Tris-HC1 pH Research Articles

PCR for the reliable detection of M. paratuberculosis in gut samples from sheep with paucimicrobial enteritis requires ribolysation of the tissue lysate

G4540 PCR FOR THE RELIABLE DETECTION OF M.PARATUBERCULOSIS IN GUT SAMPLES FROM SHEEP WITH PAUCIMICROBIAL ENTERITIS REQUIRES RIBOLYSATION OF THE TISSUE LYSATE. A. Verstocken, D. Winterbourne, J.Ford, C.Clarke, J.Hermon-Taylor. Department of Surgery, St. George's Hospital Medical School, London. U.K. M.paratuberculosis (Map) is a pathogen which causes chronic enteritis in many different animals including primates. The animal disease ranges from pluribacillary to paucimicrobial like leprosy in humans. In paucimicrobial paratuberculosis in sheep there is florid granulomatous inflammation of the intestine but Map may not be seen microscoPically or isolated by laboratory culture, and IS900 PCR for Map is frequently negative. Preliminary tests on culturable strains of bacillary-form Map from mature liquid cultures showed that boiling in detergent or chaotropic agents for 30 min, or treatment with SDS proteinase K, methods which reliably release DNA from other bacteria, were suboptimal for Map. Access to Map DNA was improved by exposure of these cultured organisms to vibration at 6.5 m/sec for 45 sec in a mixed slurry of silica and ceramic particles using Hybaid Ribolyser blue (HRB) tubes. Tests on 20-40mg samples of fresh frozen inflamed intestine from 2 sheep with paucimicrobial Map enteritis showed that tissue dissolution was best achieved by incubation in HRB tubes for 24 hours at 37°C in 800~tl lysis buffer TEN (2raM EDTA, 400mM NaCI, 10mM Tris HC1 pH 8.0) SDS 0.6% containing 20~ag proteinase K (Qiagen). After centrifugation of the lysate at 13,000 rpm for 10 rain, the supematant was removed, the pellet was ribolysed and DNA was isolated from both fractions by phenol chloroform extraction. IS900 PCR was consistently positive on both fractions. Serial dilutions, however, showed that the ribolysed pellet contained 1-2 logs more Map DNA than the supernatant suggesting that a majority of the organisms had remained intact. These findings show that digestion with SDS proteinase K alone is insufficient for assured access to Map DNA in the form that this organism exists within the intestine of sheep suffering from paucimicrobial chronic granulomatous enteritis. Reliable detection of these robust pathogens by PCR requires physical disruption by ribolysation.

HLA-DR Types in Rheumatic, Congenital and Degenerative Cardiac Valve Diseases in Saudi Patients

Subjects of Saudi origin were DNA typed for HLA-DR2, DR4 and DRw53 by amplification fragment-length polymorphism and amplification by sequence-specific primer techniques based on polymerase chain reaction. Of these subjects, 125 had sporadic heart valve disease (96 with rheumatic heart disease, 18 with degenerate and 11 with congenital degenerate valve disease) and 77 were healthy Saudi blood donors. While the frequency of individuals typed DR4 was about the same in the rheumatic heart disease as in the control category (30% versus 23%, respectively), it was found to be higher (55%; P<0.02), but below the level of marginal significance after correcting for the number of DR types, in the congenital degenerate valve category. No preferential association of any DR4 subtype could be detected. The incidence of DR2 was lower in the congenital cases compared to that in the controls (9% versus 21%) and remained about the same in the rheumatic heart disease patients as in the controls. The frequency of DRw53 in the degenerate valve categories was slightly lower than that in the controls, but the difference was not significant. The study failed to corroborate the association between HLA-DR4 and rheumatic heart disease shown in previous studies using the serotyping approach.

Analysis of the physical state of human papillomavirus type-16 in early cervical intraepithelial neoplasia

