are not expressed in the liver, but TRPM8-expressing sensory afferents innervate the hepatic portal vein, suggesting a TRPM8-mediated neuronal control of liver insulin clearance. These results demonstrate that TRPM8 is a novel regulator of serum insulin and support the role of sensory innervation in metabolic homeostasis. PMID:23651844

  5. Lack of global epigenetic methylation defects in CBS deficient mice.

    PubMed

    Lee, Hyung-Ok; Wang, Liqun; Kuo, Yin-Ming; Gupta, Sapna; Slifker, Michael J; Li, Yue-Sheng; Andrews, Andrew J; Kruger, Warren D

    2017-01-01

    Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

  6. New graduate nurses' experiences about lack of professional confidence.

    PubMed

    Ortiz, Jennifer

    2016-07-01

    Professional confidence is an essential trait for new graduate nurses to possess in order to provide quality patient care in today's complex hospital setting. However, many new graduates are entering the workforce without it and this remains to be explored. This study describes how new graduate nurses accounted for their lack of professional confidence upon entry into professional practice and how it developed during their first year of practice in the hospital setting. Two face-to-face, individual interviews of 12 participants were utilized to capture the lived experiences of new graduate nurses to gain an understanding of this phenomenon. After manual content analysis seven themes emerged: communication is huge, making mistakes, disconnect between school and practice, independence, relationship building, positive feedback is important, and gaining experience. The findings indicate that the development of professional confidence is a dynamic process that occurs throughout the first year of practice. New graduate nurses must experience both positive and negative circumstances in order to move toward the attainment of professional confidence. Knowing this, nurse educators in academia as well as in the hospital setting may better support the development of professional confidence both before and during the first year of practice.

  7. Lack of testicular seipin causes teratozoospermia syndrome in men

    PubMed Central

    Jiang, Min; Gao, Mingming; Wu, Chaoming; He, Hui; Guo, Xuejiang; Zhou, Zuomin; Yang, Hongyuan; Xiao, Xinhua; Liu, George; Sha, Jiahao

    2014-01-01

    Obesity impairs male fertility, providing evidence for a link between adipose tissue and reproductive function; however, potential consequences of adipose tissue paucity on fertility remain unknown. Lack of s.c. fat is a hallmark of Berardinelli–Seip congenital lipodystrophy type 2 (BSCL2), which is caused by mutations in BSCL2-encoding seipin. Mice with a targeted deletion of murine seipin model BSCL2 with severe lipodystrophy, insulin resistance, and fatty liver but also exhibit male sterility. Here, we report teratozoospermia syndrome in a lipodystrophic patient with compound BSCL2 mutations, with sperm defects resembling the defects of infertile seipin null mutant mice. Analysis of conditional mouse mutants revealed that adipocyte-specific loss of seipin causes progressive lipodystrophy without affecting fertility, whereas loss of seipin in germ cells results in complete male infertility and teratozoospermia. Spermatids of the human patient and mice devoid of seipin in germ cells are morphologically abnormal with large ectopic lipid droplets and aggregate in dysfunctional clusters. Elevated levels of phosphatidic acid accompanied with an altered ratio of polyunsaturated to monounsaturated and saturated fatty acids in mutant mouse testes indicate impaired phospholipid homeostasis during spermiogenesis. We conclude that testicular but not adipose tissue-derived seipin is essential for male fertility by modulating testicular phospholipid homeostasis. PMID:24778225

  8. Lack of a Benign Interpretation Bias in Social Anxiety Disorder

    PubMed Central

    Amir, Nader; Prouvost, Caroline; Kuckertz, Jennie M.

    2013-01-01

    Cognitive models of social anxiety posit that recurrent interpretation of ambiguous information as threatening maintains symptoms (e.g. Clark & Wells, 1995, pp. 69–93, Social phobia: Diagnosis, assessment, and treatment. New York: Guilford Press; Rapee & Heimberg, 1997, pp. 741–756, Behavior Research and Therapy, 35). However, biased interpretation may also be represented as a failure to make a benign interpretation of the ambiguous event. Furthermore, interpretation bias can be characterized by both an online (automatic) component and an offline (effortful) component (Hirsch & Clark, 2004, pp. 799–825, Clinical Psychology Review, 24). To measure both benign and threat biases, as well as examine the effect of social anxiety on offline versus online interpretations, Beard and Amir (2009, pp. 1135–1141, Behaviour Research and Therapy, 46) developed the Word Sentence Association Paradigm (WSAP). In the current study, we administered the WSAP to a group of participants diagnosed with social anxiety disorder (SAD) as well as to a group of non-anxious control (NAC) participants. We found that participants with SAD demonstrated a lack of benign online bias, but not an online threat bias when compared to NACs. However, when examining offline biases, SAD patients endorsed social threat interpretations and rejected benign social interpretations to a greater degree than non-anxious individuals. Our results, when taken together, clearly implicate the role of reduced bias toward benign information in SAD. PMID:22545788

  9. Lack of self-averaging and family trees

    NASA Astrophysics Data System (ADS)

    Serva, Maurizio

    2004-02-01

    We consider a large population of asexually reproducing individuals in absence of selective pressure. The population size is maintained constant by the environment. We find out that distances between individuals (time from the last common ancestor) exhibit highly non-trivial properties. In particular their distribution in a single population is random even in the thermodynamical limit, i.e., there is lack of self-averaging. As a result, not only distances are different for different pairs of individuals but also the mean distance of the individuals of a given population is different at different times. All computed quantities are parameters free and only scale linearly with the population size. Results in this paper may have some relevance in the ‘Out of Africa/Multi-regional’ debate about the origin of modern man. In fact, the recovery of mitochondrial DNA from Neandertal fossils in three different loci: Feldhofer (Germany), Mezmaiskaya (Northern Caucaso), Vinjia (Croatia), permitted to compare Neandertal/Neandertal distances with Neandertal/modern and modern/modern ones.

  10. Lack of Accessible Data on Prosthetic Heart Valves.

    PubMed

    Frank, Michelle; Ganzoni, Giulia; Starck, Christoph; Grünenfelder, Jürg; Corti, Roberto; Gruner, Christiane; Hürlimann, David; Tanner, Felix C; Jenni, Rolf; Greutmann, Matthias; Biaggi, Patric

    2016-03-01

    Incomplete information on characteristics of prosthetic heart valves (PHV) may lead to inappropriate choices for PHV implantation (patient-prosthesis-mismatch) or erroneous interpretation of PHV function after implantation. No single and easy accessible source provides all relevant information on PHV. The aim of this study was to provide a comprehensive overview of available data for the majority of PHVs and annuloplasty rings. Information was collected by reviewing articles published on www.pubmed.org up to December 2014 and by written contact to all PHV manufacturers. Four areas of interest were defined: (1) PHV image, (2) in vivo transvalvular gradients, (3) effective orifice area (EOA) calculators and (4) PHV dimensions. Available information was classified as complete (all categories), partial (two or three categories) or minimal (one category). 108 PHV (including homografts) and 34 annuloplasty rings systems were identified. The information on PHV was complete, partial or minimal in 19.5, 61.0 and 19.5% of PHV, respectively. In 91.6% a picture of the valve could be obtained, whereas normative data for transvalvular gradients and EOA calculators were available in 63.0 and 25.0% of all PHV, respectively. The available data was summarized on a new open access webpage ( www.valveguide.ch ). There is a lack of accessible data on PHV dimensions, normal transvalvular gradients and effective orifice area calculators, although such information is of crucial importance for proper PHV assessment.

  11. Lack of immunoediting in murine pancreatic cancer reversed with neoantigen

    PubMed Central

    Evans, Rebecca A.; Diamond, Mark S.; Rech, Andrew J.; Chao, Timothy; Richardson, Max W.; Lin, Jeffrey H.; Bajor, David L.; Byrne, Katelyn T.; Stanger, Ben Z.; Riley, James L.; Markosyan, Nune; Winograd, Rafael; Vonderheide, Robert H.

    2016-01-01

    In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through “immunoediting,” whereby tumors adapt to immune pressure and escape T cell–mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell–depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model — a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors. PMID:27642636

  12. Gregarina niphandrodes may lack both a plastid genome and organelle.

    PubMed

    Toso, Marc A; Omoto, Charlotte K

    2007-01-01

    Gregarines are early diverging apicomplexans that appear to be closely related to Cryptosporidium. Most apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess both plastids and corresponding plastid genomes. Cryptosporidium lacks both the organelle and the genome. To investigate the evolutionary history of plastids in the Apicomplexa, we tried to determine whether gregarines possess a plastid and/or its genome. We used PCR and dot-blot hybridization to determine whether the gregarine Gregarina niphandrodes possesses a plastid genome. We used an inhibitor of plastid function for any reduction in gregarine infection, and transmission electron microscopy to search for plastid ultrastructure. Despite an extensive search, an organelle of the appropriate ultrastructure in transmission electron microscopy, was not observed. Triclosan, an inhibitor of the plastid-specific enoyl-acyl carrier reductase enzyme, did not reduce host infection by G. niphandrodes. Plastid-specific primers produced amplicons with the DNA of Babesia equi, Plasmodium falciparum, and Toxoplasma gondii as templates, but not with G. niphandrodes DNA. Plastid-specific DNA probes, which hybridized to Babesia equi, failed to hybridize to G. niphandrodes DNA. This evidence indicates that G. niphandrodes is not likely to possess either a plastid organelle or its genome. This raises the possibility that the plastid was lost in the Apicomplexan following the divergence of gregarines and Cryptosporidium.

  13. [Evidence and Lack of Evidence in the Treatment of Tinnitus].

    PubMed

    Hesse, G

    2016-04-01

    A broad variety of therapeutic regimen is proposed, introduced and sold against tinnitus, but most of these approaches lack scientific validation and evidence. Up to date a causal, tinnitus eliminating therapy is not available. Most probably this will not be possible at all, as the mechanism of tinnitus generation are multiple and include peripheral as well as central or cortical reactions. Like in fashion and design however, therapeutic medical interventions against tinnitus come in waves again and again over the last decades, without being able to prove lasting and scientifically evident effects.This review presents, discusses and assesses almost all available therapies regarding their evidence. Evidence should include besides external evidence through publications and available data also internal evidence, e.g. including experience of the therapist and needs of the patients.Almost all interventions that try to influence the inner ear or the auditory cortex either pharmaceutically or by direct stimulation or modulation do not reach evidence. However, there are procedures that have proven to be effective and show at least certain degrees of evidence with proven strength of effect. These are habituation therapies and psychotherapeutic interventions like cognitive behavioural therapy, especially when they are combined with concrete measures to improve auditory perception like hearing-aids, cochlear implants or hearing-therapy.

  14. Special Relativity in Week One: 4) Lack of Simultaneity

    NASA Astrophysics Data System (ADS)

    Huggins, Elisha

    2011-09-01

    This is our final article on teaching special relativity in the first week of an introductory physics course.1-3 One of the profound changes in our view of the world was Einstein's discovery of the lack of simultaneity. He illustrated this result with a thought experiment in which we observe a railroad car passing by us. We see the two ends of the car struck simultaneously by lightning bolts, but to someone riding inside the car, the lightning strikes were not simultaneous. My difficulty with this thought experiment is that while doing calculations, I have to go back and forth between two imagined points of view. To avoid this, I actually perform an experiment that involves two simultaneous events. then all we have to imagine is how the experiment looks to someone moving by us. Not only does the order of the two events depend on the direction of motion of the observer, but we can demonstrate that if information could travel faster than the speed of light, we could get answers to questions that have not yet been thought of.

  15. Characterization of nonconventional hepatitis B viruses lacking the core promoter.

    PubMed

    Chang, Shau-Feng; Chang, Shih-Hsuan; Li, Bi-Chen; Will, Hans; Netter, Hans Jürgen

    2004-12-20

    The core gene (C-gene) promoter and regulatory sequences play a central role in the hepatitis B virus (HBV) life cycle. They are essential for the synthesis of the pregenomic and precore mRNA. The pregenomic RNA is the template required for replication and also the template for the synthesis of the core protein and polymerase. Here, we report the in vivo existence and functional characterization of HBV variants that lack the C-gene promoter region and the regulatory sequences located therein. HBV promoter fragments were isolated by PCR from sera of chronic carriers and characterized. Truncated promoter elements were identified, and then tested in the context of wild-type genomes in the HuH-7 cell line. The expression of the recombinant HBV genome resulted in the synthesis of surface proteins, and low level of core protein as well as a transcript pattern similar to, but smaller in size to wild-type virus. The recombinant HBV genome with the truncated promoter region produced pregenomic RNA-like transcripts. These transcripts were encapsidated and reverse transcribed when complemented by sufficient core and polymerase protein. These date provide an explanation as to why such deletion mutants of HBV can be produced at all, they highlight the functional potentials of viral sequences activated by mutations and may be of relevance for viral evolution and persistence.

