Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.

Science.gov (United States)

Mina, John G; Thye, Julie K; Alqaisi, Amjed Q I; Bird, Louise E; Dods, Robert H; Grøftehauge, Morten K; Mosely, Jackie A; Pratt, Steven; Shams-Eldin, Hosam; Schwarz, Ralph T; Pohl, Ehmke; Denny, Paul W

2017-07-21

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Deoxysphingoid bases as plasma markers in Diabetes mellitus

Directory of Open Access Journals (Sweden)

Bertea Mariana

2010-08-01

Full Text Available Abstract Background Sphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT. SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB. This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN, which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes. Results In a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction. Conclusion Our results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes.

In Vitro Palmitate Treatment of Myotubes from Postmenopausal Women Leads to Ceramide Accumulation, Inflammation and Affected Insulin Signaling

DEFF Research Database (Denmark)

Abildgaard, Julie; Henstridge, Darren C; Pedersen, Anette Tønnes

2014-01-01

Menopause is associated with an increased incidence of insulin resistance and metabolic diseases. In a chronic palmitate treatment model, we investigated the role of skeletal muscle fatty acid exposure in relation to the metabolic deterioration observed with menopause. Human skeletal muscle......, post-myotubes showed a blunted insulin stimulated phosphorylation of AS160 in response to chronic palmitate treatment compared with pre-myotubes (p = 0.02). The increased intramyocellular ceramide content in the post-myotubes was associated with a significantly higher mRNA expression of Serine...... Palmitoyltransferase1 (SPT1) after one day of palmitate treatment (p = 0.03) in post-myotubes compared with pre-myotubes. Our findings indicate that post-myotubes are more prone to develop lipid accumulation and defective insulin signaling following chronic saturated fatty acid exposure as compared to pre-myotubes....

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

International Nuclear Information System (INIS)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-01-01

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids

4500 V SPT+ IGBT optimization on static and dynamic losses

International Nuclear Information System (INIS)

Dai Qingyun; Tian Xiaoli; Zhang Wenliang; Lu Shuojin; Zhu Yangjun

2015-01-01

This paper concerns the need for improving the static and dynamic performance of the high voltage insulated gate bipolar transistor (HV IGBTs). A novel structure with a carrier stored layer on the cathode side, known as an enhanced planar IGBT of the 4500 V voltage class is investigated. With the adoption of a soft punch through (SPT) concept as the vertical structure and an enhanced planar concept as the top structure, signed as SPT + IGBT, the simulation results indicate the turn-off switching waveform of the 4500 V SPT + IGBT is soft and also realizes an improved trade-off relationship between on-state voltage drop (V on ) and turn-off loss (E off ) in comparison with the SPT IGBT. Attention is also paid to the influences caused by different carrier stored layer doping dose on static and dynamic performances, to optimize on-state and switching losses of SPT + IGBT. (paper)

Study of the thickness evolution during SPT Testing

Directory of Open Access Journals (Sweden)

David Sánchez-Ávila

2018-03-01

Full Text Available The Small Punch Test (SPT is an increasingly expanding test used to obtain different mechanical data, such as strength, fracture, creep, etc…especially when there is little material available. However, the SPT test is more complicated than the uniaxial tensile test due to its non-linearity, which makes it difficult to relate the data obtained with the tensile tests. In fact, in the literature there is no clear model linking these tests and a different calibration should be used for each material. The complication of the SPT test is that the reduction of the sample thickness is not homogeneous in its gauge volume. In this work we proceeded to determine the variation of the SPT specimen thickness at several points, especially at the center and at the rupture zone, by means of the use of finite elements in COMSOL, taking a SLM AM (selective laser melting additive manufactured 316L stainless steel as the base material for modelling. For the appropriate modelling in COMSOL, the mechanical parameters of two 316L extreme thermomechanical treatments have been implemented, one annealed to a minimum hardness and another heavily work-hardened. The sample thickness variation results allow advancing in the theoretical modeling of the SPT behavior in order to obtain more accurate correlations with tensile tests data.

Structure of the human histone chaperone FACT Spt16 N-terminal domain

Energy Technology Data Exchange (ETDEWEB)

Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

2016-01-22

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

Structure of the human histone chaperone FACT Spt16 N-terminal domain

International Nuclear Information System (INIS)

Marcianò, G.; Huang, D. T.

2016-01-01

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding

A unified CPT–SPT correlation for non-crushable and crushable cohesionless soils

Directory of Open Access Journals (Sweden)

Sayed M. Ahmed

2014-03-01

Full Text Available Despite the continuous developments in the Cone Penetration Test (CPT, the Standard Penetration Test (SPT is still used extensively in site investigations. Hence, there is a constant need to update the CPT–SPT correlations to make use of the growing experience with the CPT. Many CPT–SPT correlations have been proposed based on case histories of predominantly quartzitic/non-crushable sands; yet, more efforts are needed to enhance their reliability. Additionally, recent studies were carried out on calcareous/crushable sands have shown that the common CPT–SPT correlations for these sands are even less reliable than they are for quartzitic sands. In this study, a proposed approach is presented to define the related soil compressibility parameters of the CPT–SPT correlations. The presented methodology enhances the reliability of the CPT–SPT correlations and provides a unified approach encompassing both crushable and non-crushable sands.

VizieR Online Data Catalog: SPT-GMOS spectroscopy of gal. in massive clusters (Bayliss+, 2017)

Science.gov (United States)

Bayliss, M. B.; Zengo, K.; Ruel, J.; Benson, B. A.; Bleem, L. E.; Bocquet, S.; Bulbul, E.; Brodwin, M.; Capasso, R.; Chiu, I.-N.; McDonald, M.; Rapetti, D.; Saro, A.; Stalder, B.; Stark, A. A.; Strazzullo, V.; Stubbs, C. W.; Zenteno, A.

2017-10-01

The majority of the data set used in this analysis comes from the SPT-GMOS spectroscopic survey (Bayliss+ 2016, J/ApJS/227/3), which consists of spectroscopic follow-up of 62 galaxy clusters from the SPT-SZ survey. The full SPT-GMOS sample includes 2243 galaxy spectra, 1579 of which are cluster member galaxies. In addition to previously published galaxy spectroscopy, we also present the first publication of new spectroscopy in the fields of three SPT galaxy clusters. We observed SPT-CLJ0000-5748, SPT-CLJ0516-5430, and SPT-CLJ2337-5942 with the Inamori Magellan Areal Camera and Spectrograph (IMACS) mounted on the Magellan-I (Baade) telescope at Las Campanas Observatory on the nights of 14-15 September 2012. (2 data files).

VizieR Online Data Catalog: Full spectroscopic data release of the SPT-GMOS (Bayliss+, 2016)

Science.gov (United States)

Bayliss, M. B.; Ruel, J.; Stubbs, C. W.; Allen, S. W.; Applegate, D. E.; Ashby, M. L. N.; Bautz, M.; Benson, B. A.; Bleem, L. E.; Bocquet, S.; Brodwin, M.; Capasso, R.; Carlstrom, J. E.; Chang, C. L.; Chiu, I.; Cho, H.-M.; Clocchiatti, A.; Crawford, T. M.; Crites, A. T.; de Haan, T.; Desai, S.; Dietrich, J. P.; Dobbs, M. A.; Doucouliagos, A. N.; Foley, R. J.; Forman, W. R.; Garmire, G. P.; George, E. M.; Gladders, M. D.; Gonzalez, A. H.; Gupta, N.; Halverson, N. W.; Hlavacek-Larrondo, J.; Hoekstra, H.; Holder, G. P.; Holzapfel, W. L.; Hou, Z.; Hrubes, J. D.; Huang, N.; Jones, C.; Keisler, R.; Knox, L.; Lee, A. T.; Leitch, E. M.; von der Linden, A.; Luong-van, D.; Mantz, A.; Marrone, D. P.; McDonald, M.; McMahon, J. J.; Meyer, S. S.; Mocanu, L. M.; Mohr, J. J.; Murray, S. S.; Padin, S.; Pryke, C.; Rapetti, D.; Reichardt, C. L.; Rest, A.; Ruhl, J. E.; Saliwanchik, B. R.; Saro, A.; Sayre, J. T.; Schaffer, K. K.; Schrabback, T.; Shirokoff, E.; Song, J.; Spieler, H. G.; Stalder, B.; Stanford, S. A.; Staniszewski, Z.; Stark, A. A.; Story, K. T.; Vanderlinde, K.; Vieira, J. D.; Vikhlinin, A.; Williamson, R.; Zenteno, A.

2016-11-01

The data presented in this paper follow the same observational design described by Ruel et al. (2014ApJ...792...45R). The final Gemini-S observing allocation for SPT-GMOS concluded at the end of the 2015B semester. Over the course of the entire survey we observed 121 individual spectroscopic masks targeting 62 SPT-SZ galaxy clusters (see table 1). All final data products from SPT-GMOS are publicly released via the Harvard Dataverse Network (http://dataverse.harvard.edu/dataverse/SPT_Clusters). The galaxy clusters observed in the SPT-GMOS are all drawn from the SPT-SZ survey, completed in 2011 November. The full SPT-SZ survey covered approximately 2500deg2 of the southern sky at 95, 150, and 220GHz with an angular resolution of ~1'. (4 data files).

Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency

DEFF Research Database (Denmark)

Olpin, S E; Afifi, A; Clark, S

2003-01-01

Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients...

Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

Directory of Open Access Journals (Sweden)

Keerthi Krishnan

Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

Fabrication of Detector Arrays for the SPT-3G Receiver

Science.gov (United States)

Posada, C. M.; Ade, P. A. R.; Ahmed, Z.; Anderson, A. J.; Austermann, J. E.; Avva, J. S.; Thakur, R. Basu; Bender, A. N.; Benson, B. A.; Carlstrom, J. E.; Carter, F. W.; Cecil, T.; Chang, C. L.; Cliche, J. F.; Cukierman, A.; Denison, E. V.; de Haan, T.; Ding, J.; Divan, R.; Dobbs, M. A.; Dutcher, D.; Everett, W.; Foster, A.; Gannon, R. N.; Gilbert, A.; Groh, J. C.; Halverson, N. W.; Harke-Hosemann, A. H.; Harrington, N. L.; Henning, J. W.; Hilton, G. C.; Holzapfel, W. L.; Huang, N.; Irwin, K. D.; Jeong, O. B.; Jonas, M.; Khaire, T.; Kofman, A. M.; Korman, M.; Kubik, D.; Kuhlmann, S.; Kuo, C. L.; Lee, A. T.; Lowitz, A. E.; Meyer, S. S.; Michalik, D.; Miller, C. S.; Montgomery, J.; Nadolski, A.; Natoli, T.; Nguyen, H.; Noble, G. I.; Novosad, V.; Padin, S.; Pan, Z.; Pearson, J.; Rahlin, A.; Ruhl, J. E.; Saunders, L. J.; Sayre, J. T.; Shirley, I.; Shirokoff, E.; Smecher, G.; Sobrin, J. A.; Stan, L.; Stark, A. A.; Story, K. T.; Suzuki, A.; Tang, Q. Y.; Thompson, K. L.; Tucker, C.; Vale, L. R.; Vanderlinde, K.; Vieira, J. D.; Wang, G.; Whitehorn, N.; Yefremenko, V.; Yoon, K. W.; Young, M. R.

2018-05-01

The South Pole Telescope third-generation (SPT-3G) receiver was installed during the austral summer of 2016-2017. It is designed to measure the cosmic microwave background across three frequency bands centered at 95, 150, and 220 GHz. The SPT-3G receiver has ten focal plane modules, each with 269 pixels. Each pixel features a broadband sinuous antenna coupled to a niobium microstrip transmission line. In-line filters define the desired band-passes before the signal is coupled to six bolometers with Ti/Au/Ti/Au transition edge sensors (three bands × two polarizations). In total, the SPT-3G receiver is composed of 16,000 detectors, which are read out using a 68× frequency-domain multiplexing scheme. In this paper, we present the process employed in fabricating the detector arrays.

Supportive periodontal therapy (SPT) for maintaining the dentition in adults treated for periodontitis.

Science.gov (United States)

Manresa, Carolina; Sanz-Miralles, Elena C; Twigg, Joshua; Bravo, Manuel

2018-01-01

Periodontitis is a bacterially-induced, chronic inflammatory disease that destroys the connective tissues and bone that support teeth. Active periodontal treatment aims to reduce the inflammatory response, primarily through eradication of bacterial deposits. Following completion of treatment and arrest of inflammation, supportive periodontal therapy (SPT) is employed to reduce the probability of re-infection and progression of the disease; to maintain teeth without pain, excessive mobility or persistent infection in the long term, and to prevent related oral diseases.According to the American Academy of Periodontology, SPT should include all components of a typical dental recall examination, and importantly should also include periodontal re-evaluation and risk assessment, supragingival and subgingival removal of bacterial plaque and calculus, and re-treatment of any sites showing recurrent or persistent disease. While the first four points might be expected to form part of the routine examination appointment for periodontally healthy patients, the inclusion of thorough periodontal evaluation, risk assessment and subsequent treatment - normally including mechanical debridement of any plaque or calculus deposits - differentiates SPT from routine care.Success of SPT has been reported in a number of long-term, retrospective studies. This review aimed to assess the evidence available from randomised controlled trials (RCTs). To determine the effects of supportive periodontal therapy (SPT) in the maintenance of the dentition of adults treated for periodontitis. Cochrane Oral Health's Information Specialist searched the following databases: Cochrane Oral Health's Trials Register (to 8 May 2017), the Cochrane Central Register of Controlled Trials (CENTRAL) (the Cochrane Library, 2017, Issue 5), MEDLINE Ovid (1946 to 8 May 2017), and Embase Ovid (1980 to 8 May 2017). The US National Institutes of Health Trials Registry (ClinicalTrials.gov) and the World Health Organization

Integrated performance of a frequency domain multiplexing readout in the SPT-3G receiver

Science.gov (United States)

Bender, A. N.; Ade, P. A. R.; Anderson, A. J.; Avva, J.; Ahmed, Z.; Arnold, K.; Austermann, J. E.; Basu Thakur, R.; Benson, B. A.; Bleem, L. E.; Byrum, K.; Carlstrom, J. E.; Carter, F. W.; Chang, C. L.; Cho, H. M.; Cliche, J. F.; Crawford, T. M.; Cukierman, A.; Czaplewski, D. A.; Ding, J.; Divan, R.; de Haan, T.; Dobbs, M. A.; Dutcher, D.; Everett, W.; Gilbert, A.; Groh, J. C.; Guyser, R.; Halverson, N. W.; Harke-Hosemann, A.; Harrington, N. L.; Hattori, K.; Henning, J. W.; Hilton, G. C.; Holzapfel, W. L.; Huang, N.; Irwin, K. D.; Jeong, O.; Khaire, T.; Korman, M.; Kubik, D.; Kuo, C. L.; Lee, A. T.; Leitch, E. M.; Lendinez, S.; Meyer, S. S.; Miller, C. S.; Montgomery, J.; Nadolski, A.; Natoli, T.; Nguyen, H.; Novosad, V.; Padin, S.; Pan, Z.; Pearson, J.; Posada, C. M.; Rahlin, A.; Reichardt, C. L.; Ruhl, J. E.; Saliwanchik, B. R.; Sayre, J. T.; Shariff, J. A.; Shirley, Ian; Shirokoff, E.; Smecher, G.; Sobrin, J.; Stan, L.; Stark, A. A.; Story, K.; Suzuki, A.; Tang, Q. Y.; Thompson, K. L.; Tucker, C.; Vanderlinde, K.; Vieira, J. D.; Wang, G.; Whitehorn, N.; Yefremenko, V.; Yoon, K. W.

2016-07-01

The third generation receiver for the South Pole Telescope, SPT-3G, will make extremely deep, arcminuteresolution maps of the temperature and polarization of the cosmic microwave background. The SPT-3G maps will enable studies of the B-mode polarization signature, constraining primordial gravitational waves as well as the effect of massive neutrinos on structure formation in the late universe. The SPT-3G receiver will achieve exceptional sensitivity through a focal plane of 16,000 transition-edge sensor bolometers, an order of magnitude more than the current SPTpol receiver. SPT-3G uses a frequency domain multiplexing (fMux) scheme to read out the focal plane, combining the signals from 64 bolometers onto a single pair of wires. The fMux readout facilitates the large number of detectors in the SPT-3G focal plane by limiting the thermal load due to readout wiring on the 250 millikelvin cryogenic stage. A second advantage of the fMux system is that the operation of each bolometer can be optimized. In addition to these benefits, the fMux readout introduces new challenges into the design and operation of the receiver. The bolometers are operated at a range of frequencies up to 5 MHz, requiring control of stray reactances over a large bandwidth. Additionally, crosstalk between multiplexed detectors will inject large false signals into the data if not adequately mitigated. SPT-3G is scheduled to deploy to the South Pole Telescope in late 2016. Here, we present the pre-deployment performance of the fMux readout system with the SPT-3G focal plane.

Mengapa Perlu Menghapus SPT Tahunan 1721?

Directory of Open Access Journals (Sweden)

Yenni Mangoting

2008-01-01

Full Text Available Employers as a withholders calculate employment income tax with two ways, there are 1 calculate employment income tax in the case of periodical tax return and 2 re-count for employment income tax and combine all the income that incurred in tax year and report them in to annual income tax return form 1721. Under payment of tax liability must be paid at the latest on the 25th day of the third month after a fiscal year ended. Because of that employers actually not calculate employment income tax base on real income. This system actually make difficulties and in addition withholders have an opportunity by rule for re-count employment income tax as base for fill up annual income tax return form 1721- A1 or 1721 A2 . Employment income tax calculation in year ended was final result. Abstract in Bahasa Indonesia: Pemberi kerja yang dalam hal ini bertindak sebagai pemotong pajak, menghitung PPh pasal 21 dalam dua tahap, yaitu 1 menghitung PPh pasal 21 masa yang dilaporkan dan disetorkan setiap bulan dan 2 Perhitungan ulang PPh Pasal 21 dengan menjumlahkan semua penghasilan yang diterima dalam satu tahun pajak dan dilaporkan dalam SPT Tahunan dengan form 1721. Kekurangan pembayaran harus dilunasi sebelum tanggal 25 Maret tahun pajak berikutnya. Oleh karena itu pemberi kerja biasanya tidak mengitung PPh pasal 21 masa berdasarkan penghasilan yang sebenarnya. Selain karena secara administrasi merepotkan, pemberi kerja juga di beri kesempatan menghitung kembali PPh Pasal 21 sebagai dasar pengisian Form 1721 - A1 atau 1721 A2. Penghitungan kembali PPh Pasal 21 pada akhir tahun menjadi perhitungan final hasil revisi perhitungan PPh 21 masa. Kata kunci: pemberi kerja, SPT Tahunan PPh Pasal 21, SPT Masa PPh Pasal 21, Wajib Pajak

PERANCANGAN DAN PEMBUATAN SISTEM INFORMASI PENGGAJIAN DAN LAPORAN SPT TAHUNAN UNTUK KARYAWAN DI PERUSAHAAN KONVEKSI CV. X

Directory of Open Access Journals (Sweden)

Yulia Yulia

2009-01-01

Full Text Available CV. X is a garment company which has 55 employees. Every end of the month the company doing payroll calculations and every year make an annual tax report (Notice Letter / SPT. Currently the calculation of salaries and the making of SPT are still done manually and thus require a long time and the large possibility of miscalculation. In this research, we have designed a payroll information system and SPT reporting, including employee attendance system, payroll calculation system and the various attribute such as overtime, furlough, deductions and so forth automatically. This system is able to do the calculation and reporting of annual tax returns and tax correction automatically. Besides, the system is also able to create various reports such as salary statements, tax cuts statements, the report to the director of such employee data report, employee history report, and payroll report. From the test results of payroll information systems and SPT report, can be seen that the system is able to perform the calculation of salaries and annual tax returns correctly. Abstract in Bahasa Indonesia: CV. X merupakan sebuah perusahaan koveksi dengan total karyawan sekitar 55 orang. Setiap akhir bulan perusahaan melakukan penghitungan gaji karyawan dan tiap tahun melakukan pembuatan laporan pajak tahunan (Surat Pemberitahuan/SPT. Saat ini penghitungan gaji dan pembuatan SPT ini masih dilakukan secara manual sehingga membutuhkan waktu yang lama serta besar kemungkinan terjadinya kesalahan perhitungan. Pada penelitian ini, dirancang sebuah sistem informasi penggajian dan pembuatan laporan SPT, antara lain meliputi sistem absensi karyawan, sistem penghitungan gaji dan berbagai attributnya seperti lembur, cuti, potongan dan sebagainya secara terotomatisasi. Sistem ini juga mampu melakukan penghitungan dan pembuatan laporan SPT tahunan serta koreksi pajak secara terotomatisasi. Selain itu sistem juga mampu membuat berbagai laporan baik yang ditujukan untuk karyawan

Early-onset severe hereditary sensory and autonomic neuropathy type 1 with S331F SPTLC1 mutation.

Science.gov (United States)

Suh, Bum Chun; Hong, Young Bin; Nakhro, Khriezhanuo; Nam, Soo Hyun; Chung, Ki Wha; Choi, Byung-Ok

2014-02-01

Hereditary sensory and autonomic neuropathy type I (HSAN I) is an autosomal dominant disease characterized by prominent sensory impairment, resulting in foot ulcers or amputations and has a juvenile to adult onset. The major underlying causes of HSAN I are mutations in SPTLC1, which encodes the first subunit of serine palmitoyltransferase (SPT). To date, there have been no reports with regard to an HSAN patient of Korean origin. In this report we discussed an HSAN I patient with a missense mutation in SPTLC1 (c.992C>T: p.S331F). The patient had noticed frequent falls, lower leg weakness and hand tremors at age five. The patient also presented with foot ulcers, muscle hypotrophy, cataracts, hoarseness, vocal cord palsy and respiratory difficulties and succumbed to the condition at the age of 28 years. In accordance with previous reports, a mutation in Ser331 in the present patient was associated with early-onset and a severe phenotype. Therefore, Ser331 in SPTLC1 is a crucial amino acid, which characterizes the HSAN I phenotype.

Phosphorylation of mouse serine racemase regulates D-serine synthesis

DEFF Research Database (Denmark)

Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

2010-01-01

Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

Directory of Open Access Journals (Sweden)

Fikadu G Tafesse

2015-10-01

Full Text Available The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Science.gov (United States)

Blythe, Amanda; Gunasekara, Sanjika; Walshe, James; Mackay, Joel P; Hartzog, Grant A; Vrielink, Alice

2014-08-01

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5. Copyright © 2014 Elsevier Inc. All rights reserved.

Serine biosynthesis and transport defects.

Science.gov (United States)

El-Hattab, Ayman W

2016-07-01

l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

A 205 Hour Krypton Propellant Life Test of the SPT-100 Operating at 3 kW

Science.gov (United States)

2013-09-01

nominal xenon condition (1.35 kW), tests have validated the SPT -100 life time as exceeding 2.71 million N -s (equivalent to approximately 9,000 hours of...condition – If correlation between erosion and energy throughput holds, SPT may be able to endure • Long term life test would be required to validate Kr as...shift (to zero center of SPT at r = 0) • Small rotation to correct for linear stage sag DISTRIBUTION A: Approved for public release; distribution

Preventative effect of OMZ-SPT on lipopolysaccharide-induced acute lung injury and inflammation via nuclear factor-kappa B signaling in mice

International Nuclear Information System (INIS)

Wang, Ting; Hou, Wanru; Fu, Zhou

2017-01-01

Acute lung injury (ALI) is an early pathophysiologic change in acute respiratory distress syndrome and its management can be challenging. Omalizumab (Xolair™) is a recombinant DNA-derived, humanized antibody. OMZ-SPT is a polypeptide on the heavy chain of omalizumab monoclonal antibody. Here, we found that intramuscular administration of OMZ-SPT significantly improved survival and attenuated lung inflammation in female C57BL/6 mice suffering from lipopolysaccharide (LPS)-induced ALI. We also demonstrated that OMZ-SPT can inhibit expression of the inflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 by ELISA in mice suffering from LPS-induced ALI and a mouse macrophage line (RAW264.7 cells). In addition, we showed that OMZ-SPT inhibited LPS-induced activation of nuclear factor-kappa B (NF-κB) signaling and total expression of NF-κB by western blotting. These data suggest that OMZ-SPT could be a novel therapeutic choice for ALI. - Highlights: • OMZ-SPT is a polypeptide on the heavy chain of omalizumab monoclonal antibody. • Omalizumab (Xolair™) have anti-inflammatory effects. • OMZ-SPT can inhibit inflammatory responses and lung injury in LPS-induced ALI mice. • Protective effect of OMZ-SPT on ALI is due to inhibition of NF-κB signaling. • OMZ-SPT could be a novel therapeutic choice for ALI.

Overburden Stress Normalization and Rod Length Corrections for the Standard Penetration Test (SPT)

OpenAIRE

Deger, Tonguc Tolga

2014-01-01

The Standard Penetration Test (SPT) has been a staple of geotechnical engineering practice for more than 70 years. Empirical correlations based on in situ SPT data provide an important basis for assessment of a broad range of engineering parameters, and for empirically based analysis and design methods spanning a significant number of areas of geotechnical practice. Despite this longstanding record of usage, the test itself is relatively poorly standardized with regard to the allowable variab...

Low-Power Operation and Plasma Characterization of a Qualification Model SPT-140 Hall Thruster for NASA Science Missions

Science.gov (United States)

Garner, Charles E.; Jorns, Benjamin A.; van Derventer, Steven; Hofer, Richard R.; Rickard, Ryan; Liang, Raymond; Delgado, Jorge

2015-01-01

Hall thruster systems based on commercial product lines can potentially lead to lower cost electric propulsion (EP) systems for deep space science missions. A 4.5-kW SPT-140 Hall thruster presently under qualification testing by SSL leverages the substantial heritage of the SPT-100 being flown on Russian and US commercial satellites. The Jet Propulsion Laboratory is exploring the use of commercial EP systems, including the SPT-140, for deep space science missions, and initiated a program to evaluate the SPT-140 in the areas of low power operation and thruster operating life. A qualification model SPT-140 designated QM002 was evaluated for operation and plasma properties along channel centerline, from 4.5 kW to 0.8 kW. Additional testing was performed on a development model SPT-140 designated DM4 to evaluate operation with a Moog proportional flow control valve (PFCV). The PFCV was commanded by an SSL engineering model PPU-140 Power Processing Unit (PPU). Performance measurements on QM002 at 0.8 kW discharge power were 50 mN of thrust at a total specific impulse of 1250 s, a total thruster efficiency of 0.38, and discharge current oscillations of under 3% of the mean current. Steady-state operation at 0.8 kW was demonstrated during a 27 h firing. The SPT-140 DM4 was operated in closed-loop control of the discharge current with the PFCV and PPU over discharge power levels of 0.8-4.5 kW. QM002 and DM4 test data indicate that the SPT-140 design is a viable candidate for NASA missions requiring power throttling down to low thruster input power.

New Insights into the Regulation of Serine Palmitoyltransferase

Science.gov (United States)

2014-02-07

34 Tetrad dissection and analysis of products of meiosis ...ORF - Open reading frame PHS - Phytosphingosine P.0.M-Products of meiosis SIP - S phingosine-1-phosphate SC - Sodium citrate SD - Synthetic...analysis of products of meiosis : Yeast mating and sporulation was done according to the standard protocols (79). Yeast transformation: All yeast

Sequence Classification: 889373 [

Lifescience Database Archive (English)

Full Text Available hat stimulates the activity of serine palmitoyltransferase (Lcb1p, Lcb2p) several-fold; involved in sphingolipid biosynthesis; Tsc3p || http://www.ncbi.nlm.nih.gov/protein/14270689 ...

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Directory of Open Access Journals (Sweden)

Yuichi Hirata

Full Text Available Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2 encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Science.gov (United States)

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways

DEFF Research Database (Denmark)

2016-01-01

The present invention generally relates to the microbiological industry, and specifically to the production of L-serine using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes...... concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms....

Maps of the Southern Millimeter-wave Sky from Combined 2500 deg$^2$ SPT-SZ and Planck Temperature Data

Energy Technology Data Exchange (ETDEWEB)

Chown, R.; et al.

2018-03-28

We present three maps of the millimeter-wave sky created by combining data from the South Pole Telescope (SPT) and the Planck satellite. We use data from the SPT-SZ survey, a survey of 2540 deg$^2$ of the the sky with arcminute resolution in three bands centered at 95, 150, and 220 GHz, and the full-mission Planck temperature data in the 100, 143, and 217 GHz bands. A linear combination of the SPT-SZ and Planck data is computed in spherical harmonic space, with weights derived from the noise of both instruments. This weighting scheme results in Planck data providing most of the large-angular-scale information in the combined maps, with the smaller-scale information coming from SPT-SZ data. A number of tests have been done on the maps. We find their angular power spectra to agree very well with theoretically predicted spectra and previously published results.

Neonatal disruption of serine racemase causes schizophrenia-like behavioral abnormalities in adulthood: clinical rescue by d-serine.

Directory of Open Access Journals (Sweden)

Hiroko Hagiwara

Full Text Available D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA receptor, is synthesized from L-serine by serine racemase (SRR. Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life.Neonatal mice (7-9 days were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day, an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT, and prepulse inhibition (PPI were performed at juvenile (5-6 weeks old and adult (10-12 weeks old stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70 significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure.This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult.

Improvement of Lead Tolerance of Saccharomyces cerevisiae by Random Mutagenesis of Transcription Regulator SPT3.

Science.gov (United States)

Zhu, Liying; Gao, Shan; Zhang, Hongman; Huang, He; Jiang, Ling

2018-01-01

Bioremediation of heavy metal pollution with biomaterials such as bacteria and fungi usually suffer from limitations because of microbial sensitivity to high concentration of heavy metals. Herein, we adopted a novel random mutagenesis technique called RAISE to manipulate the transcription regulator SPT3 of Saccharomyces cerevisiae to improve cell lead tolerance. The best strain Mutant VI was selected from the random mutagenesis libraries on account of the growth performance, with higher specific growth rate than the control strain (0.068 vs. 0.040 h -1 ) at lead concentration as high as 1.8 g/L. Combined with the transcriptome analysis of S. cerevisiae, expressing the SPT3 protein was performed to make better sense of the global regulatory effects of SPT3. The data analysis revealed that 57 of S. cerevisiae genes were induced and 113 genes were suppressed, ranging from those for trehalose synthesis, carbon metabolism, and nucleotide synthesis to lead resistance. Especially, the accumulation of intracellular trehalose in S. cerevisiae under certain conditions of stress is considered important to lead resistance. The above results represented that SPT3 was acted as global transcription regulator in the exponential phase of strain and accordingly improved heavy metal tolerance in the heterologous host S. cerevisiae. The present study provides a route to complex phenotypes that are not readily accessible by traditional methods.

[Protective activity of S-PT84, a heat-killed preparation of Lactobacillus pentosus, against oral and gastric candidiasis in an experimental murine model].

Science.gov (United States)

Hayama, Kazumi; Ishijima, Sanae; Ono, Yoshiko; Izumo, Takayuki; Ida, Masayuki; Shibata, Hiroshi; Abe, Shigeru

2014-01-01

The effect of S-PT84, a heat-killed preparation of Lactobacillus pentosus on growth of Candida albicans was examined in vitro and in vivo. The mycelial growth was effectively inhibited by S-PT84 and seemed to bind to the hyphae. We assessed the potential of S-PT84 for treatment of oral and gastric candidiasis using a murine model. When 2 mg of S-PT84 was administered three times into the oral cavity of orally Candida infected mice, the score of lesions on the tongue was improved on day 2. When 50 μl and 200 μl of S-PT84 (10 mg/ml) were administered three times into the oral cavity (0.5 mg × 3) and the stomach (2 mg × 3) of the same mouse model, the number of viable Candida cells in the stomach was reduced significantly on day 2. These findings suggest the possibility that S-PT84 has potential as a food ingredient supporting anti-Candida treatment, especially for Candida infection in the gastrointestinal tract.

Fast SPT-Term Allocation and Efficient FPGA Implementation of FIR Filters for Software Defined Radio Applications

OpenAIRE

Bae, Choonghoon; Moteki, Takayuki; Kim, Minseok; Ichige, Koichi; Arai, Hiroyuki

2009-01-01

This paper presents a fast SPT-term allocation scheme and an efficient FPGA implementation of FIR filters for Software Defined Radio (SDR) applications. Direct conversion method based on RF direct sampling is nowadays widely used in SDR applications. Fast and accurate digital filters are required for RF direct sampling and processing in direct conversion, however such filters often require large digital circuit area. Signed-Power-of-Two (SPT) terms will be suitable for fast processing and eff...

The Spt-Ada-Gcn5 Acetyltransferase (SAGA complex in Aspergillus nidulans.

Directory of Open Access Journals (Sweden)

Paraskevi Georgakopoulos

Full Text Available A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

Space power technology for the twenty-first century (SPT21)

International Nuclear Information System (INIS)

Borger, W.U.; Massie, L.D.

1988-01-01

During the spring and summer months of 1987, the Aero Propulsion Laboratory of the Air Force Wright Aeronautical Laboratories, Wright-Patterson AFB, Ohio in cooperation with the Air Force Space Technology Center at Kirtland AFB, New Mexico, undertook an initiative to develop a Strategic Plan for Space Power Technology Development. The initiative was called SPT21, Space Power Technology for the Twenty-First Century. The planning process involved the participation of other Government organizations (U.S. Army, Navy, DOE and NASA) along with major aerospace companies and universities. Following an SPT21 kickoff meeting on 28 May 1987, detailed strategic planning was accomplished through seven (7) Space Power Technology Discipline Workshops commencing in June 1987 and concluding in August 1987. Technology Discipline Workshops were conducted in the following areas: (1) Solar Thermal Dynamic Power Systems (2) Solar Photovoltaic Cells and Arrays (3) Thermal Management Technology (4) Energy Storage Technology (5) Nuclear Power Systems Technology (6) Power Conditioning, Distribution and Control and (7) Systems Technology/Advanced Concepts. This technical paper summarizes the planning process and describes the salient findings and conclusions of the workshops

Glycine serine interconversion in the rooster

International Nuclear Information System (INIS)

Sugahara, Michihiro; Kandatsu, Makoto

1976-01-01

Serine was isolated by the column chromatography from the hydrolyzates of proteins of the serum, the liver and the pectoral muscle which were obtained from the roosters fed a diet containing 2- 14 C glycine for 16 - 17 days. The carbon chain of serine was cut off by treating with sodium periodate. The specific activity of each carbon (as barium carbonate) was estimated. Carboxyl carbon had little radioactivity. The specific activity of hydroxymethyl carbon was 10 - 19% of that of methylene carbon. Glycine isolated from the same hydrolyzates was degraded by ninhydrin oxidation. Formaldehyde produced from 2-C was oxidized to carbon dioxide by treating with mercuric chloride. Carboxyl carbon had little radioactivity. The specific activities of 2-C of glycine and 2-C of serine in the same tissue protein were compared. The ratio of serine 2-C/glycine 2-C was between 0.7 - 1.5. These results seem to indicate that glycine directly converts to serine in the rooster. The quantitative significance of the pathways of glycine (serine) biosynthesis is discussed. (auth.)

D-serine increases adult hippocampal neurogenesis

Directory of Open Access Journals (Sweden)

Sebastien eSultan

2013-08-01

Full Text Available Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. The neurogenic niche regulates the stem cell proliferation and the differentiation and survival of new neurons and a major contributor to the neurogenic niche are astrocytes. Among the molecules secreted by astrocytes, D-serine is an important gliotransmitter and is a co-agonist of the glutamate, N-methyl-D-aspartate (NMDA receptor. D-serine has been shown to enhance the proliferation of neural stem cells in vitro, but its effect on adult neurogenesis in vivo is unknown. Here, we tested the effect of exogenous administration of D-serine on adult neurogenesis in the mouse dentate gyrus. We found that 1 week of treatment with D-serine increased cell proliferation in vivo and in vitro and increased the density of neural stem cells and transit amplifying progenitors. Furthermore, D-serine increased the survival of newborn neurons. Together, these results indicate that D-serine treatment resulted in the improvement of several steps of adult neurogenesis in vivo.

Large arrays of dual-polarized multichroic TES detectors for CMB measurements with the SPT-3G receiver

Science.gov (United States)

Posada, Chrystian M.; Ade, Peter A. R.; Anderson, Adam J.; Avva, Jessica; Ahmed, Zeeshan; Arnold, Kam S.; Austermann, Jason; Bender, Amy N.; Benson, Bradford A.; Bleem, Lindsey; Byrum, Karen; Carlstrom, John E.; Carter, Faustin W.; Chang, Clarence; Cho, Hsiao-Mei; Cukierman, Ari; Czaplewski, David A.; Ding, Junjia; Divan, Ralu N. S.; de Haan, Tijmen; Dobbs, Matt; Dutcher, Daniel; Everett, Wenderline; Gannon, Renae N.; Guyser, Robert J.; Halverson, Nils W.; Harrington, Nicholas L.; Hattori, Kaori; Henning, Jason W.; Hilton, Gene C.; Holzapfel, William L.; Huang, Nicholas; Irwin, Kent D.; Jeong, Oliver; Khaire, Trupti; Korman, Milo; Kubik, Donna L.; Kuo, Chao-Lin; Lee, Adrian T.; Leitch, Erik M.; Lendinez Escudero, Sergi; Meyer, Stephan S.; Miller, Christina S.; Montgomery, Joshua; Nadolski, Andrew; Natoli, Tyler J.; Nguyen, Hogan; Novosad, Valentyn; Padin, Stephen; Pan, Zhaodi; Pearson, John E.; Rahlin, Alexandra; Reichardt, Christian L.; Ruhl, John E.; Saliwanchik, Benjamin; Shirley, Ian; Sayre, James T.; Shariff, Jamil A.; Shirokoff, Erik D.; Stan, Liliana; Stark, Antony A.; Sobrin, Joshua; Story, Kyle; Suzuki, Aritoki; Tang, Qing Yang; Thakur, Ritoban B.; Thompson, Keith L.; Tucker, Carole E.; Vanderlinde, Keith; Vieira, Joaquin D.; Wang, Gensheng; Whitehorn, Nathan; Yefremenko, Volodymyr; Yoon, Ki Won

2016-07-01

Detectors for cosmic microwave background (CMB) experiments are now essentially background limited, so a straightforward alternative to improve sensitivity is to increase the number of detectors. Large arrays of multichroic pixels constitute an economical approach to increasing the number of detectors within a given focal plane area. Here, we present the fabrication of large arrays of dual-polarized multichroic transition-edge-sensor (TES) bolometers for the South Pole Telescope third-generation CMB receiver (SPT-3G). The complete SPT-3G receiver will have 2690 pixels, each with six detectors, allowing for individual measurement of three spectral bands (centered at 95 GHz, 150 GHz and 220 GHz) in two orthogonal polarizations. In total, the SPT-3G focal plane will have 16140 detectors. Each pixel is comprised of a broad-band sinuous antenna coupled to a niobium microstrip transmission line. In-line filters are used to define the different band-passes before the millimeter-wavelength signal is fed to the respective Ti/Au TES sensors. Detectors are read out using a 64x frequency domain multiplexing (fMux) scheme. The microfabrication of the SPT-3G detector arrays involves a total of 18 processes, including 13 lithography steps. Together with the fabrication process, the effect of processing on the Ti/Au TES's Tc is discussed. In addition, detectors fabricated with Ti/Au TES films with Tc between 400 mK 560 mK are presented and their thermal characteristics are evaluated. Optical characterization of the arrays is presented as well, indicating that the response of the detectors is in good agreement with the design values for all three spectral bands (95 GHz, 150 GHz, and 220 GHz). The measured optical efficiency of the detectors is between 0.3 and 0.8. Results discussed here are extracted from a batch of research of development wafers used to develop the baseline process for the fabrication of the arrays of detectors to be deployed with the SPT-3G receiver. Results from

Crystal Structure of Rat Carnitine Palmitoyltransferase II (CPT-II)

Energy Technology Data Exchange (ETDEWEB)

Hsiao,Y.; Jogl, G.; Esser, V.; Tong, L.

2006-01-01

Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the {beta}-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9 Angstroms resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.

Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

Science.gov (United States)

Montefusco, David J; Newcomb, Benjamin; Gandy, Jason L; Brice, Sarah E; Matmati, Nabil; Cowart, L Ashley; Hannun, Yusuf A

2012-03-16

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

Understanding serine proteases implications on Leishmania spp lifecycle.

Science.gov (United States)

Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

2018-01-01

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased tolerance towards serine obtained by adaptive laboratory evolution

DEFF Research Database (Denmark)

Mundhada, Hemanshu; Seoane, Jose Miguel; Koza, Anna

2014-01-01

The amino acid serine has previously been identified as one of the top 30 candidates of value added chemicals, making the production of serine from glucose attractive. Production of serine have previously been attempted in E. coli and C. glutamicum, however, titers sufficient for commercial...... by glyA), the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. As expected, the resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution....... During the evolution experiment, the serine tolerance was increased substantially. Genome re-sequencing was subsequently used to analyze the genotype of a number of selected strains. These results reveal insights towards the adaptation process as well as the mechanism of serine tolerance....

Carnitine palmitoyltransferase IA polymorphism P479L is common in Greenland Inuit and is associated with elevated plasma apolipoprotein A-I

DEFF Research Database (Denmark)

Rajakumar, Chandheeb; Ban, Matthew R; Cao, Henian

2009-01-01

Carnitine palmitoyltransferase IA, encoded by CPT1A, is a key regulator of fatty acid metabolism. Previously, a loss of function mutation, namely c.1436 CT (p.P479L), was reported in CPT1A in the homozygous state in Canadian aboriginal male with presumed CPT1A deficiency. In order to determine...

SPT0346-52: NEGLIGIBLE AGN ACTIVITY IN A COMPACT, HYPER-STARBURST GALAXY AT z = 5.7

Energy Technology Data Exchange (ETDEWEB)

Ma, Jingzhe; Gonzalez, Anthony H. [Department of Astronomy, University of Florida, Gainesville, FL 32611 (United States); Vieira, J. D.; Sreevani, J. [Department of Astronomy and Department of Physics, University of Illinois, 1002 West Green St., Urbana, IL 61801 (United States); Aravena, M. [Núcleo de Astronomía, Facultad de Ingeniería, Universidad Diego Portales, Av. Ejército 441, Santiago (Chile); Ashby, M. L. N. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States); Béthermin, M.; Breuck, C. de; Gullberg, B. [European Southern Observatory, Karl Schwarzschild Straße 2, D-85748 Garching (Germany); Bothwell, M. S. [Cavendish Laboratory, University of Cambridge, JJ Thompson Ave, Cambridge CB3 0HA (United Kingdom); Brandt, W. N. [Institute for Gravitation and the Cosmos, The Pennsylvania State University, University Park, PA 16802 (United States); Carlstrom, J. E. [Kavli Institute for Cosmological Physics, University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637 (United States); Chapman, S. C. [Dalhousie University, Halifax, Nova Scotia (Canada); Hezaveh, Y. [Kavli Institute for Particle Astrophysics and Cosmology, Stanford University, Stanford, CA 94305 (United States); Litke, K.; Marrone, D. P.; Spilker, J. S. [Steward Observatory, University of Arizona, 933 North Cherry Avenue, Tucson, AZ 85721 (United States); Malkan, M. [Department of Physics and Astronomy, University of California, Los Angeles, CA 90095-1547 (United States); McDonald, M. [Kavli Institute for Astrophysics and Space Research, Massachusetts Institute of Technology, 37-582C, Cambridge, MA 02139 (United States); Murphy, E. J., E-mail: jingzhema@ufl.edu [National Radio Astronomy Observatory, 520 Edgemont Road, Charlottesville, VA 22903 (United States); and others

2016-12-01

We present Chandra ACIS-S and Australia Telescope Compact Array (ATCA) radio continuum observations of the strongly lensed dusty, star-forming galaxy SPT-S J034640-5204.9 (hereafter SPT0346-52) at z = 5.656. This galaxy has also been observed with ALMA, HST , Spitzer , Herschel , Atacama Pathfinder EXperiment, and the Very Large Telescope. Previous observations indicate that if the infrared (IR) emission is driven by star formation, then the inferred lensing-corrected star formation rate (SFR) (∼4500 M {sub ☉} yr{sup −1}) and SFR surface density Σ{sub SFR} (∼2000 M {sub ☉} yr{sup −1} kpc{sup −2}) are both exceptionally high. It remained unclear from the previous data, however, whether a central active galactic nucleus (AGN) contributes appreciably to the IR luminosity. The Chandra upper limit shows that SPT0346-52 is consistent with being star formation dominated in the X-ray, and any AGN contribution to the IR emission is negligible. The ATCA radio continuum upper limits are also consistent with the FIR-to-radio correlation for star-forming galaxies with no indication of an additional AGN contribution. The observed prodigious intrinsic IR luminosity of (3.6 ± 0.3) × 10{sup 13} L {sub ☉} originates almost solely from vigorous star formation activity. With an intrinsic source size of 0.61 ± 0.03 kpc, SPT0346-52 is confirmed to have one of the highest Σ{sub SFR} of any known galaxy. This high Σ{sub SFR}, which approaches the Eddington limit for a radiation pressure supported starburst, may be explained by a combination of very high star formation efficiency and gas fraction.