Cervical cancer is one of the most common malignant diseases in women world-wide. Infection with certain types of Human Papillomavirus (HPV) in particular HPV-16 and other related types (HPV-18, -31 and -33 ) appears to be an important etiologic factor in the development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. HPV infections causing clinically apparent lesions of the lower genital tract seem to occur more frequently during pregnancy and show a high regression rate after delivery. Schneider et al [ I ] reported the detection of HPV DNA in exfoliated cervical cells in 28% of a pregnant population compared with 12.5% for nonpregnant control patients; a higher detection level occurred in samples obtained later in pregnancy. Our previous studies [2] have shown a high rate of transmission and persistence of HPV16 and 18 from mothers to their infants. Concentrating on those mothers that were HPV16 DNA positive in groups that did and did not transmit viral DNA we investigated the physical state of the virus (integrated or episomal ) in order to determine whether this was a significant factor in the vertical transmission of HPV-16 DNA. Mothers were selected on the basis of having current or previous abnormal cervical cytology in order to increase the chance of HPV detection. It is believed that integration of HPV DNA into cellular DNA usually results in a disruption or a deletion in the E l or E2 open reading frames (ORFs) (Fig. 1.). For detection of integration two specific primers from the E2 ORF of the HPV-16 genome between nucleotides 2734 and 3872 were used in a polymerase chain reaction [3]. Primer E2-1 (5'AGGACGAGGACAAGGAAAA-3') was located between nucleotides 2734-2753 and primer E2-2 (5 ' GGATGCAGTATCAAGATTTG-3') between nucleotides 38533872. A lack of amplification of the 1139bp product using these E2-specific primers would indicate integration of the viral DNA. PCR amplification was carried out in a lOOul reaction mix containing luM of each primer, 50 mM KCI, 10 mM Tris-HC1 pH 8.3, 2.5 mM MgC12, 200 uM of each dNTP and 2 units of Taq DNA polymerase. Amplification was done for 40 cycles in an Omnigene Thermal Cycler (Hybaid).

Electrophoresis of human serum proteins following acute myocardial infarction.

Conference Article| August 01 1994 Electrophoresis of human serum proteins following acute myocardial infarction THOMAS MARSHALL; THOMAS MARSHALL 1School of Health Sciences, Fleming Building, The University of Sunderland, Sunderland SRI 3SD, UK Search for other works by this author on: This Site PubMed Google Scholar JOHN WILLIAMS; JOHN WILLIAMS *Biochemistry Department, General Hospital, Sligo, Co. Sligo, Eire Search for other works by this author on: This Site PubMed Google Scholar KATHERINE M. WILLIAMS KATHERINE M. WILLIAMS 1School of Health Sciences, Fleming Building, The University of Sunderland, Sunderland SRI 3SD, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1994) 22 (3): 312S. https://doi.org/10.1042/bst022312s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation THOMAS MARSHALL, JOHN WILLIAMS, KATHERINE M. WILLIAMS; Electrophoresis of human serum proteins following acute myocardial infarction. Biochem Soc Trans 1 August 1994; 22 (3): 312S. doi: https://doi.org/10.1042/bst022312s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: AMI, acute myocardial infarction, CK, creatine kinase, SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 2-DE, two-dimensional electrophoresis This content is only available as a PDF. © 1994 Biochemical Society1994 Article PDF first page preview Close Modal You do not currently have access to this content.

Hybridisation techniques on gridded high density DNA and in situ colony filters based on fluorescence detection.

Hybridisation techniques on gridded high density DNA and in situ colony filters based on fluorescence detection.

Mitogen-activated protein (MAP) kinase stimulation by phorbol esters and external load in the isolated perfused heart.

Mitogen-activated protein kinases are a family of serine/ threonine kinases that have been implicated in a wide variety of cellular processes [for review see 1,2]. These kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate ribosomal protein S6 and various transcription factors. In response to most growthpromoting and mitogenic factors tested, as well as to many other signals such as phorbol esters, MAP kinase phosphorylation is increased on both threonine and tyrosine residues, leading to stimulation of its kinase activity [3-51. Phosphorylation of MAP kinase involves an activator which is itself a kinase, with dual specificity for both tyrosine and serindthreonine [6]. Recent reports suggest that MAP kinase is involved in the signalling events leading to heart hypertrophy [7,8]. To evaluate this hypothesis further, we have investigated the effects of hypertrophic agents on the activity of MAP kinase in isolated perfused heart preparations. As hypertrophic agents, TPA and high load were used as they are both known to induce responses characteristic of hypertrophy in rat heart preparations. Hearts were perfused retrogradely with Krebs-Henseleit saline (1 19 mM NaCI, 25 mM NaHCO,, 4.7 mM KCl, 2.5 mM CaCI,, 1.2 mM MgSO,, 1.2 mM KH,PO, and 10 mM glucose, equilibrated with 95% 45% C02, pH 7.6 at 37°C). For control experiments, perfusions were carried out at for 15 min at 5.5 kPa. After that, hearts were either exposed to TPA or high aortic pressure for a further 5 min period. When present, TPA was added in the perfusion medium at a final concentration of 1pM. For the experiments at high aortic pressure, the perfusion pressure was increased to 16.3 kPa (120 mm Hg). Hearts were extracted with 20mM j3-glycerophosphate pH 7.5, containing 20mM NaF, 2mM EDTA, 0.2mM Na,VO,, lOmM benzamidine, 25 pg/ml leupeptin, 50 pglml PMSF and 0.3% (v/v)J?-mercaptoethanol and centrifuged for lOmin at 1oooOxg. The supernatant was applied to a Mono Q FPLC column equilibrated with 50 mM Tris-HC1 pH 7.5, containing 2 mM EDTA, 2 mM EGTA, 0.1% Jmercaptoethanol, 5% glycerol, 0.03% Brij 35, 0.3 mM Na,VO,, ImM benzamidine and 4 pg/ml leupeptin. Myelin basic protein (MBP) kinase activity was eluted with linear gradient of NaCl (00.3 M) and measured as described previously [7]. FPLC of the heart extracts revealed two peaks of MBP kinase activity that were stimulated with exposure to TPA. The first peak eluted at 0.16 M NaCl and the activity was stimulated %fold compared with control. The second peak eluted at 0.22 M NaCl and the activity was stimulated 2.3-fold (Fig. 1). Extracts of hearts exposed to high aortic pressures showed an identical elution profile but stimulation was lower (2-fold for the first peak and 1.6-fold for the second peak) (Fig. 1). Using a kinase renaturation assay method [9], we examined the peak fractions from FPLC of TPA-treated hearts for their ability to phosphorylate MBP in gels in siru. We found that the molecular masses were 42kDa and 44kDa for the first and second peak respectively (Fig. 2a). The same method was used to examine the crude extracts of control and TPA-treated hearts. MBP kinase