  16. Lack of magnetopause Kelvin Helmoltz waves in the THEMIS era

    NASA Astrophysics Data System (ADS)

    kavosi, S.; Raeder, J.

    2012-12-01

    It has been long suggested that The Kelvin Helmholtz Instability (KHI) at Earth's low latitude magnetopause plays a significant role for the entry of solar wind plasma into the magnetosphere under northward interplanetary magnetic field (IMF) conditions. In this study we analyze surface waves commonly observed by the THEMIS spacecraft at the flank magnetopause throughout the entire THEMIS mission. In select cases we perform OpenGGCM simulations of observed surface wave events in order to investigate the nature of the waves. In particular, we compare simulations driven by the full set of solar wind and IMF data with simulations in which the solar wind speed and density are held constant at appropriate average values. This allows us to compare the occurrences of surface waves caused by KHI with those caused by solar wind pressure fluctuations, and to determine the relative importance of each mechanism. Our results show that solar wind pressure fluctuations are the dominant mechanisms for generating surface waves observed during northward IMF. The KHI seems to occur much less frequently than typically expected. Since THEMIS spacecraft are often in a good position to observe KHI, the absence of KHI waves is conspicuous. In order to understand the lack of KH waves during the THEMIS era we statistically examine key quantities of the solar wind and IMF to better establish the conditions under which KHI occurs.

  17. Clinical errors as a lack of context responsiveness.

    PubMed

    Bugatti, Matteo; Boswell, James F

    2016-09-01

    Although standardized treatments have the potential to decrease clinical errors, within-session responsiveness is complicated and complementary frameworks may be needed to foster enhanced responsiveness in the context of evidence-based treatments. Recent efforts have targeted the enhancement of flexibility and responsiveness in the delivery of manualized treatments, including the development of transdiagnostic treatments (i.e., protocols that are designed to be used across different diagnoses) intended to tailor intervention principles to the needs of individual patients. Context-Responsive Psychotherapy Integration (Constantino, Boswell, Bernecker, & Castonguay, 2013) offers an if-then framework that supports the utilization of evidence-based clinical strategies in response to the identification of specific process markers. Failure to identify or appropriately respond to such markers may result in negative therapeutic process as well as outcomes. This case study uses the context-response psychotherapy integration framework to understand critical moments of clinical decision-making through examining an individual treatment case that unilaterally terminated after seven sessions of transdiagnostic treatment. This illustrative empirical case analysis focuses on three potential clinical errors, as indicated by a lack of responsiveness to three candidate process markers: (a) low outcome expectations, (b) self-strivings, and (c) outcome monitoring. For each clinical error, alternative clinical strategies are discussed. PsycINFO Database Record (c) 2016 APA, all rights reserved

  18. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  19. Lack of non-voluntary stepping responses in Parkinson's disease.

    PubMed

    Selionov, V A; Solopova, I A; Zhvansky, D S; Karabanov, A V; Chernikova, L A; Gurfinkel, V S; Ivanenko, Y P

    2013-04-03

    The majority of research and therapeutic actions in Parkinson's disease (PD) focus on the encephalic areas, however, the potential involvement of the spinal cord in its genesis has received little attention. Here we examined spinal locomotor circuitry activation in patients with PD using various types of central and peripheral tonic stimulation and compared results to those of age-matched controls. Subjects lay on their sides with both legs suspended, allowing low-friction horizontal rotation of the limb joints. Air-stepping can be used as a unique and important model for investigating human rhythmogenesis since its manifestation is largely facilitated by the absence of external resistance. In contrast to the frequent occurrence of non-voluntary stepping responses in healthy subjects, both peripheral (muscle vibration) and central (Jendrassik maneuver, mental task, Kohnstamm phenomenon) tonic influences had little if any effect on rhythmic leg responses in PD. On the other hand, a remarkable feature of voluntary air-stepping movements in patients was a significantly higher frequency of leg oscillations than in age-matched controls. A lack of non-voluntary stepping responses was also observed after dopaminergic treatment despite the presence of prominent shortening reactions (SRs) to passive movements. We argue that the state and the rhythmogenesis capacity of the spinal circuitry are impaired in patients with PD. In particular, the results suggest impaired central pattern generator (CPG) access by sensory and central activations.

  20. European Ozone Trends: Why the Lack of Decrease?

    NASA Astrophysics Data System (ADS)

    von Schneidemesser, E.; Colette, A.; Monks, P. S.

    2012-12-01

    Tropospheric ozone is a secondary air pollutant of concern for its adverse affects on human health and agricultural crops, as well as its climate impact. Formed primarily from photochemical reactions involving nitrogen oxides (NOx), non-methane volatile organic compounds (NMVOCs), and carbon monoxide (CO), emission control measures have targeted significant emission sources of these compounds for reduction. While reductions of these ozone precursors are generally observed across Europe over the past one to two decades, trends in ground-level ozone have not followed the same trajectory. Here trends are extracted from up to 15 years of data from over 600 urban, suburban, and rural sites across Europe for ozone and nitrogen dioxide. While long-term records of NMVOCs are sparse, data from London sites are included and allow for an evaluation of how the atmospheric reactivity regime has changed over the past decade. Model results based on and used in conjunction with the observed trends are used to yield insight into the reasons for the lack of decrease in surface ozone. Trends in observed data are also compared to country's emission inventory data.

  1. Hearts of some Antarctic fishes lack mitochondrial creatine kinase.

    PubMed

    O'Brien, K M; Mueller, I A; Orczewska, J I; Dullen, K R; Ortego, M

    2014-12-01

    Creatine kinase (CK; EC 2.7.3.2) functions as a spatial and temporal energy buffer, dampening fluctuations in ATP levels as ATP supply and demand change. There are four CK isoforms in mammals, two cytosolic isoforms (muscle [M-CK] and brain [B-CK]), and two mitochondrial isoforms (ubiquitous [uMtCK] and sarcomeric [sMtCK]). Mammalian oxidative muscle couples expression of sMtCK with M-CK, creating an energy shuttle between mitochondria and myofibrils. We hypothesized that the expression pattern and activity of CK would differ between hearts of red- and white-blooded Antarctic notothenioid fishes due to their striking differences in cardiac ultrastructure. Hearts of white-blooded icefishes (family Channichthyidae) have significantly higher mitochondrial densities compared to red-blooded species, decreasing the diffusion distance for ATP between mitochondria and myofibrils and potentially minimizing the need for CK. The distribution of CK isoforms was evaluated using western blotting and maximal activity of CK was measured in mitochondrial and cytosolic fractions and tissue homogenates of heart ventricles of red- and white-blooded notothenioids. Transcript abundance of sMtCK and M-CK was also quantified. Overall, CK activity is similar between hearts of red- and white-blooded notothenioids but hearts of icefishes lack MtCK and have higher activities of M-CK in the cytosol compared to red-blooded fishes. The absence of MtCK may compromise cardiac function under stressful conditions when ATP supply becomes limiting. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Respondent driven sampling of wheelchair users: A lack of traction?

    PubMed

    Bourke, John A; Schluter, Philip J; Hay-Smith, E Jean C; Snell, Deborah L

    2016-01-01

    Internationally, wheelchair users are an emerging demographic phenomenon, due to their increased prevalence and rapidly increasing life-span. While having significant healthcare implications, basic robust epidemiological information about wheelchair users is often lacking due, in part, to this population's 'hidden' nature. Increasingly popular in epidemiological research, Respondent Driven Sampling (RDS) provides a mechanism for generating unbiased population-based estimates for hard-to-reach populations, overcoming biases inherent within other sampling methods. This paper reports the first published study to employ RDS amongst wheelchair users. Between October 2015 and January 2016, a short, successfully piloted, internet-based national survey was initiated. Twenty seeds from diverse organisations were invited to complete the survey then circulate it to peers within their networks following a well-defined protocol. A predetermined reminder protocol was triggered when seeds or their peers failed to respond. All participants were entered into a draw for an iPad. Overall, 19 people participated (nine women); 12 initial seeds, followed by seven second-wave participants arising from four seeds . Completion time for the survey ranged between 7 and 36 minutes. Despite repeated reminders, no further people were recruited. While New Zealand wheelchair user numbers are unknown, an estimated 14% of people have physical impairments that limited mobility. The 19 respondents generated from adopting the RDS methodology here thus represents a negligible fraction of wheelchair users in New Zealand, and an insufficient number to ensure equilibrium required for unbiased analyses. While successful in other hard-to-reach populations, applying RDS methodology to wheelchair users requires further consideration. Formative research exploring areas of network characteristics, acceptability of RDS, appropriate incentive options, and seed selection amongst wheelchair users is needed.

  3. Cardiorespiratory Anomalies in Mice Lacking CB1 Cannabinoid Receptors

    PubMed Central

    Bastianini, Stefano; Cohen, Gary; Lo Martire, Viviana; Mazza, Roberta; Pagotto, Uberto; Quarta, Carmelo; Zoccoli, Giovanna

    2014-01-01

    Cannabinoid type 1 (CB1) receptors are expressed in the nervous and cardiovascular systems. In mice, CB1 receptor deficiency protects from metabolic consequences of a high-fat diet (HFD), increases sympathetic activity to brown fat, and entails sleep anomalies. We investigated whether sleep-wake and diet-dependent cardiorespiratory control is altered in mice lacking CB1 receptors. CB1 receptor knock-out (KO) and intact wild-type (WT) mice were fed standard diet or a HFD for 3 months, and implanted with a telemetric arterial pressure transducer and electrodes for sleep scoring. Sleep state was assessed together with arterial pressure and heart rate (home cage), or breathing (whole-body plethysmograph). Increases in arterial pressure and heart rate on passing from the light (rest) to the dark (activity) period in the KO were significantly enhanced compared with the WT. These increases were unaffected by cardiac (β1) or vascular (α1) adrenergic blockade. The breathing rhythm of the KO during sleep was also more irregular than that of the WT. A HFD increased heart rate, impaired cardiac vagal modulation, and blunted the central autonomic cardiac control during sleep. A HFD also decreased cardiac baroreflex sensitivity in the KO but not in the WT. In conclusion, we performed the first systematic study of cardiovascular function in CB1 receptor deficient mice during spontaneous wake-sleep behavior, and demonstrated that CB1 receptor KO alters cardiorespiratory control particularly in the presence of a HFD. The CB1 receptor signaling may thus play a role in physiological cardiorespiratory regulation and protect from some adverse cardiovascular consequences of a HFD. PMID:24950219

  4. Multiple Sleep Alterations in Mice Lacking Cannabinoid Type 1 Receptors

    PubMed Central

    Bastianini, Stefano; Lo Martire, Viviana; Mazza, Roberta; Pagotto, Uberto; Quarta, Carmelo; Zoccoli, Giovanna

    2014-01-01

    Cannabinoid type 1 (CB1) receptors are highly expressed in the brain and play a role in behavior control. Endogenous cannabinoid signaling is modulated by high-fat diet (HFD). We investigated the consequences of congenital lack of CB1 receptors on sleep in mice fed standard diet (SD) and HFD. CB1 cannabinoid receptor knock-out (KO) and wild-type (WT) mice were fed SD or HFD for 4 months (n = 9–10 per group). Mice were instrumented with electroencephalographic (EEG) and electromyographic electrodes. Recordings were performed during baseline (48 hours), sleep deprivation (gentle handling, 6 hours), sleep recovery (18 hours), and after cage switch (insomnia model paradigm, 6 hours). We found multiple significant effects of genotype on sleep. In particular, KO spent more time awake and less time in non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) than WT during the dark (active) period but not during the light (rest) period, enhancing the day-night variation of wake-sleep amounts. KO had slower EEG theta rhythm during REMS. REMS homeostasis after sleep deprivation was less effective in KO than in WT. Finally, KO habituated more rapidly to the arousing effect of the cage-switch test than WT. We did not find any significant effects of diet or of diet x genotype interaction on sleep. The occurrence of multiple sleep alterations in KO indicates important roles of CB1 cannabinoid receptors in limiting arousal during the active period of the day, in sleep regulation, and in sleep EEG in mice. PMID:24586776

  5. Increased Bone Mass in Mice Lacking the Adipokine Apelin

    PubMed Central

    Wattanachanya, Lalita; Lu, Wei-Dar; Kundu, Ramendra K.; Wang, Liping; Abbott, Marcia J.; O'Carroll, Dylan; Quertermous, Thomas

    2013-01-01

    Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo. PMID:23584856

  6. Natural history of amblyopia untreated owing to lack of compliance

    PubMed Central

    Simons, K.; Preslan, M.