SPT0346-52: NEGLIGIBLE AGN ACTIVITY IN A COMPACT, HYPER-STARBURST GALAXY AT z = 5.7

International Nuclear Information System (INIS)

Ma, Jingzhe; Gonzalez, Anthony H.; Vieira, J. D.; Sreevani, J.; Aravena, M.; Ashby, M. L. N.; Béthermin, M.; Breuck, C. de; Gullberg, B.; Bothwell, M. S.; Brandt, W. N.; Carlstrom, J. E.; Chapman, S. C.; Hezaveh, Y.; Litke, K.; Marrone, D. P.; Spilker, J. S.; Malkan, M.; McDonald, M.; Murphy, E. J.

2016-01-01

We present Chandra ACIS-S and Australia Telescope Compact Array (ATCA) radio continuum observations of the strongly lensed dusty, star-forming galaxy SPT-S J034640-5204.9 (hereafter SPT0346-52) at z = 5.656. This galaxy has also been observed with ALMA, HST , Spitzer , Herschel , Atacama Pathfinder EXperiment, and the Very Large Telescope. Previous observations indicate that if the infrared (IR) emission is driven by star formation, then the inferred lensing-corrected star formation rate (SFR) (∼4500 M ☉ yr −1 ) and SFR surface density Σ SFR (∼2000 M ☉ yr −1 kpc −2 ) are both exceptionally high. It remained unclear from the previous data, however, whether a central active galactic nucleus (AGN) contributes appreciably to the IR luminosity. The Chandra upper limit shows that SPT0346-52 is consistent with being star formation dominated in the X-ray, and any AGN contribution to the IR emission is negligible. The ATCA radio continuum upper limits are also consistent with the FIR-to-radio correlation for star-forming galaxies with no indication of an additional AGN contribution. The observed prodigious intrinsic IR luminosity of (3.6 ± 0.3) × 10 13 L ☉ originates almost solely from vigorous star formation activity. With an intrinsic source size of 0.61 ± 0.03 kpc, SPT0346-52 is confirmed to have one of the highest Σ SFR of any known galaxy. This high Σ SFR , which approaches the Eddington limit for a radiation pressure supported starburst, may be explained by a combination of very high star formation efficiency and gas fraction.

Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

Directory of Open Access Journals (Sweden)

Claudio Laurido

2012-01-01

Full Text Available N-methyl-D-aspartic acid receptor (NMDAr activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR. D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS and L-erythro-3-hydroxyaspartate (LEHA, among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL and LEHA (100 μg/10 μL in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

Joint measurement of lensing–galaxy correlations using SPT and DES SV data

Energy Technology Data Exchange (ETDEWEB)

Baxter, E.; Clampitt, J.; Giannantonio, T.; Dodelson, S.; Jain, B.; Huterer, D.; Bleem, L.; Crawford, T.; Efstathiou, G.; Fosalba, P.; Kirk, D.; Kwan, J.; Sánchez, C.; Story, K.; Troxel, M. A.; Abbott, T. M. C.; Abdalla, F. B.; Armstrong, R.; Benoit-Lévy, A.; Benson, B.; Bernstein, G. M.; Bernstein, R. A.; Bertin, E.; Brooks, D.; Carlstrom, J.; Rosell, A. Carnero; Carrasco Kind, M.; Carretero, J.; Chown, R.; Crocce, M.; Cunha, C. E.; da Costa, L. N.; Desai, S.; Diehl, H. T.; Dietrich, J. P.; Doel, P.; Evrard, A. E.; Fausti Neto, A.; Flaugher, B.; Frieman, J.; Gruen, D.; Gruendl, R. A.; Gutierrez, G.; de Haan, T.; Holder, G.; Honscheid, K.; Hou, Z.; James, D. J.; Kuehn, K.; Kuropatkin, N.; Lima, M.; March, M.; Marshall, J. L.; Martini, P.; Melchior, P.; Miller, C. J.; Miquel, R.; Mohr, J. J.; Nord, B.; Omori, Y.; Plazas, A. A.; Reichardt, C.; Romer, A. K.; Rykoff, E. S.; Sanchez, E.; Sevilla-Noarbe, I.; Sheldon, E.; Smith, R. C.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Stark, A.; Swanson, M. E. C.; Tarle, G.; Thomas, D.; Walker, A. R.; Wechsler, R. H.

2016-07-04

We measure the correlation of galaxy lensing and cosmic microwave background lensing with a set of galaxies expected to trace the matter density field. The measurements are performed using pre-survey Dark Energy Survey (DES) Science Verification optical imaging data and millimetre-wave data from the 2500 sq. deg. South Pole Telescope Sunyaev–Zel'dovich (SPT-SZ) survey. The two lensing–galaxy correlations are jointly fit to extract constraints on cosmological parameters, constraints on the redshift distribution of the lens galaxies, and constraints on the absolute shear calibration of DES galaxy-lensing measurements. We show that an attractive feature of these fits is that they are fairly insensitive to the clustering bias of the galaxies used as matter tracers. The measurement presented in this work confirms that DES and SPT data are consistent with each other and with the currently favoured Λ cold dark matter cosmological model. It also demonstrates that joint lensing–galaxy correlation measurement considered here contains a wealth of information that can be extracted using current and future surveys.

A MEASUREMENT OF SECONDARY COSMIC MICROWAVE BACKGROUND ANISOTROPIES FROM THE 2500 SQUARE-DEGREE SPT-SZ SURVEY

Energy Technology Data Exchange (ETDEWEB)

George, E. M.; Reichardt, C. L.; Aird, K. A.; Benson, B. A.; Bleem, L. E.; Carlstrom, J. E.; Chang, C. L.; Cho, H-M.; Crawford, T. M.; Crites, A. T.; de Haan, T.; Dobbs, M. A.; Dudley, J.; Halverson, N. W.; Harrington, N. L.; Holder, G. P.; Holzapfel, W. L.; Hou, Z.; Hrubes, J. D.; Keisler, R.; Knox, L.; Lee, A. T.; Leitch, E. M.; Lueker, M.; Luong-Van, D.; McMahon, J. J.; Mehl, J.; Meyer, S. S.; Millea, M.; Mocanu, L. M.; Mohr, J. J.; Montroy, T. E.; Padin, S.; Plagge, T.; Pryke, C.; Ruhl, J. E.; Schaffer, K. K.; Shaw, L.; Shirokoff, E.; Spieler, H. G.; Staniszewski, Z.; Stark, A. A.; Story, K. T.; van Engelen, A.; Vanderlinde, K.; Vieira, J. D.; Williamson, R.; Zahn, O.

2015-01-28

We present measurements of secondary cosmic microwave background (CMB) anisotropies and cosmic infrared background (CIB) fluctuations using data from the South Pole Telescope (SPT) covering the complete 2540 deg(2) SPT-SZ survey area. Data in the three SPT-SZ frequency bands centered at 95, 150, and 220 GHz, are used to produce six angular power spectra (three single-frequency auto-spectra and three cross-spectra) covering the multipole range 2000 < ℓ < 11, 000 (angular scales 5' gsim θ gsim 1'). These are the most precise measurements of the angular power spectra at ℓ > 2500 at these frequencies. The main contributors to the power spectra at these angular scales and frequencies are the primary CMB, CIB, thermal and kinematic Sunyaev-Zel'dovich effects (tSZ and kSZ), and radio galaxies. We include a constraint on the tSZ power from a measurement of the tSZ bispectrum from 800 deg(2) of the SPT-SZ survey. We measure the tSZ power at 143  GHz to be $D^{\\rm tSZ}_{3000} = 4.08^{+0.58}_{-0.67}\\,\\mu {\\rm K}^2{}$ and the kSZ power to be $D^{\\rm kSZ}_{3000} = 2.9 \\pm 1.3\\, \\mu {\\rm K}^2{}$. The data prefer positive kSZ power at 98.1% CL. We measure a correlation coefficient of $\\xi = 0.113^{+0.057}_{-0.054}$ between sources of tSZ and CIB power, with ξ < 0 disfavored at a confidence level of 99.0%. The constraint on kSZ power can be interpreted as an upper limit on the duration of reionization. When the post-reionization homogeneous kSZ signal is accounted for, we find an upper limit on the duration Δz < 5.4  at 95% CL.

Performance Evaluation of the SPT-140

Science.gov (United States)

Manzella, David; Sarmiento, Charles; Sankovic, John; Haag, Tom

1997-01-01

As part of an on-going cooperative program with industry, an engineering model SPT-140 Hall thruster, which may be suitable for orbit insertion and station-keeping of geosynchronous communication satellites, was evaluated with respect to thrust and radiated electromagnetic interference at the NASA Lewis Research Center. Performance measurements were made using a laboratory model propellant feed system and commercial power supplies. The engine was operated in a space simulation chamber capable of providing background pressures of 4 x 10(exp -6) Torr or less during thruster operation. Thrust was measured at input powers ranging from 1.5 to 5 kilowatts with two different output filter configurations. The broadband electromagnetic emission spectra generated by the engine was also measured for a range of frequencies from 0.01 to 18,000 Mhz. These results are compared to the noise threshold of the measurement system and MIL-STD-461C where appropriate.

Microbial Production of l-Serine from Renewable Feedstocks.

Science.gov (United States)

Zhang, Xiaomei; Xu, Guoqiang; Shi, Jinsong; Koffas, Mattheos A G; Xu, Zhenghong

2018-07-01

l-Serine is a non-essential amino acid that has wide and expanding applications in industry with a fast-growing market demand. Currently, extraction and enzymatic catalysis are the main processes for l-serine production. However, such approaches limit the industrial-scale applications of this important amino acid. Therefore, shifting to the direct fermentative production of l-serine from renewable feedstocks has attracted increasing attention. This review details the current status of microbial production of l-serine from renewable feedstocks. We also summarize the current trends in metabolic engineering strategies and techniques for the typical industrial organisms Corynebacterium glutamicum and Escherichia coli that have been developed to address and overcome major challenges in the l-serine production process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analisis Penerapan E-spt Dan E-filing Dalam Upaya Peningkatan Kepatuhan Wajib Pajak (Studi Kasus Di Kantor Pelayanan Pajak Pratama Manado)

OpenAIRE

Tumuli, Apriliani Kasandra; Sondakh, Jullie J; Wokas, Heince R.N

2016-01-01

Direktorat Jenderal Pajak melakukan berbagai inovasi baru dalam bidang perpajakan yaitu dengan pemanfaatan teknologi dan informasi sehingga terciptalah aplikasi komputer dalam sistem administrasi perpajakan seperti e-SPT dan e-Filing untuk memudahklan para wajib pajak sehingga dapat meningkatkan kepatuhan wajib pajak dalam melaporkan kewajiban perpajakannya. Penelitian ini bertujuan untuk mengetahui tingkat penggunaan e-SPT dan e-Filing, tingkat kepatuhan wajib pajak yaitu ketepatan waktu mel...

Fundamentos e métodos de medidas de energia dinâmica no SPT

Directory of Open Access Journals (Sweden)

Antonio Belincanta

2000-05-01

Full Text Available Discutem-se neste trabalho os métodos disponíveis no Brasil e no exterior para a medida da energia transferida às hastes no SPT, apresentando-se inclusive registros típicos dos eventos dinâmicos correspondentes

Crystal Structure of Serine Racemase that Produces Neurotransmitter font-variant:small-caps">d-Serine for Stimulation of the NMDA Receptor

Science.gov (United States)

Goto, Masaru

font-variant:small-caps">d-Serine is an endogenous coagonist for the N-methyl-font-variant:small-caps">d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of font-variant:small-caps">l-serine to yield font-variant:small-caps">d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-font-variant:small-caps">d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-font-variant:small-caps">d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

Serine protease inhibitors of parasitic helminths.

Science.gov (United States)

Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

2012-05-01

Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.

Evaluation of SPT energy for Donut and Safety hammers using CPT measurements in Egypt

Directory of Open Access Journals (Sweden)

Rami M. El-Sherbiny

2013-12-01

Full Text Available Standard Penetration Test (SPT blow counts require correction prior to utilization in soil characterization and determination of properties and behavior. Among the most important corrections is the energy correction required to adjust the blow counts to 60% energy efficiency. However, there are no published data supporting commonly used value in Egypt. This paper presents an evaluation of the energy efficiency of the Donut and Safety hammers commonly used in Egypt and the associated energy correction factor. The energy efficiency is estimated by comparing N-values from the SPT to back-calculated N60 values from the Cone Penetration Test (CPT using well established correlations. Results indicate that the energy efficiency of the Donut hammer based on current practice in Egypt is approximately 50%. Thus, the back-calculated energy correction factor is approximately 0.82. For the Safety hammer, results indicate that the energy efficiency is approximately 60%, and the energy correction factor is approximately 1.0.

Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

Directory of Open Access Journals (Sweden)

Craig B Bennett

2008-01-01

Full Text Available BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1 to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34 and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1. Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII carboxy terminal domain (P-CTD, phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1

Heterogeneity of D-Serine Distribution in the Human Central Nervous System

Science.gov (United States)

Suzuki, Masataka; Imanishi, Nobuaki; Mita, Masashi; Hamase, Kenji; Aiso, Sadakazu

2017-01-01

D-serine is an endogenous ligand for N-methyl-D-aspartate glutamate receptors. Accumulating evidence including genetic associations of D-serine metabolism with neurological or psychiatric diseases suggest that D-serine is crucial in human neurophysiology. However, distribution and regulation of D-serine in humans are not well understood. Here, we found that D-serine is heterogeneously distributed in the human central nervous system (CNS). The cerebrum contains the highest level of D-serine among the areas in the CNS. There is heterogeneity in its distribution in the cerebrum and even within the cerebral neocortex. The neocortical heterogeneity is associated with Brodmann or functional areas but is unrelated to basic patterns of cortical layer structure or regional expressional variation of metabolic enzymes for D-serine. Such D-serine distribution may reflect functional diversity of glutamatergic neurons in the human CNS, which may serve as a basis for clinical and pharmacological studies on D-serine modulation. PMID:28604057

Neurotoxic 1-deoxysphingolipids and paclitaxel-induced peripheral neuropathy

Science.gov (United States)

Kramer, Rita; Bielawski, Jacek; Kistner-Griffin, Emily; Othman, Alaa; Alecu, Irina; Ernst, Daniela; Kornhauser, Drew; Hornemann, Thorsten; Spassieva, Stefka

2015-01-01

Peripheral neuropathy is a major dose-limiting side effect of paclitaxel and cisplatin chemotherapy. In the current study, we tested the involvement of a novel class of neurotoxic sphingolipids, the 1-deoxysphingolipids. 1-Deoxysphingolipids are produced when the enzyme serine palmitoyltransferase uses l-alanine instead of l-serine as its amino acid substrate. We tested whether treatment of cells with paclitaxel (250 nM, 1 µM) and cisplatin (250 nM, 1 µM) would result in elevated cellular levels of 1-deoxysphingolipids. Our results revealed that paclitaxel, but not cisplatin treatment, caused a dose-dependent elevation of 1-deoxysphingolipids levels and an increase in the message and activity of serine palmitoyltransferase (P peripheral neuropathy symptoms [evaluated by the European Organization for Research and Treatment of Cancer (EORTC) QLQ-chemotherapy-induced peripheral neuropathy-20 (CIPN20) instrument] and the 1-deoxysphingolipid plasma levels (measured by mass spectrometry) in 27 patients with breast cancer who were treated with paclitaxel chemotherapy. Our results showed that there was an association between the incidence and severity of neuropathy and the levels of very-long-chain 1-deoxyceramides such as C24 (P neuropathy (P peripheral neuropathy.—Kramer, R., Bielawski, J., Kistner-Griffin, E., Othman, A., Alecu, I., Ernst, D., Kornhauser, D., Hornemann, T., Spassieva, S. Neurotoxic 1-deoxysphingolipids and paclitaxel-induced peripheral neuropathy. PMID:26198449

Accumulation of long-chain bases in yeast promotes their conversion to a long-chain base vinyl ether

OpenAIRE

Martínez-Montañés, Fernando; Lone, Museer A.; Hsu, Fong-Fu; Schneiter3, Roger

2017-01-01

Long-chain bases (LCBs) are the precursors to ceramide and sphingolipids in eukaryotic cells. They are formed by the action of serine palmitoyl-CoA transferase (SPT), a complex of integral membrane proteins located in the endoplasmic reticulum. SPT activity is negatively regulated by Orm proteins to prevent the toxic overaccumulation of LCBs. Here we show that overaccumulation of LCBs in yeast results in their conversion to a hitherto undescribed LCB derivative, an LCB vinyl ether. The ...

D-serine : The right or wrong isoform?

NARCIS (Netherlands)

Fuchs, Sabine A; Berger, Ruud; de Koning, Tom J

2011-01-01

Only recently, d-amino acids have been identified in mammals. Of these, d-serine has been most extensively studied. d-Serine was found to play an important role as a neurotransmitter in the human central nervous system (CNS) by binding to the N-methyl-d-aspartate receptor (NMDAr), similar to

The augmentation of heat transfer in a pipe flow using a swirling perforated twisted (SPT) tape insert

Science.gov (United States)

Ahmad, Shahrokh; Oishe, Sadia Noon; Rahman, Md. Lutfor

2017-12-01

The purpose of this research work is to increase the heat transfer coefficient by operating the heat exchangers at smaller revolution per minute. This signifies an achievement of reduction of pressure drop corresponding to less operating cost. This study has used two types of SPT tape insert to observe the various heat transfer coefficient, heat transfer rate and heat transfer augmentation efficiency. One tape was fully twisted and another tape was partially twisted. The shape of the SPT tape creates turbulence effect. The turbulence flow (swirl flow) generated by SPT tape promotes greater mixing and high heat transfer coefficients. An arrangement scheme has been developed for the experimental investigation. For remarking the rate of change of heat transfer, temperature has been measured numerically through the temperature sensors with various flow rates and RPM. The volume flow rate was varied from 10.3448276 LPM to 21.045574 LPM and the rotation of the perforated twisted tape was varied from 50 RPM to 400 RPM. Finally the research study demonstrates the effectiveness of the results of the proposed approaches. It is observed that the suggested method of heat transfer augmentations is much more effective than existing methods, since it results in an increase in heat transfer area and also an increase in the heat transfer coefficient and reduction of cost in the industrial sectors.

Enhancement of L-Serine Production by Corynebacterium ...

African Journals Online (AJOL)

glutamicum SYPS-062 cultivation process for efficient production of L-serine on a large scale. ... central intermediate for a number of cellular .... impeller, oxygen and pH electrodes, under the ... equation. The yield of L-serine was regressed with respect to the medium ..... is not essential for activity but is required for inhibition.

A (Small) complexity performance contest: SPT versus LBFS

DEFF Research Database (Denmark)

Nielsen, Erland Hejn

2002-01-01

When discussing the nature of loading rules relevant to continuous dynamic job/flow shop systems, the general understanding is that results obtained in simpler structures of 2 to 4 machines are invariant to scaling and can be generalised without problems to more complex structures. The SPT...... results on pure re-entrant flow shop structures emerges. It now seems that alternative loading rules as for instance the LBFS (Last Buffer First Served) due to its strong long run stabilising property attracts quite some interest. To be more precise about the complexity aspect, complexity in job...... are not entirely only of theoretical interest, as well as results from a standard serial job/flow shop set-up, but with resource limitations that prevent the independent operations of the individual stations in the system....

A (Small) Complexity Performance Contest: SPT versus LBFS

DEFF Research Database (Denmark)

Nielsen, Erland Hejn

2002-01-01

When discussing the nature of loading rules relevant to continuous dynamic job/flow shop systems, the general understanding is that results obtained in simpler structures of 2 to 4 machines are invariant to scaling and can be generalised without problems to more complex structures. The SPT...... results on pure re-entrant flow shop structures emerges. It now seems that alternative loading rules as for instance the LBFS (Last Buffer First Served) due to its strong long run stabilising property attracts quite some interest. To be more precise about the complexity aspect, complexity in job...... are not entirely only of theoretical interest, as well as results from a standard serial job/flow shop set-up, but with resource limitations that prevent the independent operations of the individual stations in the system....

Serine:glyoxylate aminotransferase mutant of barley

International Nuclear Information System (INIS)

Blackwell, R.; Murray, A.; Joy, K.; Lea, P.

1987-01-01

A photorespiratory mutant of barley (LaPr 85/84), deficient in both of the major peaks of serine:glyoxylate aminotransferase activity detected in the wild type, also lacks serine:pyruvate and asparagine:glyoxylate aminotransferase activities. Genetic analysis of the mutation demonstrated that these three activities are all carried on the same enzyme. The mutant, when placed in air, accumulated a large pool of serine, showed the expected rate (50%) of ammonia release during photorespiration but produced CO 2 at twice the wild type rate when it was fed [ 14 C] glyoxylate. Compared with the wild type, LaPr 85/84 exhibited abnormal transient changes in chlorophyll a fluorescence when the CO 2 concentration of the air was altered, indicating that the rates of the fluorescence quenching mechanisms were affected in vivo by the lack of this enzyme

Amperometric Self-Referencing Ceramic Based Microelectrode Arrays for D-Serine Detection.

Science.gov (United States)

Campos-Beltrán, Diana; Konradsson-Geuken, Åsa; Quintero, Jorge E; Marshall, Lisa

2018-03-06

D-serine is the major D-amino acid in the mammalian central nervous system. As the dominant co-agonist of the endogenous synaptic NMDA receptor, D-serine plays a role in synaptic plasticity, learning, and memory. Alterations in D-serine are linked to neuropsychiatric disorders including schizophrenia. Thus, it is of increasing interest to monitor the concentration of D-serine in vivo as a relevant player in dynamic neuron-glia network activity. Here we present a procedure for amperometric detection of D-serine with self-referencing ceramic-based microelectrode arrays (MEAs) coated with D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We demonstrate in vitro D-serine recordings with a mean sensitivity of 8.61 ± 0.83 pA/µM to D-serine, a limit of detection (LOD) of 0.17 ± 0.01 µM, and a selectivity ratio of 80:1 or greater for D-serine over ascorbic acid (mean ± SEM; n = 12) that can be used for freely moving studies.

Cross-correlation of gravitational lensing from DES Science Verification data with SPT and Planck lensing

Energy Technology Data Exchange (ETDEWEB)

Kirk, D.; Omori, Y.; Benoit-Lévy, A.; Cawthon, R.; Chang, C.; Larsen, P.; Amara, A.; Bacon, D.; Crawford, T. M.; Dodelson, S.; Fosalba, P.; Giannantonio, T.; Holder, G.; Jain, B.; Kacprzak, T.; Lahav, O.; MacCrann, N.; Nicola, A.; Refregier, A.; Sheldon, E.; Story, K. T.; Troxel, M. A.; Vieira, J. D.; Vikram, V.; Zuntz, J.; Abbott, T. M. C.; Abdalla, F. B.; Becker, M. R.; Benson, B. A.; Bernstein, G. M.; Bernstein, R. A.; Bleem, L. E.; Bonnett, C.; Bridle, S. L.; Brooks, D.; Buckley-Geer, E.; Burke, D. L.; Capozzi, D.; Carlstrom, J. E.; Rosell, A. Carnero; Kind, M. Carrasco; Carretero, J.; Crocce, M.; Cunha, C. E.; D' Andrea, C. B.; da Costa, L. N.; Desai, S.; Diehl, H. T.; Dietrich, J. P.; Doel, P.; Eifler, T. F.; Evrard, A. E.; Flaugher, B.; Frieman, J.; Gerdes, D. W.; Goldstein, D. A.; Gruen, D.; Gruendl, R. A.; Honscheid, K.; James, D. J.; Jarvis, M.; Kent, S.; Kuehn, K.; Kuropatkin, N.; Lima, M.; March, M.; Martini, P.; Melchior, P.; Miller, C. J.; Miquel, R.; Nichol, R. C.; Ogando, R.; Plazas, A. A.; Reichardt, C. L.; Roodman, A.; Rozo, E.; Rykoff, E. S.; Sako, M.; Sanchez, E.; Scarpine, V.; Schubnell, M.; Sevilla-Noarbe, I.; Simard, G.; Smith, R. C.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Thomas, D.; Wechsler, R. H.; Weller, J.

2016-03-10

We measure the cross-correlation between weak lensing of galaxy images and of the cosmic microwave background (CMB). The effects of gravitational lensing on different sources will be correlated if the lensing is caused by the same mass fluctuations. We use galaxy shape measurements from 139 deg$^{2}$ of the Dark Energy Survey (DES) Science Verification data and overlapping CMB lensing from the South Pole Telescope (SPT) and Planck. The DES source galaxies have a median redshift of $z_{\\rm med} {\\sim} 0.7$, while the CMB lensing kernel is broad and peaks at $z{\\sim}2$. The resulting cross-correlation is maximally sensitive to mass fluctuations at $z{\\sim}0.44$. Assuming the Planck 2015 best-fit cosmology, the amplitude of the DES$\\times$SPT cross-power is found to be $A = 0.88 \\pm 0.30$ and that from DES$\\times$Planck to be $A = 0.86 \\pm 0.39$, where $A=1$ corresponds to the theoretical prediction. These are consistent with the expected signal and correspond to significances of $2.9 \\sigma$ and $2.2 \\sigma$ respectively. We demonstrate that our results are robust to a number of important systematic effects including the shear measurement method, estimator choice, photometric redshift uncertainty and CMB lensing systematics. Significant intrinsic alignment of galaxy shapes would increase the cross-correlation signal inferred from the data; we calculate a value of $A = 1.08 \\pm 0.36$ for DES$\\times$SPT when we correct the observations with a simple IA model. With three measurements of this cross-correlation now existing in the literature, there is not yet reliable evidence for any deviation from the expected LCDM level of cross-correlation, given the size of the statistical uncertainties and the significant impact of systematic errors, particularly IAs. We provide forecasts for the expected signal-to-noise of the combination of the five-year DES survey and SPT-3G.

Fibrin(ogen)olytic activity of bumblebee venom serine protease

International Nuclear Information System (INIS)

Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

2011-01-01

Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

Small Subunits of Serine Palmitoyltransferase (ssSPTs) and Their Physiological Roles

Science.gov (United States)

2014-02-12

Fumonisin B1, whereas AtssSPTa knockdown lines show increased resistance compared to wild type (59). In addition to that, over expression of AtssSPTb...finding, AtssSPTa overexpression showed increased fumonisin B1 sensitivity and conversely AtssSPTa knockdown lines showed resistance. This suggests...2007. Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin - sensitive and accumulate trihydroxy-18:1 long chain base phosphate

A newly identified frontal path from fornix in septum pellucidum with 7.0T MRI track density imaging (TDI – The septum pellucidum tract (SPT

Directory of Open Access Journals (Sweden)

Zang-Hee eCho

2015-11-01

Full Text Available The high anatomical contrast achieved with the newly emerging MRI tractographic technique of super-resolution track density imaging (TDI encouraged us to search for a new fiber tract in the septum pellucidum. Although this septum pellucidum tract (SPT has been observed previously, its connections were unclear due to ambiguity and limited resolution of conventional MRI images. It is now possible to identify detailed parts of SPT with the increased resolution of TDI, which involves diffusion MRI imaging, whole-brain tractography, and voxel subdivision using the track-count information. Four healthy male subjects were included in the study. The experiment was performed with 7.0T MRI, following the guidelines of the institute’s institutional review board. Data were processed with the super-resolution TDI technique to generate a tractographic map with 0.18 mm isotropic resolution. The SPT was identified in all subjects. Based on additional seed tracking method with inter-axis correlation search, we have succeeded in identifying a new frontal lobe pathway in the SPT. We hypothesize that the tract is connected as a superior dorsal branch of the fornix that leads to the prefrontal cortex.

Impaired Uptake and/or Utilization of Leucine by Saccharomyces cerevisiae Is Suppressed by the SPT15-300 Allele of the TATA-Binding Protein Gene

DEFF Research Database (Denmark)

Baerends, RJ; Qiu, Jin-Long; Rasmussen, Simon

2009-01-01

Successful fermentations to produce ethanol require microbial strains that have a high tolerance to glucose and ethanol. Enhanced glucose/ethanol tolerance of the laboratory yeast Saccharomyces cerevisiae strain BY4741 under certain growth conditions as a consequence of the expression of a dominant...... us to examine the effect of expression of the SPT15-300 allele in various yeast species of industrial importance. Expression of SPT15-300 in leucine-prototrophic strains of S. cerevisiae, Saccharomyces bayanus, or Saccharomyces pastorianus (lager brewing yeast), however, did not improve tolerance...... to ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing the SPT15-300 mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol...

A cytotoxic serine proteinase isolated from mouse submandibular gland.

Science.gov (United States)

Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

1989-08-01

We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

Molecular analysis of carnitine palmitoyltransferase II deficiency with hepatocardiomuscular expression

Energy Technology Data Exchange (ETDEWEB)

Bonnefont, J.P.; Cepanec, C.; Leroux, J.P. [Unite INSERM, Paris (France)] [and others

1996-05-01

Carnitine palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical act phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the Ser-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, without consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency. 32 refs., 5 figs.

Integration Tests of the 4 kW-class High Voltage Hall Accelerator Power Processing Unit with the HiVHAc and the SPT-140 Hall Effect Thrusters

Science.gov (United States)

Kamhawi, Hani; Pinero, Luis; Haag, Thomas; Huang, Wensheng; Ahern, Drew; Liang, Ray; Shilo, Vlad

2016-01-01

NASAs Science Mission Directorate is sponsoring the development of a 4 kW-class Hall propulsion system for implementation in NASA science and exploration missions. The main components of the system include the High Voltage Hall Accelerator (HiVHAc), an engineering model power processing unit (PPU) developed by Colorado Power Electronics, and a xenon flow control module (XFCM) developed by VACCO Industries. NASA Glenn Research Center is performing integrated tests of the Hall thruster propulsion system. This presentation presents results from integrated tests of the PPU and XFCM with the HiVHAc engineering development thruster and a SPT-140 thruster provided by Space System Loral. The results presented in this paper demonstrate thruster discharge initiation, open-loop and closed-loop control of the discharge current with anode flow for both the HiVHAc and the SPT-140 thrusters. Integrated tests with the SPT-140 thruster indicated that the PPU was able to repeatedly initiate the thrusters discharge, achieve steady state operation, and successfully throttle the thruster between 1.5 and 4.5 kW. The measured SPT-140 performance was identical to levels reported by Space Systems Loral.

Sunyaev-Zel'dovich Effect and X-ray Scaling Relations from Weak-Lensing Mass Calibration of 32 SPT Selected Galaxy Clusters

Energy Technology Data Exchange (ETDEWEB)

Dietrich, J.P.; et al.

2017-11-14

Uncertainty in the mass-observable scaling relations is currently the limiting factor for galaxy cluster based cosmology. Weak gravitational lensing can provide a direct mass calibration and reduce the mass uncertainty. We present new ground-based weak lensing observations of 19 South Pole Telescope (SPT) selected clusters and combine them with previously reported space-based observations of 13 galaxy clusters to constrain the cluster mass scaling relations with the Sunyaev-Zel'dovich effect (SZE), the cluster gas mass $M_\\mathrm{gas}$, and $Y_\\mathrm{X}$, the product of $M_\\mathrm{gas}$ and X-ray temperature. We extend a previously used framework for the analysis of scaling relations and cosmological constraints obtained from SPT-selected clusters to make use of weak lensing information. We introduce a new approach to estimate the effective average redshift distribution of background galaxies and quantify a number of systematic errors affecting the weak lensing modelling. These errors include a calibration of the bias incurred by fitting a Navarro-Frenk-White profile to the reduced shear using $N$-body simulations. We blind the analysis to avoid confirmation bias. We are able to limit the systematic uncertainties to 6.4% in cluster mass (68% confidence). Our constraints on the mass-X-ray observable scaling relations parameters are consistent with those obtained by earlier studies, and our constraints for the mass-SZE scaling relation are consistent with the the simulation-based prior used in the most recent SPT-SZ cosmology analysis. We can now replace the external mass calibration priors used in previous SPT-SZ cosmology studies with a direct, internal calibration obtained on the same clusters.

ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

Science.gov (United States)

D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

Cerebrospinal fluid D-serine concentrations in major depressive disorder negatively correlate with depression severity.

Science.gov (United States)

Ishiwata, Sayuri; Hattori, Kotaro; Sasayama, Daimei; Teraishi, Toshiya; Miyakawa, Tomoko; Yokota, Yuuki; Matsumura, Ryo; Nishikawa, Toru; Kunugi, Hiroshi

2018-01-15

D-serine is an endogenous co-agonist of N-methyl-D-aspartate receptor (NMDAR) and plays an important role in glutamate neurotransmission. Several studies suggested the possible involvement of D-serine related in the pathophysiology of psychiatric disorders including major depression disorders (MDD). We tried to examine whether cerebrospinal fluid (CSF) or plasma D-serine concentrations are altered in MDD and whether D-serine concentrations correlated with disease severity. 26 MDD patients and 27 healthy controls matched for age, sex and ethnicity were enrolled. We measured amino acids in these samples using by high-performance liquid chromatography with fluorometric detection. D-serine and L-serine, precursor of D-serine, levels in CSF or plasma were not significantly different in patients of MDD compared to controls. Furthermore, a significant correlation between D-serine levels in CSF and Hamilton Depression Rating Scale (HAMD)-17 score was observed (r = -0.65, p = 0.006). Furthermore, we found a positive correlation between CSF D-serine and HVA concentrations in MDD patients (r = 0.54, p = 0.007). CSF D-serine concentrations were correlated with those of plasma in MDD (r = 0.61, p = 0.01) but not in controls. In CSF, we also confirmed a significant correlation between D-serine and L-serine levels in MDD (r = 0.72, p depression severity and HVA concentrations and further investigation were required to reveal the effect of medication and disease heterogeneity. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of SPT energy for Donut and Safety hammers using CPT measurements in Egypt

OpenAIRE

Rami M. El-Sherbiny; Manal A. Salem

2013-01-01

Standard Penetration Test (SPT) blow counts require correction prior to utilization in soil characterization and determination of properties and behavior. Among the most important corrections is the energy correction required to adjust the blow counts to 60% energy efficiency. However, there are no published data supporting commonly used value in Egypt. This paper presents an evaluation of the energy efficiency of the Donut and Safety hammers commonly used in Egypt and the associated energy c...

Cross genome comparisons of serine proteases in Arabidopsis and rice

Directory of Open Access Journals (Sweden)

Sowdhamini R

2006-08-01

Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

[1-14C]Glycolate metabolism and serine biosynthesis in soybean plants

International Nuclear Information System (INIS)

Calmes, J.; Viala, G.; Latche, J.C.; Cavalie, G.

1977-01-01

[1- 14 C]Glycolate metabolism was examined in leafy shoots of soybean plants (Glycine max (L.) Merr., var. Adepta). Only small amounts of 14 C were incorporated into evolved carbon dioxide and glucidic compounds. Free and protein glycine was labelled but higher levels of radioactivity were found in free serine. Changes in the distribution of 14 C with time showed that metabolic conversion glycollate → glycine → serine occurred very early and serine biosynthesis was more important in the shoot than in the leaves. Carbon dioxide labelling was always slight compared to serine labelling. These data suggest strong relations between glycollate and nitrogen metabolism

The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

DEFF Research Database (Denmark)

Jürgensen, Vibeke Würtz; Jalkanen, Karl J.

2006-01-01

The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the ......The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory...

Convergent synthesis of a deuterium-labeled serine dipeptide lipid for analysis of biological samples.

Science.gov (United States)

Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

2017-05-30

Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, (3S)-l-serine] isolated from Porphyromonas gingivalis, in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria. Copyright © 2017 John Wiley & Sons, Ltd.

L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.

Science.gov (United States)

Zhu, Qinjian; Zhang, Xiaomei; Luo, Yuchang; Guo, Wen; Xu, Guoqiang; Shi, Jinsong; Xu, Zhenghong

2015-02-01

The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the L-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of L-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity.

GALAXY CLUSTERS DISCOVERED VIA THE SUNYAEV-ZEL'DOVICH EFFECT IN THE 2500-SQUARE-DEGREE SPT-SZ SURVEY

Energy Technology Data Exchange (ETDEWEB)

Bleem, L. E.; Stalder, B.; de Haan, T.; Aird, K. A.; Allen, S. W.; Applegate, D. E.; Ashby, M. L. N.; Bautz, M.; Bayliss, M.; Benson, B. A.; Bocquet, S.; Brodwin, M.; Carlstrom, J. E.; Chang, C. L.; Chiu, I.; Cho, H. M.; Clocchiatti, A.; Crawford, T. M.; Crites, A. T.; Desai, S.; Dietrich, J. P.; Dobbs, M. A.; Foley, R. J.; Forman, W. R.; George, E. M.; Gladders, M. D.; Gonzalez, A. H.; Halverson, N. W.; Hennig, C.; Hoekstra, H.; Holder, G. P.; Holzapfel, W. L.; Hrubes, J. D.; Jones, C.; Keisler, R.; Knox, L.; Lee, A. T.; Leitch, E. M.; Liu, J.; Lueker, M.; Luong-Van, D.; Mantz, A.; Marrone, D. P.; McDonald, M.; McMahon, J. J.; Meyer, S. S.; Mocanu, L.; Mohr, J. J.; Murray, S. S.; Padin, S.; Pryke, C.; Reichardt, C. L.; Rest, A.; Ruel, J.; Ruhl, J. E.; Saliwanchik, B. R.; Saro, A.; Sayre, J. T.; Schaffer, K. K.; Schrabback, T.; Shirokoff, E.; Song, J.; Spieler, H. G.; Stanford, S. A.; Staniszewski, Z.; Stark, A. A.; Story, K. T.; Stubbs, C. W.; Vanderlinde, K.; Vieira, J. D.; Vikhlinin, A.; Williamson, R.; Zahn, O.; Zenteno, A.

2015-01-29

We present a catalog of galaxy clusters selected via their Sunyaev-Zel'dovich (SZ) effect signature from 2500 deg(2) of South Pole Telescope (SPT) data. This work represents the complete sample of clusters detected at high significance in the 2500 deg(2) SPT-SZ survey, which was completed in 2011. A total of 677 (409) cluster candidates are identified above a signal-to-noise threshold of ξ = 4.5 (5.0). Ground- and space-based optical and near-infrared (NIR) imaging confirms overdensities of similarly colored galaxies in the direction of 516 (or 76%) of the ξ > 4.5 candidates and 387 (or 95%) of the ξ > 5 candidates, the measured purity is consistent with expectations from simulations. Of these confirmed clusters, 415 were first identified in SPT data, including 251 new discoveries reported in this work. We estimate photometric redshifts for all candidates with identified optical and/or NIR counterparts, we additionally report redshifts derived from spectroscopic observations for 141 of these systems. The mass threshold of the catalog is roughly independent of redshift above z ~ 0.25 leading to a sample of massive clusters that extends to high redshift. The median mass of the sample is M (500c)(ρ(crit)) $\\sim 3.5\\times 10^{14}\\,M_\\odot \\,h_{70}^{-1}$, the median redshift is z (med) = 0.55, and the highest-redshift systems are at z > 1.4. The combination of large redshift extent, clean selection, and high typical mass makes this cluster sample of particular interest for cosmological analyses and studies of cluster formation and evolution.

Site-specific DNA Inversion by Serine Recombinases

Science.gov (United States)

2015-01-01

Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

New L-Serine Derivative Ligands as Cocatalysts for Diels-Alder Reaction

Science.gov (United States)

Sousa, Carlos A. D.; Rodríguez-Borges, José E.; Freire, Cristina

2013-01-01

New L-serine derivative ligands were prepared and tested as cocatalyst in the Diels-Alder reactions between cyclopentadiene (CPD) and methyl acrylate, in the presence of several Lewis acids. The catalytic potential of the in situ formed complexes was evaluated based on the reaction yield. Bidentate serine ligands showed good ability to coordinate medium strength Lewis acids, thus boosting their catalytic activity. The synthesis of the L-serine ligands proved to be highly efficient and straightforward. PMID:24383009

Serine integrase chimeras with activity in E. coli and HeLa cells

Directory of Open Access Journals (Sweden)

Alfonso P. Farruggio

2014-09-01

Full Text Available In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC and phiC31-TG1 (CT hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

Expression of a mutated SPT15 gene in Saccharomyces cerevisiae enhances both cell growth and ethanol production in microaerobic batch, fed-batch, and simultaneous saccharification and fermentations.

Science.gov (United States)

Seong, Yeong-Je; Park, Haeseong; Yang, Jungwoo; Kim, Soo-Jung; Choi, Wonja; Kim, Kyoung Heon; Park, Yong-Cheol

2017-05-01

The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.

Mosaic serine proteases in the mammalian central nervous system.

Science.gov (United States)

Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

Orm family proteins mediate sphingolipid homeostasis

DEFF Research Database (Denmark)

Breslow, David K; Collins, Sean R; Bodenmiller, Bernd

2010-01-01

a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression...... or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma....

Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

Directory of Open Access Journals (Sweden)

Lior Doron

Full Text Available Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

Directory of Open Access Journals (Sweden)

Nagendra S Singh

Full Text Available D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac-dehydronorketamine and (2S,6S-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S-hydroxynorketamine and (2R,6R-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

MASS CALIBRATION AND COSMOLOGICAL ANALYSIS OF THE SPT-SZ GALAXY CLUSTER SAMPLE USING VELOCITY DISPERSION σ v AND X-RAY Y X MEASUREMENTS

International Nuclear Information System (INIS)

Bocquet, S.; Saro, A.; Mohr, J. J.; Bazin, G.; Chiu, I.; Desai, S.; Aird, K. A.; Ashby, M. L. N.; Bayliss, M.; Bautz, M.; Benson, B. A.; Bleem, L. E.; Carlstrom, J. E.; Chang, C. L.; Crawford, T. M.; Crites, A. T.; Brodwin, M.; Cho, H. M.; Clocchiatti, A.; De Haan, T.

