Characterisation of the cinnamyl alcohol dehydrogenase from helicobacter pylori

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the conversion of p-hydroxy-cinnamaldehydes to their corresponding alcohols leading to the biosynthesis of lignin in plants. Outside of plants their role is less well defined. The cinnamyl alcohol dehydrogenase from H. pylori (HpCAD) has been cloned and produced in E. coli and characterised for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterised plant enzymes. The best alcohol substrate was cinnamyl alcohol with a kcat/Km value of 126 S-1.m-1. The kcat/Km values for coniferyl alcohol and benzyl alcohol were more than an order of magnitude lower. Aliphatic alcohols were poorer substrates with kcat/Km values 10-fold or more lower than the aromatic alcohols. The kcat/Km values for aldehydes were higher than those for alcohols. Of the aromatic aldehydes, cinamylaldehyde was the best substrate followed by benzaldehyde. Acetaldehyde had a 10-fold lower kcat/Km value than cimmanylaldehyde. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The degree of high substrate inhibition was dependent on the substrate employed, for example aliphatic alcohols exhibited less pronounced inhibition than aromatics alcohols. No form of inhibition was evident even at 200 mM ethanol, while high substrate inhibition became apparent at 50 mM for both propanol and butanol. In contrast, all of the aromatic aldehyde substrates employed produced inhibition in the micro molar range; above 250 uM benzaldehyde, 150 uM cinnamaldehyde and 100 uM coniferyl aldehyde. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not previously been shown for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell. An isogenic HpCAD negative mutant of H. pylori was also generated. The growth of the mutant both on plates and in liquid cultures was slower than the wild type. The growth of the mutant was further inhibited on exposure to mildly acidic conditions (pH 6.5 and 6.0)

Development and Progress of Ireland's Biobank Network: Ethical, Legal, and Social Implications (ELSI), Standardized Documentation, Sample and Data Release, and International Perspective

0info:eu-repo/semantics/publishe

Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples

info:eu-repo/semantics/publishe

Characterization of Cinnamyl Alcohol Dehydrogenase of Helicobacter Pylori. An Aldehyde Dismutating Enzyme

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p-hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell

Maintaining breast cancer specimen integrity and individual or simultaneous extraction of quality dna, rna, and proteins from allprotect-stabilized and nonstabilized tissue samples

The Saint James\u27s Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep (R) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy (R) and QIAamp (R) (both Qiagen), and (4) to examine the effectiveness of Allprotect (R) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software (R)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2 ng/mu L and 1.86, respectively, and using AllPrep were 23.2 ng/mu L and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/mu L and 8.16, respectively, and with AllPrep were 74.8 ng/mu L and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P = 0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4 degrees C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates

What GDPR and the Health Research Regulations (HRRs) mean for Ireland: a research perspective

Background: Irish Health Research Regulations (HRRs) were introduced following the European Union (EU) General Data Protection Regulation (GDPR) in 2018. The HRRs described specific supplementary regulatory requirements for research regarding governance, processes and procedure that impact on several facets of research. The numerous problems that the HRRs and particularly "explicit consent" inadvertently created were presented under the auspices of the Irish Academy of Medical Sciences (IAMS) on November 25, 2019, at the Royal College of Surgeons in Ireland. Aims: The objective of this review was to obtain feedback and to examine the impact of GDPR and the HRRs on health research in Ireland in order to determine whether the preliminary feedback, presented at the IAMS meetings, was reflected at a national level. Methods: Individuals from the research community were invited to provide feedback on the impact, if any, of the HRRs on health research. Retrospective patient recruitment and consent outside a hospital setting for a multi-institutional Breast Predict study (funded by the Irish Cancer Society) were also analysed. Results: Feedback replicated the issues presented at the IAMS with additional concerns identified. Only 20% of the original target population (n = 1987) could be included in the Breast Predict study. Conclusions: Our results confirm that the HRRs have had a significantly negative impact on health research in Ireland. Urgent meaningful engagement between patient advocate groups, the research community and legislators would help ameliorate these impacts.</p

Development and progress of Ireland's biobank network: Ethical, legal, and social implications (ELSI), Standardized documentation, sample and data release, and international perspective

Biobank Ireland Trust (BIT) was established in 2004 to promote and develop an Irish biobank network to benefit patients, researchers, industry, and the economy. The network commenced in 2008 with two hospital biobanks and currently consists of biobanks in the four main cancer hospitals in Ireland. The St. James's Hospital (SJH) Biobank coordinates the network. Procedures, based on ISBER and NCI guidelines, are standardized across the network. Policies and documents - Patient Consent Policy, Patient Information Sheet, Biobank Consent Form, Sample and Data Access Policy (SAP), and Sample Application Form have been agreed upon (after robust discussion) for use in each hospital. An optimum sequence for document preparation and submission for review is outlined. Once consensus is reached among the participating biobanks, the SJH biobank liaises with the Research and Ethics Committees, the Office of the Data Protection Commissioner, The National Cancer Registry (NCR), patient advocate groups, researchers, and other stakeholders. The NCR provides de-identified data from its database for researchers via unique biobank codes. ELSI issues discussed include the introduction of prospective consent across the network and the return of significant research results to patients. Only 4 of 363 patients opted to be re-contacted and re-consented on each occasion that their samples are included in a new project. It was decided, after multidisciplinary discussion, that results will not be returned to patients. The SAP is modeled on those of several international networks. Biobank Ireland is affiliated with international biobanking groups - Marble Arch International Working Group, ISBER, and ESBB. The Irish government continues to deliberate on how to fund and implement biobanking nationally. Meanwhile BIT uses every opportunity to promote awareness of the benefits of biobanking in events and in the media. Copyright © 2013, Mary Ann Liebert, Inc. 2013