    1999-01-01

    AIMS—A prospective study of the efficacy of amblyopia treatment in preschool children has recently been called for, requiring an untreated control group. The present study assessed data from patients with amblyopia untreated owing to lack of compliance, or with amblyopia risk factors, to determine outcome.
METHODS—Longitudinal data were obtained from 18 4-6 year old patients who had initially been screened for amblyopia, strabismus, and/or bilateral refractive error, failed to comply with prescribed treatment, and in whom amblyopia was detected at a rescreening approximately a year later. The data from three previous studies comparing outcome of patients compliant and non-compliant with amblyopia treatment were also reanalysed.
RESULTS—One child of the 18, who wore glasses sporadically, showed some improvement in visual acuity in the amblyopic eye. Otherwise, no child showed an improvement, and seven of the 17 (41%) for whom visual acuities were available at both screenings showed a deterioration of visual acuity in the amblyopic eye, including three who apparently developed amblyopia for the first time. A child with an ametropic risk factor for amblyopia whose visual acuity was not obtained at the first screening and who was largely non-compliant presented with amblyopia at the second screening. The reanalysed data from the three previous studies demonstrated a significantly poorer visual acuity outcome in the amblyopic eye in the non-compliant patient groups than in the compliant groups in each study.
CONCLUSION—Preschool children with amblyopia or its risk factors are at risk of having the current amblyopia deteriorate, or of developing amblyopia, if not treated. These results raise questions about the ethical acceptability of a prospective study of amblyopia treatment at these ages.

 PMID:10216059

  7. Synapse formation during embryogenesis on ganglion cells lacking a periphery

    PubMed Central

    Landmesser, Lynn; Pilar, G.

    1974-01-01

    1. The development of transmission was studied in chick ciliary ganglia that had been deprived of their periphery during early embryonic development. 2. Peripherally deprived neurones in the ganglion differentiate in normal numbers and send functional axons into the post-ganglionic nerve. 3. Ganglion cells lacking a periphery follow the normal developmental sequence sending out transient dendrites at the time ganglion cell synapses are formed, and later retracting them when calyces appear. 4. Synapses, which appear functionally and ultrastructurally normal, form on all ganglion cells at the normal time and transmission is normal until Stage 34. Therefore information from the periphery is not required for ganglion cell synapse formation per se. 5. From Stages 35 to 38 most cells die, so that only 8% of the original number of cells remain in the operated ganglion. Transmission fails in many cells during this same time, but precedes cell loss by only a short time, so that deafferentation probably does not contribute substantially to cell death. 6. Both ciliary and choroid cells achieve full cytologic differentiation and are distinct from each other, indicating that the periphery is not required for the elaboration of the distinctive characteristics of these cells. Presynaptic fibres also differentiate into typical bouton as well as calyciform endings. Therefore, the type of preganglionic ending does not depend on ganglion cells establishing proper peripheral contacts. 7. It has not been possible to ascertain whether ganglion cell specificity is affected by the periphery. 8. Peripheral removal affects ganglion cell migration, so that two ganglia are formed. Approximately half of the cells migrate into the remnant optic cup forming a second misplaced ganglion. Ciliary and choroid cells occur in both ganglia and these cells go through the typical sequence of events described above. ImagesPlate 1Plate 2Plate 3 PMID:4373567

  8. Respondent driven sampling of wheelchair users: A lack of traction?

    PubMed Central

    Bourke, John A.; Schluter, Philip J.; Hay-Smith, E. Jean C.; Snell, Deborah L.

    2016-01-01

    Background: Internationally, wheelchair users are an emerging demographic phenomenon, due to their increased prevalence and rapidly increasing life-span. While having significant healthcare implications, basic robust epidemiological information about wheelchair users is often lacking due, in part, to this population’s ‘hidden’ nature. Increasingly popular in epidemiological research, Respondent Driven Sampling (RDS) provides a mechanism for generating unbiased population-based estimates for hard-to-reach populations, overcoming biases inherent within other sampling methods. This paper reports the first published study to employ RDS amongst wheelchair users. Methods: Between October 2015 and January 2016, a short, successfully piloted, internet-based national survey was initiated. Twenty seeds from diverse organisations were invited to complete the survey then circulate it to peers within their networks following a well-defined protocol. A predetermined reminder protocol was triggered when seeds or their peers failed to respond. All participants were entered into a draw for an iPad. Results: Overall, 19 people participated (nine women); 12 initial seeds, followed by seven second-wave participants arising from four seeds . Completion time for the survey ranged between 7 and 36 minutes. Despite repeated reminders, no further people were recruited. Discussion: While New Zealand wheelchair user numbers are unknown, an estimated 14% of people have physical impairments that limited mobility. The 19 respondents generated from adopting the RDS methodology here thus represents a negligible fraction of wheelchair users in New Zealand, and an insufficient number to ensure equilibrium required for unbiased analyses. While successful in other hard-to-reach populations, applying RDS methodology to wheelchair users requires further consideration. Formative research exploring areas of network characteristics, acceptability of RDS, appropriate incentive options, and seed

  9. Mice Lacking Hbp1 Function Are Viable and Fertile

    PubMed Central

    Wilhelm, Dagmar; Jans, David A.; Bowles, Josephine; Koopman, Peter

    2017-01-01

    Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression. PMID:28107452

  10. Lack of familial aggregation of Parkinson disease and Alzheimer disease.

    PubMed

    Levy, Gilberto; Louis, Elan D; Mejia-Santana, Helen; Côté, Lucien; Andrews, Howard; Harris, Juliette; Waters, Cheryl; Ford, Blair; Frucht, Steven; Fahn, Stanley; Ottman, Ruth; Marder, Karen

    2004-07-01

    To investigate the risk of Alzheimer disease (AD) in first-degree relatives of patients with Parkinson disease (PD) compared with first-degree relatives of controls. Case-control study, family history method, and reconstructed cohort approach. Probands with PD without dementia and control probands, matched by age strata, sex, and ethnicity, were examined in person and enrolled without knowledge of family history of PD and other neurological disorders. Disease status in first-degree relatives of probands with PD and control probands was ascertained through a structured family history interview administered to the proband and a second informant (self-report or another informant). Cox proportional hazards models with double-censoring techniques for missing information on age of onset of AD were used to analyze the risk of AD in first-degree relatives of patients with PD compared with first-degree relatives of controls. Four hundred eighty-seven probands with PD and 409 control probands provided family history information on 4819 first-degree relatives older than 30 years (2534 relatives of probands with PD and 2285 relatives of control probands). One hundred thirteen first-degree relatives (2.3%; 61 relatives [2.4%] of patients with PD and 52 relatives [2.3%] of controls) were diagnosed with AD. The risk of AD was not increased in relatives of patients with PD compared with relatives of controls (hazard ratio, 1.1; 95% confidence interval, 0.7-1.6; P =.65). Similarly, no significantly increased risk of AD was observed when comparing relatives of patients with early-onset (< or =50 years) and late-onset (>50 years) PD with relatives of controls. The lack of familial aggregation of PD and AD does not support the hypothesis of major shared genetic contributions to the etiology of the 2 most common neurodegenerative disorders.

  11. Multiple sleep alterations in mice lacking cannabinoid type 1 receptors.

    PubMed

    Silvani, Alessandro; Berteotti, Chiara; Bastianini, Stefano; Lo Martire, Viviana; Mazza, Roberta; Pagotto, Uberto; Quarta, Carmelo; Zoccoli, Giovanna

    2014-01-01

    Cannabinoid type 1 (CB1) receptors are highly expressed in the brain and play a role in behavior control. Endogenous cannabinoid signaling is modulated by high-fat diet (HFD). We investigated the consequences of congenital lack of CB1 receptors on sleep in mice fed standard diet (SD) and HFD. CB1 cannabinoid receptor knock-out (KO) and wild-type (WT) mice were fed SD or HFD for 4 months (n = 9-10 per group). Mice were instrumented with electroencephalographic (EEG) and electromyographic electrodes. Recordings were performed during baseline (48 hours), sleep deprivation (gentle handling, 6 hours), sleep recovery (18 hours), and after cage switch (insomnia model paradigm, 6 hours). We found multiple significant effects of genotype on sleep. In particular, KO spent more time awake and less time in non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) than WT during the dark (active) period but not during the light (rest) period, enhancing the day-night variation of wake-sleep amounts. KO had slower EEG theta rhythm during REMS. REMS homeostasis after sleep deprivation was less effective in KO than in WT. Finally, KO habituated more rapidly to the arousing effect of the cage-switch test than WT. We did not find any significant effects of diet or of diet x genotype interaction on sleep. The occurrence of multiple sleep alterations in KO indicates important roles of CB1 cannabinoid receptors in limiting arousal during the active period of the day, in sleep regulation, and in sleep EEG in mice.

  12. Lack of conventional ATPase properties in CFTR chloride channel gating.

    PubMed

    Schultz, B D; Bridges, R J; Frizzell, R A

    1996-05-01

    CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for

  13. Lack of association between postactivation potentiation and subsequent jump performance.

    PubMed

    Pearson, Stephen John; Hussain, Syed Robiul

    2014-01-01

    Postactivation potentiation (PAP) is a strategy that has been used to acutely enhance the performance of explosive activities. Although, isometric maximal voluntary contractions (MVCs) have previously been shown to enhance subsequent explosive performance, no information currently exists regarding (1) the optimal variables (intensity/volume) of a MVC that best elicits a PAP response, and (2) the utilisation of evoked isometric twitch contractions in combination with performance measures to directly ascertain the presence of PAP following a MVC, and its relationship to performance. Thus, the purpose of this study was to (1) investigate the influence of isometric contraction duration on the PAP response, and (2) to determine the relationship between PAP, indicated as potentiation of muscle twitch force and subsequent jump performance following different-duration MVCs. Eight males (age: 21 ± 0.99) were assessed using performance measures [countermovement jumps] and evoked twitch contractions, before and 4 minutes after three different conditioning contractions (CCs), (1) a 3-second MVC (MVC3), (2) a 5-second MVC (MVC5) and (3) a 7-second MVC (MVC7). Following all CCs, peak twitch torque of the knee extensor muscles was found to increase (MVC3, + 3.9%; MVC5, + 9.6%; MVC7, + 5.2%), although not significantly (P > 0.05). No significant increases in jump height, jump power, rate of force development or takeoff velocity were observed following any of the CCs (P > 0.05). There was also a lack of association between the changes in PAP (twitch torque) and jump height following all CCs (MVC3, r = 0.25; MVC5, r = 0.28; MVC7, r = -0.47). These data indicate that PAP as assessed via twitch contractions is not associated with performance measures subsequent to single-set isometric CCs of varying durations.

  14. Lack of drug preparations for use in children in Brazil.

    PubMed

    Costa, Patrícia Quirino da; Rey, Luis C; Coelho, Helena Lutéscia L

    2009-01-01

    To identify drugs which are not suited for pediatric use in Brazil. A descriptive study involving the development of a national list of unlicensed and off-label medications for pediatric use (problem drugs in pediatrics, PDP) through a literature review, a comparison among sources of the Brazilian pharmaceutical industry, and a survey with pediatricians. Drugs coded at the Anatomic Therapeutic Chemical (ATC) Classification System were analyzed regarding licensing status in Brazil and recommendations/indications in pediatrics, based on the following reference sources: the list of licensed drugs of the Brazilian National Health Surveillance Agency (2005), the Brazilian Dictionary of Pharmaceutical Specialties (2005-2007) and the website www.bulas.med.br. Our literature search returned 126 PDP, but 24 drugs were excluded due to absence of national reference. To compose the final list, 24 other drugs referred by pediatricians were added. Of the 126 PDP, 23 drugs were not licensed in the country for use in children; and of the 103 licensed drugs, 24 presented age-related restrictions for pediatric use. The pharmaceutical list included 42 therapeutic groups and 68 subgroups. The groups containing larger numbers of PDP were: antibiotics for systemic use (15), antiepileptics (8), antiasthmatics (7), and analgesics (7). The most frequent problems were: inappropriate dosage (35), unlicensed for pediatric use (28), age-related restrictions (23). The lack of pediatric drug formulations in Brazil shows a profile similar to that observed in other countries, which involves a wide range of clinically important products. This study brings a contribution to the evaluation of the needs and priorities that support the development of suitable medicines for the pediatric patient.