2015-01-01

We present a velocity-dispersion-based mass calibration of the South Pole Telescope Sunyaev-Zel'dovich effect survey (SPT-SZ) galaxy cluster sample. Using a homogeneously selected sample of 100 cluster candidates from 720 deg 2 of the survey along with 63 velocity dispersion (σ v ) and 16 X-ray Y X measurements of sample clusters, we simultaneously calibrate the mass-observable relation and constrain cosmological parameters. Our method accounts for cluster selection, cosmological sensitivity, and uncertainties in the mass calibrators. The calibrations using σ v and Y X are consistent at the 0.6σ level, with the σ v calibration preferring ∼16% higher masses. We use the full SPT CL data set (SZ clusters+σ v +Y X ) to measure σ 8 (Ω m /0.27) 0.3 = 0.809 ± 0.036 within a flat ΛCDM model. The SPT cluster abundance is lower than preferred by either the WMAP9 or Planck+WMAP9 polarization (WP) data, but assuming that the sum of the neutrino masses is ∑m ν = 0.06 eV, we find the data sets to be consistent at the 1.0σ level for WMAP9 and 1.5σ for Planck+WP. Allowing for larger ∑m ν further reconciles the results. When we combine the SPT CL and Planck+WP data sets with information from baryon acoustic oscillations and Type Ia supernovae, the preferred cluster masses are 1.9σ higher than the Y X calibration and 0.8σ higher than the σ v calibration. Given the scale of these shifts (∼44% and ∼23% in mass, respectively), we execute a goodness-of-fit test; it reveals no tension, indicating that the best-fit model provides an adequate description of the data. Using the multi-probe data set, we measure Ω m = 0.299 ± 0.009 and σ 8 = 0.829 ± 0.011. Within a νCDM model we find ∑m ν = 0.148 ± 0.081 eV. We present a consistency test of the cosmic growth rate using SPT clusters. Allowing both the growth index γ and the dark energy equation-of-state parameter w to vary, we find γ = 0.73 ± 0.28 and w = –1.007 ± 0.065, demonstrating that the

Engineering of High Yield Production of L-serine in Escherichia coli

DEFF Research Database (Denmark)

Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre

2016-01-01

by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very...

NR4A nuclear receptors mediate carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600

Energy Technology Data Exchange (ETDEWEB)

Ishizawa, Michiyasu [Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610 (Japan); Kagechika, Hiroyuki [Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 (Japan); Makishima, Makoto, E-mail: makishima.makoto@nihon-u.ac.jp [Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610 (Japan)

2012-02-24

Highlights: Black-Right-Pointing-Pointer The function of RXR heterodimers with NR4 receptors remains unknown. Black-Right-Pointing-Pointer The RXR ligand HX600 induces expression of carnitine palmitoyltransferase 1A (CPT1A). Black-Right-Pointing-Pointer HX600-induced CPT1A expression is mediated by the NR4 receptors, Nur77 and NURR1. Black-Right-Pointing-Pointer CPT1A induction by HX600 is not mediated by de novo protein synthesis. Black-Right-Pointing-Pointer CPT1A could be a target of the Nur77-RXR and NURR1-RXR heterodimers. -- Abstract: Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and can be activated by 9-cis retinoic acid (9CRA). RXRs form homodimers and heterodimers with other nuclear receptors such as the retinoic acid receptor and NR4 subfamily nuclear receptors, Nur77 and NURR1. Potential physiological roles of the Nur77-RXR and NURR1-RXR heterodimers have not been elucidated. In this study, we identified a gene regulated by these heterodimers utilizing HX600, a selective RXR agonist for Nur77-RXR and NURR1-RXR. While 9CRA induced many genes, including RAR-target genes, HX600 effectively induced only carnitine palmitoyltransferase 1A (CPT1A) in human teratocarcinoma NT2/D1 cells, which express RXR{alpha}, Nur77 and NURR1. HX600 also increased CPT1A expression in human embryonic kidney (HEK) 293 cells and hepatocyte-derived HepG2 cells. Although HX600 induced CPT1A less effectively than 9CRA, overexpression of Nur77 or NURR1 increased the HX600 response to levels similar to 9CRA in NT2/D1 and HEK293 cells. A dominant-negative form of Nur77 or NURR1 repressed the induction of CPT1A by HX600. A protein synthesis inhibitor did not alter HX600-dependent CPT1A induction. Thus, the rexinoid HX600 directly induces expression of CPT1A through a Nur77 or NURR1-mediated mechanism. CPT1A, a gene involved in fatty acid {beta}-oxidation, could be a target of RXR-NR4 receptor heterodimers.

THE REDSHIFT DISTRIBUTION OF DUSTY STAR-FORMING GALAXIES FROM THE SPT SURVEY

Energy Technology Data Exchange (ETDEWEB)

Strandet, M. L.; Weiss, A. [Max-Planck-Institut für Radioastronomie, Auf dem Hügel 69 D-53121 Bonn (Germany); Vieira, J. D.; Furstenau, R. M. [European Southern Observatory, Karl Schwarzschild Straße 2, D-85748 Garching (Germany); De Breuck, C.; Béthermin, M.; Gullberg, B. [Department of Astronomy and Department of Physics, University of Illinois, 1002 West Green St., Urbana, IL 61801 (United States); Aguirre, J. E. [University of Pennsylvania, 209 South 33rd Street, Philadelphia, PA 19104 (United States); Aravena, M. [Núcleo de Astronomía, Facultad de Ingeniería, Universidad Diego Portales, Av. Ejército 441, Santiago (Chile); Ashby, M. L. N. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States); Bradford, C. M. [Jet Propulsion Laboratory, 4800 Oak Grove Drive, Pasadena, CA 91109 (United States); Carlstrom, J. E.; Crawford, T. M. [Kavli Institute for Cosmological Physics, University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637 (United States); Chapman, S. C. [Dalhousie University, Halifax, Nova Scotia (Canada); Everett, W. [Department of Astrophysical and Planetary Sciences and Department of Physics, University of Colorado, Boulder, CO 80309 (United States); Fassnacht, C. D. [Department of Physics, University of California, One Shields Avenue, Davis, CA 95616 (United States); Gonzalez, A. H. [Department of Astronomy, University of Florida, Gainesville, FL 32611 (United States); Greve, T. R. [Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT (United Kingdom); Hezaveh, Y. [Kavli Institute for Particle Astrophysics and Cosmology, Stanford University, Stanford, CA 94305 (United States); and others

2016-05-10

We use the Atacama Large Millimeter/submillimeter Array (ALMA) in Cycle 1 to determine spectroscopic redshifts of high-redshift dusty star-forming galaxies (DSFGs) selected by their 1.4 mm continuum emission in the South Pole Telescope (SPT) survey. We present ALMA 3 mm spectral scans between 84 and 114 GHz for 15 galaxies and targeted ALMA 1 mm observations for an additional eight sources. Our observations yield 30 new line detections from CO, [C i], [N ii], H{sub 2}O and NH{sub 3}. We further present Atacama Pathfinder Experiment [C ii] and CO mid- J observations for seven sources for which only a single line was detected in spectral-scan data from ALMA Cycle 0 or Cycle 1. We combine the new observations with previously published and new millimeter/submillimeter line and photometric data of the SPT-selected DSFGs to study their redshift distribution. The combined data yield 39 spectroscopic redshifts from molecular lines, a success rate of >85%. Our sample represents the largest data set of its kind today and has the highest spectroscopic completeness among all redshift surveys of high- z DSFGs. The median of the redshift distribution is z = 3.9 ± 0.4, and the highest-redshift source in our sample is at z = 5.8. We discuss how the selection of our sources affects the redshift distribution, focusing on source brightness, selection wavelength, and strong gravitational lensing. We correct for the effect of gravitational lensing and find the redshift distribution for 1.4 mm selected sources with a median redshift of z = 3.1 ± 0.3. Comparing to redshift distributions selected at shorter wavelengths from the literature, we show that selection wavelength affects the shape of the redshift distribution.

The binding mechanism of a peptidic cyclic serine protease inhibitor

DEFF Research Database (Denmark)

Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

2011-01-01

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

Cell-type specific mechanisms of D-serine uptake and release in the brain

Directory of Open Access Journals (Sweden)

Magalie eMartineau

2014-05-01

Full Text Available Accumulating evidence during the last decade established that D-serine is a key signaling molecule utilized by neurons and astroglia in the mammalian central nervous system. D-serine is increasingly appreciated as the main physiological endogenous coagonist for synaptic NMDA receptors at central excitatory synapses; it is mandatory for long-term changes in synaptic strength, memory, learning, and social interactions. Alterations in the extracellular levels of D-serine leading to disrupted cell-cell signaling are a trademark of many chronic or acute neurological (i.e. Alzheimer disease, epilepsy, stroke and psychiatric (i.e. schizophrenia disorders, and are associated with addictive behavior (i.e. cocaine addiction. Indeed, fine tuning of the extracellular levels of D-serine, achieved by various molecular machineries and signaling pathways, is necessary for maintenance of accurate NMDA receptor functions. Here, we review the experimental data supporting the notion that astroglia and neurons use different pathways to regulate levels of extracellular D-serine.

Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

International Nuclear Information System (INIS)

Sun Dejun; Liu Shanshan; Yang Chunwei; Zhao Yizhuo; Chang Shufang; Yan Weiqun

2005-01-01

Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5α. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 10 6 . Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His 41 , Asp 86 , Ser 180 ; and six disulfide bridges Cys 7 -Cys 139 , Cys 26 -Cys 42 , Cys 74 -Cys 232 , Cys 118 -Cys 186 , Cys 150 -Cys 165 , Cys 176 -Cys 201 . Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 10 6 , overtop the level of 10 5 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine

l-Serine Enhances Light-Induced Circadian Phase Resetting in Mice and Humans.

Science.gov (United States)

Yasuo, Shinobu; Iwamoto, Ayaka; Lee, Sang-Il; Ochiai, Shotaro; Hitachi, Rina; Shibata, Satomi; Uotsu, Nobuo; Tarumizu, Chie; Matsuoka, Sayuri; Furuse, Mitsuhiro; Higuchi, Shigekazu

2017-12-01

Background: The circadian clock is modulated by the timing of ingestion or food composition, but the effects of specific nutrients are poorly understood. Objective: We aimed to identify the amino acids that modulate the circadian clock and reset the light-induced circadian phase in mice and humans. Methods: Male CBA/N mice were orally administered 1 of 20 l-amino acids, and the circadian and light-induced phase shifts of wheel-running activity were analyzed. Antagonists of several neurotransmitter pathways were injected before l-serine administration, and light-induced phase shifts were analyzed. In addition, the effect of l-serine on the light-induced phase advance was investigated in healthy male students (mean ± SD age 22.2 ± 1.8 y) by using dim-light melatonin onset (DLMO) determined by saliva samples as an index of the circadian phase. Results: l-Serine administration enhanced light-induced phase shifts in mice (1.86-fold; P light-dark cycle by 6 h, l-serine administration slightly accelerated re-entrainment to the shifted cycle. In humans, l-serine ingestion before bedtime induced significantly larger phase advances of DLMO after bright-light exposure during the morning (means ± SEMs-l-serine: 25.9 ± 6.6 min; placebo: 12.1 ± 7.0 min; P light-induced phase resetting in mice and humans, and it may be useful for treating circadian disturbances. © 2017 American Society for Nutrition.

Dosage compensation of serine-4 transfer RNA in Drosophila melanogaster

International Nuclear Information System (INIS)

Birchler, J.A.; Owenby, R.K.; Jacobson, K.B.

1982-01-01

A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock were similar. Thus the transfer RNA levels conform to the expectations of dosage compensation

An ignored cause of red urine in children: rhabdomyolysis due to carnitine palmitoyltransferase II (CPT-II) deficiency.

Science.gov (United States)

Melek, Engin; Bulut, Fatma Derya; Atmış, Bahriye; Yılmaz, Berna Şeker; Bayazıt, Aysun Karabay; Mungan, Neslihan Önenli

2017-02-01

Carnitine palmitoyltransferase II (CPT-II) deficiency is an autosomal recessively inherited disorder involving the β-oxidation of long-chain fatty acids, which leads to rhabdomyolysis and subsequent acute renal failure. The clinical phenotype varies from a severe infantile form to a milder muscle form. Here, we report a 9-year-old boy referred to our hospital for the investigation of hematuria with a 2-day history of dark urine and malaise. As no erythrocytes in the microscopic examination of the urine and hemoglobinuria were present, myoglobinuria due to rhabdomyolysis was the most probable cause of dark urine. After excluding the other causes of rhabdomyolysis, with the help of metabolic investigations, the patient was suspected to have CPT-II deficiency, the most common cause of metabolic rhabdomyolysis. Our aim in presenting this case is to emphasize considering rhabdomyolysis in the differential diagnosis of dark urine in order to prevent recurrent rhabdomyolysis and renal injury.

Evaluation of oxidative stress in D-serine induced nephrotoxicity

International Nuclear Information System (INIS)

Orozco-Ibarra, Marisol; Medina-Campos, Omar Noel; Sanchez-Gonzalez, Dolores Javier; Martinez-Martinez, Claudia Maria; Floriano-Sanchez, Esau; Santamaria, Abel; Ramirez, Victoria; Bobadilla, Norma A.; Pedraza-Chaverri, Jose

2007-01-01

It has been suggested that oxidative stress is involved in D-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24 h) after a single i.p. injection of D-serine (400 mg/kg). Control rats were injected with L-serine (400 mg/kg) and sacrificed 24 h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) were measured 24 h after D-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before D-serine injection: (a) α-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl 2 ), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24 h, respectively, and KIM-1 mRNA levels increased significantly on 6-24 h. Histological analyses revealed tubular necrosis at 12 h. The activity of antioxidant enzymes

Contribution of the D-Serine-dependent pathway to the cellular mechanisms underlying cognitive aging

Directory of Open Access Journals (Sweden)

Emilie Rouaud

2010-02-01

Full Text Available An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-Methyl-D-Aspartate receptors (NMDA-R by its agonist D-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous D-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of D-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of D-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the D-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly.

The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of D-serine.

Science.gov (United States)

Sakinç, Türkân; Michalski, Nadine; Kleine, Britta; Gatermann, Sören G

2009-10-01

Human urine contains a relatively high concentration of d-serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d-serine deaminase gene (dsdA) of UPEC. We found the gene in several clinical isolates of S. saprophyticus; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii, phylogenetically close relatives of S. saprophyticus, and could also not be detected in isolates of Staphylococcus aureus, Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d-serine, whereas S. aureus, S. epidermidis and other staphylococcal species could not. The association of the dsdA gene with growth in media including d-serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d-serine utilization and detoxification may be a general property of uropathogenic bacteria. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

Directory of Open Access Journals (Sweden)

Claire N J Meunier

Full Text Available N-methyl-D-aspartate receptors (NMDARs play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

Improvement in regional CBF by L-serine contributes to its neuroprotective effect in rats after focal cerebral ischemia.

Directory of Open Access Journals (Sweden)

Tao-Jie Ren

Full Text Available To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF. Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1 reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2 improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3 increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca(2+-activated K(+ channels on the cerebral blood vessel endothelium.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Directory of Open Access Journals (Sweden)

Leah Theresa Sigle

2013-09-01

Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

Science.gov (United States)

Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi

2018-02-16

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

Science.gov (United States)

Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

2018-05-03

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

MASS CALIBRATION AND COSMOLOGICAL ANALYSIS OF THE SPT-SZ GALAXY CLUSTER SAMPLE USING VELOCITY DISPERSION σ {sub v} AND X-RAY Y {sub X} MEASUREMENTS

Energy Technology Data Exchange (ETDEWEB)

Bocquet, S.; Saro, A.; Mohr, J. J.; Bazin, G.; Chiu, I.; Desai, S. [Department of Physics, Ludwig-Maximilians-Universität, Scheinerstr. 1, D-81679 München (Germany); Aird, K. A. [University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637 (United States); Ashby, M. L. N.; Bayliss, M. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States); Bautz, M. [Kavli Institute for Astrophysics and Space Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States); Benson, B. A. [Fermi National Accelerator Laboratory, Batavia, IL 60510-0500 (United States); Bleem, L. E.; Carlstrom, J. E.; Chang, C. L.; Crawford, T. M.; Crites, A. T. [Kavli Institute for Cosmological Physics, University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637 (United States); Brodwin, M. [Department of Physics and Astronomy, University of Missouri, 5110 Rockhill Road, Kansas City, MO 64110 (United States); Cho, H. M. [NIST Quantum Devices Group, 325 Broadway Mailcode 817.03, Boulder, CO 80305 (United States); Clocchiatti, A. [Departamento de Astronomia y Astrosifica, Pontificia Universidad Catolica (Chile); De Haan, T., E-mail: bocquet@usm.lmu.de [Department of Physics, McGill University, 3600 Rue University, Montreal, Quebec H3A 2T8 (Canada); and others

2015-02-01

We present a velocity-dispersion-based mass calibration of the South Pole Telescope Sunyaev-Zel'dovich effect survey (SPT-SZ) galaxy cluster sample. Using a homogeneously selected sample of 100 cluster candidates from 720 deg{sup 2} of the survey along with 63 velocity dispersion (σ {sub v}) and 16 X-ray Y {sub X} measurements of sample clusters, we simultaneously calibrate the mass-observable relation and constrain cosmological parameters. Our method accounts for cluster selection, cosmological sensitivity, and uncertainties in the mass calibrators. The calibrations using σ {sub v} and Y {sub X} are consistent at the 0.6σ level, with the σ {sub v} calibration preferring ∼16% higher masses. We use the full SPT{sub CL} data set (SZ clusters+σ {sub v}+Y {sub X}) to measure σ{sub 8}(Ω{sub m}/0.27){sup 0.3} = 0.809 ± 0.036 within a flat ΛCDM model. The SPT cluster abundance is lower than preferred by either the WMAP9 or Planck+WMAP9 polarization (WP) data, but assuming that the sum of the neutrino masses is ∑m {sub ν} = 0.06 eV, we find the data sets to be consistent at the 1.0σ level for WMAP9 and 1.5σ for Planck+WP. Allowing for larger ∑m {sub ν} further reconciles the results. When we combine the SPT{sub CL} and Planck+WP data sets with information from baryon acoustic oscillations and Type Ia supernovae, the preferred cluster masses are 1.9σ higher than the Y {sub X} calibration and 0.8σ higher than the σ {sub v} calibration. Given the scale of these shifts (∼44% and ∼23% in mass, respectively), we execute a goodness-of-fit test; it reveals no tension, indicating that the best-fit model provides an adequate description of the data. Using the multi-probe data set, we measure Ω{sub m} = 0.299 ± 0.009 and σ{sub 8} = 0.829 ± 0.011. Within a νCDM model we find ∑m {sub ν} = 0.148 ± 0.081 eV. We present a consistency test of the cosmic growth rate using SPT clusters. Allowing both the growth index γ and the dark energy equation

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

Science.gov (United States)

Ikehara, Yukio; Pitot, Henry C.

1973-01-01

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [125I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [125I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [125I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[125I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [125I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[125I]Fab to free polysomes was also obtained. The [125I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[125I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be

Antibacterial activity of silver nanoparticles synthesized from serine

Energy Technology Data Exchange (ETDEWEB)

Jayaprakash, N. [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203 (India); Judith Vijaya, J., E-mail: jjvijayaloyola@yahoo.co.in [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); John Kennedy, L. [Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048 (India); Priadharsini, K.; Palani, P. [Department of Center for Advanced Study in Botany, University of Madras, Guindy Campus, Chennai 600 025 (India)

2015-04-01

Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV–Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443 nm. The emission spectrum of Ag NPs showed an emission band at 484 nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO{sub 3} against Gram-positive and Gram-negative bacteria. - Highlights: • Microwave irradiation method is used to synthesize silver nanoparticles. • Highly stable silver nanoparticles are produced from serine. • A detailed study of antibacterial activities is discussed. • Formation mechanism of silver microspheres has been proposed.

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Czech Academy of Sciences Publication Activity Database

Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

2010-01-01

Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

Intervention with Serine Protease Activity with Small Peptides

DEFF Research Database (Denmark)

Xu, Peng

2015-01-01

Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (u......PA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library...... before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor...

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

Energy Technology Data Exchange (ETDEWEB)

Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

2015-10-23

L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes

OpenAIRE

1988-01-01

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular ...

Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

Directory of Open Access Journals (Sweden)

Huan Zhang

2017-01-01

Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

Epigenetic Activation of ASCT2 in the Hippocampus Contributes to Depression-Like Behavior by Regulating D-Serine in Mice

Directory of Open Access Journals (Sweden)

Jiesi Wang

2017-05-01

Full Text Available The roles of D-serine in depression are raised concerned recently as an intrinsic co-agonist for the NMDA receptor. However, the mechanisms underlying its regulation are not fully elucidated. ASCT2 is a Na+-dependent D-serine transporter. We found that decreased D-serine and increased hippocampal ASCT2 levels correlated with chronic social defeat stress (CSDS in mice. Lentivirus-mediated shRNA-mediated knockdown of ASCT2 and the administration of exogenous D-serine in the hippocampus alleviated CSDS-induced social avoidance and immobility. In vivo and in vitro experiments revealed that upregulation of ASCT2 expression in CSDS was regulated through histone hyper-acetylation, not DNA methylation in its promoter region. Immunohistochemistry demonstrated the co-localization of ASCT2 and D-serine. Uptake of D-serine by ASCT2 was demonstrated by in vivo and in vitro experiments. Our results indicate that CSDS induces ASCT2 expression through epigenetic activation and decreases hippocampal D-serine levels, leading to social avoidance, and immobility. Thus, targeting D-serine transport represents an attractive new strategy for treating depression.

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

DEFF Research Database (Denmark)

Dikopoltsev, Elena; Foltyn, Veronika N; Zehl, Martin

2014-01-01

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little......, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association...... is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex...

Engineering yeast transcription machinery for improved ethanol tolerance and production.

Science.gov (United States)

Alper, Hal; Moxley, Joel; Nevoigt, Elke; Fink, Gerald R; Stephanopoulos, Gregory

2006-12-08

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe(177)Ser) and, similarly, Tyr(195)His, and Lys(218)Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

Protein kinase A phosphorylates serine 267 in the homeodomain of engrailed-2 leading to decreased DNA binding

DEFF Research Database (Denmark)

Hjerrild, Majbrit; Stensballe, Allan; Jensen, Ole N

2004-01-01

Engrailed-2 (En-2) belongs to an evolutionarily conserved family of DNA binding homeodomain-containing proteins that are expressed in mammalian brain during development. Here, we demonstrate that serine 267 in the homeodomain of En-2 is phosphorylated by protein kinase A (PKA) in forskolin......-treated COS-7 cells. Furthermore, we analyze the physiological function of En-2 phosphorylation by PKA. The nuclear localization of En-2 is not influenced by the phosphorylation of serine 267. However, substitution of serine 267 with alanine resulted in increased binding of En-2 to DNA, while replacing serine...

Constraints on Cosmological Parameters from the Angular Power Spectrum of a Combined 2500 deg$^2$ SPT-SZ and Planck Gravitational Lensing Map

Energy Technology Data Exchange (ETDEWEB)

Simard, G.; et al.

2017-12-20

We report constraints on cosmological parameters from the angular power spectrum of a cosmic microwave background (CMB) gravitational lensing potential map created using temperature data from 2500 deg$^2$ of South Pole Telescope (SPT) data supplemented with data from Planck in the same sky region, with the statistical power in the combined map primarily from the SPT data. We fit the corresponding lensing angular power spectrum to a model including cold dark matter and a cosmological constant ($\\Lambda$CDM), and to models with single-parameter extensions to $\\Lambda$CDM. We find constraints that are comparable to and consistent with constraints found using the full-sky Planck CMB lensing data. Specifically, we find $\\sigma_8 \\Omega_{\\rm m}^{0.25}=0.598 \\pm 0.024$ from the lensing data alone with relatively weak priors placed on the other $\\Lambda$CDM parameters. In combination with primary CMB data from Planck, we explore single-parameter extensions to the $\\Lambda$CDM model. We find $\\Omega_k = -0.012^{+0.021}_{-0.023}$ or $M_{\

A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen

Directory of Open Access Journals (Sweden)

M. Wadhwa

2016-12-01

Full Text Available A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, α-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen. Keywords: Metabolic engineering, Carotenoids, Isoprenoids, α-Farnesene, Rhodosporidium toruloides, SPT15

Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum

DEFF Research Database (Denmark)

østergaard, Simon; Theilgaard, Hanne Birgitte; Nielsen, Jens Bredal

1998-01-01

We have demonstrated that Penicillium chrysogenum possesses the L-cysteine biosynthetic enzyme O-acetyI-L-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates...... the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-L-serine sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-L-serine as substrate for the formation of L-cysteine....... The purified enzyme did not catalyse the formation of L-homocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold...

Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

Science.gov (United States)

Behlke, J; Heidrich, K; Naumann, M; Müller, E C; Otto, A; Reuter, R; Kriegel, T

1998-08-25

Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.

ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

International Nuclear Information System (INIS)

Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

2013-01-01

Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

Science.gov (United States)

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

Science.gov (United States)

Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

2016-08-01

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism.

Science.gov (United States)

Igamberdiev, Abir U; Kleczkowski, Leszek A

2018-01-01

Serine metabolism in plants has been studied mostly in relation to photorespiration where serine is formed from two molecules of glycine. However, two other pathways of serine formation operate in plants and represent the branches of glycolysis diverging at the level of 3-phosphoglyceric acid. One branch (the glycerate - serine pathway) is initiated in the cytosol and involves glycerate formation from 3-phosphoglycerate, while the other (the phosphorylated serine pathway) operates in plastids and forms phosphohydroxypyruvate as an intermediate. Serine formed in these pathways becomes a precursor of glycine, formate and glycolate accumulating in stress conditions. The pathways can be linked to GABA shunt via transamination reactions and via participation of the same reductase for both glyoxylate and succinic semialdehyde. In this review paper we present a hypothesis of the regulation of redox balance in stressed plant cells via participation of the reactions associated with glycerate and phosphorylated serine pathways. We consider these pathways as important processes linking carbon and nitrogen metabolism and maintaining cellular redox and energy levels in stress conditions.

Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide

Energy Technology Data Exchange (ETDEWEB)

Chung, Hsiu-Ying, E-mail: hychung@mail.fcu.edu.tw; Li, Cheng Che

2013-03-01

A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3NSS peptide solution, and finally immersed in artificial saliva for 24 h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3NSS peptide compared to samples without peptide treatment. - Highlights: Black-Right-Pointing-Pointer The degree of nanohardness recovery of enamel was 4 times greater with the aid of 3NSS peptide. Black-Right-Pointing-Pointer 3NSS peptide promoted the formation of hydroxyapatites with

Serine proteinase inhibitors from nematodes and the arms race between host and pathogen.

Science.gov (United States)

Zang, X; Maizels, R M

2001-03-01

Serine proteinase inhibitors are encoded by a large gene family of long evolutionary standing. Recent discoveries of parasite proteins that inhibit human serine proteinases, together with the complete genomic sequence from Caenorhabditis elegans, have provided a set of new serine proteinase inhibitors from more primitive metazoan animals such as nematodes. The structural features (e.g. reactive centre residues), gene organization (including intron arrangements) and inhibitory function and targets (e.g. inflammatory and coagulation pathway proteinase) all contribute important new insights into proteinase inhibitor evolution. Some parasite products have evolved that block enzymes in the mammalian host, but the human host responds with a significant immune response to the parasite inhibitors. Thus, infection produces a finely balanced conflict between host and pathogen at the molecular level, and this might have accelerated the evolution of these proteins in parasitic species as well as their hosts.

STELLAR MASSES AND STAR FORMATION RATES OF LENSED, DUSTY, STAR-FORMING GALAXIES FROM THE SPT SURVEY

Energy Technology Data Exchange (ETDEWEB)

Ma, Jingzhe; Gonzalez, Anthony H. [Department of Astronomy, University of Florida, Gainesville, FL 32611 (United States); Spilker, J. S.; Marrone, D. P. [Steward Observatory, University of Arizona, 933 North Cherry Avenue, Tucson, AZ 85721 (United States); Strandet, M. [Max-Planck-Institut für Radioastronomie, Auf dem Hügel 69 D-53121 Bonn (Germany); Ashby, M. L. N. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States); Aravena, M. [Núcleo de Astronomía, Facultad de Ingeniería, Universidad Diego Portales, Av. Ejército 441, Santiago (Chile); Béthermin, M.; Breuck, C. de; Gullberg, B. [European Southern Observatory, Karl Schwarzschild Straße 2, D-85748 Garching (Germany); Bothwell, M. S. [Cavendish Laboratory, University of Cambridge, JJ Thompson Avenue, Cambridge CB3 0HA (United Kingdom); Brodwin, M. [Department of Physics and Astronomy, University of Missouri, 5110 Rockhill Road, Kansas City, MO 64110 (United States); Chapman, S. C. [Dalhousie University, Halifax, Nova Scotia (Canada); Fassnacht, C. D. [Department of Physics, University of California, One Shields Avenue, Davis, CA 95616 (United States); Greve, T. R. [Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT (United Kingdom); Hezaveh, Y. [Kavli Institute for Particle Astrophysics and Cosmology, Stanford University, Stanford, CA 94305 (United States); Malkan, M. [Department of Physics and Astronomy, University of California, Los Angeles, CA 90095-1547 (United States); Saliwanchik, B. R., E-mail: jingzhema@ufl.edu [Department of Physics, Case Western Reserve University, Cleveland, OH 44106 (United States); and others

2015-10-10

To understand cosmic mass assembly in the universe at early epochs, we primarily rely on measurements of the stellar masses and star formation rates (SFRs) of distant galaxies. In this paper, we present stellar masses and SFRs of six high-redshift (2.8 ≤ z ≤ 5.7) dusty, star-forming galaxies (DSFGs) that are strongly gravitationally lensed by foreground galaxies. These sources were first discovered by the South Pole Telescope (SPT) at millimeter wavelengths and all have spectroscopic redshifts and robust lens models derived from Atacama Large Millimeter/submillimeter Array observations. We have conducted follow-up observations to obtain multi-wavelength imaging data using the Hubble Space Telescope (HST), Spitzer, Herschel, and the Atacama Pathfinder EXperiment. We use the high-resolution HST/Wide Field Camera 3 images to disentangle the background source from the foreground lens in Spitzer/IRAC data. The detections and upper limits provide important constraints on the spectral energy distributions (SEDs) for these DSFGs, yielding stellar masses, IR luminosities, and SFRs. The SED fits of six SPT sources show that the intrinsic stellar masses span a range more than one order of magnitude with a median value ∼5 ×10{sup 10} M{sub ⊙}. The intrinsic IR luminosities range from 4 × 10{sup 12} L{sub ⊙} to 4 × 10{sup 13} L{sub ⊙}. They all have prodigious intrinsic SFRs of 510–4800 M{sub ⊙} yr{sup −1}. Compared to the star-forming main sequence (MS), these six DSFGs have specific SFRs that all lie above the MS, including two galaxies that are a factor of 10 higher than the MS. Our results suggest that we are witnessing ongoing strong starburst events that may be driven by major mergers.

Squash inhibitor family of serine proteinases

International Nuclear Information System (INIS)

Otlewski, J.; Krowarsch, D.

1996-01-01

Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor β2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: X a and XII a , cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high k cat /K m index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)

A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region.

Science.gov (United States)

Bellacosa, A; Testa, J R; Staal, S P; Tsichlis, P N

1991-10-11

The v-akt oncogene codes for a 105-kilodalton fusion phosphoprotein containing Gag sequences at its amino terminus. Sequence analysis of v-akt and biochemical characterization of its product revealed that it codes for a protein kinase C-related serine-threonine kinase whose cellular homolog is expressed in most tissues, with the highest amount found in thymus. Although Akt is a serine-threonine kinase, part of its regulatory region is similar to the Src homology-2 domain, a structural motif characteristic of cytoplasmic tyrosine kinases that functions in protein-protein interactions. This suggests that Akt may form a functional link between tyrosine and serine-threonine phosphorylation pathways.

Hepatic beta-oxidation and carnitine palmitoyltransferase I in neonatal pigs after dietary treatments of clofibric acid, isoproterenol, and medium-chain triglycerides.

Science.gov (United States)

Peffer, Pasha Lyvers; Lin, Xi; Odle, Jack

2005-06-01

A suckling piglet model was used to study nutritional and pharmacologic means of stimulating hepatic fatty acid beta-oxidation. Newborn pigs were fed milk diets containing either long- or medium-chain triglycerides (LCT or MCT). The long-chain control diet was supplemented further with clofibric acid (0.5%) or isoproterenol (40 ppm), and growth was monitored for 10-12 days. Clofibrate increased rates of hepatic peroxisomal and mitochondrial beta-oxidation of [1-(14)C]-palmitate by 60 and 186%, respectively. Furthermore, malonyl-CoA sensitive carnitine palmitoyltransferase (CPT I) activity increased 64% (P clofibrate. Increased CPT I activity was not congruent with changes in message, as elevated abundance of CPT I mRNA was not detected (P = 0.16) when assessed by qRT-PCR. Neither rates of beta-oxidation nor CPT activities were affected by dietary MCT or by isoproterenol treatment (P > 0.1). Collectively, these findings indicate that clofibrate effectively induced hepatic CPT activity concomitant with increased fatty acid beta-oxidation.

Replacement of C305 in heart/muscle-type isozyme of human carnitine palmitoyltransferase I with aspartic acid and other amino acids.

Science.gov (United States)

Matsuo, Taisuke; Yamamoto, Atsushi; Yamamoto, Takenori; Otsuki, Kaoru; Yamazaki, Naoshi; Kataoka, Masatoshi; Terada, Hiroshi; Shinohara, Yasuo

2010-04-01

Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.

ANÁLISE DE PERFIS DE SONDAGEM SPT E CARACTERIZAÇÃO GEOTÉCNICA DE SOLOS DO MUNICÍPIO DE NATAL

Directory of Open Access Journals (Sweden)

Kadson Ranniere da Rocha Câmara

2007-12-01

Full Text Available O presente trabalho tem como objetivo a caracterização geotécnica de solos do município de Natal para fins de obtenção de parâmetros de resistência ao cisalhamento e de deformabilidade do material. Quatro zonas foram definidas para a compilação de resultados de relatórios de sondagens tipo SPT (Standard Penetration Test, buscando-se destacar aspectos que diferenciem geotecnicamente as zonas consideradas. Campanhas de ensaios de laboratório foram relizadas para a idendificação de características geotécnicas (perfil de umidades e granulometria, densidade das partículas e deformabilidade de solo definido como referência para as análises. Parâmetros de resistência ao cisalhamento e de deformabilidade dos solos encontrados foram determinados mediante emprego de equações empíricas. Os resultados encontrados indicam haver importante variabilidade nas propriedades geotécnicas dos solos analisados, tanto para solos pertencentes a uma mesma zona como para solos de zonas distintas. O posicionamento das unidades geotécnicas e o estado de compacidade das mesmas consistem em variáveis destacadas no estudo realizado. Palavras-chaves: Mecânica dos Solos; Ensaios In Situ; Perfis SPT; Caracterização Geotécnica.

Decreased levels of free D-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice.

Science.gov (United States)

Horio, Mao; Ishima, Tamaki; Fujita, Yuko; Inoue, Ran; Mori, Hisashi; Hashimoto, Kenji

2013-05-01

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding. Copyright © 2013 Elsevier Ltd. All rights reserved.

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

International Nuclear Information System (INIS)

Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S.; Rocchi, M.

1991-01-01

The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH 2 -terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH 2 -terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids

Ozone-induced airway hyperresponsiveness in patients with asthma: role of neutrophil-derived serine proteinases.

Science.gov (United States)

Hiltermann, T J; Peters, E A; Alberts, B; Kwikkers, K; Borggreven, P A; Hiemstra, P S; Dijkman, J H; van Bree, L A; Stolk, J

1998-04-01

Proteinase inhibitors may be of potential therapeutic value in the treatment of respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma. Our aim was to study the role of neutrophils, and neutrophil-derived serine proteinases in an acute model in patients with asthma. Exposure to ozone induces an acute neutrophilic inflammatory reaction accompanied by an increase in airway hyperresponsiveness. It is thought that these two effects of ozone are linked, and that neutrophil-derived serine proteinases (i.e. elastase) may play a role in the ozone-induced airway hyperresponsiveness. Therefore, we examined the effect of recombinant antileukoprotease (rALP), one of the major serine proteinase inhibitors in the lung, on ozone-induced changes in airway hyperresponsiveness in this model. We observed that 16 h after exposure to ozone, airway hyperresponsiveness to methacholine was increased both following placebo and rALP treatment. There was no significant difference between placebo and rALP treatment (change in area under the dose-response curve to methacholine: 117.3+/-59.0 vs 193.6+/-59.6 % fall x DD; p=.12). Moreover, the immediate decrease in FEV1 after ozone exposure was not significantly different between the two groups (placebo: -29.6+/-6.7%; rALP: -20.9+/-3.8%; p=.11). In addition, no significant differences were observed in plasma levels of fibrinogen degradation products generated by neutrophil serine proteinases before and after exposure to ozone. We conclude that neutrophil-derived serine proteinases are not important mediators for ozone-induced hyperresponsiveness.

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

Science.gov (United States)

Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

2017-10-01

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Fatal cerebral edema associated with serine deficiency in CSF

NARCIS (Netherlands)

Keularts, Irene M. L. W.; Leroy, Piet L. J. M.; Rubio-Gozalbo, Estela M.; Spaapen, Leo J. M.; Weber, Biene; Dorland, Bert; de Koning, Tom J.; Verhoeven-Duif, Nanda M.

2010-01-01

Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and

Negative Role of RIG-I Serine 8 Phosphorylation in the Regulatin of Interferon-beta Production

Energy Technology Data Exchange (ETDEWEB)

E Nistal-Villan; M Gack; G Martinez-Delgado; N Maharaj; K Inn; H Yang; R Wang; A Aggarwal; J Jung; A Garcia-Sastre

2011-12-31

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

Serine protease from midgut of Bombus terrestris males

Czech Academy of Sciences Publication Activity Database

Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

2013-01-01

Roč. 82, č. 3 (2013), s. 117-128 ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

Thermophysical property characterization of aqueous amino acid salt solution containing serine

International Nuclear Information System (INIS)

Navarro, Shanille S.; Leron, Rhoda B.; Soriano, Allan N.; Li, Meng-Hui

2014-01-01

Highlights: • Thermophysical properties of aqueous potassium and sodium salt solutions of serine were studied. • Density, viscosity, refractive index and electrolytic conductivity of the solution were measured. • The concentrations of amino acid salt ranges from x 1 = 0.009 to 0.07. • The temperature range studied was (298.15 to 343.15) K. • The measured data were represented satisfactorily by using the applied correlations. - Abstract: Thermophysical property characterization of aqueous potassium and sodium salt solutions containing serine was conducted in this study; specifically the system’s density, refractive index, electrical conductivity, and viscosity. Measurements were obtained over a temperature range of (298.15 to 343.15) K and at normal atmospheric pressure. Composition range from x 1 = 0.009 to 0.07 for aqueous potassium and sodium salt solutions containing serine was used. The sensitivity of the system’s thermophysical properties on temperature and composition variation were discussed and correlated based on the equations proposed for room temperature ionic liquids. The density, viscosity, and refractive index measurements of the aqueous systems were found to decrease as the temperature increases at fixed concentration and the values increase as the salt concentration increases (water composition decreases) at fixed temperature. Whereas, a different trend was observed for the electrical conductivity data; at fixed concentration, the conductivity values increase as the temperature increases and at fixed temperature, its value generally increases as the salt concentration increases but only to a certain level (specific concentration) wherein the conductivity of the solution starts to decrease when the concentration of the salt is further increased. Calculation results show that the applied models were satisfactory in representing the measured properties in the aqueous amino acid salt solution containing serine

The C-terminal sequence of several human serine proteases encodes host defense functions.

Science.gov (United States)

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Walse, Björn; Svensson, Bo; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2011-01-01

Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest. Copyright © 2011 S. Karger AG, Basel.

D-serine plasma concentration is a potential biomarker of (R,S)-ketamine antidepressant response in subjects with treatment-resistant depression.

Science.gov (United States)

Moaddel, Ruin; Luckenbaugh, David A; Xie, Ying; Villaseñor, Alma; Brutsche, Nancy E; Machado-Vieira, Rodrigo; Ramamoorthy, Anuradha; Lorenzo, Maria Paz; Garcia, Antonia; Bernier, Michel; Torjman, Marc C; Barbas, Coral; Zarate, Carlos A; Wainer, Irving W

2015-01-01

(R,S)-ketamine is a rapid and effective antidepressant drug that produces a response in two thirds of patients with treatment-resistant depression (TRD). The underlying biochemical differences between a (R,S)-ketamine responder (KET-R) and non-responder (KET-NR) have not been definitively identified but may involve serine metabolism. The aim of the study was to examine the relationship between baseline plasma concentrations of D-serine and its precursor L-serine and antidepressant response to (R,S)-ketamine in TRD patients. Plasma samples were obtained from 21 TRD patients at baseline, 60 min before initiation of the (R,S)-ketamine infusion. Patients were classified as KET-Rs (n = 8) or KET-NRs (n = 13) based upon the difference in Montgomery-Åsberg Depression Rating Scale (MADRS) scores at baseline and 230 min after infusion, with response defined as a ≥50 % decrease in MADRS score. The plasma concentrations of D-serine and L-serine were determined using liquid chromatography-mass spectrometry. Baseline D-serine plasma concentrations were significantly lower in KET-Rs (3.02 ± 0.21 μM) than in KET-NRs (4.68 ± 0.81 μM), p < 0.001. A significant relationship between baseline D-serine plasma concentrations and percent change in MADRS at 230 min was determined using a Pearson correlation, r = 0.77, p < 0.001, with baseline D-serine explaining 60 % of the variance in (R,S)-ketamine response. The baseline concentrations of L-serine (L-Ser) in KET-Rs were also significantly lower than those measured in KET-NRs (66.2 ± 9.6 μM vs 242.9 ± 5.6 μM, respectively; p < 0.0001). The results demonstrate that the baseline D-serine plasma concentrations were significantly lower in KET-Rs than in KET-NRs and suggest that this variable can be used to predict an antidepressant response following (R,S)-ketamine administration.

Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

Directory of Open Access Journals (Sweden)

Weis Robert M

2005-03-01

Full Text Available Abstract Background Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr, which contributes to an increase in the steady-state (adapted methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA and an adaptor protein (CheW, but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. Results Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493. The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively differed from those found previously for the aspartate receptor (Tar, which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately were the largest at site one, ~3 and 6-fold, respectively, and the least at site four, no change and ~2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four. This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. Conclusion The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of

Serine proteinases and their inhibitors in fertilization

Czech Academy of Sciences Publication Activity Database

Jonáková, Věra; Jelínková-Slavíčková, Petra

2004-01-01

Roč. 8, 3,4 (2004), s. 108-110 ISSN 1211-8869. [Central European Conference on Human Tumor Markers /5./. Praha, 01.10.2004-03.10.2004] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA ČR GP303/04/P070; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : serine proteinase * proteinase inhibitors * fertilization Subject RIV: CE - Biochemistry

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

Science.gov (United States)

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

2015-08-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine.