HPLC ISOENZYME PATTERNS OF GLUTATHIONE TRANSFERASE FROM MARINE FISHES WITH DIFFERENT LEVELS OF POLLUTION

Conference Article| August 01 1991 HPLC ISOENZYME PATTERNS OF GLUTATHIONE TRANSFERASE FROM MARINE FISHES WITH DIFFERENT LEVELS OF POLLUTION Plácido Pascual; Plácido Pascual 1Department of Biochemistry and Molecular Biology, University of Córdoba, 14071–Córdoba, Spain Search for other works by this author on: This Site PubMed Google Scholar Esther Martínez-Lara; Esther Martínez-Lara 1Department of Biochemistry and Molecular Biology, University of Córdoba, 14071–Córdoba, Spain Search for other works by this author on: This Site PubMed Google Scholar Fermin Toribio; Fermin Toribio 1Department of Biochemistry and Molecular Biology, University of Córdoba, 14071–Córdoba, Spain Search for other works by this author on: This Site PubMed Google Scholar Juan López-Barea; Juan López-Barea 1Department of Biochemistry and Molecular Biology, University of Córdoba, 14071–Córdoba, Spain Search for other works by this author on: This Site PubMed Google Scholar J. Antonio Bárcena J. Antonio Bárcena 1Department of Biochemistry and Molecular Biology, University of Córdoba, 14071–Córdoba, Spain Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (3): 302S. https://doi.org/10.1042/bst019302s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Plácido Pascual, Esther Martínez-Lara, Fermin Toribio, Juan López-Barea, J. Antonio Bárcena; HPLC ISOENZYME PATTERNS OF GLUTATHIONE TRANSFERASE FROM MARINE FISHES WITH DIFFERENT LEVELS OF POLLUTION. Biochem Soc Trans 1 August 1991; 19 (3): 302S. doi: https://doi.org/10.1042/bst019302s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

Characterization of polysomes from Xenopus liver synthesizing vitellogenin and translation of vitellogenin and albumin messenger RNA's in vitro.

1. Conditions are described for the isolation of polysomes from the liver of Xenopus laevis. The method involves homogenization of liver in 0.2 M Tris-HCl pH 8.5, treatment with 2% Triton X-100 and subsequent sucrose density gradient fractionation of polysomes from a 10000 X g supernatant. 2. Vitellogenin synthesis was induced in male Xenopus liver by oestradiol treatment. Polysomes were isolated and vitellogenin-synthesizing polysomes characterized by their association with monospecific 125 I-labelled rabbit anti-vitellogenin antibody and by reaction with rabbit anti-vitellogenin immunoglobulins followed by indirect immunoprecipitation with goat anti-rabbit antibody. 3. Changes in liver polysome content following oestrogen treatment of male Xenopus are correlated with the appearance of vitellogenin synthesis using an organ culture assay. 4. RNA extracted from livers of oestradiol-treated male Xenopus and from purified polysomes is shown to code for the synthesis of vitellogenin-specific immunoprecipitable polypeptides in a rabbit reticulocyte cell-free protein-synthesizing system, a major component having a molecular weight of 210000. Xanopus liver RNA is also shown to code for the synthesis of an albumin-specific immunoprecipitable polypeptide of 74000 molecular-weight which coelectrophoresed with Xenopus albumin.