  15. Thermal ablation in colorectal liver metastases: Lack of evidence or lack of capability to prove the evidence?

    PubMed Central

    Sartori, Sergio; Tombesi, Paola; Di Vece, Francesca

    2016-01-01

    Many studies suggest that combined multimodality treatments including ablative therapies may achieve better outcomes than systemic chemotherapy alone in patients with colorectal liver metastases. Nevertheless, ablative therapies are not yet considered as effective options because their efficacy has never been proved by randomized controlled trials (RCT). However, there are in literature no trials that failed in demonstrating the effectiveness of ablative treatments: what are lacking, are the trials. All the attempts to organize phase III studies on this topic failed as a result of non accrual. Just one prospective RCT comparing radiofrequency ablation combined with systemic chemotherapy vs chemotherapy alone has been published. It was designed as a phase III study, but it was closed early because of slow accrual, and was downscaled to phase II study, with the consequent limits in drawing definite conclusions on the benefit of combined treatment. However, the combination treatment met the primary end point of the study and obtained a significantly higher 3-year progression-free survival than systemic chemotherapy alone. It is very unlikely that ultimate efficacy of ablation treatments will ever be tested again, and the best available evidence points toward a benefit for the combination strategy using ablative treatments and chemotherapy. PMID:27053843

  16. Thermal ablation in colorectal liver metastases: Lack of evidence or lack of capability to prove the evidence?

    PubMed

    Sartori, Sergio; Tombesi, Paola; Di Vece, Francesca

    2016-04-07

    Many studies suggest that combined multimodality treatments including ablative therapies may achieve better outcomes than systemic chemotherapy alone in patients with colorectal liver metastases. Nevertheless, ablative therapies are not yet considered as effective options because their efficacy has never been proved by randomized controlled trials (RCT). However, there are in literature no trials that failed in demonstrating the effectiveness of ablative treatments: what are lacking, are the trials. All the attempts to organize phase III studies on this topic failed as a result of non accrual. Just one prospective RCT comparing radiofrequency ablation combined with systemic chemotherapy vs chemotherapy alone has been published. It was designed as a phase III study, but it was closed early because of slow accrual, and was downscaled to phase II study, with the consequent limits in drawing definite conclusions on the benefit of combined treatment. However, the combination treatment met the primary end point of the study and obtained a significantly higher 3-year progression-free survival than systemic chemotherapy alone. It is very unlikely that ultimate efficacy of ablation treatments will ever be tested again, and the best available evidence points toward a benefit for the combination strategy using ablative treatments and chemotherapy.

  17. Chronic exposure to ozone causes tolerance to airway hyperresponsiveness in guinea pigs: lack of SOD role.

    PubMed

    Vargas, M H; Romero, L; Sommer, B; Zamudio, P; Gustin, P; Montaño, L M

    1998-05-01

    Tolerance to respiratory effects of O3 has been demonstrated for anatomic and functional changes, but information about tolerance to O3-induced airway hyperresponsiveness (AHR) is scarce. In guinea pigs exposed to air or O3 (0.3 parts/million, 4 h/day, for 1, 3, 6, 12, 24, or 48 days, studied 16-18 h later), pulmonary insufflation pressure changes induced by intravenous substance P (SP, 0.032-3.2 micro ug/kg) were measured, then the animals were subjected to bronchoalveolar lavage (BAL). Bronchial rings with or without phosphoramidon were also evaluated 3 h after air or a single O3 exposure. O3 caused in vivo AHR (increased sensitivity) to SP after 1, 3, 6, 12, and 24 days of exposure compared with control. However, after 48 days of exposure, O3 no longer caused AHR. Total cell, macrophage, neutrophil, and eosinophil counts in BAL were increased in most O3-exposed groups. When data from all animals were pooled, we found a highly significant correlation between degree of airway responsiveness and total cells (r = 0.55), macrophages (r = 0.54), neutrophils (r = 0.47), and eosinophils (r = 0.53), suggesting that airway inflammation is involved in development of AHR to SP. Superoxide dismutase (SOD) levels in BAL fluids were increased (P < 0.05) after 1, 3, 6, and 12 days of O3 exposure and returned to basal levels after 24 and 48 days of exposure. O3 failed to induce hyperresponsiveness to SP in bronchial rings, and phosphoramidon increased responses to SP in air- and O3-exposed groups, suggesting that neutral endopeptidase inactivation was not involved in O3-induced AHR to SP in vivo. We conclude that chronic exposure to 0. 3 ppm O3, a concentration found in highly polluted cities, resulted in tolerance to AHR to SP in guinea pigs by an SOD-independent mechanism.

  18. Do some AGN lack X-ray emission?

    NASA Astrophysics Data System (ADS)

    Simmonds, C.; Bauer, F. E.; Thuan, T. X.; Izotov, Y. I.; Stern, D.; Harrison, F. A.

    2016-12-01

    Context. Intermediate-mass black holes (IMBHs) are thought to be the seeds of early supermassive black holes (SMBHs). While ≳100 IMBH and small SMBH candidates have been identified in recent years, few have been robustly confirmed to date, leaving their number density in considerable doubt. Placing firmer constraints both on the methods used to identify and confirm IMBHs/SMBHs, as well as characterizing the range of host environments that IMBHs/SMBHs likely inhabit is therefore of considerable interest and importance. Additionally, finding significant numbers of IMBHs in metal-poor systems would be particularly intriguing, since such systems may represent local analogs of primordial galaxies, and therefore could provide clues of early accretion processes. Aims: Here we study in detail several candidate active galactic nuclei (AGN) found in metal-poor hosts. Methods: We utilize new X-ray and optical observations to characterize these metal-poor AGN candidates and compare them against known AGN luminosity relations and well-characterized IMBH/SMBH samples. Results: Despite having clear broad optical emission lines that are long-lived (≳10-13 yr), these candidate AGN appear to lack associated strong X-ray and hard UV emission, lying at least 1-2 dex off the known AGN correlations. If they are IMBHs/SMBHs, our constraints imply that they either are not actively accreting, their accretion disks are fully obscured along our line-of-sight, or their accretion disks are not producing characteristic high energy emission. Alternatively, if they are not AGN, then their luminous broad emission lines imply production by extreme stellar processes. The latter would have profound implications on the applicability of broad lines for mass estimates of massive black holes. The reduced spectra (FITS files) are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/596/A64

  19. Lack of Integrative Control of Body Temperature after Capsaicin Administration

    PubMed Central

    Lee, Tai Hee; Lee, Jae Woo; Osaka, Toshimasa; Kobayashi, Akiko; Namba, Yoshio; Inoue, Shuji; Kimura, Shuichi

    2000-01-01

    Background Body temperature is usually regulated by opposing controls of heat production and heat loss. However, systemic administration of capsaicin, the pungent ingredient of hot peppers, facilitated heat production and heat loss simultaneously in rats. We recently found that the capsaicin-induced heat loss and heat production occur simultaneously and that the biphasic change in body temperature is a sum of transient heat loss and long-lasting heat production. Moreover, suppression of the heat loss response did not affect capsaicin-induced heat production and suppression of heat production did not affect capsaicin-induced heat loss. These observations suggest the independent peripheral mechanisms of capsaicin-induced thermal responses. Thus, the capsaicin-induced thermal responses apparently lack an integrated control. Methods Male Wistar rats were maintained at an ambient temperature of 24 ± 1°C on a 12 h on-off lighting schedule at least for two weeks before the experiments. They were anesthetized with urethane (1.5 g/kg, i.p.) and placed on a heating pad, which was kept between 29 and 30 °C. Skin temperature(Ts) was measured with a small thermistor, which was taped to the dorsal surface of the rat’s tail, to assess vasoactive changes indirectly. Colonic temperature(Tc) was measured with another thermistor inserted about 60 mm into the anus. O2 consumption was measured by the open-circuit method, and values were corrected for metabolic body size (kg0.75). Capsaicin (Sigma) was dissolved in a solution comprising 80% saline, 10% Tween 80, and 10% ethanol, and injected subcutaneously at a dose of 5 mg/kg. Each rat received a single injection of capsaicin because repeated administration of capsaicin renders an animal insensitive to the subsequent administration of capsaicin. Laminectomy was performed at the level of the first and second cervical vertebrae to expose the cervical spinal cord for sectioning. The brain was transected at 4-mm rostral from the

  20. Enzymatic methylation at specific altered aspartyl and asparaginyl residues in glucagon

    SciTech Connect

    Ota, I.M.; Ding, L.; Clarke, S.

    1986-05-01

    Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoasparty 1 and/or D-aspartyl residues. In order to identify the origin of these unusual residues, they studied the methylation of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. They found that glucagon could be methylated with the erythrocyte enzyme and S-adenosyl(methyl-/sup 3/H)methionine to a maximum extent of 0.004 mol methyl groups/mol glucagon. After digestion with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg c, the labelled fragments were separated by HPLC and identified. Additionally, peptides produced by protease digestions were assayed directly for methyl-acceptor activity. They found that the major site of methylation, accounting for 60% of the total, was at Asp-9. Further analysis indicated that this site probably represents an L-isoaspartyl residue. A second site of methylation, representing 23% of the total, was detected at Asn-28. Neither Asp-15 nor Asp-21 could be identified as a methyl-acceptor site. Base treatment of glucagon (0.1 M NH/sub 4/OH, 3 h, 37/sup 0/C) increased methylation at the Asn-28 site by 4 to 8 fold while methylation at the Asp-9 site remained unchanged. These studies suggest that base treatment enhances methylation at asparagine residues but not at aspartyl residues.

  1. Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.

    PubMed

    Robinson, Mark W; Tort, Jose F; Lowther, Jonathan; Donnelly, Sheila M; Wong, Emily; Xu, Weibo; Stack, Colin M; Padula, Matthew; Herbert, Ben; Dalton, John P

    2008-06-01

    Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser[downward arrow]His motif for

  2. NIH–Lacks Family Partnership Gives Henrietta Lacks’ Descendants a Seat at the Table | Poster

    Cancer.gov

    The NIH Bethesda campus recently welcomed several special guests for a presentation titled Given a Voice: Update on the NIH–Lacks Family Partnership. Henrietta Lacks’ grandchildren Jeri Lacks-Whye (who is also the Lacks family spokesperson) and David Lacks, Jr. came to speak with NIH Director Francis S. Collins, Ph.D., M.D. about the importance of protecting patients and their families.

  3. Reduced food access due to a lack of money, inability to lift and lack of access to a car for food shopping: a multilevel study in Melbourne, Victoria.

    PubMed

    Burns, Cate; Bentley, Rebecca; Thornton, Lukar; Kavanagh, Anne

    2011-06-01

    To describe associations between demographic and individual and area-level socio-economic variables and restricted household food access due to lack of money, inability to lift groceries and lack of access to a car to do food shopping. Multilevel study of three measures of restricted food access, i.e. running out of money to buy food, inability to lift groceries and lack of access to a car for food shopping. Multilevel logistic regression was conducted to examine the risk of each of these outcomes according to demographic and socio-economic variables. Random selection of households from fifty small areas in Melbourne, Australia, in 2003. The main food shoppers in each household (n 2564). A lack of money was significantly more likely among the young and in households with single adults. Difficulty lifting was more likely among the elderly and those born overseas. The youngest and highest age groups both reported reduced car access, as did those born overseas and single-adult households. All three factors were most likely among those with a lower individual or household socio-economic position. Increased levels of area disadvantage were independently associated with difficulty lifting and reduced car access. In Melbourne, households with lower individual socio-economic position and area disadvantage have restricted access to food because of a lack of money and/or having physical limitations due difficulty lifting or lack of access to a car for food shopping. Further research is required to explore the relationship between physical restrictions and food access.

  4. 42 CFR 476.90 - Lack of cooperation by a health care facility or practitioner.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Lack of cooperation by a health care facility or...) Qio Review Functions § 476.90 Lack of cooperation by a health care facility or practitioner. (a) If a health care facility or practitioner refuses to allow a QIO to enter and perform the duties and functions...

  5. 22 CFR 225.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Applications and proposals lacking definite plans for involvement of human subjects. 225.118 Section 225.118 Foreign Relations AGENCY FOR INTERNATIONAL DEVELOPMENT PROTECTION OF HUMAN SUBJECTS § 225.118 Applications and proposals lacking definite...

  6. The Lack of Motivation to Pursue Postsecondary Education among Hmong Students: A Grounded Theory Study

    ERIC Educational Resources Information Center

    Lee, Xang

    2015-01-01

    In rural areas, a lack of motivation to pursue a postsecondary degree continues to affect Hmong students at the postsecondary education level. The purpose of this qualitative grounded theory research was to create a model based on the exploration of the lack of motivation to pursue postsecondary education among Hmong high school students.…

  7. Why does the immune system of Atlantic cod lack MHC II?

    PubMed

    Star, Bastiaan; Jentoft, Sissel

    2012-08-01

    MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).

  8. Toward a Deeper Understanding of Student Interest or Lack of Interest in Science

    ERIC Educational Resources Information Center

    Yang, Li-Hsuan

    2010-01-01

    This study examined the nature of college students' interest or lack of interest in science and the factors to which they attributed their interest or lack of interest. Twenty-four college students were interviewed to gain an understanding of their ideas and experiences of science; their overall interest in science; their interest levels in four…

  9. Teacher Resistance to Frequent Rewards and Praise: Lack of Skill or a Wise Decision?

    ERIC Educational Resources Information Center

    Bear, George G.