Science.gov (United States)

Gilbert, G E; Drinkwater, D

1993-09-21

Phosphatidylserine, a negatively charged lipid, is exposed on the platelet membrane following cell stimulation, correlating with the expression of factor VIII receptors. We have explored the importance of the negative electrostatic potential of phosphatidylserine vs chemical moieties of phosphatidylserine for specific membrane binding of factor VIII. Fluorescein-labeled factor VIII bound to membranes containing 15% phosphatidic acid, a negatively charged phospholipid, with low affinity compared to phosphatidylserine-containing membranes. Binding was not specific as it was inhibited by other proteins in plasma. Factor VIII bound to membranes containing 10% phosphatidylserine in spite of a varying net charge provided by 0-15% stearylamine, a positively charged lipid. The soluble phosphatidylserine moiety, O-phospho-L-serine, inhibited factor VIII binding to phosphatidylserine-containing membranes with a Ki of 20 mM, but the stereoisomer, O-phospho-D-serine, was 5-fold less effective. Furthermore, binding of factor VIII to membranes containing synthetic phosphatidyl-D-serine was 5-fold less than binding to membranes containing phosphatidyl-L-serine. Membranes containing synthetic phosphatidyl-L-homoserine, differing from phosphatidylserine by a single methylene, supported high-affinity binding, but it was not specific as factor VIII was displaced by other plasma proteins. O-Phospho-L-serine also inhibited the binding of factor VIII to platelet-derived microparticles with a Ki of 20 mM, and the stereoisomer was 4-fold less effective. These results indicate that membrane binding of factor VIII is mediated by a stereoselective recognition O-phospho-L-serine of phosphatidylserine and that negative electrostatic potential is of lesser importance.

ANTIOXIDANT EFFECTS OF L-SERINE AGAINST FATTY STREAK FORMATION IN HYPERCHOLESTEROLEMIC ANIMALS

Directory of Open Access Journals (Sweden)

Ahmad Movahedian

2010-12-01

Full Text Available   Abstract INTRODUCTION: Peroxidation of blood lipoproteins is regarded as a key event in the development of atherosclerosis. Evidence suggests that oxidative modification of amino acids in low-density lipoprotein (LDL particles leads to its convert into an atherogenic form, which is taken up by macrophages. Therefore the reduction of oxidative modification of lipoproteins by increasing plasma antioxidant capacity may prevent cardiovascular disease. methods: In this study, the antioxidant and anti-fatty streak effects of L-serine were investigated in hypercholesterolemic rabbits. Rabbits were randomly divided into three groups which were fed high-cholesterol diet (hypercholesterolemic control group, high-cholesterol + L-serine diet (treatment group, and normal diet (control for twelve weeks and then blood samples were obtained to measure plasma cholesterol, triglyceride (TG, high-density lipoprotein (HDL, low-density lipoprotein (LDL, antioxidant capacity (AC, malondialdehyde (MDA, and conjugated dienes (CDS. Right and left coronary arteries were also obtained for histological evaluation. results: No significant difference was observed in plasma cholesterol, TG, HDL, LDL and CDS levels between treatment and hypercholesterolemic control groups (P>0.05. The levels of plasma MDA and AC were 0.29‌ µM and 56%, respectively in the treatment group which showed a significant change in comparison with hypercholesterolemic control groups (P<0.05. The mean size of produced fatty streak also showed significant reduction in the treatment group compared to the hypercholesterolemic group (P<0.05. CONCLUSIONS: The results showed that L-serine has antioxidant and anti-fatty streak effects without any influence on plasma lipid levels in hypercholesterolemic rabbits.     Keywords: Atherosclerosis, cholesterol, L-serine, antioxidant, lipids, fatty streak.

Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

Science.gov (United States)

Yasumitsu, Hidetaro

2017-01-01

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

Science.gov (United States)

Ogawara, Hiroshi

2016-09-01

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

Conserved water molecules in bacterial serine hydroxymethyltransferases.

Science.gov (United States)

Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

2015-10-01

Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174.

Science.gov (United States)

Arias, C A; Weisner, J; Blackburn, J M; Reynolds, P E

2000-07-01

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

Metabolism of serine in growing rats and chicks at various dietary protein levels

International Nuclear Information System (INIS)

Tanaka, Hideyuki; Yamaguchi, Michio; Kametaka, Masao

1976-01-01

The metabolic fate of the carbon skeleton of L-serine-U- 14 C has been investigated, in vivo and in vitro, in growing rats and chicks fed the diets with various protein calories percents (C %) at 410 kcal of metabolizable energy. The incorporation of 14 C into body protein at 12 hr after the injection of serine- 14 C was about 49% of the injected dose in rats fed the 10 or 15 PC% diet, though the value was reduced in rats fed lower and higher protein diets. The 14 CO 2 production was smaller in rats fed the 10 and 15 PC% diet, and it showed an inverse pattern to that of the 14 C incorporation into body protein. Urinary excretion of 14 C was higher in rats fed 10 and higher PC% diets, whose growth rate and net body protein retention were maximum. In contrast to the case of rats, the incorporation of 14 C into body protein of chicks at 6 hr after the injection was rather reduced in the 15 PC% group. The proportion of 14 C excreted as uric acid was remarkably increased above the 10 PC% group, and about 19% of the injected dose was recovered in the 50 PC% group. The catabolic rate of serine in the liver slices of rats and chicks was increased by high protein diets. These results support the concept that the nutritional significance of metabolism of the carbon skeleton of serine in growing rats and chicks is different from each other, especially at high protein diets. (auth.)

Incorporation of glycine and serine into sporulating cells of Bacillus subtilis

International Nuclear Information System (INIS)

Mitani, Takahiko; Kadota, Hajime

1976-01-01

The changes during growth and sporulation in activities of cells of Bacillus subtilis to incorporate various amino acids were investigated with wild-type strain and its asporogenous mutant. In the case of wild type strain the uptake of valine, phenylalanine, and proline was largest during the logarithmic growth period. The uptake of these amino acids decreased rapidly during the early stationary phase. The uptake of valine and cysteine increased again to some extent just prior to the forespore stage. The uptake of glycine and serine, however, was largest at the forespore stage at which the formation of spore coat took place. From these observed phenomena it was assumed that the remarkable incorporation of glycine and serine into the wild type strain during sporulation was closely related to the formation of spore coat. (auth.)

Dual function of a bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals.

Science.gov (United States)

Choo, Young Moo; Lee, Kwang Sik; Yoon, Hyung Joo; Kim, Bo Yeon; Sohn, Mi Ri; Roh, Jong Yul; Je, Yeon Ho; Kim, Nam Jung; Kim, Iksoo; Woo, Soo Dong; Sohn, Hung Dae; Jin, Byung Rae

2010-05-03

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus annulatuslarvae

Directory of Open Access Journals (Sweden)

Sedigheh Nabian

Full Text Available Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus annulatusis an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B. annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B. annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

Science.gov (United States)

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Serine racemase is expressed in islets and contributes to the regulation of glucose homeostasis.

Science.gov (United States)

Lockridge, Amber D; Baumann, Daniel C; Akhaphong, Brian; Abrenica, Alleah; Miller, Robert F; Alejandro, Emilyn U

2016-11-01

NMDA receptors (NMDARs) have recently been discovered as functional regulators of pancreatic β-cell insulin secretion. While these excitatory receptor channels have been extensively studied in the brain for their role in synaptic plasticity and development, little is known about how they work in β-cells. In neuronal cells, NMDAR activation requires the simultaneous binding of glutamate and a rate-limiting co-agonist, such as D-serine. D-serine levels and availability in most of the brain rely on endogenous synthesis by the enzyme serine racemase (Srr). Srr transcripts have been reported in human and mouse islets but it is not clear whether Srr is functionally expressed in β-cells or what its role in the pancreas might be. In this investigation, we reveal that Srr protein is highly expressed in primary human and mouse β-cells. Mice with whole body deletion of Srr (Srr KO) show improved glucose tolerance through enhanced insulin secretory capacity, possibly through Srr-mediated alterations in islet NMDAR expression and function. We observed elevated insulin sensitivity in some animals, suggesting Srr metabolic regulation in other peripheral organs as well. Srr expression in neonatal and embryonic islets, and adult deficits in Srr KO pancreas weight and islet insulin content, point toward a potential role for Srr in pancreatic development. These data reveal the first evidence that Srr may regulate glucose homeostasis in peripheral tissues and provide circumstantial evidence that D-serine may be an endogenous islet NMDAR co-agonist in β-cells.

The Mini-SPT (Space Particle Telescope) for dual use: Precision flux measurement of low energy proton electron and heavy ion with tracking capability and A compact, low-cost realtime local radiation hazard/alarm detector to be used on board a satellite

Science.gov (United States)

Alpat, Behcet; Ergin, Tulun; Kalemci, Emrah

2016-07-01

The Mini-SPT project is the first, and most important, step towards the ambitious goal of creating a low-cost, compact, radiation hardened and high performance space particle telescope that can be mounted, in the near future, as standard particle detector on any satellite. Mini-SPT will be capable of providing high quality physics data on local space environment. In particular high precision flux measurement and tracking of low energy protons and electrons on different orbits with same instrumentation is of paramount importance for studies as geomagnetically trapped fluxes and space weather dynamics, dark matter search, low energy proton anisotropy and its effects on ICs as well as the solar protons studies. In addition, it will provide real-time "differentiable warnings" about the local space radiation hazard to other electronics systems on board the hosting satellite, including different criticality levels and alarm signals to activate mitigation techniques whenever this is strictly necessary to protect them from temporary/permanent failures. A real-time warning system will help satellite subsystems to save significant amount of power and memory with respect to other conventional techniques where the "mitigation" solutions are required to be active during entire mission life. The Mini-SPT will combine the use of technologies developed in cutting-edge high energy physics experiments (including technology from CMS experiments at CERN) and the development of new charged particle detecting systems for their use for the first time in space. The Mini-SPT essential objective is, by using for the first time in space SIPMs (Silicon Photomultipliers) technology for TOF and energy measurements, the production of high quality data with a good time, position and energy resolutions. The mini-SPT will consists of three main sub-units: a- A tracking and dE/dX measuring sub-detector which will be based on silicon pixel detectors (SPD) coupled to the rad-hard chip ROC-DIG (Read

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Science.gov (United States)

Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

2016-08-01

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

Science.gov (United States)

Arias, C A; Martín-Martinez, M; Blundell, T L; Arthur, M; Courvalin, P; Reynolds, P E

1999-03-01

Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper Leaves Using Response Surface Methodology

Directory of Open Access Journals (Sweden)

Md. Zaidul Islam Sarker

2011-11-01

Full Text Available Response surface methodology (RSM using a central composite design (CCD was employed to optimize the conditions for extraction of serine protease from kesinai (Streblus asper leaves. The effect of independent variables, namely temperature (42.5,47.5, X1, mixing time (2–6 min, X2, buffer content (0–80 mL, X3 and buffer pH (4.5–10.5, X4 on specific activity, storage stability, temperature and oxidizing agent stability of serine protease from kesinai leaves was investigated. The study demonstrated that use of the optimum temperature, mixing time, buffer content and buffer pH conditions protected serine protease during extraction, as demonstrated by low activity loss. It was found that the interaction effect of mixing time and buffer content improved the serine protease stability, and the buffer pH had the most significant effect on the specific activity of the enzyme. The most desirable conditions of 2.5 °C temperature, 4 min mixing time, 40 mL buffer at pH 7.5 was established for serine protease extraction from kesinai leaves.

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis.

Science.gov (United States)

Waern, Ida; Karlsson, Iulia; Thorpe, Michael; Schlenner, Susan M; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Åbrink, Magnus; Hellman, Lars; Pejler, Gunnar; Wernersson, Sara

2012-12-01

Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Directory of Open Access Journals (Sweden)

Nicolas Faller

Full Text Available Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Apical serine protease activity is necessary for assembly of a high-resistance renal collecting duct epithelium

DEFF Research Database (Denmark)

Steensgaard, Mette; Svenningsen, Per; Tinning, Anne R

2010-01-01

Abstract AIM: We hypothesized that the serine protease prostasin is necessary for differentiation of a high resistance renal collecting duct epithelium governed by glucocorticoid. METHODS: Postnatal rat kidney and adult human kidney was used to study expression and localization of prostasin......-cadherin distribution did not change. CONCLUSION: Apical, GPI-anchored, lipid raft-associated serine protease activity, compatible with prostasin, is necessary for development of a high-resistance collecting duct epithelium....

Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

Science.gov (United States)

Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

2012-09-01

The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases. Published by Elsevier Inc.

Unlike pregnant adult women, pregnant adolescent girls cannot maintain glycine flux during late pregnancy because of decreased synthesis from serine.

Science.gov (United States)

Hsu, Jean W; Thame, Minerva M; Gibson, Raquel; Baker, Tameka M; Tang, Grace J; Chacko, Shaji K; Jackson, Alan A; Jahoor, Farook

2016-03-14

During pregnancy, glycine and serine become more important because they are the primary suppliers of methyl groups for the synthesis of fetal DNA, and more glycine is required for fetal collagen synthesis as pregnancy progresses. In an earlier study, we reported that glycine flux decreased by 39% from the first to the third trimester in pregnant adolescent girls. As serine is a primary precursor for glycine synthesis, the objective of this study was to measure and compare glycine and serine fluxes and inter-conversions in pregnant adolescent girls and adult women in the first and third trimesters. Measurements were made after an overnight fast by continuous intravenous infusions of 2H2-glycine and 15N-serine in eleven adolescent girls (17·4 (se 0·1) years of age) and in ten adult women (25·8 (se 0·5) years of age) for 4 h. Adolescent girls had significantly slower glycine flux and they made less glycine from serine in the third (Padolescent girls (P=0·04) and was significantly associated with third trimester glycine flux. These findings suggest that the pregnant adolescent cannot maintain glycine flux in late pregnancy compared with early pregnancy because of decreased synthesis from serine. It is possible that the inability to maintain glycine synthesis makes her fetus vulnerable to impaired cartilage synthesis, and thus linear growth.

Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

Science.gov (United States)

Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

2016-01-01

Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

Characterization of a serine protease-mediated cell death program activated in human leukemia cells

International Nuclear Information System (INIS)

O'Connell, A.R.; Holohan, C.; Torriglia, A.; Lee, B.F.; Stenson-Cox, C.

2006-01-01

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis

Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

Directory of Open Access Journals (Sweden)

Little Tom J

2009-06-01

Full Text Available Abstract Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the An. gambiae species complex in both East and West Africa. Results Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes. Conclusion It is well known that phylogenetic and population history in the An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

Science.gov (United States)

Meziane-Cherif, Djalal; Stogios, Peter J; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl-D-alanine target of peptidoglycan precursors with D-alanyl-D-lactate or D-alanyl-D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the L-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the D-alanine ending precursors in cell wall

Detection of a Double Relic in the Torpedo Cluster: SPT-CL J0245-5302

Science.gov (United States)

Zheng, Q.; Johnston-Hollitt, M.; Duchesne, S. W.; Li, W. T.

2018-06-01

The Torpedo cluster, SPT-CL J0245-5302 (S0295) is a massive, merging cluster at a redshift of z = 0.300, which exhibits a strikingly similar morphology to the Bullet cluster 1E 0657-55.8 (z = 0.296), including a classic bow shock in the cluster's intra-cluster medium revealed by Chandra X-ray observations. We present Australia Telescope Compact Array data centred at 2.1 GHz and Murchison Widefield Array data at frequencies between 72 MHz and 231 MHz which we use to study the properties of the cluster. We characterise a number of discrete and diffuse radio sources in the cluster, including the detection of two previously unknown radio relics on the cluster periphery. The average spectral index of the diffuse emission between 70 MHz and 3.1 GHz is α =-1.63_{-0.10}^{+0.10} and a radio-derived Mach number for the shock in the west of the cluster is calculated as M = 2.04. The Torpedo cluster is thus a double relic system at moderate redshift.

Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis

DEFF Research Database (Denmark)

Hein, Jamin B; Hertz, Emil P T; Garvanska, Dimitriya H

2017-01-01

Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit. Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains ...

Oxytocin analogues with O-glycosylated serine and threonine in position 4

Czech Academy of Sciences Publication Activity Database

Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

2007-01-01

Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at C-terminal serine residues

DEFF Research Database (Denmark)

Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B

2014-01-01

heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4....... Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser...

Optimization of serine protease purification from mango (Mangifera indica cv. Chokanan) peel in polyethylene glycol/dextran aqueous two phase system.

Science.gov (United States)

Mehrnoush, Amid; Mustafa, Shuhaimi; Sarker, Md Zaidul Islam; Yazid, Abdul Manap Mohd

2012-01-01

Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.

Characterization of a membrane-associated serine protease in Escherichia coli

International Nuclear Information System (INIS)

Palmer, S.M.; St John, A.C.

1987-01-01

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

The Hunger Games: p53 regulates metabolism upon serine starvation.

Science.gov (United States)

Tavana, Omid; Gu, Wei

2013-02-05

Cancer cells reprogram their metabolism to support a high proliferative rate. A new study shows that, upon serine starvation, the tumor suppressor p53 activates p21 to shift metabolic flux from purine biosynthesis to glutathione production, which enhances cellular proliferation and viability by combating ROS (Maddocks et al., 2013). Copyright © 2013 Elsevier Inc. All rights reserved.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

Directory of Open Access Journals (Sweden)

José Andrés Morgado-Díaz

2005-07-01

Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Generation of serine/threonine check points in HN(C)N spectra

Indian Academy of Sciences (India)

Administrator

ing to generate alanine. 6 and serine/threonine specific peak patterns. 7 have enhanced the speed of assign- ment quite substantially. These developments involved a simple modification to the pulse sequence. Continuing such efforts for rapid resonance as- signments, we have implemented here the tuning ideas.

Deficiency in L-serine deaminase interferes with one-carbon metabolism and cell wall synthesis in Escherichia coli K-12.

Science.gov (United States)

Zhang, Xiao; El-Hajj, Ziad W; Newman, Elaine

2010-10-01

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C(1) units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

International Nuclear Information System (INIS)

Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

2004-01-01

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

Long-term increased carnitine palmitoyltransferase 1A expression in ventromedial hypotalamus causes hyperphagia and alters the hypothalamic lipidomic profile.

Directory of Open Access Journals (Sweden)

Paula Mera

Full Text Available Lipid metabolism in the ventromedial hypothalamus (VMH has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT 1A is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH, long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT and reduced vesicular glutamate transporter 2 (VGLUT2 expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH.

Sol-gel immobilization of serine proteases for application in organic solvents

NARCIS (Netherlands)

van Unen, D.J.; Engbersen, Johannes F.J.; Reinhoudt, David

2001-01-01

The serine proteases α-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of α-chymotrypsin and the resulting specific enzyme activity in

A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate.

Science.gov (United States)

Shrestha, Ritu; Lockless, Steve W; Sorg, Joseph A

2017-06-23

Clostridium difficile has become one of the most common bacterial pathogens in hospital-acquired infections in the United States. Although C. difficile is strictly anaerobic, it survives in aerobic environments and transmits between hosts via spores. C. difficile spore germination is triggered in response to certain bile acids and glycine. Although glycine is the most effective co-germinant, other amino acids can substitute with varying efficiencies. Of these, l-alanine is an effective co-germinant and is also a germinant for most bacterial spores. Many endospore-forming bacteria embed alanine racemases into their spore coats, and these enzymes are thought to convert the l-alanine germinant into d-alanine, a spore germination inhibitor. Although the C. difficile Alr2 racemase is the sixth most highly expressed gene during C. difficile spore formation, a previous study reported that Alr2 has little to no role in germination of C. difficile spores in rich medium. Here, we hypothesized that Alr2 could affect C. difficile l-alanine-induced spore germination in a defined medium. We found that alr2 mutant spores more readily germinate in response to l-alanine as a co-germinant. Surprisingly, d-alanine also functioned as a co-germinant. Moreover, we found that Alr2 could interconvert l- and d-serine and that Alr2 bound to l- and d-serine with ∼2-fold weaker affinity to that of l- and d-alanine. Finally, we demonstrate that l- and d-serine are also co-germinants for C. difficile spores. These results suggest that C. difficile spores can respond to a diverse set of amino acid co-germinants and reveal that Alr2 can accommodate serine as a substrate. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

Science.gov (United States)

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12▿

Science.gov (United States)

Zhang, Xiao; El-Hajj, Ziad W.; Newman, Elaine

2010-01-01

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide. PMID:20729359

Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila

Directory of Open Access Journals (Sweden)

Philip Yuk Kwong Yung

2015-06-01

Full Text Available Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28 whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

Crystal structure of the NADP+ and tartrate-bound complex of L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis.

Science.gov (United States)

Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

2018-05-01

A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP + /sulfate ion at 1.18 Å and the structure in complex with NADP + /L-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP + and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.

Characterization of the Usage of the Serine Metabolic Network in Human Cancer

Directory of Open Access Journals (Sweden)

Mahya Mehrmohamadi

2014-11-01

Full Text Available The serine, glycine, one-carbon (SGOC metabolic network is implicated in cancer pathogenesis, but its general functions are unknown. We carried out a computational reconstruction of the SGOC network and then characterized its expression across thousands of cancer tissues. Pathways including methylation and redox metabolism exhibited heterogeneous expression indicating a strong context dependency of their usage in tumors. From an analysis of coexpression, simultaneous up- or downregulation of nucleotide synthesis, NADPH, and glutathione synthesis was found to be a common occurrence in all cancers. Finally, we developed a method to trace the metabolic fate of serine using stable isotopes, high-resolution mass spectrometry, and a mathematical model. Although the expression of single genes didn’t appear indicative of flux, the collective expression of several genes in a given pathway allowed for successful flux prediction. Altogether, these findings identify expansive and heterogeneous functions for the SGOC metabolic network in human cancer.

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

Science.gov (United States)

Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

1999-01-01

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

Analysis of L-serine-O-phosphate in cerebrospinal spinal fluid by derivatization-liquid chromatography/mass spectrometry.

Science.gov (United States)

McNaney, Colleen A; Benitex, Yulia; Luchetti, David; Labasi, Jeffrey M; Olah, Timothy V; Morgan, Daniel G; Drexler, Dieter M

2014-05-01

L-serine-O-phosphate (L-SOP), the precursor of L-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of L-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine L-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization-LC/MS, d-LC/MS). Copyright © 2014 Elsevier Inc. All rights reserved.

Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

DEFF Research Database (Denmark)

Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

2005-01-01

processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Czech Academy of Sciences Publication Activity Database

Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

2017-01-01

Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein - protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016

A cyclic peptidic serine protease inhibitor

DEFF Research Database (Denmark)

Zhao, Baoyu; Xu, Peng; Jiang, Longguang

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes

NARCIS (Netherlands)

Boerrigter, Ingrid J.; Buist, Girbe; Haandrikman, Alfred J.; Nijhuis, Monique; Reuver, Marjon B. de; Siezen, Roland J.; Venema, Gerhardus; Vos, Willem M. de; Kok, Jan

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were

Single residue mutation in active site of serine acetyltransferase isoform 3 from Entamoeba histolytica assists in partial regaining of feedback inhibition by cysteine.

Directory of Open Access Journals (Sweden)

Sudhir Kumar

Full Text Available The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT and O-acetylserine sulfhydrylase (OASS are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated in vitro, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by K(m, compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3 shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor.

Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling.

Science.gov (United States)

Chen, Xiaoyu; Bai, Bo; Tian, Yanjun; Du, Hui; Chen, Jing

2014-11-07

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

Science.gov (United States)

Rojas, Ana; Doolittle, Russell F.

2003-01-01

Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

Science.gov (United States)

He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

2008-10-01

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

Realizing Serine/Threonine Ligation: Scope and Limitations and Mechanistic Implication Thereof

Directory of Open Access Journals (Sweden)

Clarence T. T. Wong

2014-05-01

Full Text Available Serine/Threonine ligation (STL has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the serine/threonine ligation strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky β-branched amino acid (Thr, Val and Ile is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.

Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

Directory of Open Access Journals (Sweden)

Tanveer Majeed

2013-01-01

Full Text Available A putative protease gene (aprE from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10. In addition, the enzyme had an elevated optimum temperature (60°C. The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.

Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of β-oxidation.

Directory of Open Access Journals (Sweden)

Cong-Hui Yao

2018-03-01

Full Text Available It has been suggested that some cancer cells rely upon fatty acid oxidation (FAO for energy. Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1 with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 μM have an off-target effect of inhibiting complex I of the electron transport chain. Surprisingly, however, when FAO was reduced further by genetic knockdown of CPT1, the proliferation rate of these same cells decreased nearly 2-fold and could not be restored by acetate or octanoic acid supplementation. Moreover, CPT1 knockdowns had altered mitochondrial morphology and impaired mitochondrial coupling, whereas cells in which CPT1 had been approximately 90% inhibited by etomoxir did not. Lipidomic profiling of mitochondria isolated from CPT1 knockdowns showed depleted concentrations of complex structural and signaling lipids. Additionally, expression of a catalytically dead CPT1 in CPT1 knockdowns did not restore mitochondrial coupling. Taken together, these results suggest that transport of at least some long-chain fatty acids into the mitochondria by CPT1 may be required for anabolic processes that support healthy mitochondrial function and cancer cell proliferation independent of FAO.

Detection systems for carbapenemase gene identification should include the SME serine carbapenemase.

Science.gov (United States)

Bush, Karen; Pannell, Megan; Lock, John L; Queenan, Anne Marie; Jorgensen, James H; Lee, Ryan M; Lewis, James S; Jarrett, Deidre

2013-01-01

Carbapenemase detection has become a major problem in hospitals that encounter outbreaks of infections caused by carbapenem-resistant Gram-negative bacteria. Rapid detection systems have been reported using multiplex PCR analyses and DNA microarray assays. Major carbapenemases that are detected by these systems include the KPC and OXA serine carbapenemases, and the IMP, VIM and NDM families of metallo-β-lactamases. However, increasing numbers of the SME serine carbapenemase are being reported from Serratia marcescens, especially from North and South America. These organisms differ from many of the other carbapenemase-producing pathogens in that they are generally susceptible to the expanded-spectrum cephalosporins ceftazidime and cefepime while retaining resistance to almost all other β-lactam antibiotics. Thus, multiplex PCR assays or DNA microarray testing of carbapenem-resistant S. marcescens isolates should include analyses for production of the SME carbapenemase. Confirmation of the presence of this enzyme may provide reassurance that oxyimino-cephalosporins can be considered for treatment of infections caused by these carbapenem-resistant pathogens. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

Directory of Open Access Journals (Sweden)

Lei eShi

2014-09-01

Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

Short hydrogen bonds in the catalytic mechanism of serine proteases

Directory of Open Access Journals (Sweden)

VLADIMIR LESKOVAC

2008-04-01

Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Bregenholt, S; Johansen, B

1999-01-01

B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine...

Studies of Environmental Risk Factors in Amyotrophic Lateral Sclerosis (ALS) and a Phase I Clinical Trial of L-Serine.

Science.gov (United States)

Bradley, Walter G; Miller, R X; Levine, T D; Stommel, E W; Cox, P A

2018-01-01

β-N-Methylamino-L-alanine (BMAA) has been linked to Guam ALS/PDC and shown to produce neurodegeneration in vitro and in vivo (Drosophila, mice, rats, primates). BMAA misincorporation into neuroproteins produces protein misfolding and is inhibited by L-serine. Case-control studies in Northern New England indicate that living near to water-bodies with cyanobacterial blooms increases the risk of developing amyotrophic lateral sclerosis (ALS). The distribution of addresses of ALS cases in New Hampshire, Vermont, and Florida was compared to that of controls. Areas of statistically significantly increased numbers of ALS cases were examined for sources of environmental toxins. A phase I trial of oral L-serine was performed in 20 ALS patients (0.5 to 15 g twice daily). Safety and tolerability were assessed by comparing the rate of deterioration with 430 matched placebo controls. The distribution of residential addresses of ALS cases in New England and Florida revealed many areas where the age- and gender-adjusted frequency of ALS was greater than expected (P ALS patients suggests that residential exposure to environmental pollutants may play an important role in the etiology of ALS. L-Serine in doses up to 15 g twice daily appears to be safe in patients with ALS. Exploratory studies of efficacy suggested that L-serine might slow disease progression. A phase II trial is planned.

Skeletal muscle PLIN3 and PLIN5 are serine phosphorylated at rest and following lipolysis during adrenergic or contractile stimulation

Science.gov (United States)

MacPherson, Rebecca E K; Vandenboom, Rene; Roy, Brian D; Peters, Sandra J

2013-01-01

In adipose tissue, access of adipose triglyceride and hormone-sensitive lipases (ATGL and HSL) to the lipid droplet depends on PLIN1 phosphorylation, however, PLIN1 is not expressed in skeletal muscle and the phosphorylation of the expressed PLINs has yet to be investigated. Further, direct interactions between skeletal muscle PLINs and HSL are unknown. We investigated the isolated and combined effects of epinephrine and contraction on PLIN-to-lipase interactions as well as phosphorylation. Isolated rat solei were assigned to one of four 30 min in vitro conditions (25°C): (1) rest; (2) intermittent tetanic stimulation (60 Hz for 150 msec; train rate 20/min); (3) 5 nmol/L epinephrine; (4) intermittent tetanic stimulation and 5 nmol/L epinephrine. Immunoprecipitation of serine phosphorylated proteins followed by Western blotting for PLIN2, PLIN3, PLIN5, revealed that only PLIN2 is not phosphorylated under any of the experimental conditions. This is the first study to show that in whole rat skeletal muscle PLIN3 and PLIN5 are serine phosphorylated. The degree of serine phosphorylation remained unchanged following adrenergic and/or contractile stimulation. Oil red O staining of muscle sections for lipid content shows a significant decrease following each condition, confirming lipolysis occurred (P < 0.05). PLIN2, 3, and 5 all interact with HSL and ATGL, but these interactions were unchanged following treatments. Our results show that in skeletal muscle, PLIN2 is not serine phosphorylated at rest or with lipolytic stimulation and that while PLIN3, PLIN5 are serine phosphorylated at rest, the degree of phosphorylation does not change with lipolytic stimulation. PMID:24303154

Identification of B cell recognized linear epitopes in a snake venom serine proteinase from the central American bushmaster Lachesis stenophrys.

Science.gov (United States)

Madrigal, M; Alape-Girón, A; Barboza-Arguedas, E; Aguilar-Ulloa, W; Flores-Díaz, M

2017-12-15

Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikrein-kinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surface-located epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex

International Nuclear Information System (INIS)

Onischenko, Evgeny A.; Crafoord, Ellinor; Hallberg, Einar

2007-01-01

The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo. An alanine substitution mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. Our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC

Efficacy of Glutamate Modulators in Tic Suppression: A Double-Blind, Randomized Control Trial of D-serine and Riluzole in Tourette Syndrome.

Science.gov (United States)

Lemmon, Monica E; Grados, Marco; Kline, Tina; Thompson, Carol B; Ali, Syed F; Singer, Harvey S

2015-06-01

It has been hypothesized that glutamatergic transmission may be altered in Tourette syndrome. In this study, we explored the efficacy of a glutamate agonist (D-serine) and antagonist (riluzole) as tic-suppressing agents in children with Tourette syndrome. We performed a parallel three-arm, 8-week, double-blind, randomized placebo-controlled treatment study in children with Tourette syndrome. Each child received 6 weeks of treatment with D-serine (maximum dose 30 mg/kg/day), riluzole (maximum dose 200 mg/day), or placebo, followed by a 2-week taper. The primary outcome measure was effective tic suppression as determined by the differences in the Yale Global Tic Severity Scale score; specifically, the total tic score and the combined score (total tic score + global impairment) between treatment arms after 6 weeks of treatment. Mann-Whitney U tests were performed to analyze differences between each group and the placebo group. Twenty-four patients (males = 21, ages 9-18) enrolled in the study; one patient dropped out before completion. Combined Yale Global Tic Severity Scale score and total tic scores improved in all groups. The 6-week mean percent improvement of the riluzole (n = 10), D-serine (n = 9), and placebo (n = 5) groups in the combined Yale Global Tic Severity Scale score were 43.7, 39.5, and 30.2 and for total tic scores were 38.0, 25.0, and 34.0, respectively. There were no significant differences in Yale Global Tic Severity Scale score or total tic score, respectively, between the riluzole and placebo (P = 0.35, 0.85) or D-serine and placebo (P = 0.50, 0.69) groups. Tics diminished by comparable percentages in the riluzole, D-serine, and placebo groups. These preliminary data suggest that D-serine and riluzole are not effective in tic suppression. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

International Nuclear Information System (INIS)

Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

1988-01-01

A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

Science.gov (United States)

Canova, Marc J.; Molle, Virginie

2014-01-01

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

Bacterial serine/threonine protein kinases in host-pathogen interactions.

Science.gov (United States)

Canova, Marc J; Molle, Virginie

2014-04-04

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

Inhibition of Human Serine Racemase, an Emerging Target for Medicinal Chemistry

Czech Academy of Sciences Publication Activity Database

Jirásková-Vaníčková, Jana; Ettrich, Rüdiger; Vorlová, Barbora; Hoffman, Hillary Elizabeth; Lepšík, Martin; Jansa, Petr; Konvalinka, Jan

2011-01-01

Roč. 12, č. 7 (2011), s. 1037-1055 ISSN 1389-4501 R&D Projects: GA MŠk 1M0508; GA ČR GA203/08/0114 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z60870520 Keywords : amino acid analogs * L-erythro-3-hydroxyaspartate (L-EHA) * D-serine * neurodegenerative diseases * NMDA receptors * pyridoxal-5´-phosphate (PLP) Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.553, year: 2011

An Approach to Flooding Inundation Combining the Streamflow Prediction Tool (SPT) and Downscaled Soil Moisture

Science.gov (United States)

Cotterman, K. A.; Follum, M. L.; Pradhan, N. R.; Niemann, J. D.

2017-12-01

Flooding impacts numerous aspects of society, from localized flash floods to continental-scale flood events. Many numerical flood models focus solely on riverine flooding, with some capable of capturing both localized and continental-scale flood events. However, these models neglect flooding away from channels that are related to excessive ponding, typically found in areas with flat terrain and poorly draining soils. In order to obtain a holistic view of flooding, we combine flood results from the Streamflow Prediction Tool (SPT), a riverine flood model, with soil moisture downscaling techniques to determine if a better representation of flooding is obtained. This allows for a more holistic understanding of potential flood prone areas, increasing the opportunity for more accurate warnings and evacuations during flooding conditions. Thirty-five years of near-global historical streamflow is reconstructed with continental-scale flow routing of runoff from global land surface models. Elevation data was also obtained worldwide, to establish a relationship between topographic attributes and soil moisture patterns. Derived soil moisture data is validated against observed soil moisture, increasing confidence in the ability to accurately capture soil moisture patterns. Potential flooding situations can be examined worldwide, with this study focusing on the United States, Central America, and the Philippines.

Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

Directory of Open Access Journals (Sweden)

S. S. Hasson

2010-01-01

Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

Directory of Open Access Journals (Sweden)

Evan L Pannkuk

Full Text Available White nose syndrome (WNS is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1 was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE, broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

Science.gov (United States)

Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

2015-01-01

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

Energy Technology Data Exchange (ETDEWEB)

Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

2016-02-01

The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

Science.gov (United States)

Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

2015-01-15

Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Science.gov (United States)

Kostyleva, E V; Sereda, A S; Velikoretskaya, I A; Nefedova, L I; Sharikov, A Yu; Tsurikova, N V; Lobanov, N S; Semenova, M V; Sinitsyn, A P

2016-07-01

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley

DEFF Research Database (Denmark)

Roberts, T.H.; Marttila, S.; Rasmussen, S.K.

2003-01-01

centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression...... their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.......Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (

Effect of methylglyoxal bis(guanylhydrazone) on hepatic, heart and skeletal muscle mitochrondrial carnitine palmitoyltransferase and. beta. -oxidation of fatty acids

Energy Technology Data Exchange (ETDEWEB)

Brady, L.J.; Brady, P.S.; Gandour, R.D.

1986-05-01

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antileukemic agent and polyamine analog which inhibits S-adenosyl methionine decarboxylase. However, MGBG also produces mitochondrial structural damage and inhibition of ..beta..-oxidation. The present experiments were designed to determine if MGBG acts via carnitine palmitoyltransferase-A (CPT-A) inhibition. Liver, heart and skeletal muscle mitochondria were isolated from rats following a 24 h fast. MGBG was competitive with 1-carnitine. The MGBG CPT-A Ki were (mM): liver, 5.0 +/- 0.6 (n = 15); heart, 3.2 +/- 1.2 (n = 3); skeletal muscle, 2.8 +/- 1.0 (n = 3). Lysis of hepatic mitochondria with Triton X-100 yielded a Ki of 4.0 +/- 2.0. Purified hepatic CPT was also sensitive to MGBG inhibition (Ki = 4.5 mM). Spermine and spermidine, which are structurally similar to MGBG, did not inhibit CPT or acid-soluble product formation from 1-(/sup 14/C)-palmitoyl-CoA. MGBG inhibited mitochondrial state 3 oxidation rates of palmitoyl-CoA and palmitoylcarnitine, as well as of glutamate. However, the fatty acid substrates were considerably more sensitive than glutamate to MGBG inhibition. MGBG also increased hepatic mitochondrial aggregation which was reversed by 1-carnitine. Fluorescence polarization, using diphenylhexatriene as a probe, indicated that MGBG increased membrane rigidity in a dose dependent manner. This effect was not reversed by 1-carnitine. The authors conclude that MGBG exhibits competitive competition with 1-carnitine for CPT. However, MGBG also exhibits a number of effects which may be mediated through membrane interaction and which are not necessarily reversed by carnitine.

Effect of methylglyoxal bis(guanylhydrazone) on hepatic, heart and skeletal muscle mitochrondrial carnitine palmitoyltransferase and β-oxidation of fatty acids

International Nuclear Information System (INIS)

Brady, L.J.; Brady, P.S.; Gandour, R.D.

1986-01-01

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antileukemic agent and polyamine analog which inhibits S-adenosyl methionine decarboxylase. However, MGBG also produces mitochondrial structural damage and inhibition of β-oxidation. The present experiments were designed to determine if MGBG acts via carnitine palmitoyltransferase-A (CPT-A) inhibition. Liver, heart and skeletal muscle mitochondria were isolated from rats following a 24 h fast. MGBG was competitive with 1-carnitine. The MGBG CPT-A Ki were (mM): liver, 5.0 +/- 0.6 (n = 15); heart, 3.2 +/- 1.2 (n = 3); skeletal muscle, 2.8 +/- 1.0 (n = 3). Lysis of hepatic mitochondria with Triton X-100 yielded a Ki of 4.0 +/- 2.0. Purified hepatic CPT was also sensitive to MGBG inhibition (Ki = 4.5 mM). Spermine and spermidine, which are structurally similar to MGBG, did not inhibit CPT or acid-soluble product formation from 1-[ 14 C]-palmitoyl-CoA. MGBG inhibited mitochondrial state 3 oxidation rates of palmitoyl-CoA and palmitoylcarnitine, as well as of glutamate. However, the fatty acid substrates were considerably more sensitive than glutamate to MGBG inhibition. MGBG also increased hepatic mitochondrial aggregation which was reversed by 1-carnitine. Fluorescence polarization, using diphenylhexatriene as a probe, indicated that MGBG increased membrane rigidity in a dose dependent manner. This effect was not reversed by 1-carnitine. The authors conclude that MGBG exhibits competitive competition with 1-carnitine for CPT. However, MGBG also exhibits a number of effects which may be mediated through membrane interaction and which are not necessarily reversed by carnitine

Evidence for possible involvement of an elastolytic serine protease in aspergillosis.

OpenAIRE

Kolattukudy, P E; Lee, J D; Rogers, L M; Zimmerman, P; Ceselski, S; Fox, B; Stein, B; Copelan, E A

1993-01-01

A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalyzed by a single 33-kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33-kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infect...

L-serine capped ZnS:Mn nanocrystals for plant cell biological studies and as a growth enhancing agent for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae)

Science.gov (United States)

Augustine, M. Sajimol; Mathew, Lizzy; Alex, Roselin; Deepa, G. D.; Jayalekshmi, S.

2014-01-01

In the present work, the prospects of ZnS:Mn nanocrystals capped with L- serine, a bio-compatible amino acid, synthesized by wet chemical route, as efficient fluorescent probes for plant cell biological studies have been investigated. The present synthesis route using bio-compatible material is a low cost and easy to control method. The colloidal stability of the capped nano crystals is very good as they remain stable without settling down for long time. It is observed that L- serine significantly modifies the structural and optical characteristics of the ZnS:Mn nanocrystals and hence is suitable as a bio-compatible capping agent. The structural properties of L- serine capped nanocrystals were investigated by XRD technique. The size of the L- serine capped ZnS:Mn nanocrystals is found to be around 2 nm . The optical characterization of the nanocrystals was carried out on the basis of photoluminescence (PL) spectroscopic studies. The intense photoluminescence emission observed around 597nm for L-serine capped ZnS:Mn offers high prospects of applications in bio-imaging fields. The unique optical properties of nanoparticles make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations. In the present study, L-serine capped ZnS:Mn nanocrystals were used as a staining dye in fluorescent microscope for observing cell division, cell structure etc. These nanocrystals were also incorporated into the culture media along with the normal auxin- cytokinin hormone combinations in Murashige and Skoog (MS) medium for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae), an Ayurvedic medicine. The results suggest that L-serine capped ZnS:Mn nanocrystals can act as efficient enhancers towards quick callusing and shoot proliferation.

L-serine capped ZnS:Mn nanocrystals for plant cell biological studies and as a growth enhancing agent for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae)

International Nuclear Information System (INIS)

Augustine, M. Sajimol; Mathew, Lizzy; Alex, Roselin; Deepa, G. D.; Jayalekshmi, S.

2014-01-01

In the present work, the prospects of ZnS:Mn nanocrystals capped with L- serine, a bio-compatible amino acid, synthesized by wet chemical route, as efficient fluorescent probes for plant cell biological studies have been investigated. The present synthesis route using bio-compatible material is a low cost and easy to control method. The colloidal stability of the capped nano crystals is very good as they remain stable without settling down for long time. It is observed that L- serine significantly modifies the structural and optical characteristics of the ZnS:Mn nanocrystals and hence is suitable as a bio-compatible capping agent. The structural properties of L- serine capped nanocrystals were investigated by XRD technique. The size of the L- serine capped ZnS:Mn nanocrystals is found to be around 2 nm . The optical characterization of the nanocrystals was carried out on the basis of photoluminescence (PL) spectroscopic studies. The intense photoluminescence emission observed around 597nm for L-serine capped ZnS:Mn offers high prospects of applications in bio-imaging fields. The unique optical properties of nanoparticles make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations. In the present study, L-serine capped ZnS:Mn nanocrystals were used as a staining dye in fluorescent microscope for observing cell division, cell structure etc. These nanocrystals were also incorporated into the culture media along with the normal auxin- cytokinin hormone combinations in Murashige and Skoog (MS) medium for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae), an Ayurvedic medicine. The results suggest that L-serine capped ZnS:Mn nanocrystals can act as efficient enhancers towards quick callusing and shoot proliferation

L-serine capped ZnS:Mn nanocrystals for plant cell biological studies and as a growth enhancing agent for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae)

Energy Technology Data Exchange (ETDEWEB)

Augustine, M. Sajimol, E-mail: sajimollazar@gmail.com [Department of Physics, St.Teresa' s College , Kochi-11, Kerala (India); Mathew, Lizzy [Department of Botany, St.Teresa' s College , Kochi-11, Kerala (India); Alex, Roselin [Department of Biotechnology, Cochin University of Science and Technology, Kochi-22 (India); Deepa, G. D. [NCAAH, Cochin University of Science and Technology,Kochi-22, Kerala (India); Jayalekshmi, S., E-mail: jayalekshmi@cusat.ac.in [Department of Physics, Cochin University of Science and Technology, Kochi-22 (India)

2014-01-28

In the present work, the prospects of ZnS:Mn nanocrystals capped with L- serine, a bio-compatible amino acid, synthesized by wet chemical route, as efficient fluorescent probes for plant cell biological studies have been investigated. The present synthesis route using bio-compatible material is a low cost and easy to control method. The colloidal stability of the capped nano crystals is very good as they remain stable without settling down for long time. It is observed that L- serine significantly modifies the structural and optical characteristics of the ZnS:Mn nanocrystals and hence is suitable as a bio-compatible capping agent. The structural properties of L- serine capped nanocrystals were investigated by XRD technique. The size of the L- serine capped ZnS:Mn nanocrystals is found to be around 2 nm . The optical characterization of the nanocrystals was carried out on the basis of photoluminescence (PL) spectroscopic studies. The intense photoluminescence emission observed around 597nm for L-serine capped ZnS:Mn offers high prospects of applications in bio-imaging fields. The unique optical properties of nanoparticles make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations. In the present study, L-serine capped ZnS:Mn nanocrystals were used as a staining dye in fluorescent microscope for observing cell division, cell structure etc. These nanocrystals were also incorporated into the culture media along with the normal auxin- cytokinin hormone combinations in Murashige and Skoog (MS) medium for micropropagation of Bacopa monnieri Linn. (Brahmi:Scrophulariaceae), an Ayurvedic medicine. The results suggest that L-serine capped ZnS:Mn nanocrystals can act as efficient enhancers towards quick callusing and shoot proliferation.