    2013-01-01

    Resistance and the lack of fidelity or integrity in the use of rewards and praise are commonly cited in the behavioral consultation literature, particularly when teachers are asked to manage student behavior using frequent rewards and praise in a systematic manner. There are multiple potential reasons for resistance and lack of implementation…

  10. 38 CFR 16.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... proposals lacking definite plans for involvement of human subjects. 16.118 Section 16.118 Pensions, Bonuses... and proposals lacking definite plans for involvement of human subjects. Certain types of applications... grants when selection of specific projects is the institution's responsibility; research training...

  11. 38 CFR 16.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... proposals lacking definite plans for involvement of human subjects. 16.118 Section 16.118 Pensions, Bonuses... and proposals lacking definite plans for involvement of human subjects. Certain types of applications... grants when selection of specific projects is the institution's responsibility; research training...

  12. 38 CFR 16.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... proposals lacking definite plans for involvement of human subjects. 16.118 Section 16.118 Pensions, Bonuses... and proposals lacking definite plans for involvement of human subjects. Certain types of applications... grants when selection of specific projects is the institution's responsibility; research training...

  13. 38 CFR 16.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... proposals lacking definite plans for involvement of human subjects. 16.118 Section 16.118 Pensions, Bonuses... and proposals lacking definite plans for involvement of human subjects. Certain types of applications... grants when selection of specific projects is the institution's responsibility; research training...

  14. Teacher Resistance to Frequent Rewards and Praise: Lack of Skill or a Wise Decision?

    ERIC Educational Resources Information Center

    Bear, George G.

    2013-01-01

    Resistance and the lack of fidelity or integrity in the use of rewards and praise are commonly cited in the behavioral consultation literature, particularly when teachers are asked to manage student behavior using frequent rewards and praise in a systematic manner. There are multiple potential reasons for resistance and lack of implementation…

  15. 49 CFR 11.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Applications and proposals lacking definite plans for involvement of human subjects. 11.118 Section 11.118 Transportation Office of the Secretary of Transportation PROTECTION OF HUMAN SUBJECTS § 11.118 Applications and proposals lacking definite plans...

  16. The Lack of Motivation to Pursue Postsecondary Education among Hmong Students: A Grounded Theory Study

    ERIC Educational Resources Information Center

    Lee, Xang

    2015-01-01

    In rural areas, a lack of motivation to pursue a postsecondary degree continues to affect Hmong students at the postsecondary education level. The purpose of this qualitative grounded theory research was to create a model based on the exploration of the lack of motivation to pursue postsecondary education among Hmong high school students.…

  17. 42 CFR 476.90 - Lack of cooperation by a health care facility or practitioner.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Lack of cooperation by a health care facility or...) Qio Review Functions § 476.90 Lack of cooperation by a health care facility or practitioner. (a) If a health care facility or practitioner refuses to allow a QIO to enter and perform the duties and functions...

  18. In vitro and in vivo pharmacological profile of PL-3994, a novel cyclic peptide (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)) natriuretic peptide receptor-A agonist that is resistant to neutral endopeptidase and acts as a bronchodilator.

    PubMed

    Edelson, Jeffrey D; Makhlina, Marie; Silvester, Kevin R; Vengurlekar, Shailesh S; Chen, Xiaomei; Zhang, Jie; Koziol-White, Cynthia J; Cooper, Philip R; Hallam, Trevor J; Hay, Douglas W P; Panettieri, Reynold A

    2013-04-01

    The pharmacological and airways relaxant profiles of PL-3994 (Hept-cyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)), a novel natriuretic peptide receptor-A (NPR-A) agonist, were evaluated. PL-3994, a full agonist, has high affinity for recombinant human (h), dog, or rat NPR-As (K(i)s of 1, 41, and 10 nm, respectively), and produced concentration-dependent cGMP generation in human, dog and rat NPR-As (respective EC(50)s of 2, 3 and 14 nm). PL-3994 has a K(i) of 7 nm for hNPR-C but was without effect on cGMP generation in hNPR-B. PL-3994 (1 μm) was without significant effect against 75 diverse molecular targets. PL-3994 or BNP, a natural NPR ligand, produced concentration-dependent relaxation of pre-contracted guinea-pig trachea (IC(50)s of 42.7 and 10.7 nm, respectively). PL-3994, and also BNP, (0.1 nm-100 μm) elicited a potent, concentration-dependent but small relaxation of pre-contracted human precision-cut lung slices (hPCLS). Intratracheal PL-3994 (1-1000 μg/kg) produced a dose-dependent inhibition of the bronchoconstrictor response evoked by aerosolized methacholine, but was without significant effect on cardiovascular parameters. PL-3994 was resistant to degradation by human neutral endopeptidase (hNEP) (92% remaining after 2 h), whereas the natural ligands, ANP and CNP, were rapidly metabolized (≤1% remaining after 2 h). PL-3994 is a potent, selective NPR agonist, resistant to NEP, with relaxant effects in guinea-pig and human airway smooth muscle systems. PL-3994 has the profile predictive of longer clinical bronchodilator activity than observed previously with ANP, and suggests its potential utility in the treatment of asthma, in addition to being a useful research tool to evaluate NPR biology.

  19. Lack of Research Amongst Undergraduate Medical Students in India: It's time to Act and Act Now.

    PubMed

    Garg, Rajesh; Goyal, Shobha; Singh, Kamaljit

    2017-05-15

    Participation in research is important in producing doctors with an understanding of evidence-based medicine. Though a mandatory part in post-graduate medical course, research has largely been invisible from the under graduation medical course in India. Very few research opportunities are available at under graduate level. The reason behind this is lack of encouragement, lack of basic infrastructure, facilities and structured mentorship programs, no extra incentives to researchers and the long journey to get academic acclaim. Another additional aspect is of lack of writing skills for biomedical publication. Additional incentives to students as well faculty members are required to foster the research environment in India.

  20. Report: Lack of Final Guidance on Vapor Intrusion Impedes Efforts to Address Indoor Air Risks

    EPA Pesticide Factsheets

    Report #10-P-0042, December 14, 2009. EPA’s efforts to protect human health at sites where vapor intrusion risks may occur have been impeded by the lack of final Agency guidance on vapor intrusion risks.

  1. Many Parts of the World Lack Soap for Hand-Washing

    MedlinePlus

    ... news/fullstory_167056.html Many Parts of the World Lack Soap for Hand-Washing Areas with lowest ... that occur annually in young children around the world. Never before has hand-washing been systematically measured ...

  2. Relation Between Lack of Forgiveness and Depression: The Moderating Effect of Self-Compassion.

    PubMed

    Chung, Myung-Sun

    2016-12-01

    Although an association between lack of forgiveness and poor mental health is known, prior studies have reported mixed findings of the relationship between lack of forgiveness and depressive symptoms. In an attempt to explain the strength differences between lack of forgiveness and depressive symptoms, this study examined the moderating effect of self-compassion. A total of 311 Korean teachers (89 men, 222 women; M age = 39.3 year, SD = 9.1) were asked to complete self-report questionnaires, including the Korean versions of the Trait Forgivingness Scale, the Self-Compassion Scale, and the Center for Epidemiologic Studies Depression Scale. Moderated multiple regression was used for analysis, and a buffering interaction of self-compassion was discovered. Specifically, self-compassion moderated the relationship between lack of forgiveness and depression; the relationship was stronger for those low on self-compassion. © The Author(s) 2016.

  3. Experimental research of fluorescence spectra of watercress stressed by lack or excess of watering

    NASA Astrophysics Data System (ADS)

    Bullo, O. A.; Fedotov, Yu. V.; Belov, M. L.; Gorodnichev, V. A.

    2015-11-01

    Experimental laboratory investigations of the laser-induced fluorescence spectra of watercress were conducted. The fluorescence spectra were excited by a YAG:Nd laser emitting at 532 nm. The laboratory setup was described and fluorescence spectra of watercress in stressed states caused by lack and excess of water were presented. It was established that the influence of stress caused by lack and excess of watering is manifested in changes of fluorescence spectra.

  4. Economic stress and lack of internal health locus of control: a life course approach.

    PubMed

    Lindström, Martin; Rosvall, Maria

    2014-02-01

    To investigate associations between economic stress in childhood and adulthood, and lack of internal health locus of control (HLC), testing the accumulation and critical period life course hypotheses. A cross-sectional public health (postal) survey was conducted in Skåne in 2008, based on a random sample with 28,198 participants in the age interval 18-80 years, with 55% participation. Logistic regressions analyzed associations between childhood and current economic stress, and lack of internal HLC. A 33.7% prevalence of men and 31.8% of women lack internal HLC, which was significantly associated with the covariates included. The accumulation hypothesis was partly supported because combined childhood and adulthood economic stress exposures were significantly associated with lack of internal HLC in a graded manner. The critical period hypothesis was not supported since the association between economic stress in childhood and lack of internal HLC was partly significant in the final model, and the association with adult (current) economic stress was also significant. The accumulation hypothesis was partly supported. The critical period hypothesis was not supported since both childhood and current economic stress experience were significantly associated with lack of internal HLC. Economic conditions in childhood as well as adulthood are plausibly of relevance for HLC.

  5. The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation.

    PubMed

    Cunningham, P R; Clark, D P

    1986-12-01

    Mutants of Escherichia coli resistant to chloroethanol or to chloroacetaldehyde were selected. Such mutants were found to lack the fermentative coenzyme A (CoA) linked acetaldehyde dehydrogenase activity. Most also lacked the associated fermentative enzyme alcohol dehydrogenase. Both types of mutants, those lacking acetaldehyde dehydrogenase alone or lacking both enzymes, mapped close to the regulatory adhC gene at 27 min on the E. coli genetic map. The previously described acd mutants which lack acetaldehyde dehydrogenase and which map at 63 min were shown to be pleiotropic, affecting respiration and growth on a variety of substrates. It therefore seems likely that the structural genes for both the acetaldehyde and alcohol dehydrogenases lie in the adhCE operon. This interpretation was confirmed by the isolation of temperature sensitive chloracetaldehyde-resistant mutants, some of which produced thermolabile acetaldehyde dehydrogenase and alcohol dehydrogenase and were also found to map at the adh locus. Reversion analysis indicated that mutants lacking one or both enzymes carried single mutations. The gene order in the adh region was determined by three point crosses to be trp-zch::Tn10-adh-galU-bglY-tyrT-chlC.

  6. Predictors and Consequences of Perceived Lack of Choice in Becoming an Informal Caregiver

    PubMed Central

    Schulz, Richard; Beach, Scott R.; Cook, Thomas B.; Martire, Lynn M.; Tomlinson, Jennifer M.; Monin, Joan K.

    2012-01-01

    Objectives Using data from a national sample of informal caregivers to older adults, we identify predictors of lack of choice and the consequences of lack of choice in taking on the caregiving role. Methods A national telephone survey with 1397 caregivers was carried out to assess whether respondents had a choice in taking on the caregiving role, their demographic characteristics, the nature and duration of their caregiving experience, and its impact on their physical and psychological well-being. We compare caregivers who felt they had no choice in taking on the caregiving role to those who did. Results Forty-four percent of caregivers reported a lack of choice in taking on the caregiving role. Highly educated, older caregivers caring for a younger care recipient with emotional or behavioral problems were most likely to report that they had no choice in taking on the caregiving role. Lack of choice is associated with higher levels of emotional stress, physical strain, and negative health impacts, after controlling for multiple confounds including level of care provided, relationship type, primary health condition of the care recipient, and demographic characteristics. Conclusion Lack of choice is an independent risk factor for the negative effects of caregiving, and clinicians should be vigilant to lack of choice as a marker of caregiver distress. PMID:22360296

  7. Intranasal Vaccination against Cutaneous Leishmaniasis with a Particulated Leishmanial Antigen or DNA Encoding LACK

    PubMed Central

    Pinto, Eduardo Fonseca; Pinheiro, Roberta Olmo; Rayol, Alice; Larraga, Vicente; Rossi-Bergmann, Bartira

    2004-01-01

    We have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis (LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 μg of LaAg and were challenged 1 week postvaccination with L. amazonensis developed delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-γ) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 μg/dose) had no effect. Interestingly, LACK DNA (30 μg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-γ. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA. PMID:15271911

  8. Lack of Energy: An Important and Distinct Component of HIV-Related Fatigue and Daytime Function

    PubMed Central

    Aouizerat, Bradley E.; Gay, Caryl L.; Lerdal, Anners; Portillo, Carmen J.; Lee, Kathryn A.

    2012-01-01

    Context Fatigue is a prevalent symptom among adults living with human immunodeficiency virus (HIV). There is increasing evidence that fatigue and energy are related, yet distinct constructs. Although HIV-related fatigue has been well studied, little is known about perceived energy and how it relates to fatigue, individual characteristics, and other symptoms. Objectives To describe the experience of perceived energy in adults with HIV and evaluate its relationship to demographic and clinical characteristics as well as symptoms of fatigue, sleep disturbance, anxiety, depression, and daytime function. Methods The design was descriptive, comparative, and correlational. The sample of 318 adults with HIV completed a demographic questionnaire, the Memorial Symptom Assessment Scale, and measures of fatigue, sleep disturbance, anxiety, depressive symptoms, and daytime function. Medical records were reviewed for disease and treatment data. Participants who reported a lack of energy were compared with those who did not on demographic, clinical, and symptom variables. Regression models of perceived energy and its interference with daytime function also were evaluated. Results Perceived lack of energy was highly prevalent (65%) and more strongly related to interference with daytime function than more general measures of fatigue severity, even when controlling for other characteristics and symptoms. Like other aspects of fatigue, lack of energy was associated with sleep disturbance, anxiety, and depressive symptoms. Lack of energy was more strongly related to morning fatigue than to evening fatigue. Conclusion Lack of energy interferes with daytime function and is not just the inverse of fatigue but a distinct perception that differs from fatigue. PMID:22917712

  9. Millions of mothers lack health insurance coverage in the United States. Most uninsured mothers lack access both to employer-based coverage and to publicly subsidized health insurance.

    PubMed

    Guyer, Jocelyn; Broaddus, Matthew; Dude, Annie

    2002-01-01

    Some 5.9 million American mothers caring for young or school-aged children lack health insurance. Although nearly nine in ten uninsured mothers are members of working families, most lack access to affordable coverage through their job or a spouse's job. Most are ineligible for publicly subsidized coverage unless their incomes are far below the poverty line. The millions of uninsured mothers are at high risk of going without needed preventive and primary care. If they become seriously ill, their families can face the prospect of a financial crisis. The nation has made significant progress in extending health care coverage to children in low-income families through Medicaid and the State Children's Health Insurance Program (SCHIP), but no comparable effort has been made to insure the mothers of these children. A few states have started to address the problem by transforming their SCHIPs into family-based programs that also cover low-income parents. Bipartisan legislation under consideration, known as FamilyCare, would encourage this trend by providing more federal funding to states that could be used to extend health insurance to the parents of children already covered by publicly funded programs.

  10. Analysis of the lack of scientific and technological talents of high-level women in China

    NASA Astrophysics Data System (ADS)

    Lin, Wang

    2017-08-01

    The growth and development of high-level female scientific and technological talents has become a global problem, facing severe challenges. The lack of high-level women in science and technology has become a global problem. How to recruit and help female scientists and technological talents grow raises awareness from the industry. To find out the main reasons for the lack of high-level female scientific and technological talent. This paper analyses the impact of gender discrimination on the lack of high-level female scientific and technological talents, the impact of disciplinary differences on female roles. The main reasons are: women’s natural disadvantage of mathematical thinking; female birth, the traditional culture on the role of women and the impact of values.

  11. A syndrome with lack of udder developments in a high-yielding Jersey herd.

    PubMed

    Simesen, M G; Basse, A; Christensen, K A; Christensen, T

    1979-09-01

    A syndrome with lack of udder development in a high-yielding jersey herd is described. The syndrome first appeared in the herd about 10 years ago. To try to elucidate this syndrome the endocrine pattern visualized by serum estrogen (Fig. 1) and progesterone (Fig. 2) levels was followed in 12 parturient cows. One case of typical lack of udder development appeared and was followed up by slaughter and pathological and histological examinations (Fig. 3 and 4). Pedigree investigations indicated that genetics could be involved in the syndrome. The endocrine pattern observed combined with the lack of udder development seems to indicate a complex disorder in the hormonal regulation which trigger the maternal preparation and initiation of parturition.

  12. Lack of anticipated support for care for community-dwelling older adults.

    PubMed

    Oliveira, Déborah Cristina de; Neri, Anita Liberalesso; D'Elboux, Maria José

    2016-06-01

    to identify the factors associated with lack of anticipated support for care for community-dwelling older adults. this study presents comparison and logistic regression analyses of data from 671 individuals who took part of the multicentric study entitled "Frailty in older Brazilians" - a quantitative, epidemiologic and transversal investigation carried out between 2008 and 2009. the subjective evaluation of anticipated support for care for community-dwelling older adults was a good indicator of risk for lack of anticipated support for care in women, older adults who live alone and those with poor self-rated health. it is necessary to reflect upon the formal support system currently available for older people in Brazil, considering that those who most frequently presented lack of anticipated support for care are an increasing population. The study also highlights the importance of using subjective methods for the evaluation of the adequacy of older adults' support network.

  13. Increased brain-derived neurotrophic factor (BDNF) protein concentrations in mice lacking brain serotonin.

    PubMed

    Kronenberg, Golo; Mosienko, Valentina; Gertz, Karen; Alenina, Natalia; Hellweg, Rainer; Klempin, Friederike

    2016-04-01

    The interplay between BDNF signaling and the serotonergic system remains incompletely understood. Using a highly sensitive enzyme-linked immunosorbent assay, we studied BDNF concentrations in hippocampus and cortex of two mouse models of altered serotonin signaling: tryptophan hydroxylase (Tph)2-deficient (Tph2 (-/-)) mice lacking brain serotonin and serotonin transporter (SERT)-deficient (SERT(-/-)) mice lacking serotonin re-uptake. Surprisingly, hippocampal BDNF was significantly elevated in Tph2 (-/-) mice, whereas no significant changes were observed in SERT(-/-) mice. Furthermore, BDNF levels were increased in the prefrontal cortex of Tph2 (-/-) but not of SERT(-/-) mice. Our results emphasize the interaction between serotonin signaling and BDNF. Complete lack of brain serotonin induces BDNF expression.

  14. Universality class of XY -like spin glasses lacking time-reversal symmetry

    SciTech Connect

    Gingras, M.J.P. )

    1991-10-01

    {ital XY}-like spin-glass models without time-reversal symmetry are considered, with attention focused on the gauge glass and the Dzyaloshinsky-Moriya {ital XY} spin glass. A mean-field approximation of the replicated Ginzburg-Landau free energy of these models is used to show that, due to the lack of time-reversal symmetry, these models have two soft (massless) modes at the transition. A consequence of this result is that all {ital XY}-like spin glasses lacking time-reversal symmetry belong to the same universality class, but which is a different one from that found for Ising and isotropic vector spin glasses.

  15. One case of rare gastric cancer combined with lack of factor XI.

    PubMed

    Hou, Xianming; Han, Xianlin; You, Lei; Chen, Ge

    2016-07-01

    The patient, a 50-year-old woman, was admitted to hospital diagnosed with gastric cancer. While performing preoperative examination, we found that this patient had a lack of factor XI. This is the first case diagnosed with gastric cancer combined with lack of emitted factor XI, at least in China. We used only fresh frozen plasma to correct the blood coagulation disorder to perform an operation. Firstly, we think this is a rare case that should be reported. Secondly, what we have done to help this patient may help other doctors to make decisions when they meet any similar cases.

  16. Desert Emergency of Lack of Water; How to Find and Collect Water.

    DTIC Science & Technology

    1984-03-01

    D-ie991 DESERT EMERGENCY OF LACK OF WATER; HOW TO FIND AND 1 COLLECT WATER(U) BEN-GURION UNIY’ OF THE NEGEV SEDE ROOER (ISRAEL) JACOB BLAUST. . Y...Desert Emergency - Lack of Water - How to Find and Collect Water. Plants and Human Survival In the Desert 00 The Principal Investigator and Contractor0...desert conditions. The alms of the second part of S this study were 1) to find out if there is, and If so, what is, the "" connection between the

  17. Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

    PubMed Central

    Biel, S W; Biel, A J

    1990-01-01

    A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway. Images FIG. 3 FIG. 5 PMID:2155198

  18. Best interests of adults who lack capacity part 2: key considerations.

    PubMed

    Griffith, Richard

    Last month's article discussed the key concepts underpinning the notion of best interests. In this article the author discusses the requirements for determining the best interests of an adult who lacks capacity under the provisions of the Mental Capacity Act 2005 and its code of practice (Department for Constitutional Affairs 2007).

  19. 40 CFR 26.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in Human Research Conducted or Supported by EPA § 26.118 Applications and proposals lacking definite... projects is the institution's responsibility; research training grants in which the activities involving... completion of instruments, prior animal studies, or purification of compounds. These applications need not be...

  20. [Psychosocial functioning in non-psychiatric acute and chronic inpatients: depression, alexithymia and lack of assertiveness].

    PubMed

    Arancibia, Marcelo; Behar, Rosa; Marín, Sofía; Inzunza, Nicolás; Madrid, Eva

    2016-11-01

    Depression, alexithymia, and lack of assertiveness interfere with individual psychosocial functioning and may result in longer hospitalization stay and poorer therapeutic results. To analyze the psychosocial functioning in acute and chronic patients and its association with psychological, clinical and sociodemographic variables. We performed a cross-sectional study that included 80 inpatients of both sexes with organic pathology, aged between 18 to 70 years old, without any current psychiatric disorder. Clinical and sociodemographic data were collected from a semi-structured interview and hospital records. Beck Depression Inventory-IA, Toronto Alexithymia Scale-20 and Rathus Assertiveness Scale were administered. Fifty five percent of patients had some degree of depression, 33% alexithymia and 34% lack of assertiveness. The levels of depression, alexithymia and lack of assertiveness in chronic patients were significantly higher than those observed in acute patients. Women and participants older than 60 years exhibited the highest degrees of depression. Alexithymia and lack of assertiveness were associated with a lower educational level. A negative significant correlation between alexithymia and assertiveness scores was observed among acute patients. Participants with chronic diseases had a lower psychosocial functioning. Less educated patients showed more alexithymic and less assertive features. We emphasized the need of a better management of these aspects by the health team, since social functioning might interfere with the outcome of physical illnesses.

  1. Barriers to Faculty Pedagogical Change: Lack of Training, Time, Incentives, and. . .Tensions with Professional Identity?

    ERIC Educational Resources Information Center

    Brownell, Sara E.; Tanner, Kimberly D.

    2012-01-01

    A substantial body of literature has highlighted many factors that impede faculty change, the most common of which are a lack of training, time, and incentives. However, there may be other barriers--unacknowledged and unexamined barriers--that might prove to be equally important. In particular, the tensions between a scientist's professional…

  2. 30 CFR 721.14 - Failure to give notice and lack of reasonable belief.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... belief. 721.14 Section 721.14 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT... and lack of reasonable belief. No notice of violation or cessation order may be vacated by reason of... create a reasonable belief that a violation had occurred....

  3. Accessible medical equipment for patients with disabilities in primary care clinics: why is it lacking?

    PubMed

    Pharr, Jennifer

    2013-04-01

    Previous research has identified inaccessible medical equipment as a barrier to health care services encountered by people with disabilities. However, no research has been conducted to understand why medical practices lack accessible equipment. The purpose of this study was to examine practice administrators' knowledge of accessible medical equipment and cost of accessible medical equipment to understand why medical practices lack such equipment. Hypotheses were: 1) Practice administrators lacked knowledge about accessible medical equipment and 2) The cost of accessible medical equipment was too great compared to standard equipment for the clinic. This study was a mixed methods survey of primary care practice administrators. The sixty-three participates were members of a medical management organization. Data were collected between December 20, 2011 and January 17, 2012. Proportions, Guttman scalogram, and Spearman's Rho correlation analyses were utilized. For this sample, less than half of the administrators knew that accessible equipment existed and a fourth knew what accessible equipment existed. There was a significant (p < 0.01), positive correlation between knowledge of accessible equipment and pieces of accessible equipment in the clinics. Because less than half of the administrators had ever considered purchasing accessible equipment, it was inconclusive if cost of accessible equipment was too great. Practice administrators' lack of knowledge of accessible medical equipment emphasizes the need not only for more education about the availability of accessible equipment but also about the importance of accessible equipment for their patients with disabilities and for physicians who provide them care. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. 34 CFR 97.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Applications and proposals lacking definite plans for involvement of human subjects. 97.118 Section 97.118 Education Office of the Secretary, Department of Education PROTECTION OF HUMAN SUBJECTS Federal Policy for the Protection of Human Subjects (Basic ED...

  5. 34 CFR 97.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Applications and proposals lacking definite plans for involvement of human subjects. 97.118 Section 97.118 Education Office of the Secretary, Department of Education PROTECTION OF HUMAN SUBJECTS Federal Policy for the Protection of Human Subjects (Basic ED...

  6. 34 CFR 97.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 1 2013-07-01 2013-07-01 false Applications and proposals lacking definite plans for involvement of human subjects. 97.118 Section 97.118 Education Office of the Secretary, Department of Education PROTECTION OF HUMAN SUBJECTS Federal Policy for the Protection of Human Subjects (Basic ED...