HOMOLOGY MODELING AND PROTEIN ENGINEERING STRATEGY OF SUBTILASES, THE FAMILY OF SUBTILISIN-LIKE SERINE PROTEINASES

NARCIS (Netherlands)

SIEZEN, RJ; DEVOS, WM; LEUNISSEN, JAM

1991-01-01

Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase

Activation of liver carnitine palmitoyltransferase-1 and mitochondrial acetoacetyl-CoA thiolase is associated with elevated ketone body levels in the elasmobranch Squalus acanthias.

Science.gov (United States)

Treberg, Jason R; Crockett, Elizabeth L; Driedzic, William R

2006-01-01

Elasmobranch fishes are an ancient group of vertebrates that have unusual lipid metabolism whereby storage lipids are mobilized from the liver for peripheral oxidation largely as ketone bodies rather than as nonesterified fatty acids under normal conditions. This reliance on ketones, even when feeding, implies that elasmobranchs are chronically ketogenic. Compared to specimens sampled within 2 d of capture (recently captured), spiny dogfish Squalus acanthias that were held for 16-33 d without apparent feeding displayed a 4.5-fold increase in plasma concentration of d- beta -hydroxybutyrate (from 0.71 to 3.2 mM) and were considered ketotic. Overt activity of carnitine palmitoyltransferase-1 in liver mitochondria from ketotic dogfish was characterized by an increased apparent maximal activity, a trend of increasing affinity (reduced apparent K(m); P=0.09) for l-carnitine, and desensitization to the inhibitor malonyl-CoA relative to recently captured animals. Acetoacetyl-CoA thiolase (ACoAT) activity in isolated liver mitochondria was also markedly increased in the ketotic dogfish compared to recently captured fish, whereas no difference in 3-hydroxy-3-methylglutaryl-CoA synthase activity was found between these groups, suggesting that ACoAT plays a more important role in the activation of ketogenesis in spiny dogfish than in mammals and birds.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

Science.gov (United States)

Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

2010-06-01

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanine

International Nuclear Information System (INIS)

Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru; Kobayashi, Michihiko; Shimizu, Sakayu

2003-01-01

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable β-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of β-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various β-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the β-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the β-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed β-cyano-L-alanine synthase. Heat stable β-cyano-L-alanine synthase can be applied to the synthesis of [4- 11 C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

Energy Technology Data Exchange (ETDEWEB)

Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

2003-10-01

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carnea.

NARCIS (Netherlands)

Patel, A.K.; Oosterwijk, N. van; Singh, V.K.; Rozeboom, H.J.; Kalk, K.H.; Siezen, R.J.; Jagannadham, M.V.; Dijkstra, B.W.

2009-01-01

Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants

Mannan-binding lectin and mannan-binding lectin-associated serine protease 2 in acute pancreatitis

DEFF Research Database (Denmark)

Novovic, Srdan; Andersen, Anders; Ersbøll, Annette Kjær

2011-01-01

Complement activation may play a prominent role in acute pancreatitis (AP). Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) participate in complement activation. The objective of the present study was to evaluate the role of MBL and MASP-2 as markers in AP with regard...

D-Serine rescues the deficits of hippocampal long-term potentiation and learning and memory induced by sodium fluoroacetate.

Science.gov (United States)

Han, Huili; Peng, Yan; Dong, Zhifang

2015-06-01

It is well known that bidirectional glia-neuron interactions play important roles in the neurophysiological and neuropathological processes. It is reported that impairing glial functions with sodium fluoroacetate (FAC) impaired hippocampal long-term depression (LTD) and spatial memory retrieval. However, it remains unknown whether FAC impairs hippocampal long-term potentiation (LTP) and learning and/or memory, and if so, whether pharmacological treatment with exogenous d-serine can recuse the impairment. Here, we reported that systemic administration of FAC (3mg/kg, i.p.) before training resulted in dramatic impairments of spatial learning and memory in water maze and fear memory in contextual fear conditioning. Furthermore, the behavioral deficits were accompanied by impaired LTP induction in the hippocampal CA1 area of brain slices. More importantly, exogenous d-serine treatment succeeded in recusing the deficits of hippocampal LTP and learning and memory induced by FAC. Together, these results suggest that astrocytic d-serine may be essential for hippocampal synaptic plasticity and memory, and that alteration of its levels may be relevant to the induction and potentially treatment of psychiatric and neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis.

Science.gov (United States)

Amel, Kadi-Saci; Fatima, Laraba-Djebari

2015-08-01

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway. © 2015 Wiley Periodicals, Inc.

Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

Science.gov (United States)

Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

2013-01-01

After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival.

Science.gov (United States)

de Magalhães, Mariana T Q; Mambelli, Fábio S; Santos, Bruno P O; Morais, Suellen B; Oliveira, Sergio C

2018-03-31

Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

NARCIS (Netherlands)

Siezen, Roland J.; Vos, Willem M. de; Leunissen, Jack A.M.; Dijkstra, Bauke W.

1991-01-01

Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and

Highly potent fibrinolytic serine protease from Streptomyces.

Science.gov (United States)

Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

2011-01-05

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Thrombocytin, a serine protease from Bothrops atrox venom. 1. Purification and characterization of the enzyme

Energy Technology Data Exchange (ETDEWEB)

Kirby, E.P. (Temple Univ. Health Sciences Center, Philadelphia, PA); Niewiarowski, S.; Stocker, K.; Kettner, C.; Shaw, E.; Brudzynsi, T.M.

1979-08-07

Thrombocytin, a platelet-activating enzyme from Bothrops atrox venom, has been purified to homogeneity by precipitation with sodium salicylate and chromatography on heparin-agarose. Thrombocytin is a single-chain glycoprotein with a molecular weight of 36,000 which contains 5.6% carbohydrate. It causes platelet aggregation, release of platelet serotonin, and activation of factor XIII. The most sensitive substrate for the amidolytic activity of thrombocytin was Tos-Gly-Pro-Arg-p-nitroanilide hydrochloride. The activity of thrombocytin on this substrate and on platelets was inhibited by diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, and several arginine chloromethyl ketones. Active site titration with nitrophenyl guanidinobenzoate demonstrated that approximately 86% of the preparation was in the active form. These experiments demonstrate the presence of serine and histidine in the active site of thrombocytin and suggest that thrombocytin is a classical serine protease with a platelet-activating activity similar to thrombin.

Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

International Nuclear Information System (INIS)

Vanderslice, P.; Ballinger, S.M.; Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H.

1990-01-01

Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

Serine proteases as candidates for proteolytic processing of angiotensin-I converting enzyme.

Science.gov (United States)

Aragão, Danielle S; de Andrade, Maria Claudina C; Ebihara, Fabiana; Watanabe, Ingrid K M; Magalhães, Dayane C B P; Juliano, Maria Aparecida; Hirata, Izaura Yoshico; Casarini, Dulce Elena

2015-01-01

Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. N-domain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension. Copyright © 2014 Elsevier B.V. All rights reserved.

Functional insight into the C-terminal extension of halolysin SptA from haloarchaeon Natrinema sp. J7.

Directory of Open Access Journals (Sweden)

Zhisheng Xu

Full Text Available Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE and collagen binding domain (CBD, and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems.

Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

Directory of Open Access Journals (Sweden)

Kellie Burnside

2010-06-01

Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

Analysis of binding properties and specificity through identification of the interface forming residues (IFR for serine proteases in silico docked to different inhibitors

Directory of Open Access Journals (Sweden)

da Silveira Carlos H

2010-10-01

Full Text Available Abstract Background Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR. We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. Results We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI, ecotine and ovomucoid third domain inhibitor. The table (matrix of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. Conclusions The serine proteases interfaces prefer polar (including glycine residues (with some exceptions. Charged residues were found to be uniquely prevalent at the

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors.

Science.gov (United States)

Ribeiro, Cristina; Togawa, Roberto C; Neshich, Izabella A P; Mazoni, Ivan; Mancini, Adauto L; Minardi, Raquel C de Melo; da Silveira, Carlos H; Jardine, José G; Santoro, Marcelo M; Neshich, Goran

2010-10-20

Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes. The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily

The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

OpenAIRE

Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

2014-01-01

The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

DEFF Research Database (Denmark)

Studdert, C A; Herrera Seitz, M K; Plasencia, I

2001-01-01

A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rat......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.......5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from...... other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests...

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

Science.gov (United States)

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

Serine protease immunohistochemistry and lectin histochemistry in the small intestine of weaned and unweaned pigs

DEFF Research Database (Denmark)

Brown, P J; Poulsen, Steen Seier; Wells, M

1991-01-01

The distribution of goblet cells containing serine protease and of those binding the lectin Ulex europaeus agglutinin-1 (UEA-1) in the pig small intestine is altered during the period after weaning. Goblet cells exhibiting binding of other lectins were not altered. These alterations and other...

The Effect of Serine Protease Inhibitors on Airway Inflammation in a Chronic Allergen-Induced Asthma Mouse Model

Directory of Open Access Journals (Sweden)

Chih-Che Lin

2014-01-01

Full Text Available Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT, gabexate mesilate (FOY, and ulinastatin (UTI on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50 μL, 1 mg/mL at one-week intervals. Therapeutic doses of FUT (0.0625 mg/kg, FOY (20 mg/kg, or UTI (10,000 U/kg were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR, remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF-κB activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-κB activation in lung tissue.

A cyclohexanecarboxamide derivative with inhibitory effects on Schistosoma mansoni cercarial serine protease and penetration of mice skin by the parasite.

Science.gov (United States)

Bahgat, Mahmoud; Aboul-Enein, Mohamed N; El Azzouny, Aida A; Maghraby, Amany; Ruppel, Andreas; Soliman, Wael M

2009-01-01

A cyclohexanecarboxamide derivative, N-phenyl-N-[1-(piperidine-1-carbonyl)cyclohexyl] benzamide (MNRC-5), was evaluated for its inhibitory effects on Schistosoma mansoni cercarial serine protease activity and cercarial penetration. MNRC-5 exerted an inhibitory effect on S. mansoni cercarial serine protease at serial concentrations of the specific chromogenic substrate Boc-Val-Leu-Gly-Arg-PNA for such enzyme family and the inhibitory coefficient (Ki) value was deduced. Moreover, topical treatment of mice tails with the most potent inhibitory concentration of MNRC-5 formulated in jojoba oil successfully blocked cercarial penetration as demonstrated by a significant reduction (75%; p jojoba oil base containing no MNRC-5. In addition, the IgM and IgG reactivities to crude S. mansoni cercarial, worm and egg antigens were generally lower in sera from treated infected mice than untreated infected mice. In conclusion, we report on a new serine protease inhibitor capable for blocking penetration of host skin by S. mansoni cercariae as measured by lowering worm burden and decrease in the levels of both IgM and IgG towards different bilharzial antigens upon topical treatment.

Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53.

Science.gov (United States)

Riscal, Romain; Schrepfer, Emilie; Arena, Giuseppe; Cissé, Madi Y; Bellvert, Floriant; Heuillet, Maud; Rambow, Florian; Bonneil, Eric; Sabourdy, Frédérique; Vincent, Charles; Ait-Arsa, Imade; Levade, Thierry; Thibaut, Pierre; Marine, Jean-Christophe; Portais, Jean-Charles; Sarry, Jean-Emmanuel; Le Cam, Laurent; Linares, Laetitia K

2016-06-16

The mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD(+)/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatin-bound MDM2 in cancer cell metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

Energy Technology Data Exchange (ETDEWEB)

Naffin-Olivos, Jacqueline L.; Daab, Andrew; White, Andre; Goldfarb, Nathan E.; Milne, Amy C.; Liu, Dali; Baikovitz, Jacqueline; Dunn, Ben M.; Rengarajan, Jyothi; Petsko, Gregory A.; Ringe, Dagmar

2017-04-07

The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

Science.gov (United States)

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

Science.gov (United States)

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

A clip domain serine protease involved in moulting in the silkworm, Bombyx mori: cloning, characterization, expression patterns and functional analysis.

Science.gov (United States)

Liu, H-W; Wang, L-L; Meng, Z; Tang, X; Li, Y-S; Xia, Q-Y; Zhao, P

2017-10-01

Clip domain serine proteases (CLIPs), characterized by one or more conserved clip domains, are essential components of extracellular signalling cascades in various biological processes, especially in innate immunity and the embryonic development of insects. Additionally, CLIPs may have additional non-immune functions in insect development. In the present study, the clip domain serine protease gene Bombyx mori serine protease 95 (BmSP95), which encodes a 527-residue protein, was cloned from the integument of B. mori. Bioinformatics analysis indicated that BmSP95 is a typical CLIP of the subfamily D and possesses a clip domain at the N terminus, a trypsin-like serine protease (tryp_spc) domain at the C terminus and a conserved proline-rich motif between these two domains. At the transcriptional level, BmSP95 is expressed in the integument during moulting and metamorphosis, and the expression pattern is consistent with the fluctuating 20-hydroxyecdysone (20E) titre in B. mori. At the translational level, BmSP95 protein is synthesized in the epidermal cells, secreted as a zymogen and activated in the moulting fluid. Immunofluorescence revealed that BmSP95 is distributed into the old endocuticle in the moulting stage. The expression of BmSP95 was upregulated by 20E. Moreover, expression of BmSP95 was downregulated by pathogen infection. RNA interference-mediated silencing of BmSP95 led to delayed moulting from pupa to moth. These results suggest that BmSP95 is involved in integument remodelling during moulting and metamorphosis. © 2017 The Royal Entomological Society.

Genetic polymorphisms in carnitine palmitoyltransferase 1A gene are associated with variation in body composition and fasting lipid traits in Yup'ik Eskimos[S

Science.gov (United States)

Lemas, Dominick J.; Wiener, Howard W.; O'Brien, Diane M.; Hopkins, Scarlett; Stanhope, Kimber L.; Havel, Peter J.; Allison, David B.; Fernandez, Jose R.; Tiwari, Hemant K.; Boyer, Bert B.

2012-01-01

Variants of carnitine palmitoyltransferase 1A (CPT1A), a key hepatic lipid oxidation enzyme, may influence how fatty acid oxidation contributes to obesity and metabolic outcomes. CPT1A is regulated by diet, suggesting interactions between gene variants and diet may influence outcomes. The objective of this study was to test the association of CPT1A variants with body composition and lipids, mediated by consumption of polyunsaturated fatty acids (PUFA). Obesity phenotypes and fasting lipids were measured in a cross-sectional sample of Yup'ik Eskimo individuals (n = 1141) from the Center of Alaska Native Health Research (CANHR) study. Twenty-eight tagging CPT1A SNPs were evaluated with outcomes of interest in regression models accounting for family structure. Several CPT1A polymorphisms were associated with HDL-cholesterol and obesity phenotypes. The P479L (rs80356779) variant was associated with all obesity-related traits and fasting HDL-cholesterol. Interestingly, the association of P479L with HDL-cholesterol was still significant after correcting for body mass index (BMI), percentage body fat (PBF), or waist circumference (WC). Our findings are consistent with the hypothesis that the L479 allele of the CPT1A P479L variant confers a selective advantage that is both cardioprotective (through increased HDL-cholesterol) and associated with reduced adiposity. PMID:22045927

Discovery of an Unexplored Protein Structural Scaffold of Serine Protease from Big Blue Octopus (Octopus cyanea): A New Prospective Lead Molecule.

Science.gov (United States)

Panda, Subhamay; Kumari, Leena

2017-01-01

Serine proteases are a group of enzymes that hydrolyses the peptide bonds in proteins. In mammals, these enzymes help in the regulation of several major physiological functions such as digestion, blood clotting, responses of immune system, reproductive functions and the complement system. Serine proteases obtained from the venom of Octopodidae family is a relatively unexplored area of research. In the present work, we tried to effectively utilize comparative composite molecular modeling technique. Our key aim was to propose the first molecular model structure of unexplored serine protease 5 derived from big blue octopus. The other objective of this study was to analyze the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the aid of different bioinformatic tools. In the present study, molecular model has been generated with the help of I-TASSER suite. Afterwards the refined structural model was validated with standard methods. For functional annotation of protein molecule we used Protein Information Resource (PIR) database. Serine protease 5 of big blue octopus was analyzed with different bioinformatical algorithms for the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. The molecular model data in cooperation to other pertinent post model analysis data put forward molecular insight to proteolytic activity of serine protease 5, which helps in the clear understanding of procoagulant and anticoagulant characteristics of this natural lead molecule. Our approach was to investigate the octopus

A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

Directory of Open Access Journals (Sweden)

Abirami Kugadas

2016-08-01

Full Text Available AbstractThe ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP, the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophage and MAC-T cells and coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increase bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5 conditions. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

Directory of Open Access Journals (Sweden)

Thomas A Prohaska

Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Identification and activity of a lower eukaryotic serine proteinase inhibitor (serpin) from Cyanea capillata: analysis of a jellyfish serpin, jellypin.

Science.gov (United States)

Cole, Elisabeth B; Miller, David; Rometo, David; Greenberg, Robert M; Brömme, Dieter; Cataltepe, Sule; Pak, Stephen C; Mills, David R; Silverman, Gary A; Luke, Cliff J

2004-09-21

Delineating the phylogenetic relationships among members of a protein family can provide a high degree of insight into the evolution of domain structure and function relationships. To identify an early metazoan member of the high molecular weight serine proteinase inhibitor (serpin) superfamily, we initiated a cDNA library screen of the cnidarian, Cyanea capillata. We identified one serpin cDNA encoding for a full-length serpin, jellypin. Phylogenetic analysis using the deduced amino acid sequence showed that jellypin was most similar to the platyhelminthe Echinococcus multiocularis serpin and the clade P serpins, suggesting that this serpin evolved approximately 1000 million years ago (MYA). Modeling of jellypin showed that it contained all the functional elements of an inhibitory serpin. In vitro biochemical analysis confirmed that jellypin was an inhibitor of the S1 clan SA family of serine proteinases. Analysis of the interactions between the human serine proteinases, chymotrypsin, cathepsin G, and elastase, showed that jellypin inhibited these enzymes in the classical serpin manner, forming a SDS stable enzyme/inhibitor complex. These data suggest that the coevolution of serpin structure and inhibitory function date back to at least early metazoan evolution, approximately 1000 MYA.

Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

International Nuclear Information System (INIS)

Rantanen, Mika K.; Lehtiö, Lari; Rajagopal, Lakshmi; Rubens, Craig E.; Goldman, Adrian

2006-01-01

Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail

Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

Energy Technology Data Exchange (ETDEWEB)

Rantanen, Mika K.; Lehtiö, Lari [Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Helsinki (Finland); Rajagopal, Lakshmi; Rubens, Craig E. [Division of Infectious Disease, Children’s Hospital and Regional Medical Center, Seattle, Washington 98105 (United States); Goldman, Adrian, E-mail: adrian.goldman@helsinki.fi [Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Helsinki (Finland)

2006-09-01

Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.

Auditory-motor interaction revealed by fMRI: speech, music, and working memory in area Spt.

Science.gov (United States)

Hickok, Gregory; Buchsbaum, Bradley; Humphries, Colin; Muftuler, Tugan

2003-07-01

The concept of auditory-motor interaction pervades speech science research, yet the cortical systems supporting this interface have not been elucidated. Drawing on experimental designs used in recent work in sensory-motor integration in the cortical visual system, we used fMRI in an effort to identify human auditory regions with both sensory and motor response properties, analogous to single-unit responses in known visuomotor integration areas. The sensory phase of the task involved listening to speech (nonsense sentences) or music (novel piano melodies); the "motor" phase of the task involved covert rehearsal/humming of the auditory stimuli. A small set of areas in the superior temporal and temporal-parietal cortex responded both during the listening phase and the rehearsal/humming phase. A left lateralized region in the posterior Sylvian fissure at the parietal-temporal boundary, area Spt, showed particularly robust responses to both phases of the task. Frontal areas also showed combined auditory + rehearsal responsivity consistent with the claim that the posterior activations are part of a larger auditory-motor integration circuit. We hypothesize that this circuit plays an important role in speech development as part of the network that enables acoustic-phonetic input to guide the acquisition of language-specific articulatory-phonetic gestures; this circuit may play a role in analogous musical abilities. In the adult, this system continues to support aspects of speech production, and, we suggest, supports verbal working memory.

Influence of Microheterogeneous Environments of Sodium Dodecyl Sulfate on the Kinetics of Oxidation of l-Serine by Chloro and Chlorohydroxo Complexes of Gold(III).

Science.gov (United States)

Maiti, Krishnendu; Sen, Pratik K; Barik, Anil K; Pal, Biswajit

2018-06-21

The oxidation of l-serine by chloro and chlorohydroxo complexes of gold(III) was spectrophotometrically investigated in acidic buffer media in the absence and presence of the anionic surfactant sodium dodecyl sulfate (SDS). The oxidation rate decreases with increase in either [H + ] or [Cl - ]. Gold(III) complex species react with the zwitterionic form of serine to yield acetaldehyde (principal reaction product) through oxidative decarboxylation and subsequent deamination processes. A reaction pathway involving one electron transfer from serine to Au(III) followed by homolytic cleavage of α-C-C bond with the concomitant formation of iminic cation intermediate has been proposed where Au(III) is initially reduced to Au(II). The surfactant in the submicellar region exhibits a catalytic effect on the reaction rate at [SDS] ≤ 4 mM; however, in the postmicellar region an inhibitory effect was prominent at [SDS] ≥ 4 mM. The catalytic effect below the critical micelle concentration (cmc) may be attributable to the electrostatic attraction between serine and SDS that, in turn, enhances the nucleophilicity of the carboxylate ion of the amino acid. The inhibition effect beyond cmc has been explained by considering the distribution of the reactant species between the aqueous and the micellar pseudophases that restricts the close association of the reactant species. The thermodynamic parameters Δ H 0 and Δ S 0 associated with the binding between serine and SDS micelle were calculated to be -14.4 ± 2 kJ mol -1 and -6.3 ± 0.5 J K -1 mol -1 , respectively. Water structure rearrangement and micelle-substrate binding play instrumental roles during the transfer of the reactant species from aqueous to micellar pseudophase.

Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus

International Nuclear Information System (INIS)

Nakazawa, Hiroshi; Tsuneishi, Eiko; Ponnuvel, Kangayam M.; Furukawa, Seiichi; Asaoka, Ai; Tanaka, Hiromitsu; Ishibashi, Jun; Yamakawa, Minoru

2004-01-01

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. The molecular mass of this protein was 24 271 Da. Partial N-terminal amino acid sequence of the protein was determined and cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 94% identity with B. mori serine protease so the protein was designated B. mori serine protease-2 (BmSP-2). Analysis of BmSP-2 gene expression showed that this gene is expressed in the midgut but not in other tissues. In addition, BmSP-2 gene was shown to not be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that BmSP-2, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection

Molecular cloning, expression and characterization of a serine proteinase inhibitor gene from Entamoeba histolytica.

Science.gov (United States)

Riahi, Yael; Siman-Tov, Rama; Ankri, Serge

2004-02-01

Serine proteinase inhibitors (serpins) are irreversible suicide inhibitors of proteinases that regulate a wide range of biological processes, including pathogen evasion of the host defence system. We report the cloning and characterization of a gene encoding a serpin from the protozoan parasite Entamoeba histolytica (Ehserp) that may function in this manner. The protein encoded by Ehserp contains 371 amino acids with a predicted mass of 42.6 kDa. Antibodies to a 42 kDa recombinant Ehserp react specifically with two bands of 42 and 49 kDa in trophozoite extracts. Ehserp has a cytoplasmic localization and is secreted by trophozoites incubated in the presence of mammalian cells, but not by resting trophozoites. A panel of mammalian serine proteinases was screened, but none of them was inhibited by the recombinant Ehserp. In contrast, the 49 kDa Ehserp present in the secretion product (SP) of activated macrophages interacted with human neutrophil cathepsin G to form a complex resistant to sodium dodecyl sulphate. We discuss the nature of the 42 and 49 kDa Ehserp and the possible roles that Ehserp may play in the survival of the parasite inside the host.

Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine.

Science.gov (United States)

Noda, Kyoko; Murata, Masatsune

2017-02-01

Pyrrolothiazolate formed by the Maillard reaction between l-cysteine and d-glucose has a pyrrolothiazole skeleton as a chromophore. We searched for a Maillard pigment having a pyrrolooxazole skeleton formed from l-threonine or l-serine instead of l-cysteine in the presence of d-glucose. As a result, two novel yellow pigments, named pyrrolooxazolates A and B, were isolated from model solutions of the Maillard reaction containing l-threonine and d-glucose, and l-serine and d-glucose, respectively, and identified as (2R,3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-2,5,7a-trimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid and (3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-5,7a-dimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid by instrumental analyses. These compounds were pyrrolooxazole derivatives carrying a carboxy group, and showed the absorption maxima at 300-360 nm under acidic and neutral conditions and at 320-390 nm under alkaline conditions.

Viral kinetics in patients with chronic hepatitis C treated with the serine protease inhibitor BILN 2061

NARCIS (Netherlands)

Herrmann, Eva; Zeuzem, Stefan; Sarrazin, Christoph; Hinrichsen, Holger; Benhamou, Yves; Manns, Michael P.; Reiser, Markus; Reesink, Henk; Calleja, José L.; Forns, Xavier; Steinmann, Gerhard G.; Nehmiz, Gerhard

2006-01-01

We analysed viral kinetics from a 2-day treatment with BILN 2061, a serine protease inhibitor of hepatitis C virus, in patients chronically infected with genotype 1 hepatitis C virus. The efficiency (E), describing inhibition of viral production, was above 99.45% in all patients with minor or

IDENTIFICATION OF SERINE CARBAPENEMASE AND METALLOCARBAPENEMASE ENZYMES IN PSEUDOMONAS AERUGINOSA IN GEMS MEDICAL COLLEGE, RAGOLU, SRIKAKULAM

Directory of Open Access Journals (Sweden)

Radhika

2016-06-01

Full Text Available Various carbapenems have been reported in Pseudomonas aeruginosa such as VIM, NDM & OXA-48, etc. In addition, carbapenemase producers are usually associated with many other non–β-lactam resistance determinants which give rise to multidrug and pan drug resistant isolates. Detection of these enzymes in infected patients and in carriers are the two main approaches for prevention of their spread. Potential carbapenemase producers are currently screened first by susceptibility testing, using breakpoint values for carbapenems. However, many carbapenemase producers do not confer obvious resistance levels to carbapenems. So there is need for Laboratories to search for carbapenemase producers. In such instance, phenotypic based test such as Modified Hodge Test (MHT is very much useful in confirming in vitro production of carbapenemase enzymes. But this test does not differentiate serine carbapenemase enzyme (i.e. Ambler class A & C from metallocarbapenemase (i.e. Ambler class B. To differentiate these two enzymes, MHT positive isolates can be subjected to Disc Synergy test. These two tests are highly sensitive and specific and adaptable to any laboratory in the world. Out of 100 ceftazidime resistant Pseudomonas aeruginosa, 75(75% were sensitive, 7(7% were intermediate sensitive and 18(18% were resistant to imipenem. When the 18 imipenem resistant strains were subjected to Modified Hodge test, 15 gave positive results. When the 15 MHT positive strains subjected to disc synergy test, 8 were positive and 7 were negative showing that 8 were producing metallocarbapenemases and 7 were producing serine carbapenemases. Out of 7 intermediately imipenem sensitive isolates, 2 were producing metallocarbapenemase and 3 were producing serine carbapenemase. Hence, total number of imipenem resistant Pseudomonas aeruginosa isolates were 23.

Hereditary sensory and autonomic neuropathy type I in a Chinese family: British C133W mutation exists in the Chinese.

Science.gov (United States)

Bi, Hongyan; Gao, Yunying; Yao, Sheng; Dong, Mingrui; Headley, Alexander Peter; Yuan, Yun

2007-10-01

Hereditary sensory and autonomic neuropathy type I (HSAN I) is an autosomal dominant disorder of the peripheral nervous system characterized by marked progressive sensory loss, with variable autonomic and motor involvement. The HSAN I locus maps to chromosome 9q22.1-22.3 and is caused by mutations in the gene coding for serine palmitoyltransferase long chain base subunit 1 (SPTLC1). Sequencing in HSAN I families have previously identified mutations in exons 5, 6 and 13 of this gene. Here we report the clinical, electrophysiological and pathological findings of a proband in a Chinese family with HSAN I. The affected members showed almost typical clinical features. Electrophysiological findings showed an axonal, predominantly sensory, neuropathy with motor and autonomic involvement. Sural nerve biopsy showed loss of myelinated and unmyelinated fibers. SPTLC1 mutational analysis revealed the C133W mutation, a mutation common in British HSAN I families.

The solvation of L-serine in mixtures of water with some aprotic solvents at 298.15 K

Science.gov (United States)

Mezhevoi, I. N.; Badelin, V. G.

2009-03-01

The integral enthalpies of solution Δsol H m of L-serine in mixtures of water with acetonitrile, 1,4-dioxane, dimethylsulfoxide (DMSO), and acetone were measured by solution calorimetry at organic component concentrations up to 0.31 mole fractions. The standard enthalpies of solution (Δsol H°), transfer (Δtr H°), and solvation (Δsolv H°) of L-serine from water into mixed solvents were calculated. The dependences of Δsol H°, Δsolv H°, and Δtr H° on the composition of aqueous-organic solvents contained extrema. The calculated enthalpy coefficients of pair interactions of the amino acid with cosolvent molecules were positive and increased in the series acetonitrile, 1,4-dioxane, DMSO, acetone. The results obtained were interpreted from the point of view of various types of interactions in solutions and the influence of the nature of organic solvents on the thermochemical characteristics of solutions.

Trypsin- and Chymotrypsin-Like Serine Proteases in Schistosoma mansoni - 'The Undiscovered Country'

Czech Academy of Sciences Publication Activity Database

Horn, Martin; Fajtová, Pavla; Arreola, L. R.; Ulrychová, Lenka; Bartošová-Sojková, Pavla; Franta, Zdeněk; Protasio, A. V.; Opavský, David; Vondrášek, Jiří; McKerrow, J. H.; Mareš, Michael; Caffrey, C. R.; Dvořák, Jan

2014-01-01

Roč. 8, č. 3 (2014), e2766/1-e2766/13 ISSN 1935-2735 R&D Projects: GA ČR(CZ) GAP302/11/1481; GA MŠk(CZ) ME10011 EU Projects: European Commission(XE) 248642 - SCHISTOSOMA PROTEASE Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:60077344 Keywords : schistosomiasis * blood fluke * serine protease Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (UMG-J); FN - Epidemiology, Contagious Diseases ; Clinical Immunology (BC-A) Impact factor: 4.446, year: 2014 http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0002766

Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

Directory of Open Access Journals (Sweden)

Daniel J White

Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

Malonate-based inhibitors of mammalian serine racemase: Kinetic characterization and structure-based computational study

Czech Academy of Sciences Publication Activity Database

Vorlová, Barbora; Nachtigallová, Dana; Jirásková-Vaníčková, Jana; Ajani, Haresh; Jansa, Petr; Řezáč, Jan; Fanfrlík, Jindřich; Otyepka, M.; Hobza, Pavel; Konvalinka, Jan; Lepšík, Martin

2015-01-01

Roč. 89, Jan 7 (2015), s. 189-197 ISSN 0223-5234 R&D Projects: GA ČR GBP208/12/G016 Grant - others:GA MŠk(CZ) ED2.1.00/03.0058 Program:ED Institutional support: RVO:61388963 Keywords : NMDA receptor * pyridoxal-5 '-phosphate-dependent enzyme * human/mouse serine racemase * malonate-based inhibitors * semiempirical quantum mechanical calculations Subject RIV: CE - Biochemistry Impact factor: 3.902, year: 2015

Mutations in serine protease inhibitor Kazal type 1 are strongly associated with chronic pancreatitis

OpenAIRE

Drenth, J P H; te Morsche, R; Jansen, J B M J

2002-01-01

Background: Although chronic pancreatitis is associated with risk factors such as alcoholism, hyperparathyroidism, and hypertriglyceridaemia, little is known of the actual aetiology of the disease. It is thought that inappropriate activation of trypsinogen causes pancreatitis, and indeed in cases of hereditary pancreatitis mutations of cationic trypsinogen (PRSS1) have been described. As serine protease inhibitor Kazal type 1 (SPINK1) is a potent natural inhibitor of pancreatic trypsin activi...

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

Energy Technology Data Exchange (ETDEWEB)

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of); Chang, Hyo-Ihl [College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of); Ha, Chang Hoon, E-mail: chhoonha@amc.seoul.kr [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of)

2016-04-22

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

International Nuclear Information System (INIS)

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae; Chang, Hyo-Ihl; Ha, Chang Hoon

2016-01-01

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis (X5B).

Science.gov (United States)

Ghafoori, Hossein; Askari, Mansoure; Sarikhan, Sajjad

2016-03-01

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48-50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50% of activity at 2.5 M NaCl and about 70% of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca(2+). The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K m and V max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k cat was obtained as 3.284 × 10(-2) s(-1). These special and important characteristics make this serine protease as valuable tool for industrial applications.

Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

Directory of Open Access Journals (Sweden)

Ann C Smigocki

Full Text Available Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica*

Science.gov (United States)

Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.

2012-01-01

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

Characterization of Toxoplasma DegP, a rhoptry serine protease crucial for lethal infection in mice.

Directory of Open Access Journals (Sweden)

Gaelle Lentini

Full Text Available During the infection process, Apicomplexa discharge their secretory organelles called micronemes, rhoptries and dense granules to sustain host cell invasion, intracellular replication and to modulate host cell pathways and immune responses. Herein, we describe the Toxoplasma gondii Deg-like serine protein (TgDegP, a rhoptry protein homologous to High temperature requirement A (HtrA or Deg-like family of serine proteases. TgDegP undergoes processing in both types I and II strains as most of the rhoptries proteins. We show that genetic disruption of the degP gene does not impact the parasite lytic cycle in vitro but affects virulence in mice. While in a type I strain DegPI appears dispensable for the establishment of an infection, removal of DegPII in a type II strain dramatically impairs the virulence. Finally, we show that KO-DegPII parasites kill immunodeficient mice as efficiently as the wild-type strain indicating that the protease might be involved in the complex crosstalk that the parasite engaged with the host immune response. Thus, this study unravels a novel rhoptry protein in T. gondii important for the establishment of lethal infection.

Study of astrophysically important resonant states in 30 S using the 32S(p,t30 S reaction

Directory of Open Access Journals (Sweden)

Wrede C.

2010-03-01

Full Text Available A small fraction (< 1% of presolar SiC grains is suggested to have been formed in the ejecta of classical novae. The 29P(p,γ30S reaction plays an important role in understanding the Si isotopic abundances in such grains, which in turn provide us with information on the nature of the probable white dwarf progenitor’s core, as well as the peak temperatures achieved during nova outbursts, and thus the nova nucleosynthetic path. The 29P(p,γ30S reaction rate at nova temperatures is determined by two low-lying 3+ and 2+ resonances above the proton threshold at 4399 keV in 30S. Despite several experimental studies in the past, however, only one of these two states has only been observed very recently. We have studied the 30S nuclear structure via the 32S(p,t 30S reaction at 5 laboratory angles between 9° to 62°. We have observed 14 states, eleven of which are above the proton threshold, including two levels at 4692.7 ± 4.5 keV and 4813.8 ± 3.4 keV that are candidates for the 3+ and the previously “issing” 2+ state, respectively.

Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway.

Science.gov (United States)

Alecu, Irina; Othman, Alaa; Penno, Anke; Saied, Essa M; Arenz, Christoph; von Eckardstein, Arnold; Hornemann, Thorsten

2017-01-01

The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic β-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

Science.gov (United States)

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Fragoso, Rodrigo R; Silva, Rodrigo O; Gomes, Eliane A; Franco, Octávio L; Dias, Simoni C; Cordeiro, Célia M T; Monnerat, Rose G; Grossi-De-Sá, Maria F

2004-09-01

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated. Copyright 2004 Elsevier Ltd.

Syntheses of sulphurated amino-acids from cystein, serine and phosphoserine using pyridoxal and a metal as catalysts (1961)

International Nuclear Information System (INIS)

Ratsisalovanina, O.; Chapeville, F.; Fromageot, P.

1961-01-01

Pyridoxal or pyridoxal phosphate in the presence of certain metals catalyzes the substitution of the -SH, -OH, or -O-PO 3 H 2 groups of cysteine, serine or phosphoserine by a -SH or -SO 3 H group brought by mineral sulfide or sulfite. (authors) [fr

The Serine Protease Inhibitor Neuroserpin Is Required for Normal Synaptic Plasticity and Regulates Learning and Social Behavior

Science.gov (United States)

Reumann, Rebecca; Vierk, Ricardo; Zhou, Lepu; Gries, Frederice; Kraus, Vanessa; Mienert, Julia; Romswinkel, Eva; Morellini, Fabio; Ferrer, Isidre; Nicolini, Chiara; Fahnestock, Margaret; Rune, Gabriele; Glatzel, Markus; Galliciotti, Giovanna

2017-01-01

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the…

An Epithelial Serine Protease, AgESP, Is Required for Plasmodium Invasion in the Mosquito Anopheles gambiae

Czech Academy of Sciences Publication Activity Database

Rodrigues, J.; Oliveira, G. A.; Kotsyfakis, Michalis; Dixit, R.; Molina-Cruz, A.; Jochim, R.; Barillas-Mury, C.

2012-01-01

Roč. 7, č. 4 (2012), e35210 E-ISSN 1932-6203 Institutional support: RVO:60077344 Keywords : malaria * mosquito * serine protease * sporozoites * ookinetes * gene silencing * midgut * salivary glands * Plasmodium falciparum * Anopheles gambiae Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035210

Urinary serine proteases and activation of ENaC in kidney

DEFF Research Database (Denmark)

Svenningsen, Per; Andersen, Henrik; Nielsen, Lise Hald

2015-01-01

with albuminuria compatible with impaired renal Na(+) excretion: hypertension and volume retention is secondary to proteinuria in, e.g., preeclampsia and nephrotic syndrome; plasma concentrations of renin, angiotensin II, and aldosterone are frequently suppressed in proteinuric conditions, e.g., preeclampsia......Serine proteases, both soluble and cell-attached, can activate the epithelial sodium channel (ENaC) proteolytically through release of a putative 43-mer inhibitory tract from the ectodomain of the γ-subunit. ENaC controls renal Na(+) excretion and loss-of-function mutations lead to low blood...... pressure, while gain-of-function mutations lead to impaired Na(+) excretion, hypertension, and hypokalemia. We review an emerging pathophysiological concept that aberrant glomerular filtration of plasma proteases, e.g., plasmin, prostasin, and kallikrein, contributes to proteolytic activation of ENaC, both...

Non-Maxwellian electron energy probability functions in the plume of a SPT-100 Hall thruster

Science.gov (United States)

Giono, G.; Gudmundsson, J. T.; Ivchenko, N.; Mazouffre, S.; Dannenmayer, K.; Loubère, D.; Popelier, L.; Merino, M.; Olentšenko, G.

2018-01-01

We present measurements of the electron density, the effective electron temperature, the plasma potential, and the electron energy probability function (EEPF) in the plume of a 1.5 kW-class SPT-100 Hall thruster, derived from cylindrical Langmuir probe measurements. The measurements were taken on the plume axis at distances between 550 and 1550 mm from the thruster exit plane, and at different angles from the plume axis at 550 mm for three operating points of the thruster, characterized by different discharge voltages and mass flow rates. The bulk of the electron population can be approximated as a Maxwellian distribution, but the measured distributions were seen to decline faster at higher energy. The measured EEPFs were best modelled with a general EEPF with an exponent α between 1.2 and 1.5, and their axial and angular characteristics were studied for the different operating points of the thruster. As a result, the exponent α from the fitted distribution was seen to be almost constant as a function of the axial distance along the plume, as well as across the angles. However, the exponent α was seen to be affected by the mass flow rate, suggesting a possible relationship with the collision rate, especially close to the thruster exit. The ratio of the specific heats, the γ factor, between the measured plasma parameters was found to be lower than the adiabatic value of 5/3 for each of the thruster settings, indicating the existence of non-trivial kinetic heat fluxes in the near collisionless plume. These results are intended to be used as input and/or testing properties for plume expansion models in further work.

A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

Science.gov (United States)

Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

2012-07-01

A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis of block copolymers derived from N-trityl-(S)-serine and pyrene end-labeled poly(methyl methacrylate) or poly(N-isopropylacrylamide) via ATRP

International Nuclear Information System (INIS)

Buruiana, Emil C.; Podasca, Viorica; Buruiana, Tinca

2012-01-01

A new monomer bearing N-trityl-L-serine methyl ester in structure, methacryloyloxyethyl carbamoyloxy–N-trityl methyl serine (MTS), was prepared to be further polymerized by atom transfer radical polymerization (ATRP) with pyrene-endcapped poly(methyl methacrylate) (Py–PMMA–Br) or poly(N-isopropylacrylamide) (Py–PNIPA–Br). The resulting block copolymers, poly(methyl methacrylate–block–methacryloyloxyethyl carbamoyloxy–N-trityl methyl serine) (Py–PMMA–b–PMTS) and poly(N-isopropylacrylamide–block–methacryloyloxyethyl carbamoyloxy–N-trityl methyl serine (Py–PNIPA–b–PMTS) were characterized by 1 H ( 13 C) NMR, ultraviolet, FTIR and fluorescence spectroscopy, thermal analysis, differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), and gel permeation chromatography (GPC) measurements. The chemical composition in Py–PMMA–b–PMTS was estimated from the 1 H NMR analysis that indicated a ratio of the repeating units of 46:19 (MMA:MTS). For the Py–PNIPA–b–PMTS the composition rate in the copolymer was 61:25 (NIPA:MTS). Quenching of the pyrene species with N,N-diethylaniline, nitrobenzene, nitrophenol, potassium iodide, p-nitrotoluene and tetracyanoquinodimethane (TCNQ) in DMF solution excited at 348 nm was evidenced, more efficiently being nitrophenol and TCNQ. In this case, the monomer emission at 388–409 nm underwent a significant decrease caused of an electron transfer from the electron-reach photoexcited pyrene molecule to the electron-deficient quenchers. - Highlights: ► Diblock copolymers combine the fluorescence of pyrene–PMMA (PNIPA) with the characteristics of PMTS. ► Such copolymers could be used for nitroderivatives detecting. ► UV/vis and fluorescence measurements give a good correlation for LCST of Py–PNIPA–Br.