  7. 34 CFR 97.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Applications and proposals lacking definite plans for involvement of human subjects. 97.118 Section 97.118 Education Office of the Secretary, Department of Education PROTECTION OF HUMAN SUBJECTS Federal Policy for the Protection of Human Subjects (Basic ED...

  8. Lack of Emotional Support from Parents Early in Life and Alcohol Abuse Later in Life

    ERIC Educational Resources Information Center

    Shaw, Benjamin A.

    2006-01-01

    The purpose of this study is to examine the association between lacking emotional support from parents early in life and adult alcohol abuse. A series of logistic regression models were run with data collected from a nationally representative sample of over 2,500 adults ages 25-74. The findings reveal a linear relationship between level of…

  9. 40 CFR 26.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in Human Research Conducted or Supported by EPA § 26.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants, cooperative... application or proposal. These include activities such as institutional type grants when selection of specific...

  10. 45 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Research Subjects § 46.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants, cooperative agreements, or contracts are submitted to... such as institutional type grants when selection of specific projects is the institution's...

  11. 17 CFR 14.8 - Lack of requisite qualifications, character and integrity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... qualifications, character and integrity. 14.8 Section 14.8 Commodity and Securities Exchanges COMMODITY FUTURES... requisite qualifications, character and integrity. In addition to those matters specifically referred to in... represent others; or (b) To be lacking in character or integrity; or (c) To have engaged in unethical or...

  12. The Lack of an Association between Adolescent Friends' Prejudices and Stereotypes.

    ERIC Educational Resources Information Center

    Ritchey, P. Neal; Fishbein, Harold D.

    2001-01-01

    Studied the extent to which friends influenced the prejudices and stereotypes of white adolescents by evaluating race, homosexuality, HIV/AIDS, fat prejudice, and sex-role stereotyping. Found friends' prejudices and stereotypes were not associated. Examined possible explanations including discussion among friends, lack of questioning of apparent…

  13. 30 CFR 721.14 - Failure to give notice and lack of reasonable belief.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... belief. 721.14 Section 721.14 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT... and lack of reasonable belief. No notice of violation or cessation order may be vacated by reason of... create a reasonable belief that a violation had occurred. ...

  14. 30 CFR 721.14 - Failure to give notice and lack of reasonable belief.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... belief. 721.14 Section 721.14 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT... and lack of reasonable belief. No notice of violation or cessation order may be vacated by reason of... create a reasonable belief that a violation had occurred. ...

  15. 30 CFR 721.14 - Failure to give notice and lack of reasonable belief.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... belief. 721.14 Section 721.14 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT... and lack of reasonable belief. No notice of violation or cessation order may be vacated by reason of... create a reasonable belief that a violation had occurred. ...

  16. Quantitative trait loci for a neurocranium deformity, lack of operculum, in gilthead seabream (Sparus aurata L.).

    PubMed

    Negrín-Báez, D; Navarro, A; Afonso, J M; Toro, M A; Zamorano, M J

    2016-04-01

    Lack of operculum, a neurocranial deformity, is the most common external abnormality to be found among industrially produced gilthead seabream (Sparus aurata L.), and this entails significant financial losses. This study conducts, for the first time in this species, a quantitative trait loci (QTL) analysis of the lack of operculum. A total of 142 individuals from a paternal half-sibling family (six full-sibling families) were selected for QTL mapping. They had previously shown a highly significant association with the prevalence of lack of operculum in a segregation analysis. All the fish were genotyped for 106 microsatellite markers using a set of multiplex PCRs (ReMsa1-ReMsa13). A linear regression methodology was used for the QTL analysis. Four QTL were detected for this deformity, two of which (QTLOP1 and QTLOP2) were significant. They were located at LG (linkage group) nine and LG10 respectively. Both QTL showed a large effect (about 27%), and furthermore, the association between lack of operculum and sire allelic segregation observed was statistically significant in the QTLOP1 analysis. These results represent a significant step towards including marker-assisted selection for this deformity in genetic breeding programmes to reduce the incidence of the deformity in the species.

  17. When the YA Authors Are the Students: Learning from Cissy Lacks.

    ERIC Educational Resources Information Center

    McCracken, Nancy

    1996-01-01

    Reviews the case of Cissy Lacks, who was summarily dismissed from her teaching job after her principal went into a locked closet and found a videotape of students reading their creative compositions, which contained profanity. Discusses the effect of censorship on teaching. (TB)

  18. Resident Characteristics Related to the Lack of Morning Care Provision in Long-Term Care

    ERIC Educational Resources Information Center

    Simmons, Sandra F.; Durkin, Daniel W.; Rahman, Anna N.; Choi, Leena; Beuscher, Linda; Schnelle, John F.

    2013-01-01

    Purpose: The purpose of this study was to examine usual long-term care (LTC) practices related to 3 aspects of morning care and determine if there were resident characteristics related to the lack of care. Design and Methods: Participants were 169 long-stay residents in 4 community LTC facilities who required staff assistance with either transfer…

  19. Special Deliveries: Certified Nurse-Midwifery Programs Lacking in New England

    ERIC Educational Resources Information Center

    Franzosa, Alyssa

    2012-01-01

    With Boston serving as a hub of both educational and medical excellence, it's no wonder that New England has a high reputation to uphold in both of these areas. However, Boston and the rest of the region lack a specific degree program that is putting New England below the radars of potential midwives. Certified nurse-midwifery is a popular field…

  20. 14 CFR 1230.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Applications and proposals lacking definite plans for involvement of human subjects. 1230.118 Section 1230.118 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION PROTECTION OF HUMAN SUBJECTS § 1230.118 Applications and...

  1. 14 CFR 1230.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 5 2012-01-01 2012-01-01 false Applications and proposals lacking definite plans for involvement of human subjects. 1230.118 Section 1230.118 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION PROTECTION OF HUMAN SUBJECTS § 1230.118 Applications and...

  2. 14 CFR 1230.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Applications and proposals lacking definite plans for involvement of human subjects. 1230.118 Section 1230.118 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION PROTECTION OF HUMAN SUBJECTS § 1230.118 Applications and...

  3. 14 CFR 1230.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 5 2013-01-01 2013-01-01 false Applications and proposals lacking definite plans for involvement of human subjects. 1230.118 Section 1230.118 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION PROTECTION OF HUMAN SUBJECTS § 1230.118 Applications and...

  4. Relation of Neuroticism and Negative Career Thoughts and Feelings to Lack of Information

    ERIC Educational Resources Information Center

    Kelly, Kevin R.; Shin, Yun-Jeong

    2009-01-01

    The purpose of this study was to explore correlates of chronic career indecision with multivariate modeling. We examined the effects of neuroticism and negative career thoughts and feelings on lack of information, which is one of the core elements of chronic career indecision. The sample included 310 first-semester students who had entered…

  5. Barriers to Faculty Pedagogical Change: Lack of Training, Time, Incentives, and. . .Tensions with Professional Identity?

    ERIC Educational Resources Information Center

    Brownell, Sara E.; Tanner, Kimberly D.

    2012-01-01

    A substantial body of literature has highlighted many factors that impede faculty change, the most common of which are a lack of training, time, and incentives. However, there may be other barriers--unacknowledged and unexamined barriers--that might prove to be equally important. In particular, the tensions between a scientist's professional…

  6. Mind Maps to Modify Lack of Attention among Saudi Kindergarten Children

    ERIC Educational Resources Information Center

    Daghistan, Bulquees Ismail Abdul Majid

    2016-01-01

    This research study aims at investigating the impact of Mind Maps on modifying the lack of attention in Arabic language class among Saudi Kindergarten children. To achieve the goals of this study the researcher used an experimental design with a random sample from AlRae'd Kindergarten's children in Riyadh -Saudi Arabia for the academic year…

  7. Special Deliveries: Certified Nurse-Midwifery Programs Lacking in New England

    ERIC Educational Resources Information Center

    Franzosa, Alyssa

    2012-01-01

    With Boston serving as a hub of both educational and medical excellence, it's no wonder that New England has a high reputation to uphold in both of these areas. However, Boston and the rest of the region lack a specific degree program that is putting New England below the radars of potential midwives. Certified nurse-midwifery is a popular field…

  8. Teaching from "The Immortal Life of Henrietta Lacks": Student Perspectives on Health Disparities and Medical Ethics

    ERIC Educational Resources Information Center

    Dimaano, Christian; Spigner, Clarence

    2017-01-01

    Objective: "The Immortal Life of Henrietta Lacks" by Rebecca Skloot is an award-winning biography engaging its readers on important topics ranging from race, science and ethics to the social determinants of health. However, the multiple pedagogic impacts of this book on the public health classroom setting have yet to be comprehensively…

  9. Lack of lipogenesis in parasitoids: a review of physiological mechanisms and evolutionary implications.

    PubMed

    Visser, Bertanne; Ellers, Jacintha

    2008-09-01

    The ability of organisms to adapt to fluctuating food conditions is essential for their survival and reproduction. Accumulating energy reserves, such as lipids, in anticipation of harsh conditions, will reduce negative effects of a low food supply. For Hymenoptera and Diptera, several parasitoid species lack adult lipogenesis, and are unable to store excess energy in the form of lipid reserves. The aim of this review is to provide a synthesis of current knowledge regarding the inability to accumulate lipids in parasitoids, leading to new insights and prospects for further research. We will emphasize physiological mechanisms underlying lack of lipogenesis, the evolution of this adaptation in parasitoids and its biological implications with regard to life history traits. We suggest the occurrence of lack of lipogenesis in parasitoids to be dependent on the extent of host exploitation through metabolic manipulation. Currently available data shows lack of lipogenesis to have evolved independently at least twice, in parasitic Hymenoptera and Diptera. The underlying genetic mechanism, however, remains to be solved. Furthermore, due to the inability to replenish adult fat reserves, parasitoids are severely constrained in resource allocation strategies, in particular the trade-off between survival and reproduction.

  10. 10 CFR 745.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... involvement of human subjects. 745.118 Section 745.118 Energy DEPARTMENT OF ENERGY PROTECTION OF HUMAN SUBJECTS § 745.118 Applications and proposals lacking definite plans for involvement of human subjects... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  11. 32 CFR 219.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... plans for involvement of human subjects. 219.118 Section 219.118 National Defense Department of Defense....118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types... institutional type grants when selection of specific projects is the institution's responsibility;...

  12. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  13. 45 CFR 690.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... for involvement of human subjects. 690.118 Section 690.118 Public Welfare Regulations Relating to... Applications and proposals lacking definite plans for involvement of human subjects. Certain types of... grants when selection of specific projects is the institution's responsibility; research training...

  14. 32 CFR 219.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... plans for involvement of human subjects. 219.118 Section 219.118 National Defense Department of Defense....118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types... institutional type grants when selection of specific projects is the institution's responsibility;...

  15. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  16. 32 CFR 219.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... plans for involvement of human subjects. 219.118 Section 219.118 National Defense Department of Defense....118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types... institutional type grants when selection of specific projects is the institution's responsibility;...

  17. 40 CFR 26.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... definite plans for involvement of human subjects. 26.118 Section 26.118 Protection of Environment... in Human Research Conducted or Supported by EPA § 26.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types of applications for grants,...

  18. 10 CFR 745.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... involvement of human subjects. 745.118 Section 745.118 Energy DEPARTMENT OF ENERGY PROTECTION OF HUMAN SUBJECTS § 745.118 Applications and proposals lacking definite plans for involvement of human subjects... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  19. 10 CFR 745.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... involvement of human subjects. 745.118 Section 745.118 Energy DEPARTMENT OF ENERGY PROTECTION OF HUMAN SUBJECTS § 745.118 Applications and proposals lacking definite plans for involvement of human subjects... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  20. 10 CFR 745.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... involvement of human subjects. 745.118 Section 745.118 Energy DEPARTMENT OF ENERGY PROTECTION OF HUMAN SUBJECTS § 745.118 Applications and proposals lacking definite plans for involvement of human subjects... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  1. 10 CFR 745.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... involvement of human subjects. 745.118 Section 745.118 Energy DEPARTMENT OF ENERGY PROTECTION OF HUMAN SUBJECTS § 745.118 Applications and proposals lacking definite plans for involvement of human subjects... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  2. 32 CFR 219.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... plans for involvement of human subjects. 219.118 Section 219.118 National Defense Department of Defense....118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types... institutional type grants when selection of specific projects is the institution's responsibility;...

  3. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  4. 7 CFR 1c.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... involvement of human subjects. 1c.118 Section 1c.118 Agriculture Office of the Secretary of Agriculture PROTECTION OF HUMAN SUBJECTS § 1c.118 Applications and proposals lacking definite plans for involvement of... responsibility; research training grants in which the activities involving subjects remain to be selected;...