Synthesis of block copolymers derived from N-trityl-(S)-serine and pyrene end-labeled poly(methyl methacrylate) or poly(N-isopropylacrylamide) via ATRP

Energy Technology Data Exchange (ETDEWEB)

Buruiana, Emil C., E-mail: emilbur@icmpp.ro [Petru Poni Institute of Macromolecular Chemistry, 41A Gr. Ghica Voda Alley 700487, Iasi (Romania); Podasca, Viorica; Buruiana, Tinca [Petru Poni Institute of Macromolecular Chemistry, 41A Gr. Ghica Voda Alley 700487, Iasi (Romania)

2012-10-15

A new monomer bearing N-trityl-L-serine methyl ester in structure, methacryloyloxyethyl carbamoyloxy-N-trityl methyl serine (MTS), was prepared to be further polymerized by atom transfer radical polymerization (ATRP) with pyrene-endcapped poly(methyl methacrylate) (Py-PMMA-Br) or poly(N-isopropylacrylamide) (Py-PNIPA-Br). The resulting block copolymers, poly(methyl methacrylate-block-methacryloyloxyethyl carbamoyloxy-N-trityl methyl serine) (Py-PMMA-b-PMTS) and poly(N-isopropylacrylamide-block-methacryloyloxyethyl carbamoyloxy-N-trityl methyl serine (Py-PNIPA-b-PMTS) were characterized by {sup 1}H ({sup 13}C) NMR, ultraviolet, FTIR and fluorescence spectroscopy, thermal analysis, differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), and gel permeation chromatography (GPC) measurements. The chemical composition in Py-PMMA-b-PMTS was estimated from the {sup 1}H NMR analysis that indicated a ratio of the repeating units of 46:19 (MMA:MTS). For the Py-PNIPA-b-PMTS the composition rate in the copolymer was 61:25 (NIPA:MTS). Quenching of the pyrene species with N,N-diethylaniline, nitrobenzene, nitrophenol, potassium iodide, p-nitrotoluene and tetracyanoquinodimethane (TCNQ) in DMF solution excited at 348 nm was evidenced, more efficiently being nitrophenol and TCNQ. In this case, the monomer emission at 388-409 nm underwent a significant decrease caused of an electron transfer from the electron-reach photoexcited pyrene molecule to the electron-deficient quenchers. - Highlights: Black-Right-Pointing-Pointer Diblock copolymers combine the fluorescence of pyrene-PMMA (PNIPA) with the characteristics of PMTS. Black-Right-Pointing-Pointer Such copolymers could be used for nitroderivatives detecting. Black-Right-Pointing-Pointer UV/vis and fluorescence measurements give a good correlation for LCST of Py-PNIPA-Br.

Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA

Science.gov (United States)

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise ...

Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Directory of Open Access Journals (Sweden)

Priscila Praxedes-Garcia

2012-01-01

Full Text Available Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (Km 55.7 μM in an optimum pH of 7.1, and this activity is effectively retained until 50∘C. CeSP remained stable in the presence of kosmotropic anions (PO4 3−, SO4 2−, and CH3COO− or chaotropic cations (K+ and Na+. It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

Energy Technology Data Exchange (ETDEWEB)

Yang, Chao-Yu; Chin, Ko-Hsin [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China); Chou, Chia-Cheng; Wang, Andrew H.-J. [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Chou, Shan-Ho, E-mail: shchou@nchu.edu.tw [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China)

2006-06-01

The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

Science.gov (United States)

Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Maintained activity of glycogen synthase kinase-3β despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

International Nuclear Information System (INIS)

Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

2010-01-01

Glycogen synthase kinase-3β (GSK3β) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3β. However, the inactive form of GSK3β which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3β substrates, such as β-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3β at serine-9 and other substrates including tau, β-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3β inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3β may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3β inhibitors could be a valuable drug candidate in AD.

Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

Directory of Open Access Journals (Sweden)

Sonja Rakette

Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure

Directory of Open Access Journals (Sweden)

M. Klein

2008-06-01

Full Text Available Inhibition of tyrosine kinases can reverse pulmonary hypertension but little is known about the role of serine-/threonine kinases in vascular and myocardial remodeling. We investigated the effects of sorafenib, an inhibitor of the tyrosine kinases VEGFR, PDGFR and c-kit as well as the serine-/threonine kinase Raf-1, in pulmonary hypertension and right ventricular (RV pressure overload. In monocrotaline treated rats, sorafenib (10 mg·kg–1·d–1 p.o. reduced pulmonary arterial pressure, pulmonary artery muscularization and RV hypertrophy, and improved systemic hemodynamics (table 1. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of downstream signaling pathways (Erk 1/2. After pulmonary banding, sorafenib, but not the PDGFR/c-KIT/ABL-inhibitor imatinib reduced RV mass and RV filling pressure significantly. Congruent with these results, sorafenib only prevented ERK phosphorylation and vasopressin induced hypertrophy of the cardiomyocyte cell line H9c2 dose dependently (IC50 = 300 nM. Combined inhibition of tyrosine and serine-/threonine kinases by sorafenib prevents vascular and cardiac remodeling in pulmonary hypertension, which is partly mediated via inhibition of the Raf kinase pathway.

Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer.

Science.gov (United States)

Martin, Matthew; Hua, Laiqing; Wang, Benlian; Wei, Han; Prabhu, Lakshmi; Hartley, Antja-Voy; Jiang, Guanglong; Liu, Yunlong; Lu, Tao

2017-02-24

Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

Science.gov (United States)

Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

2016-11-01

The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP. Copyright © 2016 Elsevier B.V. All rights reserved.

MBL-associated serine proteases (MASPs) and infectious diseases.

Science.gov (United States)

Beltrame, Marcia H; Boldt, Angelica B W; Catarino, Sandra J; Mendes, Hellen C; Boschmann, Stefanie E; Goeldner, Isabela; Messias-Reason, Iara

2015-09-01

The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication.

Directory of Open Access Journals (Sweden)

Annekatrin Krause

Full Text Available Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2 is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.

The kinase domain residue serine 173 of Schizosaccharomyces pombe Chk1 kinase is critical for the response to DNA replication stress

Directory of Open Access Journals (Sweden)

Naomi Coulton

2017-12-01

Full Text Available While mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10 and is epistatic with a mutation in DNA polymerase delta (cdc6.23 when DNA is alkylated by methyl-methanesulfate (MMS. The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50 is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

Selective inhibition reveals cyclin-dependent kinase 2 as another kinase that phosphorylates the androgen receptor at serine 81

Czech Academy of Sciences Publication Activity Database

Jorda, Radek; Bučková, Zuzana; Řezníčková, Eva; Bouchal, J.; Kryštof, Vladimír

2018-01-01

Roč. 1865, č. 2 (2018), s. 354-363 ISSN 0167-4889 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : Androgen receptor * Cyclin-dependent kinase * Inhibitor * Phosphorylation * Serine 81 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.521, year: 2016

[Effects of fatigue and restraint stress on the expression of carnitine palmitoyltransferase-I and 5-hydroxytryptamine receptors in aorta of rats].

Science.gov (United States)

Wei, Cong; Han, Jian-ke; Wang, Hong-tao; Jia, Zhen-hua; Chang, Li-Ping; Wu, Yi-ling

2011-04-05

To investigate the effect of fatigue and restraint stress on the expressions of CPT (carnitine palmitoyltransferase)-I, PPAR (peroxisome proliferator-activated receptor) δ, 5-HT (hydroxytryptamine) 1D and 5-HT2A receptors in aorta of rats. A total of 45 healthy male Wistar rats were randomly divided into control group, excessive fatigue group and restraint stress group (n = 15 each). The general condition, morphological changes of aortic endothelium cell and the blood levels of ET-1 (endothelin) and NO (nitric oxide) were observed. The real-time reverse transcription PCR (polymerase chain reaction) and Western blot were used to detect the gene and protein expressions of CPT-I, PPAR δ, 5-HT1D and 5-HT2A receptors in aorta. Compared with control group, the structural damages of endothelial cell were induced by excessive fatigue and restraint stress. The plasma levels of ET-1 increased [(124 ± 18) ng/L vs (161 ± 18) ng/L, (154 ± 17) ng/L] (P fatigue rats, [(1.23 ± 0.21) vs (0.42 ± 0.05)], [(1.09 ± 0.10) vs (0.25 ± 0.07)] (P fatigue rats, [(1.32 ± 0.07) vs (0.83 ± 0.04)], [(1.41 ± 0.05) vs. (0.75 ± 0.06)]; the mRNA and protein expressions of 5-HT1D receptor decreased in excessive fatigue rats and restraint stress rats, [(1.10 ± 0.15) vs (0.46 ± 0.13), (0.45 ± 0.02)], [(1.19 ± 0.05) vs (0.71 ± 0.06), (0.70 ± 0.05)] (P fatigue rats and restraint stress rats, [(0.99 ± 0.08) vs (6.73 ± 0.46), (7.01 ± 1.56)], [(0.64 ± 0.03) vs (0.79 ± 0.05), (0.82 ± 0.03)] (P fatigue and restraint stress can injure the structure and function of endothelial cell. The changes in energy of abnormal carnitine metabolism and 5-HT receptors may play important roles.

Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain.

Science.gov (United States)

Barnard, G F; Staniunas, R J; Puder, M; Steele, G D; Chen, L B

1994-08-02

Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species.

Topical effects of N-acetyl-L-hydroxyproline on ceramide synthesis and alleviation of pruritus

Directory of Open Access Journals (Sweden)

Hashizume E

2013-02-01

Full Text Available Erika Hashizume,1 Tetsuo Nakano,2 Ayako Kamimura,1 Koji Morishita31Healthcare Products Development Center, Kyowa Hakko Bio, Tsukuba, Ibaraki, 2Technical Research Laboratories, Kyowa Hakko Bio, Hofu, Yamaguchi, 3Technology Development and Research Department, Kyowa Hakko Bio, Tokyo, JapanPurpose: N-acetyl-l-hydroxyproline (AHYP is an acetylated form of l-hydroxyproline that is used to treat skin ulcers and porphyria cutanea tarda. Its other biological and physiological effects on the skin have not been elucidated. We investigated the effects of AHYP on the skin-barrier function, focusing on ceramide synthesis and the effects of topical AHYP on atopic dermatitis.Materials and methods: AHYP was applied to a three-dimensional cultured skin model. Ceramides were quantified by high-performance thin-layer chromatography. Serine palmitoyltransferase (SPT is the rate-limiting enzyme in de novo ceramide synthesis, and the mRNA of its long-chain base subunit 1 (SPTLC1 was evaluated by quantitative reverse-transcription polymerase chain reaction. A clinical trial in the form of an intraindividual, comparative, double-blind, randomized, vehicle-controlled test involving 15 female subjects suffering from slight atopic dermatitis was performed. Subjects applied 1% (w/w AHYP cream to one forearm and a control cream to the other forearm twice daily for 4 weeks. Skin condition was evaluated by measuring transepidermal water loss (TEWL. Dermatological observations were made by a dermatologist, and subjects evaluated their own pruritus intensity before beginning treatment and 4 weeks after the start of treatment.Results: SPTLC1 expression and ceramide synthesis were significantly increased in an AHYP-treated skin model (P < 0.05. In the clinical trial, no adverse effects were observed in any subjects. TEWL was increased in the control-treated region of the forearm (P < 0.05 after 4 weeks' application, whereas there was no change in the AHYP-treated region of the

Catalysis of the Oligomerization of O-Phospho-Serine, Aspartic Acid, or Glutamic Acid by Cationic Micelles

Science.gov (United States)

Bohler, Christof; Hill, Aubrey R., Jr.; Orgel, Leslie E.

1996-01-01

Treatment of relatively concentrated aqueous solutions of 0-phospho-serine (50 mM), aspartic acid (100 mM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at their surfaces and catalyze efficient oligomerization even from dilute solutions.

Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

Science.gov (United States)

Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

2012-01-15

Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structural, Linear, and Nonlinear Optical and Mechanical Properties of New Organic L-Serine Crystal

Directory of Open Access Journals (Sweden)

K. Rajesh

2014-01-01

Full Text Available Nonlinear optical single crystal of organic amino acid L-Serine (LS was grown by slow evaporation technique. Solubility study of the compound was measured and metastable zone width was found. Single crystal X-ray diffraction study was carried out for the grown crystal. The linear and nonlinear optical properties of the crystal were confirmed by UV-Vis analysis and powder SHG tester. FT-IR spectrum was recorded and functional groups were analyzed. Vickers’ microhardness studies showed the mechanical strength of the grown crystal. Laser damage threshold value of the crystal was calculated. Photoconductivity studies reveal the conductivity of the crystal.

The action of neutrophil serine proteases on elastin and its precursor

DEFF Research Database (Denmark)

Heinz, Andrea; Jung, Michael C; Jahreis, Günther

2012-01-01

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass...... spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three...... proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr...

Serine protease inhibitor A3K suppressed the formation of ocular surface squamous metaplasia in a mouse model of experimental dry eye.

Science.gov (United States)

Lin, Zhirong; Zhou, Yueping; Wang, Yuqian; Zhou, Tong; Li, Jie; Luo, Pingping; He, Hui; Wu, Huping; Liu, Zuguo

2014-08-07

To investigate the effects and possible mechanisms of serine protease inhibitor A3K (SERPINA3K) on the formation of ocular surface squamous metaplasia in a mouse dry eye model induced by topical benzalkonium chloride (BAC). The eye drops containing SERPINA3K were topically administered during the induction of BAC-induced dry eye. The clinical indications of dry eye were evaluated on day (D)16, including tear break-up time (BUT), tear volume, corneal fluorescein staining, and inflammatory index. Global specimens were collected on D16 and the following examinations were performed: histologic investigation, immunostaining of cytokeratin 10 (K10), p63 and Ki67 in the cornea, and Western blot analysis of tumor necrosis factor-α (TNF-α). Serine protease inhibitor A3K suppressed the formation of BAC-induced dry eye, presenting with longer BUTs, lower corneal fluorescein staining scores, and inflammatory index, while no significant changes in tear volume. It also reduced the severity of abnormal differentiation and proliferation on ocular surface with lower expressions of K10, p63, and Ki67, and retained the number of goblet cells in the conjunctival fornix. Serine protease inhibitor A3K significantly decreased the levels of TNF-α in the cornea. Topical application of SERPINA3K ameliorated the severity of ocular surface squamous metaplasia and suppressed the formation of BAC-induced dry eye. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Stromal serine protein kinase activity in spinach chloroplasts

International Nuclear Information System (INIS)

Cortez, N.; Lucero, H.A.; Vallejos, R.H.

1987-01-01

At least twelve 32 P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32 Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma- 32 P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

DEFF Research Database (Denmark)

Peters, Diane E; Szabo, Roman; Friis, Stine

2014-01-01

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. ...

Site-SpecificCu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N), Using Copper Free Click Chemistry

DEFF Research Database (Denmark)

Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H

2018-01-01

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migrati...

Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

Science.gov (United States)

Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

2016-01-01

Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

Multimerization of GPIHBP1 and Familial Chylomicronemia from a Serine-to-Cysteine Substitution in GPIHBP1's Ly6 Domain

DEFF Research Database (Denmark)

Plengpanich, Wanee; Young, Stephen G; Khovidhunkit, Weerapan

2014-01-01

-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in GPIHBP1's Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were...

The SPT+Herschel+ALMA+Spitzer Legacy Survey: The stellar content of high redshift strongly lensed systems

Science.gov (United States)

Vieira, Joaquin; Ashby, Matt; Carlstrom, John; Chapman, Scott; DeBreuck, Carlos; Fassnacht, Chris; Gonzalez, Anthony; Phadke, Kedar; Marrone, Dan; Malkan, Matt; Reuter, Cassie; Rotermund, Kaja; Spilker, Justin; Weiss, Axel

2018-05-01

The South Pole Telescope (SPT) has systematically identified 90 high-redshift strongly gravitationally lensed submillimeter galaxies (SMGs) in a 2500 square-degree cosmological survey of the millimeter (mm) sky. These sources are selected by their extreme mm flux, which is largely independent of redshift and lensing configuration. We are undertaking a comprehensive and systematic followup campaign to use these "cosmic magnifying glasses" to study the infrared background in unprecedented detail, inform the condition of the interstellar medium in starburst galaxies at high redshift, and place limits on dark matter substructure. Here we ask for 115.4 hours of deep Spitzer/IRAC imaging to complete our survey of 90 systems to a uniform depth of 30min integrations at 3.6um and 60min at 4.5um. In our sample of 90 systems, 16 have already been fully observed, 30 have been partially observed, and 44 have not been observed at all. Our immediate goals are to: 1) constrain the specific star formation rates of the background high-redshift submillimeter galaxies by combining these Spitzer observations with our APEX, Herschel, and ALMA data, 2) robustly determine the stellar masses and mass-to-light ratios of all the foreground lensing galaxies in the sample by combining these observations with our VLT and Gemini data, the Dark Energy Survey, and ALMA; and 3) provide complete, deep, and uniform NIR coverage of our entire sample of lensed systems to characterize the environments of high redshift SMGs, maximize the discovery potential for additional spectacular and rare sources, and prepare for JWST. This program will provide the cornerstone data set for two PhD theses: Kedar Phadke at Illinois will lead the analysis of stellar masses for the background SMGs, and Kaja Rotermund at Dalhousie will lead the analysis of stellar masses for the foreground lenses.

Endothelin-1 stimulates catalase activity through the PKCδ mediated phosphorylation of Serine 167

Science.gov (United States)

Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Hou, Yali; Kangath, Archana; Pardo, Daniel; Fineman, Jeffrey R.; Black, Stephen M.

2013-01-01

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells (PAEC) to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be PKCδ dependent. Mass spectrometry identified serine167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from E.coli or transiently transfected COS-7 cells, demonstrated that S167D-catalase had an increased ability to degrade H2O2 compared to the wildtype enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist, tezosentan. S167 is being located on the dimeric interface suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel-filtration to examine the multimeric structure of recombinant wildtype- and S167D-catalase. We found that recombinant wildtype catalase was present as a mixture of monomers and dimers while S167D catalase was primarily tetrameric. Further, the incubation of wildtype catalase with PKCδ was sufficient to convert wildtype catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. PMID:24211614

The story of an exceptional serine protease, tissue-type plasminogen activator (tPA).

Science.gov (United States)

Hébert, M; Lesept, F; Vivien, D; Macrez, R

2016-03-01

The only acute treatment of ischemic stroke approved by the health authorities is tissue recombinant plasminogen activator (tPA)-induced thrombolysis. Under physiological conditions, tPA, belonging to the serine protease family, is secreted by endothelial and brain cells (neurons, astrocytes, microglia, oligodendrocytes). Although revascularisation induced by tPA is beneficial during a stroke, research over the past 20 years shows that tPA can also be deleterious for the brain parenchyma. Thus, in this review of the literature, after a brief history on the discovery of tPA, we reviewed current knowledge of mechanisms by which tPA can influence brain function in physiological and pathological conditions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Serine/Threonine protein kinases from bacteria, archaea and eukarya share a common evolutionary origin deeply rooted in the tree of life

DEFF Research Database (Denmark)

Stancik, Ivan Andreas; Šestak, Martin Sebastijan; Ji, Boyang

2018-01-01

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988. It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reaso...

Distribution of serine/threonine kinase SAD-B in mouse peripheral nerve synapse.

Science.gov (United States)

Hagiwara, Akari; Harada, Kenu; Hida, Yamato; Kitajima, Isao; Ohtsuka, Toshihisa

2011-05-11

The serine/threonine kinase SAD regulates neural functions such as axon/dendrite polarization and neurotransmitter release. In the vertebrate central nervous system, SAD-B, a homolog of Caenorhabditis elegans SAD-1, is associated with synaptic vesicles and the active zone cytomatrix in nerve terminals. However, the distribution of SAD-B in the peripheral nervous system remains elusive. Here, we show that SAD-B is specifically localized to neuromuscular junctions. Although the active zone protein bassoon showed a punctated signal indicating its localization to motor end plates, SAD-B shows relatively diffuse localization indicating its association with both the active zone and synaptic vesicles. Therefore, SAD kinase may regulate neurotransmitter release from motor end plates in a similar manner to its regulation of neurotransmitter release in the central nervous system.

Serine 62-Phosphorylated MYC Associates with Nuclear Lamins and Its Regulation by CIP2A Is Essential for Regenerative Proliferation

Directory of Open Access Journals (Sweden)

Kevin Myant

2015-08-01

Full Text Available An understanding of the mechanisms determining MYC’s transcriptional and proliferation-promoting activities in vivo could facilitate approaches for MYC targeting. However, post-translational mechanisms that control MYC function in vivo are poorly understood. Here, we demonstrate that MYC phosphorylation at serine 62 enhances MYC accumulation on Lamin A/C-associated nuclear structures and that the protein phosphatase 2A (PP2A inhibitor protein CIP2A is required for this process. CIP2A is also critical for serum-induced MYC phosphorylation and for MYC-elicited proliferation induction in vitro. Complementary transgenic approaches and an intestinal regeneration model further demonstrated the in vivo importance of CIP2A and serine 62 phosphorylation for MYC activity upon DNA damage. However, targeting of CIP2A did not influence the normal function of intestinal crypt cells. These data underline the importance of nuclear organization in the regulation of MYC phosphorylation, leading to an in vivo demonstration of a strategy for inhibiting MYC activity without detrimental physiological effects.

Serine proteases SP1 and SP13 mediate the melanization response of Asian corn borer, Ostrinia furnacalis, against entomopathogenic fungus Beauveria bassiana.

Science.gov (United States)

Chu, Yuan; Liu, Yang; Shen, Dongxu; Hong, Fang; Wang, Guirong; An, Chunju

2015-06-01

Exposure to entomopathogenic fungi is one approach for insect pest control. Little is known about the immune interactions between fungus and its insect host. Melanization is a prominent immune response in insects in defending against pathogens such as bacteria and fungi. Clip domain serine proteases in insect plasma have been implicated in the activation of prophenoloxidase, a key enzyme in the melanization. The relationship between host melanization and the infection by a fungus needs to be established. We report here that the injection of entomopathogenic fungus Beauveria bassiana induced both melanin synthesis and phenoloxidase activity in its host insect, the Asian corn borer, Ostrinia furnacalis (Guenée). qRT-PCR analysis showed several distinct patterns of expression of 13 clip-domain serine proteases in response to the challenge of fungi, with seven increased, two decreased, and four unchanged. Of special interest among these clip-domain serine protease genes are SP1 and SP13, the orthologs of Manduca sexta HP6 and PAP1 which are involved in the prophenoloxidase activation pathway. Recombinant O. furnacalis SP1 was found to activate proSP13 and induce the phenoloxidase activity in corn borer plasma. Additionally, SP13 was determined to directly cleave prophenoloxidase and therefore act as the prophenoloxidase activating protease. Our work thus reveals a biochemical mechanism in the melanization in corn borer associated with the challenge by B. bassiana injection. These insights could provide valuable information for better understanding the immune responses of Asian corn borer against B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine.

Directory of Open Access Journals (Sweden)

Fiona Hey

Full Text Available The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3 is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

C. elegans serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation

Directory of Open Access Journals (Sweden)

Cohen Stephen

2005-04-01

Full Text Available Background In C. elegans there are two well-defined TGFβ-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFβ signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. Results kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. Conclusion kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFβ outputs, such as tail morphogenesis.

Fiber specific changes in sphingolipid metabolism in skeletal muscles of hyperthyroid rats.

Science.gov (United States)

Chabowski, A; Zendzian-Piotrowska, M; Mikłosz, A; Łukaszuk, B; Kurek, K; Górski, J

2013-07-01

Thyroid hormones (T3, T4) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T3 on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, with diverse fiber compositions: soleus (slow-twitch oxidative), red (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. We demonstrated that T3 induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingomyelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, hyperthyroidism induced fiber specific changes in the content of sphingolipids that were relatively more related to de novo synthesis of ceramide rather than to its generation via hydrolysis of sphingomyelin.

D-Serine exposure resulted in gene expression changes indicative of activation of fibrogenic pathways and down-regulation of energy metabolism and oxidative stress response

International Nuclear Information System (INIS)

Soto, Armando; DelRaso, Nicholas J.; Schlager, John J.; Chan, Victor T.

2008-01-01

Renal toxicity can commonly occur after exposure to xenobiotics, pharmaceutical agents or environmental pollutants. Changes in the gene expression in kidney parenchymal cells that precede and/or accompany renal injury may be hallmark critical events in the onset of pathologic changes of renal functions. Over the last several years, transcriptomic analysis has evolved to enable simultaneous analysis of the expression profiles of tens of thousands of genes in response to various endogenous and exogenous stimuli. In this study, we investigated gene expression changes in the kidney after acute exposure to a nephrotoxin, D-serine, which targets the proximal tubule of the kidney. Male F-344 rats injected intraperitoneally with a single dose of D-serine (5, 20, 50, 200 or 500 mg/kg), and gene expression profiles in the kidney were determined using the Affymetrix RAE230A gene arrays at 96 h post-dosing. D-Serine treatment resulted in the up- and down-regulation of 1158 and 749 genes, respectively, over the entire dose range based on the intersection of the results of t-test, p < 0.01 over two consecutive doses, and ANOVA with Bonferonni correction for multiple testing. Interestingly, both the up-and down-regulated genes show a unified dose response pattern as revealed in the self-organized map clustering analysis using the expression profiles of the 1907 differentially expressed genes as input data. There appears to be minimal changes in the expression level of these genes in the dose range of 5-50 mg/kg, while the most prominent changes were observed at the highest doses tested, i.e. 200 and 500 mg/kg. Pathway analysis of the differentially expressed genes showed perturbation of a large number of biological processes/pathways after D-serine exposure. Among the up-regulated pathways are actin cytoskeleton biogenesis and organization, apoptosis, cell cycle regulation, chromatin assembly, excision repair of damaged DNA, DNA replication and packaging, protein biosynthesis

Portulaca oleracea L. as a Prospective Candidate Inhibitor of Hepatitis C Virus NS3 Serine Protease.

Science.gov (United States)

Noreen, Sobia; Hussain, Ishtiaq; Tariq, Muhammad Ilyas; Ijaz, Bushra; Iqbal, Shahid; Qamar-ul-Zaman; Ashfaq, Usman Ali; Husnain, Tayyab

2015-06-01

Hepatitis C virus (HCV) infection is a worldwide health problem affecting about 300 million individuals. HCV causes chronic liver disease, liver cirrhosis, hepatocellular carcinoma, and death. Many side effects are associated with the current treatment options. Natural products that can be used as anti-HCV drugs are thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for the screening of antiviral activity against HCV. The present work explores plants with anti-HCV potential, isolating possible lead compounds. Ten plants, used for medicinal purposes against different infections in rural areas of Pakistan, were collected. The cellular toxicity effects of methanolic extracts of the plants on the viability of Huh-7 cells were studied through the Trypan blue dye exclusion method. Following this, the anti-HCV potential of phytoextracts was assessed by infecting liver cells with HCV-3a-infected serum inoculum. Only the methanolic extract of Portulaca oleracea L. (PO) exhibited more than 70% inhibition. Four fractions were obtained through bioassay-guided extraction of PO. Subsequent inhibition of all organic extract fractions against NS3 serine protease was checked to track the specific target in the virus. The results showed that the PO methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. Hence, PO extract and its constituents either alone or with interferon could offer a future option to treat chronic HCV.

The identification and biochemical properties of the catalytic specificity of a serine peptidase secreted by Aspergillus fumigatus Fresenius.

Science.gov (United States)

da Silva, Ronivaldo Rodrigues; Caetano, Renato Cesar; Okamoto, Debora Nona; de Oliveira, Lilian Caroline Goncalves; Bertolin, Thiago Carlos; Juliano, Maria Aparecida; Juliano, Luiz; de Oliveira, Arthur H C; Rosae, Jose C; Cabral, Hamilton

2014-07-01

Aspergillus fumigatus is a saprophytic fungus as well as a so-called opportunist pathogen. Its biochemical potential and enzyme production justify intensive studies about biomolecules secreted by this microorganism. We describe the alkaline serine peptidase production, with optimum activity at 50°C and a pH of 7.5 and a reduction in proteolytic activity in the presence of the Al(+3) ions. When using intramolecularly quenched fluorogenic substrates, the highest catalytic efficiency was observed with the amino acid leucine on subsite S'(3) (60,000 mM(-1)s(-1)) and preference to non-polar amino acids on subsite S(3). In general, however, the peptidase shows non-specificity on other subsites studied. According to the biochemical characteristics, this peptidase may be an important biocatalyst for the hydrolysis of an enormous variety of proteins and can constitute an essential molecule for the saprophytic lifestyle or invasive action of the opportunistic pathogen. The peptidase described herein exhibits an estimated molecular mass of 33 kDa. Mass spectrometry analysis identified the sequence GAPWGLGSISHK displaying similarities to that of serine peptidase from Aspergillus fumigatus. These data may lead to a greater understanding of the advantageous biochemical potential, biotechnological interest, and trends of this fungus in spite of being an opportunist pathogen.

Identification of a serine proteinase homolog (Sp-SPH) involved in immune defense in the mud crab Scylla paramamosain.

Science.gov (United States)

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, Pparahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

Oxygen-18 as a tool for studying photorespiration. Oxygen uptake and incorporation into glycolate, glycine and serine

International Nuclear Information System (INIS)

Gerster, R.; Dimon, B.; Tournier, P.; Peybernes, A.

1977-01-01

The intensity of photosynthesis and photorespiration has been determined by measuring 16 O 2 evolvement and 18 O 2 uptake on algae and leaves. In the case of algae, there is still an important oxygen uptake even when ribulose diphosphate oxygenase is inhibited by 10 -3 M cyanide. Oxygen-18 incorporation into glycolate, glycine and serine of photorespiring algae and leaves exposed to atmospheres containing 18 O 2 has also been measured. Only one of the two atoms present in molecular oxygen was incorporated into the carboxyl group of the glycolate excreted from algae; the rate of 18 O incorporation was important (65 to 80% according to experimental conditions), even in the presence of 10 -3 M cyanide. Thus, oxidation of ribulose diphosphate is not the sole reaction leading to 18 O glycolate synthesis. In the case of maize, there was a rapid and important 18 O incorporation into the carboxyl group of glycine and serine, the kinetics of which was determined as a function of CO 2 presence in the atmosphere. These results suggest that photorespiration is also operating in C 4 species. Furthermore, in vitro experiments showed that phosphorylated ceto-acids of the Calvin cycle were very sensitive to H 2 O 2 ; the corresponding reaction can explain O 2 uptake and 18 O labelling of glycolate. (author)

An analysis of 3D solvation structure in biomolecules: application to coiled coil serine and bacteriorhodopsin.

Science.gov (United States)

Hirano, Kenji; Yokogawa, Daisuke; Sato, Hirofumi; Sakaki, Shigeyoshi

2010-06-17

Three-dimensional (3D) solvation structure around coiled coil serine (Coil-Ser) and inner 3D hydration structure in bacteriorhodopsin (bR) were studied using a recently developed method named multicenter molecular Ornstein-Zernike equation (MC-MOZ) theory. In addition, a procedure for analyzing the 3D solvent distribution was proposed. The method enables us to calculate the coordination number of solvent water as well as the strength of hydrogen bonding between the water molecule and the protein. The results for Coil-Ser and bR showed very good agreement with the experimental observations.

The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids

OpenAIRE

Kreuzer, Johannes

2015-01-01

The target proteins of acivicin and structure derived probes in tumor cells were identified using activity-based protein profiling. The target proteins were further characterized and their relation to the antitumor activity of acivicin pointed out. In a further project, the activity of serine hydrolases in myoma and myometrium was examined from tissue samples. This revealed a different activity of mast cell proteases. Mittels Activity-based Protein Profiling wurde eine Identifikation der Z...

Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

Science.gov (United States)

Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T; Bailowitz, Zachary; De Filippis, Elena A; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J

2010-10-01

The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

Synthesis of novel chitosan resin derivatized with serine moiety for the column collection/concentration of uranium and the determination of uranium by ICP-MS

International Nuclear Information System (INIS)

Oshita, Koji; Oshima, Mitsuko; Gao Yunhua; Lee, Kyue-Hyung; Motomizu, Shoji

2003-01-01

A chitosan resin derivatized with serine moiety (serine-type chitosan) was newly developed by using the cross-linked chitosan as a base material. The adsorption behavior of trace amounts of metal ions on the serine-type chitosan resin was systematically examined by packing it in a mini-column, passing a metal solution through it and measuring metal ions in the effluent by ICP-MS. The resin could adsorb a number of metal cations at pH from neutral to alkaline region, and several oxoanionic metals at acidic pH region by an anion exchange mechanism. Uranium and Cu could be adsorbed selectively at pH from acidic to alkaline region by a chelating mechanism; U could be adsorbed quantitatively even at pH 3-4. Uranium adsorbed on the resin was easily eluted with 1 M nitric acid: the preconcentration (5-, 10-, 50- and 100-fold) of U was possible. The column treatment method was used prior to the ICP-MS measurement of U in natural river, sea and tap waters; R.S.D. were 2.63, 1.13 and 1.37%, respectively. Uranium in tap water could be determined by 10-fold preconcentration: analytical result was 1.46±0.02 ppt. The resin also was applied to the recovery of U in sea water: the recovery tests for artificial and natural sea water were 97.1 and 93.0%, respectively

Appearance and distribution of regioisomers in metallo- and serine-protease-catalysed acylation of sucrose in N,N-dimethylformamide

DEFF Research Database (Denmark)

Lie, Aleksander; Meyer, Anne S.; Pedersen, Lars Haastrup

2014-01-01

laurate was obtained in yields from 12 to 53% after 48 h under different catalytic conditions. The serine protease ALP-901, derived from a Streptomyces sp., produced the highest yield at this reaction time, while reaction with the zinc-protease thermolysin achieved the overall highest yield (63%) after 6...

IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas tranplant recipient

DEFF Research Database (Denmark)

Bouzakri, K; Karlsson, HRK; Vestergaard, Henrik

2006-01-01

Insulin-dependent diabetic recipients of successful pancreas allografts achieve self-regulatory insulin secretion and discontinue exogenous insulin therapy; however, chronic hyperinsulinemia and impaired insulin sensitivity generally develop. To determine whether insulin resistance is accompanied....... In conclusion, peripheral insulin resistance in pancreas-kidney transplant recipients may arise from a negative feedback regulation of the canonical insulin-signaling cascade from excessive serine phosphorylation of IRS-1, possibly as a consequence of immunosuppressive therapy and hyperinsulinemia....... insulin receptor substrate (IRS)-1 Ser (312) and Ser (616) phosphorylation, IRS-1-associated phosphatidylinositol 3-kinase activity, and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation were elevated in pancreas-kidney transplant recipients, coincident with fasting hyperinsulinemia. Basal...

Carnitine palmitoyltransferase 1B 531K allele carriers sustain a higher respiratory quotient after aerobic exercise, but β3-adrenoceptor 64R allele does not affect lipolysis: a human model.

Directory of Open Access Journals (Sweden)

Eduardo Gómez-Gómez

Full Text Available Carnitine palmitoyltransferase IB (CPT1B and adrenoceptor beta-3 (ADRB3 are critical regulators of fat metabolism. CPT1B transports free acyl groups into mitochondria for oxidation, and ADRB3 triggers lipolysis in adipocytes, and their respective polymorphisms E531K and W64R have been identified as indicators of obesity in population studies. It is therefore important to understand the effects of these mutations on ADRB3 and CPT1B function in adipose and skeletal muscle tissue, respectively. This study aimed to analyze the rate of lipolysis of plasma indicators (glycerol, free fatty acids, and beta hydroxybutyrate and fat oxidation (through the non-protein respiratory quotient. These parameters were measured in 37 participants during 30 min of aerobic exercise at approximately 62% of maximal oxygen uptake, followed by 30 min of recovery. During recovery, mean respiratory quotient values were higher in K allele carriers than in non-carriers, indicating low post-exercise fatty acid oxidation rates. No significant differences in lipolysis or lipid oxidation were observed between R and W allele carriers of ADRB3 at any time during the aerobic load. The substitution of glutamic acid at position 531 by lysine in the CPT1B protein decreases the mitochondrial beta-oxidation pathway, which increases the non-protein respiratory quotient value during recovery from exercise. This may contribute to weight gain or reduced weight-loss following exercise.

LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

Energy Technology Data Exchange (ETDEWEB)

Kawamoto, Eiji [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Takagi, Yoshimi [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Honda, Goichi [Medical Affairs Department, Asahi Kasei Pharma Corporation, 1-105 Kanda Jinbo-cho, Chiyoda-ku, Tokyo 101-8101 (Japan); Suzuki, Koji [Faculty of Pharmaceutical Science, Suzuka University of Medical Science, 3500-3, Minamitamagaki-cho, Suzuka, Mie 513-8679 (Japan); Imai, Hiroshi [Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Shimaoka, Motomu, E-mail: shimaoka@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan)

2016-05-13

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

Directory of Open Access Journals (Sweden)

Takehiro Ko

2010-01-01

Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint

OpenAIRE

Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T.C.; Fu, Li; Xu, Bo

2011-01-01

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in...

Antiketogenic and hypoglycemic effects of aminocarnitine and acylaminocarnitines

International Nuclear Information System (INIS)

Jenkins, D.L.; Griffith, O.W.

1986-01-01

DL-Aminocarnitine (DL-3-amino-4-trimethyl-aminobutyrate) is a potent, noncovalent inhibitor of carnitine palmitoyltransferase (palmitoyl-CoA:L-carinite O-palmitoyltransferase, EC 2.3.1.21). Here the authors show that decanoyl-DL-aminocarnitine and palmitoyl-DL-aminocarnitine inhibit carnitine palmitoyltransferase in vitro about 7-fold and 100-fold more effectively than does aminocarnitine. Aminocarnitine and its decanoyl and palmitoyl derivatives are active in vivo following oral or parenteral administration and, at doses of 0.3 mmol/kg or less, inhibit the oxidation of [ 14 C]palmitate to 14 CO 2 by 45-70% in mice. Larger doses do not significantly increase the extent of inhibition, a finding suggesting that substantial carnitine palmitoyltransferase-independent long-chain fatty acid oxidation may occur in vivo. Small doses of aminocarnitine and palmitoylaminocarnitine prevent the development of ketoacidemia in fasted, normal mice and reverse the ketoacidemia observed in diabetic mice. Aminocarnitine has a strong hypoglycemic effect in fasted diabetic mice; a single dose (0.3 mmol/kg) normalizes plasma glucose levels within 4-8 hr and remains effective for at least 12 hr

Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic N-15 chemical shielding anisotropy tensors

Czech Academy of Sciences Publication Activity Database

Emmer, J.; Vavrinská, A.; Sychrovský, Vladimír; Benda, Ladislav; Kříž, Z.; Koča, J.; Boelens, R.; Sklenář, V.; Trantírek, L.

2013-01-01

Roč. 55, č. 1 (2013), s. 59-70 ISSN 0925-2738 R&D Projects: GA ČR GAP205/10/0228 Grant - others:CEITEC(XE) CZ.1.05/1.1.00/02.0068 Institutional support: RVO:61388963 Keywords : CSA * phosphorylation * amidic nitrogen * serine * threonine * tyrosine * protein * NMR Subject RIV: CE - Biochemistry Impact factor: 3.305, year: 2013

Effects of Blocked and Random Practice Schedule on Outcomes of Sound Production Treatment for Acquired Apraxia of Speech: Results of a Group Investigation.

Science.gov (United States)

Wambaugh, Julie L; Nessler, Christina; Wright, Sandra; Mauszycki, Shannon C; DeLong, Catharine; Berggren, Kiera; Bailey, Dallin J

2017-06-22

The purpose of this investigation was to compare the effects of schedule of practice (i.e., blocked vs. random) on outcomes of Sound Production Treatment (SPT; Wambaugh, Kalinyak-Fliszar, West, & Doyle, 1998) for speakers with chronic acquired apraxia of speech and aphasia. A combination of group and single-case experimental designs was used. Twenty participants each received SPT administered with randomized stimuli presentation (SPT-R) and SPT applied with blocked stimuli presentation (SPT-B). Treatment effects were examined with respect to accuracy of articulation as measured in treated and untreated experimental words produced during probes. All participants demonstrated improved articulation of treated items with both practice schedules. Effect sizes were calculated to estimate magnitude of change for treated and untreated items by treatment condition. No significant differences were found for SPT-R and SPT-B relative to effect size. Percent change over the highest baseline performance was also calculated to provide a clinically relevant indication of improvement. Change scores associated with SPT-R were significantly higher than those for SPT-B for treated items but not untreated items. SPT can result in improved articulation regardless of schedule of practice. However, SPT-R may result in greater gains for treated items. https://doi.org/10.23641/asha.5116831.

Comparative Mitogenomics of Plant Bugs (Hemiptera: Miridae): Identifying the AGG Codon Reassignments between Serine and Lysine

Science.gov (United States)

Wang, Pei; Song, Fan; Cai, Wanzhi

2014-01-01

Insect mitochondrial genomes are very important to understand the molecular evolution as well as for phylogenetic and phylogeographic studies of the insects. The Miridae are the largest family of Heteroptera encompassing more than 11,000 described species and of great economic importance. For better understanding the diversity and the evolution of plant bugs, we sequence five new mitochondrial genomes and present the first comparative analysis of nine mitochondrial genomes of mirids available to date. Our result showed that gene content, gene arrangement, base composition and sequences of mitochondrial transcription termination factor were conserved in plant bugs. Intra-genus species shared more conserved genomic characteristics, such as nucleotide and amino acid composition of protein-coding genes, secondary structure and anticodon mutations of tRNAs, and non-coding sequences. Control region possessed several distinct characteristics, including: variable size, abundant tandem repetitions, and intra-genus conservation; and was useful in evolutionary and population genetic studies. The AGG codon reassignments were investigated between serine and lysine in the genera Adelphocoris and other cimicomorphans. Our analysis revealed correlated evolution between reassignments of the AGG codon and specific point mutations at the antidocons of tRNALys and tRNASer(AGN). Phylogenetic analysis indicated that mitochondrial genome sequences were useful in resolving family level relationship of Cimicomorpha. Comparative evolutionary analysis of plant bug mitochondrial genomes allowed the identification of previously neglected coding genes or non-coding regions as potential molecular markers. The finding of the AGG codon reassignments between serine and lysine indicated the parallel evolution of the genetic code in Hemiptera mitochondrial genomes. PMID:24988409

Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1.

Science.gov (United States)

Sakaguchi, Masayoshi; Takezawa, Makoto; Nakazawa, Rie; Nozawa, Kazutaka; Kusakawa, Taro; Nagasawa, Takeshi; Sugahara, Yasusato; Kawakita, Masao

2008-05-01

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40 degrees C in terms of k(cat) value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90 degrees C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7 degrees C and 86.9 degrees C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

Science.gov (United States)

Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

2013-01-01

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

Inhibition of growth hormone and prolactin secretion by a serine proteinase inhibitor

International Nuclear Information System (INIS)

Rappay, G.; Nagy, I.; Makara, G.B.; Horvath, G.; Karteszi, M.; Bacsy, E.; Stark, E.

1984-01-01

The action of the tripeptide aldehyde t-butyloxycarbonyl-DPhe-Pro-Arg-H (boc-fPR-H), belonging to a family of serine proteinase inhibitors, on the release of immunoreactive prolactin (iPRL) and growth hormone (iGH) has been studied. In rat anterior pituitary cell cultures and pituitary quarters 1 mM boc-fPR-H inhibited basal iPRL and iGH release. Thyroliberin-induced iPRL release by cultured cells was also markedly inhibited with a concomitant accumulation of intracellular iPRL. During the short- and long-term exposure of cells to boc-fPR-H there were no changes in total cell protein contents and in activities of some lysosomal marker enzymes. The marked inhibition of basal as well as stimulated hormone release in the presence of the enzyme inhibitor might suggest that at least a portion of the hormones is released via a proteolytic enzyme-dependent process

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

Science.gov (United States)

Iijima, N; Tanaka, M; Mitsui, S; Yamamura, Y; Yamaguchi, N; Ibata, Y

1999-03-20

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons. Copyright 1999 Elsevier Science B.V.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

Directory of Open Access Journals (Sweden)

A.V Pérez

2008-01-01

Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg and fibrinogen (minimum coagulant dose = 4.2 µg in vitro, and promotes defibrin(ogenation in vivo (minimum defibrin(ogenating dose = 1.0 µg. In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain

Science.gov (United States)

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, Pparahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain. PMID:23724001

Pdx1 is post-translationally modified in vivo and serine 61 is the principal site of phosphorylation

DEFF Research Database (Denmark)

Frogne, Thomas; Sylvestersen, Kathrine Beck; Kubicek, Stefan

2012-01-01

signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We...... alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis...