  5. Private Pre-University Education in Romania: Mixing Control with Lack of Strategy

    ERIC Educational Resources Information Center

    Stanus, Cristina

    2014-01-01

    This paper approaches private provision of pre-university education in Romania, exploring available data on the sector's size and main characteristics and evaluating the extent to which the current regulatory framework enables positive effects in terms of freedom of choice, quality, equity, and social cohesion. The paper argues that the lack of a…

  6. 30 CFR 721.14 - Failure to give notice and lack of reasonable belief.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 3 2011-07-01 2011-07-01 false Failure to give notice and lack of reasonable belief. 721.14 Section 721.14 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT, DEPARTMENT OF THE INTERIOR INITIAL PROGRAM REGULATIONS FEDERAL INSPECTIONS § 721.14 Failure to give notice...

  7. Relation of Neuroticism and Negative Career Thoughts and Feelings to Lack of Information

    ERIC Educational Resources Information Center

    Kelly, Kevin R.; Shin, Yun-Jeong

    2009-01-01

    The purpose of this study was to explore correlates of chronic career indecision with multivariate modeling. We examined the effects of neuroticism and negative career thoughts and feelings on lack of information, which is one of the core elements of chronic career indecision. The sample included 310 first-semester students who had entered…

  8. Non-Native Student's Communication Is Affected Due to the Lack of Pragmatic Competence

    ERIC Educational Resources Information Center

    Latha, V. G.; Rajan, Premalatha

    2012-01-01

    This paper aims at focusing how the lack of pragmatic competence affects student's communication in L2 (Second language) at tertiary level. The city based Indian students learn English which is their second language from 3 years onwards whereas the rural based students learn English only from 6 years onwards. This exposure of the L2 shows the…

  9. Who Lacks Support and Why? An Examination of Mothers' Personal Safety Nets

    ERIC Educational Resources Information Center

    Harknett, Kristen S.; Hartnett, Caroline Sten

    2011-01-01

    We use data from the Fragile Families and Child Wellbeing Study (N = 12,140 person-waves) to identify characteristics associated with mothers' having or lacking "personal safety net" support from family and friends. We focus on characteristics that are likely to increase the importance of having support available but may also interfere with the…

  10. 28 CFR 541.30 - Lack of verification of need for protection.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Lack of verification of need for protection. 541.30 Section 541.30 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE... verification of need for protection. If a staff investigation fails to verify your need for placement in the...

  11. 28 CFR 541.30 - Lack of verification of need for protection.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Lack of verification of need for protection. 541.30 Section 541.30 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE... verification of need for protection. If a staff investigation fails to verify your need for placement in the...

  12. 28 CFR 541.30 - Lack of verification of need for protection.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Lack of verification of need for protection. 541.30 Section 541.30 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE... verification of need for protection. If a staff investigation fails to verify your need for placement in the...

  13. Resident Characteristics Related to the Lack of Morning Care Provision in Long-Term Care

    ERIC Educational Resources Information Center

    Simmons, Sandra F.; Durkin, Daniel W.; Rahman, Anna N.; Choi, Leena; Beuscher, Linda; Schnelle, John F.

    2013-01-01

    Purpose: The purpose of this study was to examine usual long-term care (LTC) practices related to 3 aspects of morning care and determine if there were resident characteristics related to the lack of care. Design and Methods: Participants were 169 long-stay residents in 4 community LTC facilities who required staff assistance with either transfer…

  14. 32 CFR 219.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... plans for involvement of human subjects. 219.118 Section 219.118 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS PROTECTION OF HUMAN SUBJECTS § 219.118 Applications and proposals lacking definite plans for involvement of human subjects. Certain types...

  15. 38 CFR 16.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in which the activities involving subjects remain to be selected; and projects in which human... proposals lacking definite plans for involvement of human subjects. 16.118 Section 16.118 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS PROTECTION OF HUMAN SUBJECTS § 16.118 Applications...

  16. Who Lacks Support and Why? An Examination of Mothers' Personal Safety Nets

    ERIC Educational Resources Information Center

    Harknett, Kristen S.; Hartnett, Caroline Sten

    2011-01-01

    We use data from the Fragile Families and Child Wellbeing Study (N = 12,140 person-waves) to identify characteristics associated with mothers' having or lacking "personal safety net" support from family and friends. We focus on characteristics that are likely to increase the importance of having support available but may also interfere with the…

  17. 45 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... SERVICES GENERAL ADMINISTRATION PROTECTION OF HUMAN SUBJECTS Basic HHS Policy for Protection of Human Research Subjects § 46.118 Applications and proposals lacking definite plans for involvement of human... responsibility; research training grants in which the activities involving subjects remain to be selected; and...

  18. 45 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... SERVICES GENERAL ADMINISTRATION PROTECTION OF HUMAN SUBJECTS Basic HHS Policy for Protection of Human Research Subjects § 46.118 Applications and proposals lacking definite plans for involvement of human... responsibility; research training grants in which the activities involving subjects remain to be selected; and...

  19. 40 CFR 26.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY GENERAL PROTECTION OF HUMAN SUBJECTS Basic EPA Policy for Protection of Subjects in Human Research Conducted or Supported by EPA § 26.118 Applications and proposals lacking definite... projects is the institution's responsibility; research training grants in which the activities involving...

  20. 34 CFR 97.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Education PROTECTION OF HUMAN SUBJECTS Federal Policy for the Protection of Human Subjects (Basic ED Policy for Protection of Human Research Subjects) § 97.118 Applications and proposals lacking definite plans... projects is the institution's responsibility; research training grants in which the activities involving...

  1. 40 CFR 26.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ENVIRONMENTAL PROTECTION AGENCY GENERAL PROTECTION OF HUMAN SUBJECTS Basic EPA Policy for Protection of Subjects in Human Research Conducted or Supported by EPA § 26.118 Applications and proposals lacking definite... projects is the institution's responsibility; research training grants in which the activities involving...

  2. 45 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Services GENERAL ADMINISTRATION PROTECTION OF HUMAN SUBJECTS Basic HHS Policy for Protection of Human Research Subjects § 46.118 Applications and proposals lacking definite plans for involvement of human... responsibility; research training grants in which the activities involving subjects remain to be selected; and...

  3. 45 CFR 46.118 - Applications and proposals lacking definite plans for involvement of human subjects.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... SERVICES GENERAL ADMINISTRATION PROTECTION OF HUMAN SUBJECTS Basic HHS Policy for Protection of Human Research Subjects § 46.118 Applications and proposals lacking definite plans for involvement of human... responsibility; research training grants in which the activities involving subjects remain to be selected; and...

  4. 40 CFR 52.1118 - Approval of bubbles in nonattainment areas lacking approved demonstrations: State assurances.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 4 2011-07-01 2011-07-01 false Approval of bubbles in nonattainment... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1118 Approval of bubbles in nonattainment areas lacking approved...

  5. 40 CFR 52.1118 - Approval of bubbles in nonattainment areas lacking approved demonstrations: State assurances.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 4 2012-07-01 2012-07-01 false Approval of bubbles in nonattainment... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1118 Approval of bubbles in nonattainment areas lacking approved...

  6. 40 CFR 52.1118 - Approval of bubbles in nonattainment areas lacking approved demonstrations: State assurances.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 4 2013-07-01 2013-07-01 false Approval of bubbles in nonattainment... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1118 Approval of bubbles in nonattainment areas lacking approved...

  7. 40 CFR 52.1118 - Approval of bubbles in nonattainment areas lacking approved demonstrations: State assurances.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 4 2010-07-01 2010-07-01 false Approval of bubbles in nonattainment... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS (CONTINUED) Maryland § 52.1118 Approval of bubbles in nonattainment areas lacking approved...

  8. 49 CFR 821.26 - Motion to dismiss petition for review for lack of standing.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Motion to dismiss petition for review for lack of standing. 821.26 Section 821.26 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL TRANSPORTATION SAFETY BOARD RULES OF PRACTICE IN AIR SAFETY PROCEEDINGS Special Rules Applicable...

  9. 49 CFR 821.26 - Motion to dismiss petition for review for lack of standing.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Motion to dismiss petition for review for lack of standing. 821.26 Section 821.26 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL TRANSPORTATION SAFETY BOARD RULES OF PRACTICE IN AIR SAFETY PROCEEDINGS Special Rules Applicable...

  10. 49 CFR 821.26 - Motion to dismiss petition for review for lack of standing.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Motion to dismiss petition for review for lack of standing. 821.26 Section 821.26 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL TRANSPORTATION SAFETY BOARD RULES OF PRACTICE IN AIR SAFETY PROCEEDINGS Special Rules Applicable...

  11. 49 CFR 821.26 - Motion to dismiss petition for review for lack of standing.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Motion to dismiss petition for review for lack of standing. 821.26 Section 821.26 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL TRANSPORTATION SAFETY BOARD RULES OF PRACTICE IN AIR SAFETY PROCEEDINGS Special Rules Applicable...

  12. 49 CFR 821.26 - Motion to dismiss petition for review for lack of standing.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Motion to dismiss petition for review for lack of standing. 821.26 Section 821.26 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL TRANSPORTATION SAFETY BOARD RULES OF PRACTICE IN AIR SAFETY PROCEEDINGS Special Rules Applicable...

  13. 42 CFR 476.90 - Lack of cooperation by a provider or practitioner.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 4 2014-10-01 2014-10-01 false Lack of cooperation by a provider or practitioner. 476.90 Section 476.90 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... cooperation by a provider or practitioner. (a) If a provider or practitioner refuses to allow a QIO to...

  14. Lack of energy: an important and distinct component of HIV-related fatigue and daytime function.

    PubMed

    Aouizerat, Bradley E; Gay, Caryl L; Lerdal, Anners; Portillo, Carmen J; Lee, Kathryn A

    2013-02-01

    Fatigue is a prevalent symptom among adults living with HIV. There is increasing evidence that fatigue and energy are related, yet distinct constructs. Although HIV-related fatigue has been well studied, little is known about perceived energy and how it relates to fatigue, individual characteristics, and other symptoms. To describe the experience of perceived energy in adults with HIV and evaluate its relationship to demographic and clinical characteristics as well as symptoms of fatigue, sleep disturbance, anxiety, depression, and daytime function. The design was descriptive, comparative, and correlational. The sample of 318 adults with HIV completed a demographic questionnaire; the Memorial Symptom Assessment Scale; and measures of fatigue, sleep disturbance, anxiety, depressive symptoms, and daytime function. Medical records were reviewed for disease and treatment data. Participants who reported a lack of energy were compared with those who did not on demographic, clinical, and symptom variables. Regression models of perceived energy and its interference with daytime function also were evaluated. Perceived lack of energy was highly prevalent (65%) and more strongly related to interference with daytime function than more general measures of fatigue severity, even when controlling for other characteristics and symptoms. Like other aspects of fatigue, lack of energy was associated with sleep disturbance, anxiety, and depressive symptoms. Lack of energy was more strongly related to morning fatigue than to evening fatigue. Lack of energy interferes with daytime function and is not just the inverse of fatigue but a distinct perception that differs from fatigue. Copyright © 2013 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved.

  15. 37 CFR 1.489 - Protest to lack of unity of invention before the International Preliminary Examining Authority.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false Protest to lack of unity of... Protest to lack of unity of invention before the International Preliminary Examining Authority. (a) If the applicant disagrees with the holding of lack of unity of invention by the International...

  16. 37 CFR 1.489 - Protest to lack of unity of invention before the International Preliminary Examining Authority.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Protest to lack of unity of... Protest to lack of unity of invention before the International Preliminary Examining Authority. (a) If the applicant disagrees with the holding of lack of unity of invention by the International...

  17. 37 CFR 1.477 - Protest to lack of unity of invention before the International Searching Authority.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Protest to lack of unity of... PATENT CASES International Processing Provisions Unity of Invention § 1.477 Protest to lack of unity of... lack of unity of invention by the International Searching Authority, additional fees may be paid...

  18. 37 CFR 1.489 - Protest to lack of unity of invention before the International Preliminary Examining Authority.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2014-07-01 2014-07-01 false Protest to lack of unity of... Protest to lack of unity of invention before the International Preliminary Examining Authority. (a) If the applicant disagrees with the holding of lack of unity of invention by the International...

  19. 37 CFR 1.477 - Protest to lack of unity of invention before the International Searching Authority.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Protest to lack of unity of... PATENT CASES International Processing Provisions Unity of Invention § 1.477 Protest to lack of unity of... lack of unity of invention by the International Searching Authority, additional fees may be paid...

  20. 37 CFR 1.489 - Protest to lack of unity of invention before the International Preliminary Examining Authority.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Protest to lack of unity of... Protest to lack of unity of invention before the International Preliminary Examining Authority. (a) If the applicant disagrees with the holding of lack of unity of invention by the International...