Mice, double deficient in lysosomal serine carboxypeptidases Scpep1 and Cathepsin A develop the hyperproliferative vesicular corneal dystrophy and hypertrophic skin thickenings.

Directory of Open Access Journals (Sweden)

Xuefang Pan

Full Text Available Vasoactive and mitogenic peptide, endothelin-1 (ET-1 plays an important role in physiology of the ocular tissues by regulating the growth of corneal epithelial cells and maintaining the hemodynamics of intraocular fluids. We have previously established that ET-1 can be degraded in vivo by two lysosomal/secreted serine carboxypeptidases, Cathepsin A (CathA and Serine Carboxypeptidase 1 (Scpep1 and that gene-targeted CathAS190A /Scpep1-/- mice, deficient in CathA and Scpep1 have a prolonged half-life of circulating ET-1 associated with systemic hypertension. In the current work we report that starting from 6 months of age, ~43% of CathAS190A /Scpep1-/- mice developed corneal clouding that eventually caused vision impairment. Histological evaluation of these mice demonstrated a selective fibrotic thickening and vacuolization of the corneas, resembling human hyperproliferative vesicular corneal stromal dystrophy and coexisting with a peculiar thickening of the skin epidermis. Moreover, we found that cultured corneal epithelial cells, skin fibroblasts and vascular smooth muscle cells derived from CathA/Scpep1-deficient mice, demonstrated a significantly higher proliferative response to treatment with exogenous ET-1, as compared with cells from wild type mice. We also detected increased activation level of ERK1/2 and AKT kinases involved in cell proliferation in the ET-1-treated cultured cells from CathA/Scpep1 deficient mice. Together, results from our experimental model suggest that; in normal tissues the tandem of serine carboxypeptidases, Scpep1 and CathA likely constitutes an important part of the physiological mechanism responsible for the balanced elimination of heightened levels of ET-1 that otherwise would accumulate in tissues and consequently contribute to development of the hyper-proliferative corneal dystrophy and abnormal skin thickening.

Nucleocytoplasmic shuttling of the HSV-2 serine/threonine kinase Us3

International Nuclear Information System (INIS)

Finnen, Renee L.; Johnston, Susan M.; Neron, Casey E.; Banfield, Bruce W.

2011-01-01

The alphaherpesvirus serine/threonine kinase Us3 plays diverse roles in virus multiplication and modifies both nuclear and cytoplasmic substrates. We recently reported that treatment of HSV-2 Us3-transfected and HSV-2-infected cells with leptomycin B, an inhibitor of nuclear export mediated by interaction of chromosomal regional maintenance protein (CRM1) with leucine rich nuclear export signals (NESs), resulted in nuclear trapping of Us3. Here, we utilized fluorescence loss in photobleaching to monitor nuclear export of HSV-2 Us3 and confirm that this process proceeds solely via a CRM1-mediated mechanism. Analysis of deletion derivatives of HSV-2 Us3 fused to a nuclear export reporter protein implicated the involvement of NES-like sequences in nuclear export. However, nuclear trapping of HSV-2 Us3 proteins carrying mutations in these potential NESs was not observed, indicating that these sequences are not functional in the context of full-length protein. Our analyses also revealed previously unidentified regions of HSV-2 Us3 that contribute to its kinase activity.

Distribution and classification of Serine β-lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes deposited in Public Databases

Directory of Open Access Journals (Sweden)

Adriana Fróes

2016-11-01

Full Text Available β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South -SZ- and North Zone -NZ, presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data, exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST against conserved domain database profiles (CDD, Pfam, and COG followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%, while Class D is predominant in NZ (55%. CfxA (65% and ACC (47% types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%, CfxA (28%, ACC (21%, CEPA (20% and FOX (19%. Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of

Distribution and Classification of Serine β-Lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes Deposited in Public Databases.

Science.gov (United States)

Fróes, Adriana M; da Mota, Fábio F; Cuadrat, Rafael R C; Dávila, Alberto M R

2016-01-01

β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South Zone, SZ and North Zone, NZ), presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data), exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST) against conserved domain database profiles (CDD, Pfam, and COG) followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%), while Class D is predominant in NZ (55%). CfxA (65%) and ACC (47%) types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%), CfxA (28%), ACC (21%), CEPA (20%), and FOX (19%). Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of

Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

Directory of Open Access Journals (Sweden)

Rebrikov Denis V

2004-01-01

Full Text Available Abstract Background In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. Results The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa and 226 amino acid residues (Mcalc 23.5 kDa, respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70% and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%. The phylogenetic tree of these enzymes was constructed. Conclusions Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases.

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.

Science.gov (United States)

Calderon, L A; Teles, R C L; Leite, J R S A; Franco, O L; Grossi-de-Sá, M F; Medrano, F J; Bloch, C; Freitas, S M

2005-08-01

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

Comparison of F-18-FDG PET/CT findings between pancreatic solid pseudopapillary tumor and pancreatic ductal adenocarcinoma

International Nuclear Information System (INIS)

Kim, Yong-il; Kim, Seok-ki; Paeng, Jin Chul; Lee, Ho-Young

2014-01-01

Objective: Pancreatic solid pseudopapillary tumor (SPT) is a rare benign tumor. Little data are available on positron emission tomographic/computed tomographic (PET/CT) characteristics of this tumor. Therefore, we analyzed the metabolic characteristics of SPT using F-18-FDG PET/CT and compared the results with those of pancreatic ductal adenocarcinoma. Methods: We retrospectively reviewed the records of 11 SPT patients and 46 patients with ductal adenocarcinoma. Ten SPT patients had primary tumors and 1 patient had metastatic SPT. Maximum standardized uptake value (max SUV), mean SUV, metabolic tumor volume (MTV), total lesion glycolysis (TLG), and tumor-to-background ratio (TBR) were evaluated. Mann–Whitney U test between pancreatic SPT and ductal adenocarcinoma was performed. In addition, age, gender and tumor size-adjusted analysis of covariance (ANCOVA) was done between pancreatic SPT and ductal adenocarcinoma. Results: Compared with pancreatic ductal adenocarcinomas, SPTs had significantly higher tumor size-adjusted MTV and TLG. MTV and TLG values were significantly correlated with T-stage of the SPTs. In 1 SPT patient, metastases in the liver and mesentery were revealed by intense uptake of FDG on F-18-FDG PET/CT, and after PET/CT had suggested the presence of pancreatic SPT. Conclusion: We recommend that SPT be considered when a solid pancreatic mass with increased FDG metabolism is encountered on PET/CT. F-18-FDG PET/CT may be useful in detecting subtle metastases of SPT

Aminocarnitine and acylaminocarnitines: Carnitine acyltransferase inhibitors affecting long-chain fatty acid and glucose metabolism

International Nuclear Information System (INIS)

Clark, D.J.

1989-01-01

DL-Aminocarnitine (DL-3-amino-4-trimethylaminobutyrate) and the acylaminocarnitines acetyl-, decanoyl- and palmitoyl-DL-aminocarnitine have been synthesized and tested as inhibitors of carnitine palmitoyl-transferase and carnitine acetyltransferase in vitro and in vivo. Acetyl-DL-aaminocarnitine is the most potent reversible inhibitor of carnitine acetyltransferase reported to date, and is competitive with respect to acetyl-L-carnitine. Mice given acetyl-DL-aminocarnitine metabolize [U- 14 C]acetyl-L-carnitine at about 60% of the rate of control mice. Palmitoyl-DL-aminocarnitine is the most potent reversible inhibitor of carnitine palmitoyltransferase reported to date. Decanoyl-DL-aminocarnitine and DL-aminocarnitine are also very potent inhibitors; all compounds inhibit the catabolism of [ 14 C]palmitate to 14 CO 2 in intact mice by at least 50%. Carnitine palmitoyltransferase controls the entry of long-chain fatty acids into the mitochondrial matrix for β-oxidation. The inhibition of carnitine palmitoyltransferase by aminocarnitine or acylaminocarnitines in vivo prevents or reverses ketogenesis in fasted mice, and causes the reversible accumulation of triglycerides in liver, kidney and plasma. Administration of DL-aminocarnitine to streptozotocindiabetic mice lowers plasma glucose levels and improves the glucose tolerance test

Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

Directory of Open Access Journals (Sweden)

Zhuo Zhan

Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

Novel Two-Component System of Streptococcus sanguinis Affecting Functions Associated with Viability in Saliva and Biofilm Formation.

Science.gov (United States)

Camargo, Tarsila M; Stipp, Rafael N; Alves, Lívia A; Harth-Chu, Erika N; Höfling, José F; Mattos-Graner, Renata O

2018-04-01

Streptococcus sanguinis is a pioneer species of teeth and a common opportunistic pathogen of infective endocarditis. In this study, we identified a two-component system, S. sanguinis SptRS (SptRS Ss ), affecting S. sanguinis survival in saliva and biofilm formation. Isogenic mutants of sptR Ss (SKsptR) and sptS Ss (SKsptS) showed reduced cell counts in ex vivo assays of viability in saliva compared to those of parent strain SK36 and complemented mutants. Reduced counts of the mutants in saliva were associated with reduced growth rates in nutrient-poor medium (RPMI) and increased susceptibility to the deposition of C3b and the membrane attach complex (MAC) of the complement system, a defense component of saliva and serum. Conversely, sptR Ss and sptS Ss mutants showed increased biofilm formation associated with higher levels of production of H 2 O 2 and extracellular DNA. Reverse transcription-quantitative PCR (RT-qPCR) comparisons of strains indicated a global role of SptRS Ss in repressing genes for H 2 O 2 production (2.5- to 15-fold upregulation of spxB , spxR , vicR , tpk , and ackA in sptR Ss and sptS Ss mutants), biofilm formation, and/or evasion of host immunity (2.1- to 11.4-fold upregulation of srtA , pcsB , cwdP , iga , and nt5e ). Compatible with the homology of SptR Ss with AraC-type regulators, duplicate to multiple conserved repeats were identified in 1,000-bp regulatory regions of downstream genes, suggesting that SptR Ss regulates transcription by DNA looping. Significant transcriptional changes in the regulatory genes vicR , spxR , comE , comX , and mecA in the sptR Ss and sptS Ss mutants further indicated that SptRS Ss is part of a regulatory network that coordinates cell wall homeostasis, H 2 O 2 production, and competence. This study reveals that SptRS Ss is involved in the regulation of crucial functions for S. sanguinis persistence in the oral cavity. Copyright © 2018 American Society for Microbiology.

Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants

OpenAIRE

Silvia eTavares; Silvia eTavares; Markus eWirtz; Marcel Pascal Beier; Jochen eBogs; Jochen eBogs; Jochen eBogs; Ruediger eHell; Sara eAmâncio

2015-01-01

In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT protein fam...

Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants

OpenAIRE

Tavares, Sílvia; Wirtz, Markus; Beier, Marcel P.; Bogs, Jochen; Hell, Rüdiger; Amâncio, Sara

2015-01-01

In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family...

Multipolar electrostatics based on the Kriging machine learning method: an application to serine.

Science.gov (United States)

Yuan, Yongna; Mills, Matthew J L; Popelier, Paul L A

2014-04-01

A multipolar, polarizable electrostatic method for future use in a novel force field is described. Quantum Chemical Topology (QCT) is used to partition the electron density of a chemical system into atoms, then the machine learning method Kriging is used to build models that relate the multipole moments of the atoms to the positions of their surrounding nuclei. The pilot system serine is used to study both the influence of the level of theory and the set of data generator methods used. The latter consists of: (i) sampling of protein structures deposited in the Protein Data Bank (PDB), or (ii) normal mode distortion along either (a) Cartesian coordinates, or (b) redundant internal coordinates. Wavefunctions for the sampled geometries were obtained at the HF/6-31G(d,p), B3LYP/apc-1, and MP2/cc-pVDZ levels of theory, prior to calculation of the atomic multipole moments by volume integration. The average absolute error (over an independent test set of conformations) in the total atom-atom electrostatic interaction energy of serine, using Kriging models built with the three data generator methods is 11.3 kJ mol⁻¹ (PDB), 8.2 kJ mol⁻¹ (Cartesian distortion), and 10.1 kJ mol⁻¹ (redundant internal distortion) at the HF/6-31G(d,p) level. At the B3LYP/apc-1 level, the respective errors are 7.7 kJ mol⁻¹, 6.7 kJ mol⁻¹, and 4.9 kJ mol⁻¹, while at the MP2/cc-pVDZ level they are 6.5 kJ mol⁻¹, 5.3 kJ mol⁻¹, and 4.0 kJ mol⁻¹. The ranges of geometries generated by the redundant internal coordinate distortion and by extraction from the PDB are much wider than the range generated by Cartesian distortion. The atomic multipole moment and electrostatic interaction energy predictions for the B3LYP/apc-1 and MP2/cc-pVDZ levels are similar, and both are better than the corresponding predictions at the HF/6-31G(d,p) level.

Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

International Nuclear Information System (INIS)

Liu, Lingling; Wang, Yong-Dong; Wu, Jing; Cui, Jimmy; Chen, Taosheng

2012-01-01

Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein’s transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR–mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility. We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR–mediated cell migration and metastasis. Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

Science.gov (United States)

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato.

Science.gov (United States)

Stork, Ines; Gartemann, Karl-Heinz; Burger, Annette; Eichenlaub, Rudolf

2008-09-01

Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

Sound production treatment for acquired apraxia of speech: Effects of blocked and random practice on multisyllabic word production.

Science.gov (United States)

Wambaugh, Julie; Nessler, Christina; Wright, Sandra; Mauszycki, Shannon; DeLong, Catharine

2016-10-01

This study was designed to examine the effects of practice schedule, blocked vs random, on outcomes of a behavioural treatment for acquired apraxia of speech (AOS), Sound Production Treatment (SPT). SPT was administered to four speakers with chronic AOS and aphasia in the context of multiple baseline designs across behaviours and participants. Treatment was applied to multiple sound errors within three-to-five syllable words. All participants received both practice schedules: SPT-Random (SPT-R) and SPT-Blocked (SPT-B). Improvements in accuracy of word production for trained items were found for both treatment conditions for all participants. One participant demonstrated better maintenance effects associated with SPT-R. Response generalisation to untreated words varied across participants, but was generally modest and unstable. Stimulus generalisation to production of words in sentence completion was positive for three of the participants. Stimulus generalisation to production of phrases was positive for two of the participants. Findings provide additional efficacy data regarding SPT's effects on articulation of treated items and extend knowledge of the treatment's effects when applied to multiple targets within multisyllabic words.

The mediating effects of childhood neglect on the association between schizotypal and autistic personality traits and depression in a non-clinical sample.

Science.gov (United States)

Liu, Jianbo; Gong, Jingbo; Nie, Guanghui; He, Yuqiong; Xiao, Bo; Shen, Yanmei; Luo, Xuerong

2017-10-25

Autistic personality traits (APT) and schizotypal personality traits (SPT) are associated with depression. However, mediating factors within these relationships have not yet been explored. Thus, the focus of the current study was to examine the effects of childhood neglect on the relationship between APT/SPT and depression. This cross-sectional study was conducted on first-year students (N = 2469) at Hunan University of Chinese Medicine and Hengyang Normal College (Changsha, China). Participants completed surveys on APT, SPT, childhood neglect, abuse and depression. Through correlational analyses, APT and SPT traits were positively correlated with childhood neglect and depression (p childhood maltreatment, emotional neglect (β = 0.112, p Childhood neglect did not account for the relationships between APT/SPT and depression. Further analysis found that childhood neglect mediated the relationship between SPT and depression but not APT and depression. Among types of childhood maltreatment, neglect was the strongest predicting factor for depression. Neglect did not account for the relationship between APT/SPT and depression but was a strong mediating factor between SPT and depression.

Volumetric properties for glycine and L-serine in aqueous solutions of 1-Ethyl-3-methylimidazolium hydrogen sulfate ([Emim][HSO4]) at T = (293.15–313.15) K and ambient pressure

International Nuclear Information System (INIS)

Rafiee, Hamid Reza; Frouzesh, Farshid

2016-01-01

Highlights: • Ternary systems (amino acid + ionic liquid + H 2 O) are considered. • Volumetric properties including density, V φ , ΔV φ 0 and E φ 0 are determined. • Glycine and L-serine were amino acids and [Emim][HSO 4 ] was IL studied. • V φ values for amino acids increased with both concentration of amino acids and temperature. • Both studied amino acids act as structure maker in ternary systems. - Abstract: By using the volumetric properties, the solute–solvent interactions are studied in the ternary (glycine + 1-Ethyl-3-methylimidazolium hydrogen sulfate [Emim][HSO 4 ] + water) and (L-serine + 1-Ethyl-3-methylimidazolium hydrogen sulfate [Emim][HSO 4 ] + water) systems. For this purpose, the apparent molar volumes, V φ , are calculated from the experimental density data. To obtain limiting apparent molar volumes V φ 0 , the apparent molar volume values are fitted to the Redlich-Mayer type equation. Then the limiting apparent molar volumes of transfer, ΔV φ 0 , for studied amino acids from water to aqueous solutions of ionic liquid (IL) are calculated. The results showed that the ΔV φ 0 values are negative for both ternary systems at all temperatures. The limiting apparent molar expansibility E φ 0 values have been obtained from the first derivative of limiting apparent molar volumes with respect to temperature. The values of second derivative of limiting apparent molar volumes respect to temperature show the structure making or breaking ability of glycine and L-serine in studied IL aqueous solutions. The results indicated that glycine and L-serine act as structure maker in studied solutions. McMillan–Mayer theory is used for evaluating the interaction parameters. Also the hydration number, n H , for both amino acids in ternary solutions has been reported.

Clinical relevance is associated with allergen-specific wheal size in skin prick testing

DEFF Research Database (Denmark)

Haahtela, T.; Burbach, G. J.; Bachert, C.

2014-01-01

, asthma, atopic dermatitis, food allergy). The effects of age, gender, and geographical area on SPT results were assessed. For each allergen, the wheal size in mm with an 80% positive predictive value (PPV) for being clinically relevant was calculated. ResultsDepending on the allergen, from 40% (blatella...... by providing quantitative decision points. MethodsThe GA(2)LEN SPT study with 3068 valid data sets was used to investigate the relationship between SPT results and patient-reported clinical relevance for each of the 18 inhalant allergens as well as SPT wheal size and physician-diagnosed allergy (rhinitis...... SPT reactions had a smaller risk of sensitizations being clinically relevant compared with adults. The 80% PPV varied from 3 to 10mm depending on the allergen. ConclusionThese reading keys' for 18 inhalant allergens can help interpret SPT results with respect to their clinical significance. A SPT form...

ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage

Science.gov (United States)

Maya, Ruth; Balass, Moshe; Kim, Seong-Tae; Shkedy, Dganit; Leal, Juan-Fernando Martinez; Shifman, Ohad; Moas, Miri; Buschmann, Thomas; Ronai, Ze'ev; Shiloh, Yosef; Kastan, Michael B.; Katzir, Ephraim; Oren, Moshe

2001-01-01

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258–260 and 393–395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2. PMID:11331603

Identification of a serine proteinase homolog (Sp-SPH involved in immune defense in the mud crab Scylla paramamosain.

Directory of Open Access Journals (Sweden)

Qiu-xia Zhang

Full Text Available Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH, originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus, bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN, and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05, and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05. Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness.

Science.gov (United States)

Cybulski, Larisa Estefanía; Ballering, Joost; Moussatova, Anastassiia; Inda, Maria Eugenia; Vazquez, Daniela B; Wassenaar, Tsjerk A; de Mendoza, Diego; Tieleman, D Peter; Killian, J Antoinette

2015-05-19

DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.

Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen*

Science.gov (United States)

Sharma, Aditya K.; Arora, Divya; Singh, Lalit K.; Gangwal, Aakriti; Sajid, Andaleeb; Molle, Virginie; Singh, Yogendra; Nandicoori, Vinay Kumar

2016-01-01

Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events. PMID:27758870

Long-term repeatability of the skin prick test is high when supported by history or allergen-sensitivity tests

DEFF Research Database (Denmark)

Bødtger, Uffe; Jacobsen, C R; Poulsen, L K

2003-01-01

subjects. An SPT was positive when > or =3 mm, and repeatable if either persistently positive or negative. Clinical sensitivity to birch pollen was used as model for inhalation allergy, and was investigated at inclusion and at study termination by challenge tests, intradermal test, titrated SPT and Ig......E measurements. Birch pollen symptoms were confirmed in diaries. RESULTS: The repeatability of a positive SPT was 67%, increasing significantly to 100% when supported by the history. When not supported by history, the presence of specific IgE was significantly associated with a repeatable SPT. Allergen....... CONCLUSION: SPT changes are clinically relevant. Further studies using other allergens are needed. Long-term repeatability of SPT is high in the presence of a supportive history....

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

Science.gov (United States)

Nicolas, A; Egmond, M; Verrips, C T; de Vlieg, J; Longhi, S; Cambillau, C; Martinez, C

1996-01-16

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

Science.gov (United States)

Zheng, Yanhua; Lu, Zhimin

2013-01-01

Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis

DEFF Research Database (Denmark)

Tarlton, J F; Whiting, C V; Tunmore, D

2000-01-01

, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues....... Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium...

Gravitational spreading, bookshelf faulting, and tectonic evolution of the South Polar Terrain of Saturn's moon Enceladus

Science.gov (United States)

Yin, An; Pappalardo, Robert T.

2015-11-01

Despite a decade of intense research the mechanical origin of the tiger-stripe fractures (TSF) and their geologic relationship to the hosting South Polar Terrain (SPT) of Enceladus remain poorly understood. Here we show via systematic photo-geological mapping that the semi-squared SPT is bounded by right-slip, left-slip, extensional, and contractional zones on its four edges. Discrete deformation along the edges in turn accommodates translation of the SPT as a single sheet with its transport direction parallel to the regional topographic gradient. This parallel relationship implies that the gradient of gravitational potential energy drove the SPT motion. In map view, internal deformation of the SPT is expressed by distributed right-slip shear parallel to the SPT transport direction. The broad right-slip shear across the whole SPT was facilitated by left-slip bookshelf faulting along the parallel TSF. We suggest that the flow-like tectonics, to the first approximation across the SPT on Enceladus, is best explained by the occurrence of a transient thermal event, which allowed the release of gravitational potential energy via lateral viscous flow within the thermally weakened ice shell.

FACT, the Bur kinase pathway, and the histone co-repressor HirC have overlapping nucleosome-related roles in yeast transcription elongation.

Directory of Open Access Journals (Sweden)

Jennifer R Stevens

Full Text Available Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of 'cryptic' promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4-Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4-Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the

The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

Directory of Open Access Journals (Sweden)

Ashis K Mukherjee

Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

Science.gov (United States)

Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

2013-09-01

We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

The probe rules in single particle tracking

DEFF Research Database (Denmark)

Clausen, Mathias P.; Lagerholm, B. Christoffer

2011-01-01

techniques and probes that have made historically very demanding and specialized bio-imaging techniques more easily accessible and achievable. SPT has in particular found extensive use for analyzing the molecular organization of biological membranes. From these and other studies using complementary...... techniques it has been determined that the organization of native plasma membranes is heterogeneous over a very large range of spatial and temporal scales. The observed heterogeneities in the organization have the practical consequence that the SPT results in investigations of native plasma membranes...... are time dependent. Furthermore, because the accessible time dynamics, and also the spatial resolution, in an SPT experiment is mainly dependent on the luminous brightness and photostability of the particular SPT probe that is used, available SPT results are ultimately dependent on the SPT probes...

Preparation and functional evaluation of collagen oligopeptide-rich hydrolysate from fish skin with the serine collagenolytic protease from Pseudoalteromonas sp. SM9913.

Science.gov (United States)

Chen, Xiu-Lan; Peng, Ming; Li, Jing; Tang, Bai-Lu; Shao, Xuan; Zhao, Fang; Liu, Chang; Zhang, Xi-Ying; Li, Ping-Yi; Shi, Mei; Zhang, Yu-Zhong; Song, Xiao-Yan

2017-11-16

Although several serine collagenolytic proteases from bacteria were reported, none has been used to prepare bioactive collagen peptides. MCP-01 is the most abundant extracellular protease of deep-sea Pseudoalteromonas sp. SM9913 and is a serine collagenolytic protease with high efficiency on fish collagen hydrolysis. Here, we set up a pilot scale process to ferment SM9913 for extracellular protease production. With SM9913 extracellular protease as a tool, a process to prepare collagen oligopeptide-rich hydrolysate from codfish skin was set up, which was further scaled up to pilot (100 L) and plant (2000 L) levels with yields >66%. The hydrolysates from laboratory-, pilot- and plant-scales had quite similar quality, containing ~95% peptides with molecular weights lower than 3000 Da and approximately 60% lower than 1000 Da, in which collagen oilgopeptides account for approximately 95%. Bioactivity analyses showed that the hydrolysate had moisture-retention ability, antioxidant activity, and promoting effect on cell viability of human dermal fibroblasts. Safety evaluation showed that the hydrolysate was nontoxic and nonirritating to skin. Therefore, SM9913 extracellular protease is a good enzyme to prepare bioactive oligopeptides from fish skin. The results also suggest that the collagen oligopeptides-rich hydrolysate may have potentials in biomedical, functional food, pharmaceutical and cosmetic industries.

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Directory of Open Access Journals (Sweden)

Garabaya Cecilia

2006-03-01

Full Text Available Abstract Background We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. Results Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

Directory of Open Access Journals (Sweden)

Olivier Barré

Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

The incidence and features of systemic reactions to skin prick tests.

Science.gov (United States)

Sellaturay, Priya; Nasser, Shuaib; Ewan, Pamela

2015-09-01

Skin prick testing (SPT) has been regarded as a safe procedure with few systemic reactions. To evaluate the rate of systemic reactions and their associations after SPT in the largest population to date. In this study reactions were recorded prospectively in a specialist UK allergy clinic for 6 years (2007-2013). An estimated 31,000 patients underwent SPT. Twenty-four patients (age range 7 months to 56 years, mean 23.5 years, 17 female patients, 12 with asthma) had systemic reactions. The rate of systemic reactions to SPT was 0.077%. The likely allergens causing the reaction were foods (18; peanut, 7; walnut, 1; Brazil nut, 2; pistachio, 1; lupin, 1; cow's milk, 2; shrimp, 1; spinach, 1; legume, 1; soy, 1), aeroallergens (4; rabbit, 1; rat, 1; ragwort, 1; grass pollen, 1), wasp venom (1), and Tazocin (1). The causative SPT wheal was larger than 8 mm in 75%. The reaction to Tazocin was severe, with anaphylaxis occurring minutes after SPT. Reactions were treated immediately in the clinic and did not require further medical care. In this largest single-center study, the rate of systemic reactions after SPT was 77 per 100,000 patients. It is the first study to identify foods as a common and important cause (75%), with nuts posing the highest risk. This study reports the first systemic reaction to venom SPT and the first anaphylactic reaction after drug SPT. There was an association with a history of severe reactions and large skin test reaction. There are risks, albeit small, when undertaking SPT. Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Stability of a sweet product of tamarillo (Cyphomandra betacea preserved by combined methods

Directory of Open Access Journals (Sweden)

Villegas-Ruíz Xadeni

2013-06-01

Full Text Available The aim of this study was the use of the hurdle technology for preparing a sweet product (SPT of tamarillo (Cyphomandra betacea and assess its storage stability. SPT was prepared at the fruit natural pH (3.72, lowering the water activity (aw (0.901 by adding sucrose and sodium benzoate (0.1% as antimicrobial. SPT was stored under refrigeration (4 °C conditions protected from light and it was analyzed every third day for 60 days. Physicochemical (color, aw, pH, and titratable acidity as citric acid, antioxidant (antioxidant capacity, total phenolics, and anthocyanins, and microbiological (aerobic mesophilic bacteria, molds, and yeasts, were determined. SPT was mixed with plain yogurt to evaluate the overall sensory acceptance during storage. The evaluation took place every seventh day during 49 days. The storage time had no effect neither on aw, pH or acidity of the SPT. It was observed an increase in the net change of color (0 to 4.9 and a decrease in total phenolic compounds (1.213-0.816 mg gallic acid/g SPT, total monomeric anthocyanins (0.910-0.611 mg cyanidin-3-glucoside/g SPT g, and antioxidant capacity (5.816-3.576 µmole Trolox/g SPT. The aerobic mesophilic bacteria ( 0.05 during storage.

Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

International Nuclear Information System (INIS)

Qasim, Mohammad A.; Song, Jikui; Markley, John L.; Laskowski, Michael

2010-01-01

Research highlights: → Large pK shifts in ionizable groups when buried in the protein interior. → Substrate dependent shifts in pH optimum for serine proteases. → Lys side chain is a stronger acid in serine protease S 1 pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S 1 pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

DEFF Research Database (Denmark)

Teillet, F; Lacroix, M; Thiel, Steffen

2007-01-01

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To ident......Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2....... To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19...... and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3...

Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K

Directory of Open Access Journals (Sweden)

Peng Sang

2016-02-01

Full Text Available To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

International Nuclear Information System (INIS)

Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

2008-01-01

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects

Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

Science.gov (United States)

LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

2012-01-01

SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

The Serine Hydrolase ABHD6 Is a Critical Regulator of the Metabolic Syndrome

Directory of Open Access Journals (Sweden)

Gwynneth Thomas

2013-10-01

Full Text Available The serine hydrolase α/β hydrolase domain 6 (ABHD6 has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6’s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

Phosphorylation of serine 248 of C/EBPα is dispensable for myelopoiesis but its disruption leads to a low penetrant myeloid disorder with long latency.

Directory of Open Access Journals (Sweden)

Marie S Hasemann

Full Text Available BACKGROUND: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A animals display a competitive advantage compared to wild type cells in a transplantation assay. CONCLUSIONS/SIGNIFICANCE: Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to

Phosphorylation of serine 248 of C/EBPα is dispensable for myelopoiesis but its disruption leads to a low penetrant myeloid disorder with long latency.

Science.gov (United States)

Hasemann, Marie S; Schuster, Mikkel B; Frank, Anne-Katrine; Theilgaard-Mönch, Kim; Pedersen, Thomas Å; Nerlov, Claus; Porse, Bo T

2012-01-01

Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences

Agreement between the Skin Prick Test and Specific Serum IgE for Egg White and Cow's Milk Allergens in Young Infant with Atopic Dermatitis

Directory of Open Access Journals (Sweden)

Hyeon-Jong Yang

2014-01-01

Conclusions: The results of SPT agreed well with those of sIgE. This finding provides a rationale for using SPT, and suggests that SPT can be used along with sIgE to detect food sensitization in young infants with AD.

A Comparison of Cosmological Parameters Determined from CMB Temperature Power Spectra from the South Pole Telescope and the Planck Satellite

Science.gov (United States)

Aylor, K.; Hou, Z.; Knox, L.; Story, K. T.; Benson, B. A.; Bleem, L. E.; Carlstrom, J. E.; Chang, C. L.; Cho, H.-M.; Chown, R.; Crawford, T. M.; Crites, A. T.; de Haan, T.; Dobbs, M. A.; Everett, W. B.; George, E. M.; Halverson, N. W.; Harrington, N. L.; Holder, G. P.; Holzapfel, W. L.; Hrubes, J. D.; Keisler, R.; Lee, A. T.; Leitch, E. M.; Luong-Van, D.; Marrone, D. P.; McMahon, J. J.; Meyer, S. S.; Millea, M.; Mocanu, L. M.; Mohr, J. J.; Natoli, T.; Omori, Y.; Padin, S.; Pryke, C.; Reichardt, C. L.; Ruhl, J. E.; Sayre, J. T.; Schaffer, K. K.; Shirokoff, E.; Staniszewski, Z.; Stark, A. A.; Vanderlinde, K.; Vieira, J. D.; Williamson, R.

2017-11-01

The Planck cosmic microwave background temperature data are best fit with a ΛCDM model that mildly contradicts constraints from other cosmological probes. The South Pole Telescope (SPT) 2540 {\\deg }2 SPT-SZ survey offers measurements on sub-degree angular scales (multipoles 650≤slant {\\ell }≤slant 2500) with sufficient precision to use as an independent check of the Planck data. Here we build on the recent joint analysis of the SPT-SZ and Planck data in Hou et al. by comparing ΛCDM parameter estimates using the temperature power spectrum from both data sets in the SPT-SZ survey region. We also restrict the multipole range used in parameter fitting to focus on modes measured well by both SPT and Planck, thereby greatly reducing sample variance as a driver of parameter differences and creating a stringent test for systematic errors. We find no evidence of systematic errors from these tests. When we expand the maximum multipole of SPT data used, we see low-significance shifts in the angular scale of the sound horizon and the physical baryon and cold dark matter densities, with a resulting trend to higher Hubble constant. When we compare SPT and Planck data on the SPT-SZ sky patch to Planck full-sky data but keep the multipole range restricted, we find differences in the parameters n s and {A}s{e}-2τ . We perform further checks, investigating instrumental effects and modeling assumptions, and we find no evidence that the effects investigated are responsible for any of the parameter shifts. Taken together, these tests reveal no evidence for systematic errors in SPT or Planck data in the overlapping sky coverage and multipole range and at most weak evidence for a breakdown of ΛCDM or systematic errors influencing either the Planck data outside the SPT-SZ survey area or the SPT data at {\\ell }> 2000.

Hormone-sensitive lipase serine phosphorylation and glycerol exchange across skeletal muscle in lean and obese subjects

DEFF Research Database (Denmark)

Jocken, Johan We; Roepstorff, Carsten; Goossens, Gijs H.

2008-01-01

from the vastus lateralis muscle before and during ISO to investigate hormone-sensitive lipase (HSL) protein expression and serine phosphorylation. Results: Baseline total glycerol release across the forearm was significantly blunted in obese compared with lean subjects (P=0.045). This was accompanied......Objective: Increased intramuscular triacylglycerol (IMTG) storage is a characteristic of the obese insulin resistant state. We aimed to investigate whether a blunted fasting or beta-adrenergically mediated lipolysis contributes to this increased IMTG storage in obesity. Research design and Methods......: Forearm skeletal muscle (SM) lipolysis was investigated in thirteen lean and ten obese men using [(2)H(5)]-glycerol combined with the measurement of arterio-venous differences before and during beta-adrenergic stimulation using the non-selective beta-agonist isoprenaline (ISO). Muscle biopsies were taken...

Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops.

Science.gov (United States)

Wu, Yongzhong; Fox, Tim W; Trimnell, Mary R; Wang, Lijuan; Xu, Rui-Ji; Cigan, A Mark; Huffman, Gary A; Garnaat, Carl W; Hershey, Howard; Albertsen, Marc C

2016-03-01

We have developed a novel hybridization platform that utilizes nuclear male sterility to produce hybrids in maize and other cross-pollinating crops. A key component of this platform is a process termed Seed Production Technology (SPT). This process incorporates a transgenic SPT maintainer line capable of propagating nontransgenic nuclear male-sterile lines for use as female parents in hybrid production. The maize SPT maintainer line is a homozygous recessive male sterile transformed with a SPT construct containing (i) a complementary wild-type male fertility gene to restore fertility, (ii) an α-amylase gene to disrupt pollination and (iii) a seed colour marker gene. The sporophytic wild-type allele complements the recessive mutation, enabling the development of pollen grains, all of which carry the recessive allele but with only half carrying the SPT transgenes. Pollen grains with the SPT transgenes exhibit starch depletion resulting from expression of α-amylase and are unable to germinate. Pollen grains that do not carry the SPT transgenes are nontransgenic and are able to fertilize homozygous mutant plants, resulting in nontransgenic male-sterile progeny for use as female parents. Because transgenic SPT maintainer seeds express a red fluorescent protein, they can be detected and efficiently separated from seeds that do not contain the SPT transgenes by mechanical colour sorting. The SPT process has the potential to replace current approaches to pollen control in commercial maize hybrid seed production. It also has important applications for other cross-pollinating crops where it can unlock the potential for greater hybrid productivity through expanding the parental germplasm pool. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

The role of spectrophotometry in the diagnosis of melanoma

Science.gov (United States)

2010-01-01

Background Spectrophotometry (SPT) could represent a promising technique for the diagnosis of cutaneous melanoma (CM) at earlier stages of the disease. Starting from our experience, we further assessed the role of SPT in CM early detection. Methods During a health campaign for malignant melanoma at National Cancer Institute of Naples, we identified a subset of 54 lesions to be addressed to surgical excision and histological examination. Before surgery, all patients were investigated by clinical and epiluminescence microscopy (ELM) screenings; selected lesions underwent spectrophotometer analysis. For SPT, we used a video spectrophotometer imaging system (Spectroshade® MHT S.p.A., Verona, Italy). Results Among the 54 patients harbouring cutaneous pigmented lesions, we performed comparison between results from the SPT screening and the histological diagnoses as well as evaluation of both sensitivity and specificity in detecting CM using either SPT or conventional approaches. For all pigmented lesions, agreement between histology and SPT classification was 57.4%. The sensitivity and specificity of SPT in detecting melanoma were 66.6% and 76.2%, respectively. Conclusions Although SPT is still considered as a valuable diagnostic tool for CM, its low accuracy, sensitivity, and specificity represent the main hamper for the introduction of such a methodology in clinical practice. Dermoscopy remains the best diagnostic tool for the preoperative diagnosis of pigmented skin lesions. PMID:20707921

Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella (L.).

Science.gov (United States)

Lin, Hailan; Xia, Xiaofeng; Yu, Liying; Vasseur, Liette; Gurr, Geoff M; Yao, Fengluan; Yang, Guang; You, Minsheng

2015-12-10

Serine proteases (SPs) are crucial proteolytic enzymes responsible for digestion and other processes including signal transduction and immune responses in insects. Serine protease homologs (SPHs) lack catalytic activity but are involved in innate immunity. This study presents a genome-wide investigation of SPs and SPHs in the diamondback moth, Plutella xylostella (L.), a globally-distributed destructive pest of cruciferous crops. A total of 120 putative SPs and 101 putative SPHs were identified in the P. xylostella genome by bioinformatics analysis. Based on the features of trypsin, 38 SPs were putatively designated as trypsin genes. The distribution, transcription orientation, exon-intron structure and sequence alignments suggested that the majority of trypsin genes evolved from tandem duplications. Among the 221 SP/SPH genes, ten SP and three SPH genes with one or more clip domains were predicted and designated as PxCLIPs. Phylogenetic analysis of CLIPs in P. xylostella, two other Lepidoptera species (Bombyx mori and Manduca sexta), and two more distantly related insects (Drosophila melanogaster and Apis mellifera) showed that seven of the 13 PxCLIPs were clustered with homologs of the Lepidoptera rather than other species. Expression profiling of the P. xylostella SP and SPH genes in different developmental stages and tissues showed diverse expression patterns, suggesting high functional diversity with roles in digestion and development. This is the first genome-wide investigation on the SP and SPH genes in P. xylostella. The characterized features and profiled expression patterns of the P. xylostella SPs and SPHs suggest their involvement in digestion, development and immunity of this species. Our findings provide a foundation for further research on the functions of this gene family in P. xylostella, and a better understanding of its capacity to rapidly adapt to a wide range of environmental variables including host plants and insecticides.

Expression, purification, crystallization and preliminary X-ray diffraction studies of the human keratin 4-binding domain of serine-rich repeat protein 1 from Streptococcus agalactiae

International Nuclear Information System (INIS)

Sundaresan, Ramya; Samen, Ulrike; Ponnuraj, Karthe

2011-01-01

Expression, purification and crystallization of Srr-1-K4BD, a human keratin 4-binding domain of serine-rich repeat protein 1 from S. agalactiae, was carried out. Native crystals of Srr-1-K4BD diffracted to 3.8 Å resolution using synchrotron radiation. Serine-rich repeat protein 1 (Srr-1) is a surface protein from Streptococcus agalactiae. A 17 kDa region of this protein has been identified to bind to human keratin 4 (K4) and is termed the Srr-1 K4-binding domain (Srr-1-K4BD). Recombinant Srr-1-K4BD was overexpressed in Escherichia coli BL21 (DE3) cells. Native and selenomethionine-substituted proteins were prepared using Luria–Bertani (LB) and M9 minimal media, respectively. A two-step purification protocol was carried out to obtain a final homogenous sample of Srr-1-K4BD. Crystals of native Srr-1-K4BD were obtained using PEG 3350 as a precipitant. The crystals diffracted to 3.8 Å resolution using synchrotron radiation and belonged to space group P2 1 , with unit-cell parameters a = 47.56, b = 59.48, c = 94.71 Å, β = 93.95°

The Role of Social Perspective-Taking in Developing Students' Leadership Capacities

Science.gov (United States)

Dugan, John P.; Bohle, Christopher W.; Woelker, Lindsey R.; Cooney, Matthew A.

2014-01-01

This study examined relationships between social perspective-taking (SPT) and the individual, group, and societal domains of socially responsible leadership. SPT is a higher-order cognitive skill linked to moral development and social coordination, but never empirically connected to leadership development. Analyses determined SPT has a strong…

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor*

Science.gov (United States)

Collier, Mary E. W.; Ettelaie, Camille

2011-01-01

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF. PMID:21310953

Interactions of "bora-penicilloates" with serine β-lactamases and DD-peptidases.

Science.gov (United States)

Dzhekieva, Liudmila; Adediran, S A; Pratt, R F

2014-10-21

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

Analysis of serine proteases from marine sponges by 2-D zymography.

Science.gov (United States)

Wilkesman, Jeff G; Schröder, Heinz C

2007-02-01

Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.

Isolation, cDNA cloning, and structure-based functional characterization of oryctin, a hemolymph protein from the coconut rhinoceros beetle, Oryctes rhinoceros, as a novel serine protease inhibitor.

Science.gov (United States)

Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

2010-09-24

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant (13)C,(15)N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K(i) values of 3.9 × 10(-10) m, 6.2 × 10(-10) m, 1.4 × 10(-9) m, and 1.2 × 10(-8) m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.

Army Sustainment. Volume 47, Number 6. November-December 2015

Science.gov (United States)

2015-11-01

SP TBD AWT/ AVN T: SPT Convoy GOLD / 13B SP 1315 / 1415 T: SPT Convoy Turani SP 1115 T: SPT Convoy GOLD / 13B SP 1315 / 1415 T: SPT Convoy Turani SP...white Ford observed). Legend A/DACG = Arrival/departure airfield control group AWT/ AVN = Air weapons teams/aviation BSA = Brigade support area...Tech AED Engineer AMCOM LAR AMCOM LAR AMCOM LAR AMCOM UAS LAR PM UAS Lead TACOM LAR AMCOM LAR AMCOM LAR CECOM AVN LAR AMCOM LAR PM UAS CECOM AVN LAR

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

Science.gov (United States)

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

Science.gov (United States)

Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V

2015-07-01

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

Energetically benign synthesis of lanthanum silicate through “silica garden” route and its characterization

Energy Technology Data Exchange (ETDEWEB)

Parmar, Kavita [Central University of Jharkhand, Ranchi (India); Bhattacharjee, Santanu, E-mail: santanu@nmlindia.org [CSIR-National Metallurgical Laboratory, Jamshedpur (India)

2017-06-15

Lanthanum silicate synthesis through “silica garden” route has been reported as an alternative to energy intensive milling procedure. Under optimum conditions lanthanum chloride crystals react with water glass (sodium silicate) to produce self generating hollow lanthanum silicate precipitation tube(s) (LaSPT). The micro tubes are irregular, thick, white coloured and amorphous but are hierarchically built from smaller tubules of 10–20 nm diameters. They retain their amorphous nature on being heated up to 600 °C beyond which crystallization starts. The major phase in the LaSPT heated at 900 °C is La{sub 2}Si{sub 2}O{sub 7}. “As synthesized” LaSPT is heterogeneous and comprises non stoichiometric phases. The exterior and interior surfaces of these tubes are remarkably different in their morphology and chemical composition. LaSPT sintered at 1200 and 1300 °C show fair amount of ionic conductivity. - Graphical abstract: Lanthanum silicate precipitation tube (LaSPT) produced through ‘silica garden’ route offers a green alternative to energy intensive milling procedure. - Highlights: • La-silicate precipitation tube (LaSPT) synthesized via silica garden route. • The microtubes are irregular, thick, white coloured and amorphous. • They are hierarchically built from smaller tubules of 10–20 nm diameters. • The major phase in the LaSPT heated at 900 °C is La{sub 2}Si{sub 2}O{sub 7}. • LaSPT sintered at 1200 °C is fairly conducting.

Energetically benign synthesis of lanthanum silicate through “silica garden” route and its characterization

International Nuclear Information System (INIS)

Parmar, Kavita; Bhattacharjee, Santanu

2017-01-01

Lanthanum silicate synthesis through “silica garden” route has been reported as an alternative to energy intensive milling procedure. Under optimum conditions lanthanum chloride crystals react with water glass (sodium silicate) to produce self generating hollow lanthanum silicate precipitation tube(s) (LaSPT). The micro tubes are irregular, thick, white coloured and amorphous but are hierarchically built from smaller tubules of 10–20 nm diameters. They retain their amorphous nature on being heated up to 600 °C beyond which crystallization starts. The major phase in the LaSPT heated at 900 °C is La_2Si_2O_7. “As synthesized” LaSPT is heterogeneous and comprises non stoichiometric phases. The exterior and interior surfaces of these tubes are remarkably different in their morphology and chemical composition. LaSPT sintered at 1200 and 1300 °C show fair amount of ionic conductivity. - Graphical abstract: Lanthanum silicate precipitation tube (LaSPT) produced through ‘silica garden’ route offers a green alternative to energy intensive milling procedure. - Highlights: • La-silicate precipitation tube (LaSPT) synthesized via silica garden route. • The microtubes are irregular, thick, white coloured and amorphous. • They are hierarchically built from smaller tubules of 10–20 nm diameters. • The major phase in the LaSPT heated at 900 °C is La_2Si_2O_7. • LaSPT sintered at 1200 °C is fairly conducting.

Beyond the Dopamine Receptor: Regulation and Roles of Serine/Threonine Protein Phosphatases

Directory of Open Access Journals (Sweden)

Sven I Walaas

2011-08-01

Full Text Available Dopamine plays an important modulatory role in the central nervous system, helping to control critical aspects of motor function and reward learning. Alteration in normal dopaminergic neurotransmission underlies multiple neurological diseases including schizophrenia, Huntington's disease and Parkinson's disease. Modulation of dopamine-regulated signaling pathways is also important in the addictive actions of most drugs of abuse. Our studies over the last 30 years have focused on the molecular actions of dopamine acting on medium spiny neurons, the predominant neurons of the neostriatum. Striatum-enriched phosphoproteins, particularly DARPP-32, RCS (Regulator of Calmodulin Signaling and ARPP-16, mediate pleiotropic actions of dopamine. Notably, each of these proteins, either directly or indirectly, regulates the activity of one of the three major subclasses of serine/threonine protein phosphatases, PP1, PP2B and PP2A, respectively. For example, phosphorylation of DARPP-32 at Thr34 by protein kinase A results in potent inhibition of PP1, leading to potentiation of dopaminergic signaling at multiple steps from the dopamine receptor to the nucleus. The discovery of DARPP-32 and its emergence as a critical molecular integrator of striatal signaling will be discussed, as will more recent studies that highlight novel roles for RCS and ARPP-16 in dopamine-regulated striatal signaling pathways.

The prevalence of skin-test-positive allergic rhinitis in Danish adults

DEFF Research Database (Denmark)

Linneberg, A; Jørgensen, T; Nielsen, N H

2000-01-01

BACKGROUND: It is disputed whether increases in self-reported respiratory allergy represent a true increase or merely increased recognition. We aimed to investigate whether the prevalence of skin-prick-test (SPT)-positive allergic rhinitis had increased in an adult general population in Copenhagen...... (participation rate 74.6%) and 482 (participation rate 53.4%) subjects were examined in 1990 and 1998, respectively. Diagnoses of SPT-positive allergic rhinitis were based on a history of nasal symptoms on exposure to allergens and SPT positivity to allergens. RESULTS: The prevalence of a diagnosis of SPT...

HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

International Nuclear Information System (INIS)

Devroe, Eric; Silver, Pamela A.; Engelman, Alan

2005-01-01

Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells

The expression of a motoneuron-specific serine protease, motopsin (PRSS12), after facial nerve axotomy in mice.

Science.gov (United States)

Numajiri, Toshiaki; Mitsui, Shinichi; Hisa, Yasuo; Ishida, Toshihiro; Nishino, Kenichi; Yamaguchi, Nozomi

2006-01-01

Motopsin (PRSS12) is a mosaic serine protease that is preferentially expressed in motor neurons. To study the relationship between motopsin and motoneuron function, we investigated the expression of motopsin mRNA in facial nerve nuclei after facial nerve axotomy at the anterior margin of the parotid gland in mice. Neuronal function was monitored by assessing vibrissal motion in 3 months. Vibrissal behaviour on the injured side disappeared until the day 14 post-operation, and then recovered between the day 21 and 35. Motopsin expression decreased at the day 14, but markedly recovered by the day 21. In contrast, expression of growth-associated protein-43 (GAP-43) was induced at the day 3. These results suggest that the recovery of motopsin expression is correlated with the recovery of the facial motor neuronal function.

Smurf1 Inhibits Osteoblast Differentiation, Bone Formation, and Glucose Homeostasis through Serine 148

Directory of Open Access Journals (Sweden)

Junko Shimazu

2016-04-01

Full Text Available The E3 ubiquitin ligase Smurf1 targets the master regulator of osteoblast differentiation, Runx2, for degradation, yet the function of Smurf1, if any, during osteoblast differentiation in vivo is ill defined. Here, we show that Smurf1 prevents osteoblast differentiation by decreasing Runx2 accumulation in osteoblasts. Remarkably, mice harboring a substitution mutation at serine 148 (S148 in Smurf1 that prevents its phosphorylation by AMPK (Smurf1ki/ki display a premature osteoblast differentiation phenotype that is equally severe as that of Smurf1−/− mice, as well as a high bone mass, and are also hyperinsulinemic and hypoglycemic. Consistent with the fact that Smurf1 targets the insulin receptor for degradation, there is, in Smurf1ki/ki mice, an increase in insulin signaling in osteoblasts that triggers a rise in the circulating levels of osteocalcin, a hormone that favors insulin secretion. These results identify Smurf1 as a determinant of osteoblast differentiation during the development of bone formation and glucose homeostasis post-natally and demonstrate the necessity of S148 for these functions.

Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

Science.gov (United States)

Ma, Guang Xu; Zhou, Rong Qiong; Hu, Shi Jun; Huang, Han Cheng; Zhu, Tao; Xia, Qing You

2014-06-01

Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction. Copyright © 2014 Elsevier Inc. All rights reserved.

Measurement of Galaxy Cluster Integrated Comptonization and Mass Scaling Relations with the South Pole Telescope

Energy Technology Data Exchange (ETDEWEB)

Saliwanchik, B. R.; et al.

2015-01-22

We describe a method for measuring the integrated Comptonization (Y (SZ)) of clusters of galaxies from measurements of the Sunyaev-Zel'dovich (SZ) effect in multiple frequency bands and use this method to characterize a sample of galaxy clusters detected in the South Pole Telescope (SPT) data. We use a Markov Chain Monte Carlo method to fit a β-model source profile and integrate Y (SZ) within an angular aperture on the sky. In simulated observations of an SPT-like survey that include cosmic microwave background anisotropy, point sources, and atmospheric and instrumental noise at typical SPT-SZ survey levels, we show that we can accurately recover β-model parameters for inputted clusters. We measure Y (SZ) for simulated semi-analytic clusters and find that Y (SZ) is most accurately determined in an angular aperture comparable to the SPT beam size. We demonstrate the utility of this method to measure Y (SZ) and to constrain mass scaling relations using X-ray mass estimates for a sample of 18 galaxy clusters from the SPT-SZ survey. Measuring Y (SZ) within a 0.'75 radius aperture, we find an intrinsic log-normal scatter of 21% ± 11% in Y (SZ) at a fixed mass. Measuring Y (SZ) within a 0.3 Mpc projected radius (equivalent to 0.'75 at the survey median redshift z = 0.6), we find a scatter of 26% ± 9%. Prior to this study, the SPT observable found to have the lowest scatter with mass was cluster detection significance. We demonstrate, from both simulations and SPT observed clusters that Y (SZ) measured within an aperture comparable to the SPT beam size is equivalent, in terms of scatter with cluster mass, to SPT cluster detection significance.

VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum.

Science.gov (United States)

Croxatto, Antony; Chalker, Victoria J; Lauritz, Johan; Jass, Jana; Hardman, Andrea; Williams, Paul; Cámara, Miguel; Milton, Debra L

2002-03-01

Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum Delta vanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the Delta vanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an L-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum Delta vanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

Why Sustainers Should Care About the Targeting Process

Science.gov (United States)

2016-02-29

1200 T: Recon GOLD / 13B SP TBD AWT/ AVN T: SPT Convoy GOLD / 13B SP 1315 / 1415 T: SPT Convoy Turani SP 1115 T: SPT Convoy GOLD / 13B SP 1315 / 1415 T...on the lookout for a white Ford observed). Legend A/DACG = Arrival/departure airfield control group AWT/ AVN = Air weapons teams/aviation BSA

A sandwiched piezoelectric transducer with flex end-caps for energy harvesting in large force environments

International Nuclear Information System (INIS)

Kuang, Yang; Daniels, Alice; Zhu, Meiling

2017-01-01

This paper presents a sandwiched piezoelectric transducer (SPT) for energy harvesting in large force environments with increased load capacity and electric power output. The SPT uses (1) flex end-caps to amplify the applied load force so as to increase its power output and (2) a sandwiched piezoelectric-substrate structure to reduce the stress concentration in the piezoelectric material so as to increase the load capacity. A coupled piezoelectric-circuit finite element model (CPC-FEM) was developed, which is able to directly predict the electric power output of the SPT connected to a load resistor. The CPC-FEM was used to study the effects of various parameters of the SPT on the performance to obtain an optimal design. These parameters included the substrate thickness, the end-cap material and thickness, the electrode length, the joint length, the end-cap internal angle and the PZT thickness. A prototype with optimised parameters was tested on a loading machine, and the experimental results were compared with simulation. A good agreement was observed between simulation and experiment. When subjected to a 1 kN 2 Hz sinusoidal force applied by the loading machine, the SPT produced an average power of 4.68 mW. The application of the SPT as a footwear energy harvester was demonstrated by fitting the SPT into a boot and performing the tests on a treadmill, and the SPT generated an average power of 2.5 mW at a walking speed of 4.8 km h −1 . (paper)

Computational Power of Symmetry-Protected Topological Phases.

Science.gov (United States)

Stephen, David T; Wang, Dong-Sheng; Prakash, Abhishodh; Wei, Tzu-Chieh; Raussendorf, Robert

2017-07-07

We consider ground states of quantum spin chains with symmetry-protected topological (SPT) order as resources for measurement-based quantum computation (MBQC). We show that, for a wide range of SPT phases, the computational power of ground states is uniform throughout each phase. This computational power, defined as the Lie group of executable gates in MBQC, is determined by the same algebraic information that labels the SPT phase itself. We prove that these Lie groups always contain a full set of single-qubit gates, thereby affirming the long-standing conjecture that general SPT phases can serve as computationally useful phases of matter.

Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications.

Science.gov (United States)

Yang, Zhang-Min; Yu, Hui; Liu, Zhen-Zhen; Pei, Jian-Zhu; Yang, Yu-E; Yan, Su-Xian; Zhang, Cui; Zhao, Wen-Long; Wang, Zhe-Zhi; Wang, Ying-Ming; Tsai, Inn-Ho

2017-07-05

Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. Full sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and

Isolation, cDNA Cloning, and Structure-based Functional Characterization of Oryctin, a Hemolymph Protein from the Coconut Rhinoceros Beetle, Oryctes rhinoceros, as a Novel Serine Protease Inhibitor*

Science.gov (United States)

Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru

2010-01-01

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant 13C,15N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with Ki values of 3.9 × 10−10 m, 6.2 × 10−10 m, 1.4 × 10−9 m, and 1.2 × 10−8 m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections. PMID:20630859

Flexibility of cold- and heat-adapted subtilisin-like serine proteinases evaluated with fluorescence quenching and molecular dynamics

DEFF Research Database (Denmark)

Sigtryggsdóttir, Asta Rós; Papaleo, Elena; Thorbjarnardóttir, Sigríður H.

2014-01-01

activity of cold adapted enzymes when compared to homologues from thermophiles, reflects their higher molecular flexibility. To assess a potential difference in molecular flexibility between the two homologous proteinases, we have measured their Trp fluorescence quenching by acrylamide at different......The subtilisin-like serine proteinases, VPR, from a psychrotrophic Vibrio species and aqualysin I (AQUI) from the thermophile Thermus aquaticus, are structural homologues, but differ significantly with respect to stability and catalytic properties. It has been postulated that the higher catalytic...... to Trp (Y191W). A lower quenching effect of acrylamide on the intrinsic fluorescence of the thermophilic AQUI_Y191W was observed at all temperatures measured (10-55°C), suggesting that it possesses a more rigid structure than VPR. The MD analysis (Cα rmsf profiles) showed that even though VPR and AQUI...

Purification and characterization of a serine protease (CESP) from mature coconut endosperm

Science.gov (United States)

Panicker, Leelamma M; Usha, Rajamma; Roy, Samir; Mandal, Chhabinath

2009-01-01

Background In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm. Findings We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. Studies relating to substrate specificity and pattern of inhibition by various protease inhibitors indicated that CESP is a serine protease with cleavage specificity to peptide bonds after arginine. Purified CESP was often autolysed to two polypeptides of 41.6 kDa (CESP1) and 26.7 kDa (CESP2) and is confirmed by immunochemistry. We have shown the expression of CESP in all varieties of coconut and in all stages of coconut endosperm development with maximum amount in fully matured coconut. Conclusion Since the involvement of proteases in the processing of pre-proteins and maintenance of intracellular protein levels in seeds are well known, we suspect this CESP might play an important role in the coconut endosperm development. However this need to be confirmed using further studies. PMID:19426537

Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

Science.gov (United States)

Ishida, Ken; Kuboshima, Megumi; Morita, Hiroto; Maeda, Hiroshi; Okamoto, Ayako; Takeuchi, Michio; Yamagata, Youhei

2014-01-01

Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

[The role of the basophil activation test (BAT) in qualification for specific immunotherapy with inhalant allergens].

Science.gov (United States)

Bulanda, Małgorzata; Dyga, Wojciech; Rusinek, Barbara; Czarnobilska, Ewa

Qualification for specific immunotherapy (SIT) according to the guidelines of the European Academy of Allergy and Clinical Immunology (EAACI) includes medical history, skin prik tests (SPT) and/or measuring the concentration of sIgE. It is necessary to perform additional diagnostic tests in case of discrepancies between the history and the results of SPT/sIgE or differences between SPT and sIgE. Basophil activation test (BAT) assesses the expression of activation markers of these cells, eg. CD63 and CD203c after stimulation. The aim of our study was to evaluate the usefulness of BAT in the qualification for the SIT in comparison to the SPT and sIgE and in case of discrepancies between the results of SPT and sIgE. The study included 30 patients with allergic rhinitis (AR) caused by allergy to house dust mite (Dermatophagoides pteronyssinus, Dp) or birch pollen qualified for SIT. All patients had SPT, sIgE and BAT determination. The group of patients with allergy to birch was a control group for Dp allergic and vice versa. BAT with CD63 antigen expression was performed using a Flow2CAST test. Basophils were stimulated with allergen preparation (50, 500, and 5000 SBU/ml concentrations). BAT results were expressed as a stimulation index (SI). For optimal concentrations of 50 and 500 SBU/ml parameters comparing BAT to SPT and sIgE as the gold standards were consecutively: sensitivity 82-100% and 93-100%, specificity 50-94% and 47-89%, positive predictive value 65- 94% and 61-87%, negative predictive value 86-100% and 93-100%. Correlation BAT - SPT and BAT - sIgE ranged within 0.59 to 0.84 and 0.51 to 0.72. BAT was helpful in 2 of 30 patients with incompatible results of SPT and sIgE. Optimal concentrations for basophil stimulation are 50 and 500 SBU/ ml. BAT may be useful diagnostic tool in the qualification for the SIT in case of discrepancies between the results of SPT and sIgE.

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate.

Science.gov (United States)

Pillai, Priya; Archana, G

2008-03-01

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45-50 degrees C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65 degrees C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation.

Science.gov (United States)

Kurat, Christoph F; Lambert, Jean-Philippe; Petschnigg, Julia; Friesen, Helena; Pawson, Tony; Rosebrock, Adam; Gingras, Anne-Claude; Fillingham, Jeffrey; Andrews, Brenda

2014-09-30

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase-specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APC(Cdh1)) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation.

Deficient symbol processing in Alzheimer disease.

Science.gov (United States)

Toepper, Max; Steuwe, Carolin; Beblo, Thomas; Bauer, Eva; Boedeker, Sebastian; Thomas, Christine; Markowitsch, Hans J; Driessen, Martin; Sammer, Gebhard

2014-01-01

Symbols and signs have been suggested to improve the orientation of patients suffering from Alzheimer disease (AD). However, there are hardly any studies that confirm whether AD patients benefit from signs or symbols and which symbol characteristics might improve or impede their symbol comprehension. To address these issues, 30 AD patients and 30 matched healthy controls performed a symbol processing task (SPT) with 4 different item categories. A repeated-measures analysis of variance was run to identify impact of different item categories on performance accuracy in both the experimental groups. Moreover, SPT scores were correlated with neuropsychological test scores in a broad range of other cognitive domains. Finally, diagnostic accuracy of the SPT was calculated by a receiver-operating characteristic curve analysis. Results revealed a global symbol processing dysfunction in AD that was associated with semantic memory and executive deficits. Moreover, AD patients showed a disproportional performance decline at SPT items with visual distraction. Finally, the SPT total score showed high sensitivity and specificity in differentiating between AD patients and healthy controls. The present findings suggest that specific symbol features impede symbol processing in AD and argue for a diagnostic benefit of the SPT in neuropsychological assessment.

Plant Outage Time Savings Provided by Subcritical Physics Testing at Vogtle Unit 2

International Nuclear Information System (INIS)

Cupp, Philip; Heibel, M.D.

2006-01-01

The most recent core reload design verification physics testing done at Southern Nuclear Company's (SNC) Vogtle Unit 2, performed prior to initial power operations in operating cycle 12, was successfully completed while the reactor was at least 1% ΔK/K subcritical. The testing program used was the first application of the Subcritical Physics Testing (SPT) program developed by the Westinghouse Electric Company LLC. The SPT program centers on the application of the Westinghouse Subcritical Rod Worth Measurement (SRWM) methodology that was developed in cooperation with the Vogtle Reactor Engineering staff. The SRWM methodology received U. S. Nuclear Regulatory Commission (NRC) approval in August of 2005. The first application of the SPT program occurred at Vogtle Unit 2 in October of 2005. The results of the core design verification measurements obtained during the SPT program demonstrated excellent agreement with prediction, demonstrating that the predicted core characteristics were in excellent agreement with the actual operating characteristics of the core. This paper presents an overview of the SPT Program used at Vogtle Unit 2 during operating cycle 12, and a discussion of the critical path outage time savings the SPT program is capable of providing. (authors)

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation

Science.gov (United States)

Kurat, Christoph F.; Lambert, Jean-Philippe; Petschnigg, Julia; Friesen, Helena; Pawson, Tony; Rosebrock, Adam; Gingras, Anne-Claude; Fillingham, Jeffrey; Andrews, Brenda

2014-01-01

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase–specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APCCdh1) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation. PMID:25228766

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis

DEFF Research Database (Denmark)

Skjødt, Mikkel-Ole; Palarasah, Yaseelan; Rasmussen, Karina Juhl

2010-01-01

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intest...... protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway....

Moessbauer studies of the structural phase transitions in RbFeF4

International Nuclear Information System (INIS)

Baumeler, Hp.; Keller, H.; Kuendig, W.; Savic, I.M.; Wanklyn, B.M.

1984-01-01

RbFeF 4 exhibits two structural phase transitions (SPT), namely a first-order transition at 381 K and a second-order transition at 417 K. A detailed 57 Fe Moessbauer investigation of these SPT is presented. At pronounced discontinuities in the quadrupole splitting and the recoil-free fraction are observed, whereas at 417 K both quantities vary continuously with temperature. Both SPT are also seen in the area ratio of the single crystal quadrupole lines which reflects the tilting of the FeF 6 octahedra with respect to the c-axis. However, no noticeable indication of the SPT is found in the center shift. (Auth.)

Moessbauer studies of the structural phase transitions in RbFeF/sub 4/

Energy Technology Data Exchange (ETDEWEB)

Baumeler, Hp.; Keller, H.; Kuendig, W.; Savic, I.M.; Wanklyn, B.M.

1984-03-20

RbFeF/sub 4/ exhibits two structural phase transitions (SPT), namely a first-order transition at 381 K and a second-order transition at 417 K. A detailed /sup 57/Fe Moessbauer investigation of these SPT is presented. At pronounced discontinuities in the quadrupole splitting and the recoil-free fraction are observed, whereas at 417 K both quantities vary continuously with temperature. Both SPT are also seen in the area ratio of the single crystal quadrupole lines which reflects the tilting of the FeF/sub 6/ octahedra with respect to the c-axis. However, no noticeable indication of the SPT is found in the center shift.

Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

Directory of Open Access Journals (Sweden)

Aysha Kamran

2015-06-01

Full Text Available Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2+, Co2+, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80 and other commercial detergents (SDS, Triton X-100.

A Case Study On Pile Relaxation In Dilative Silts

Science.gov (United States)

2011-12-20

2 Table 2-1 Correlation of Uncorrected SPT N -Value with Total Unit Weight and Friction Angle (Kulhawy and Mayne, 1990...Effective internal friction angle. N = Uncorrected SPT blow count. σ’v = Effective vertical stress. cn = Overburden correction factor...shear strength of the soil. The unit weight of a soil can be determined from correlations to SPT N values, as shown in Table 2.1. 10

Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

Science.gov (United States)

Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

2014-09-15

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A Comparison of Maps and Power Spectra Determined from South Pole Telescope and Planck Data

Energy Technology Data Exchange (ETDEWEB)

Hou, Z.; Aylor, K.; Benson, B. A.; Bleem, L. E.; Carlstrom, J. E.; Chang, C. L.; Cho, H-M.; Chown, R.; Crawford, T. M.; Crites, A. T.; de Haan, T.; Dobbs, M. A.; Everett, W. B.; Follin, B.; George, E. M.; Halverson, N. W.; Harrington, N. L.; Holder, G. P.; Holzapfel, W. L.; Hrubes, J. D.; Keisler, R.; Knox, L.; Lee, A. T.; Leitch, E. M.; Luong-Van, D.; Marrone, D. P.; McMahon, J. J.; Meyer, S. S.; Millea, M.; Mocanu, L. M.; Mohr, J. J.; Natoli, T.; Omori, Y.; Padin, S.; Pryke, C.; Reichardt, C. L.; Ruhl, J. E.; Sayre, J. T.; Schaffer, K. K.; Shirokoff, E.; Staniszewski, Z.; Stark, A. A.; Story, K. T.; Vanderlinde, K.; Vieira, J. D.; Williamson, R.

2018-01-17

We study the consistency of 150 GHz data from the South Pole Telescope (SPT) and 143 GHz data from the \\textit{Planck} satellite over the 2540 $\\text{deg}^2$ patch of sky covered by the SPT-SZ survey. We first visually compare the maps and find that the map residuals appear consistent with noise after we account for differences in angular resolution and filtering. To make a more quantitative comparison, we calculate (1) the cross-spectrum between two independent halves of SPT 150 GHz data, (2) the cross-spectrum between two independent halves of \\textit{Planck} 143 GHz data, and (3) the cross-spectrum between SPT 150 GHz and \\textit{Planck} 143 GHz data. We find the three cross-spectra are well-fit (PTE = 0.30) by the null hypothesis in which both experiments have measured the same sky map up to a single free parameter characterizing the relative calibration between the two. As a by-product of this analysis, we improve the calibration of SPT data by nearly an order of magnitude, from 2.6\\% to 0.3\\% in power; the best-fit power calibration factor relative to the most recent published SPT calibration is $1.0174 \\pm 0.0033$. Finally, we compare all three cross-spectra to the full-sky \\textit{Planck} $143 \\times 143$ power spectrum and find a hint ($\\sim$1.5$\\sigma$) for differences in the power spectrum of the SPT-SZ footprint and the full-sky power spectrum, which we model and fit as a power law in the spectrum. The best-fit value of this tilt is consistent between the three cross-spectra in the SPT-SZ footprint, implying that the source of this tilt---assuming it is real---is a sample variance fluctuation in the SPT-SZ region relative to the full sky. Despite the precision of our tests, we find no evidence for systematic errors in either data set. The consistency of cosmological parameters derived from these datasets is discussed in a companion paper.

A novel nonionic surfactant- and solvent-stable alkaline serine protease from Serratia sp. SYBC H with duckweed as nitrogen source: production, purification, characteristics and application.

Science.gov (United States)

Li, G Y; Cai, Y J; Liao, X R; Yin, J

2011-07-01

A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn(2+) and Na(+) (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn(2+) enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.

Human eosinophils constitutively express a unique serine protease, PRSS33.

Science.gov (United States)

Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

2017-07-01

Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities.

Science.gov (United States)

Witschel, Matthias C; Rottmann, Matthias; Schwab, Anatol; Leartsakulpanich, Ubolsree; Chitnumsub, Penchit; Seet, Michael; Tonazzi, Sandro; Schwertz, Geoffrey; Stelzer, Frank; Mietzner, Thomas; McNamara, Case; Thater, Frank; Freymond, Céline; Jaruwat, Aritsara; Pinthong, Chatchadaporn; Riangrungroj, Pinpunya; Oufir, Mouhssin; Hamburger, Matthias; Mäser, Pascal; Sanz-Alonso, Laura M; Charman, Susan; Wittlin, Sergio; Yuthavong, Yongyuth; Chaiyen, Pimchai; Diederich, François

2015-04-09

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Robust model-based analysis of single-particle tracking experiments with Spot-On

Science.gov (United States)

Grimm, Jonathan B; Lavis, Luke D

2018-01-01

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce ‘Spot-On’, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants. PMID:29300163

Skin prick test in patients with chronic allergic skin disorders

Directory of Open Access Journals (Sweden)

Pooja Bains

2015-01-01

Full Text Available Background: Chronic allergic skin disorders are the inflammatory and proliferative conditions in which both genetic and environmental factors play important roles. Chronic idiopathic urticaria (CIU and atopic dermatitis (AD are among the most common chronic allergic skin disorders. These can be provoked by various food and aeroallergens. Skin prick tests (SPTs represent the cheapest and most effective method to diagnose type I hypersensitivity. Positive skin tests with a history suggestive of clinical sensitivity strongly incriminate the allergen as a contributor to the disease process. Aim s and Objectives: To determine the incidence of positive SPT in patients with chronic allergic skin disorders and to identify the various allergens implicated in positive SPT. Methods: Fifty patients of chronic allergic disorders were recruited in this study. They were evaluated by SPT with both food and aeroallergens. Results: In our study, SPT positivity in patients of CIU was 63.41% and in AD was 77.78%. Out of the 41 patients of CIU, the most common allergen groups showing SPT positivity were dust and pollen, each comprising 26.83% patients. SPT reaction was positive with food items (21.6%, insects (17.07%, fungus (12.20%, and Dermatophagoides farinae, that is, house dust mite (HDM (7.32%. The allergen which showed maximum positivity was grain dust wheat (19.51%. Among nine patients of AD, maximum SPT positivity was seen with Dermatophagoides farinae, pollen Amaranthus spinosus, grain dust wheat, and cotton mill dust; each comprising 22.22% of patients. Conclusion: Our study showed that a significant number of patients of CIU and AD showed sensitivity to dust, pollen, insects, Dermatophagoides farinae, and fungi on SPT. Thus, it is an important tool in the diagnosis of CIU and AD.

The role of Ca2+ and Mg2+ in the cytotoxic T lymphocyte reaction and in the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-serine esterase by human T cell clones

NARCIS (Netherlands)

Blanchard, D.; Aubry, J. P.; de Vries, J. E.; Spits, H.

1989-01-01

Human T cell clones contain enzymes that can cleave the substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). All CTL clones tested in this study secreted BLT-serine esterase activity, whereas only one of three tested non-cytolytic T cell clones secreted this enzymatic activity upon

Empirical Comparison of Two Psychological Therapies: Self Psychology and Cognitive Orientation in the Treatment of Anorexia and Bulimia

Science.gov (United States)

Bachar, Eytan; Latzer, Yael; Kreitler, Shulamit; Berry, Elliot M.

1999-01-01

The authors investigated the applicability of self psychological treatment (SPT) and cognitive orientation treatment (COT) to the treatment of anorexia and bulimia. Thirty-three patients participated in this study. The bulimic patients (n = 25) were randomly assigned either to SPT, COT, or control/nutritional counseling only (C/NC). The anorexic patients (n = 8) were randomly assigned to either SPT or COT. Patients were administered a battery of outcome measures assessing eating disorders symptomatology, attitudes toward food, self structure, and general psychiatric symptoms. After SPT, significant improvement was observed. After COT, slight but nonsignificant improvement was observed. After C/NC, almost no changes could be detected.(The Journal of Psychotherapy Practice and Research 1999; 8:115–128) PMID:10079459

A comparative study of the adsorption and oxidation of L-alanine and L-serine on Au(1 0 0), Au(1 1 1) and gold thin film electrodes in acid media

International Nuclear Information System (INIS)

Sandoval, Andrea P.; Orts, José Manuel; Rodes, Antonio; Feliu, Juan M.

2013-01-01

The adsorption and oxidation of L-alanine and L-serine at Au(1 1 1) and Au(1 0 0) single crystal and evaporated thin-film electrodes with preferential (1 1 1) orientation was studied in perchloric acid solutions. For this purpose, cyclic voltammetry experiments were combined with external reflection infrared spectroscopy (gold single crystals) and surface-enhanced infrared reflection–absorption spectroscopy under attenuated total reflection conditions (ATR-SEIRAS) (gold thin films). In addition, theoretical harmonic vibrational frequencies, obtained from B3LYP/LANL2DZ, 6-31+G(d) calculations for the zwitterionic species adsorbed on Au clusters with (1 1 1) orientation, were used to interpret the experimental spectra. The optimized geometry obtained from DFT calculations for the corresponding zwitterion plus a water molecule, under the application of an external electric field of 0.01 a.u. corresponds to a bidentate asymmetrical bridge adsorption configuration. The absence of an adsorbate band for the asymmetric OCO stretching in the experimental infrared spectra confirms the bidentate bonding of the adsorbed zwitterion through the oxygen atoms of the carboxylate group irrespective of the crystallographic orientation of the electrode surface, the adsorbate coverage and the electrode potential. In addition to typical interfacial water bands associated to perchlorate anions, which are co-adsorbed in order to compensate the positive charge of the ammonium group, the ATR-SEIRA spectra also show bands around 2950 cm −1 that can be related to the formation of hydrogen bonds between interfacial water and the ammonium group of the adsorbed zwitterion. The voltammetric experiments have shown that, as in the case of platinum electrodes, L-serine oxidizes at lower potentials than L-alanine. Under these conditions, the in situ infrared experiments show the formation of carbon dioxide and adsorbed cyanide as oxidation products of L-serine. In the case of L-alanine, only

Gas-breakdown effects associated with the self-pinched transport of intense light-ion beams

International Nuclear Information System (INIS)

Ottinger, P.F.; Olson, C.L.; Welch, D.R.; Oliver, B.V.

1997-01-01

Self-pinched transport (SPT) of intense light-ion beams is being considered for delivering energy to a high-gain, high-yield inertial confinement fusion target. Proton beam SPT experiments are underway on the Gamble II generators at the Naval Research Laboratory. The physics of SPT in low-pressure gas is being analyzed with analytic theory and numerical simulations. A 1-D theory estimates the net current fraction necessary for stable transport as a function of gas density for a given beam profile. SPT simulations using the 3-D hybrid particle-in-cell (PIC) code IPROP determine the beam profile. Important to both theory and simulations is the inclusion of gas-breakdown physics. A comparison between the theory and the self-consistent simulations using IPROP is made. Additional SPT simulations have been carried out using the 2-D hybrid PIC code SOLENZ which assumes a pre-ionized plasma. This simulation model enables the investigation of long time scale beam propagation issues. A comparison between IPROP and SOLENZ will be presented. SOLENZ simulations with the Gamble I beam parameters demonstrate SPT but point to the need to study the injection conditions to improve beam confinement. Simulations examining beam-to-wall distance and injection conditions will be presented

Lupin sensitization and clinical allergy in food allergic children in Norway.

Science.gov (United States)

Lindvik, Helene; Holden, Lise; Løvik, Martinus; Cvancarova, Milada; Halvorsen, Ragnhild

2008-01-01

The aim of the present pilot study was to investigate to what extent children in Norway sensitized to lupin had clinical lupin allergy, and to compare sensitization to lupin with sensitization to other legumes. Thirty-five children with food allergy referred to a national referral hospital were evaluated with skin prick test (SPT) and analysis of serum-specific IgE to lupin, peanut, pea and soy. The children with positive SPTs to lupin were offered oral food challenges with lupin flour. Fifteen children (43%) had positive SPT and 17 children (49%) had serum-specific IgE to lupin. Ten SPT-positive children underwent oral food challenges and one experienced an allergic reaction to lupin flour. This child was one of six challenged children with IgE antibodies to peanut >15 kU(A)/L. There was a strong relationship between positive SPT to lupin flour and positive SPT to soy and between positive SPT to lupin and specific IgE to soy, pea and peanut. Children with sensitization to lupin are not likely to have a clinical lupin allergy. Avoidance of lupin on the basis of lupin sensitization or peanut allergy would lead to unnecessarily strict diets. Food challenge is currently necessary to diagnose lupin allergy.

Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

Science.gov (United States)

Vivas, Jeilyn; Ibarra, Carlos; Salazar, Ana M; Neves-Ferreira, Ana G C; Sánchez, Elda E; Perales, Jonás; Rodríguez-Acosta, Alexis; Guerrero, Belsy

2016-01-01

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of autologous serum skin test and skin prick test reactivity to house dust mite in patients with chronic spontaneous urticaria.

Directory of Open Access Journals (Sweden)

Zhiqiang Song

Full Text Available BACKGROUND: Chronic spontaneous urticaria (CSU is a common skin disorder with etiology that is not well understood. METHODS: In this study, we evaluated the prevalence of autologous serum skin test (ASST and skin prick testing (SPT to house dust mite (HDM in 862 CSU cases in China. Clinical features, courses and treatment responses were also recorded. RESULTS: The prevalence of positive ASST was 46.3%, and patients aged 30-39 years had the highest positive rate (52.1%. Positive SPT to HDM was seen in 153 patients (17.7% with the highest positive rate (34.2% in patients aged 20 or less. Patients with positive ASST had higher urticaria activity scores (UAS (4.18±0.65 vs. 3.67±0.53 but lower positive rates of HDM (24.6% vs. 37.6%, as compared with those with negative ASST (odds ratio (OR 1.84, 95% CI 1.38-2.47. Patients could be categorized into four groups based on the results of ASST and SPT to HDM and patients with positive ASST and positive SPT to HDM had the highest disease activity scores, experienced higher frequencies of angioedema, diseases duration, and required higher dosage of loratadine every month, compared with other subgroups (P<0.0001. CONCLUSIONS: Patients with CSU showed varied responses of positive ASST and varied sensitivity to HDM, Patients with positive ASST and/or positive SPT had more disease activity compared with patients with negative ASST and/or negative SPT. Further classification can be made based on the result of SPT and ASST.

Circulating levels of mannan-binding lectin (MBL) and MBL-associated serine protease 2 in endemic pemphigus foliaceus

DEFF Research Database (Denmark)

Messias-Reason, I; Bosco, DG; Nisihara, RM

2008-01-01

Endemic pemphigus foliaceus (EPF) is an autoimmune disease, which occurs in Brazil and other regions of South America. Mannose-binding lectin (MBL) and MBL-associated serine protease (MASP-2) play a key role in innate immunity, and its deficiency has been related to increased susceptibility...... to infection and autoimmune diseases. MBL and MASP-2 serum levels were measured in 114 patients with EPF and in 100 healthy individuals in Brazil. MBL and MASP-2 levels were measured by sandwich assays (time-resolved immunofluorimetic assay) using monoclonal antibodies. No difference was observed in the MBL...... level in patients with EPF compared with controls [mean +/- SEM 1230.07 +/- 132.18 ng/mL (median 789.0 ng/mL) vs. 1036.98 +/- 117.99 ng/mL (median 559.5 ng/mL), P = 0.32]. Non-significant lower MASP-2 levels were observed in EPF [274.34 +/- 15.66 ng/mL (median 239.5 ng/mL ) vs. 304.72 +/- 15.28 ng...

AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

Science.gov (United States)

Xu, Feng; Previti, Mary Lou; Nieman, Marvin T; Davis, Judianne; Schmaier, Alvin H; Van Nostrand, William E

2009-04-29

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

Science.gov (United States)

Molloy, M P; Andrews, P C

2001-11-15

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.

The Mycobacterium tuberculosis transcriptional repressor EthR is negatively regulated by Serine/Threonine phosphorylation.

Science.gov (United States)

Leiba, Jade; Carrère-Kremer, Séverine; Blondiaux, Nicolas; Dimala, Martin Moune; Wohlkönig, Alexandre; Baulard, Alain; Kremer, Laurent; Molle, Virginie

2014-04-18

Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb. Copyright © 2014 Elsevier Inc. All rights reserved.

A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility

DEFF Research Database (Denmark)

Lund, F. W.; Wustner, D.

2013-01-01

Single particle tracking (SPT) is becoming a standard method to extract transport parameters from time-lapse image sequences of fluorescent vesicles in living cells. Another method to obtain these data is temporal image correlation spectroscopy (TICS), but this method is less often used for measu......Single particle tracking (SPT) is becoming a standard method to extract transport parameters from time-lapse image sequences of fluorescent vesicles in living cells. Another method to obtain these data is temporal image correlation spectroscopy (TICS), but this method is less often used...... for measurement of intracellular vesicle transport. Here, we present an extensive comparison of SPT and TICS. First we examine the effect of photobleaching, shading and noise on SPT and TICS analysis using simulated image sequences. To this end, we developed a simple photophysical model, which relates spatially...... varying illumination intensity to the bleaching propensity and fluorescence intensity of the moving particles. We found that neither SPT nor TICS are affected by photobleaching per se, but the transport parameters obtained by both methods are sensitive to the signal-to-noise ratio. In addition, the number...

[Clinical investigation of basophil activation test as a complementary test for house dust mite allergen].

Science.gov (United States)

Ren, H L; Li, J D; Miao, Y H; Xu, T

2018-03-01

Objective: To investigate the clinical application of glass micro fiber basophil activation test (BAT) used as a complementary test for house dust mite allergen. Method: Forty patients with clinical diagnosed allergic rhinitis was test by three methods for house dust mite allergen, skin prick test(SPT),Immuno CAP sIgE, and BAT in vitro. The sensitivity and specificity of glass micro fiber were accessed, and the consistency between BAT, SPT, and Immuno sIgE was analyzed. As in vivo provocation was not performed, gold standard is regarded as the combination of medical history and positive reports of SPT and/or ImmunoCAP sIgE test. Result: Twentythree patients are diagnosed as house dust mite allergic rhinitis by gold standard. The sensitivity and specificity of glass micro fiber BAT were 60.9% and 88.2%, the sensitivity of SPT and sIgE was 87.0% and sIgE 73.9%. The correlation rates between BAT with SPT is 0.67( P house dust mite allergic rhinitis, BAT have a good consistency with SPT and sIgE, while as it has only moderate consistency with "gold standard", further studies are needed to prove its clinical significance. Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.

Cosmological data and indications for new physics

Energy Technology Data Exchange (ETDEWEB)

Benetti, Micol [Physics Department and ICRA, Università di Roma ' ' La Sapienza' ' , Ple. Aldo Moro 2, 00185, Rome (Italy); Gerbino, Martina; Melchiorri, Alessandro; Pagano, Luca [Physics Department and INFN, Università di Roma ' ' La Sapienza' ' , Ple Aldo Moro 2, 00185, Rome (Italy); Kinney, William H. [Department of Physics, University at Buffalo, the State University of New York, Buffalo, NY 14260-1500 (United States); Kolb, Edward W. [Department of Astronomy and Astrophysics, Enrico Fermi Institute, and Kavli Institute for Cosmological Physics, University of Chicago, Chicago, Illinois 60637-1433 (United States); Lattanzi, Massimiliano [Dipartimento di Fisica e Science della Terra, Università di Ferrara and INFN, sezione di Ferrara, Polo Scientifico e Tecnologico - Edificio C Via Saragat, 1, I-44122 Ferrara Italy (Italy); Riotto, Antonio, E-mail: micol.benetti@roma1.infn.it, E-mail: martina.gerbino@roma1.infn.it, E-mail: whkinney@buffalo.edu, E-mail: Rocky.Kolb@uchicago.edu, E-mail: lattanzi@fe.infn.it, E-mail: alessandro.melchiorri@roma1.infn.it, E-mail: luca.pagano@roma1.infn.it, E-mail: antonio.riotto@unige.ch [Department of Theoretical Physics and Center for Astroparticle Physics (CAP) 24 quai E. Ansermet, CH-1211 Geneva 4 (Switzerland)

2013-10-01

Data from the Atacama Cosmology Telescope (ACT) and the South Pole Telescope (SPT), combined with the nine-year data release from the WMAP satellite, provide very precise measurements of the cosmic microwave background (CMB) angular anisotropies down to very small angular scales. Augmented with measurements from Baryonic Acoustic Oscillations surveys and determinations of the Hubble constant, we investigate whether there are indications for new physics beyond a Harrison-Zel'dovich model for primordial perturbations and the standard number of relativistic degrees of freedom at primordial recombination. All combinations of datasets point to physics beyond the minimal Harrison-Zel'dovich model in the form of either a scalar spectral index different from unity or additional relativistic degrees of freedom at recombination (e.g., additional light neutrinos). Beyond that, the extended datasets including either ACT or SPT provide very different indications: while the extended-ACT (eACT) dataset is perfectly consistent with the predictions of standard slow-roll inflation, the extended-SPT (eSPT) dataset prefers a non-power-law scalar spectral index with a very large variation with scale of the spectral index. Both eACT and eSPT favor additional light degrees of freedom on top of the Harrison-Zel'dovich model. eACT is consistent with zero neutrino masses, while eSPT favors nonzero neutrino masses at more than 95% confidence.