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A J Turner

Publications and source records attributed to A J Turner.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme.

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Amino Acid Sequence↗
PubMed2000

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P.

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Amino Acid Sequence↗
PubMed2000

A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase.

A novel human zinc metalloprotease that has considerable homology to human angiotensin-converting enzyme (ACE) (40% identity and 61% similarity) has been identified. This metalloprotease (angiotensin-converting enzyme homolog (ACEH)) contains a single HEXXH zinc-binding domain and conserves other critical residues typical of the ACE family. The predicted protein sequence consists of 805 amino acids, including a potential 17-amino acid N-terminal signal peptide sequence and a putative C-terminal membrane anchor. Expression in Chinese hamster ovary cells of a soluble, truncated form of ACEH, lacking the transmembrane and cytosolic domains, produces a glycoprotein of 120 kDa, which is able to cleave angiotensin I and angiotensin II but not bradykinin or Hip-His-Leu. In the hydrolysis of the angiotensins, ACEH functions exclusively as a carboxypeptidase. ACEH activity is inhibited by EDTA but not by classical ACE inhibitors such as captopril, lisinopril, or enalaprilat. Identification of the genomic sequence of ACEH has shown that the ACEH gene contains 18 exons, of which several have considerable size similarity with the first 17 exons of human ACE. The gene maps to chromosomal location Xp22. Northern blotting analysis has shown that the ACEH mRNA transcript is approximately 3. 4 kilobase pairs and is most highly expressed in testis, kidney, and heart. This is the first report of a mammalian homolog of ACE and has implications for our understanding of cardiovascular and renal function.

Amino Acid Sequence↗
PubMed2000

The neprilysin family in health and disease.

The mammalian neprilysin (NEP) family comprises at least seven members: NEP itself, Kell blood group antigen (KELL), the endothelin-converting enzymes (ECE-1 and ECE-2), the enzyme PEX, associated with X-linked hypophosphataemia, "X-converting enzyme" (XCE) a CNS-expressed orphan peptidase and a soluble, secreted endopeptidase (SEP). These zinc metallopeptidases are all type II integral membrane proteins. Where identified, these enzymes have roles in the processing or metabolism of regulatory peptides and therefore represent potential therapeutic targets. A distinct feature of ECE-1 species is their existence as distinct isoforms differing in their N-terminal cytoplasmic tails. These tails play a role in enzyme targeting and turnover with di-leucine and tyrosine-based motifs affecting localization. Additional anchorage of these enzymes can also occur through palmitoylation. Bacterial homologues of the neprilysin family exist, for example the products of the pepO genes from L. lactis and S. parasanguis, and a recently described gene product of P. gingivalis which is an ECE-1 homologue that can catalyse the conversion of big endothelin to endothelin. A genomics based approach to understanding the functions of this proteinase family is aided by the completion of the C. elegans and Drosophila genomes, both of which encode multiple copies of NEP-like enzymes.

Acylation↗
PubMed2000

The role of proteolysis in Alzheimer's disease.

Alzheimer's disease is characterised by the progressive deposition of the 4 kDa beta-amyloid peptide (A beta) in extracellular senile plaques in the brain. A beta is derived by proteolytic cleavage of the amyloid precursor protein (APP) by various proteinases termed secretases. alpha-Secretase is inhibited by hydroxamate-based zinc metalloproteinase inhibitors such as batimastat with I50 values in the low micromolar range, and displays many properties in common with the secretase that releases angiotensin converting enzyme. A cell impermeant biotinylated derivative of one such inhibitor completely blocked the release of APP from the surface of neuronal cells, indicating that alpha-secretase cleaves APP at the cell-surface. A range of hydroxamate-based compounds have been used to distinguish between alpha-secretase and tumour necrosis factor-alpha convertase, a member of the ADAMs (a disintegrin and metalloproteinase-like) family of zinc metalloproteinases. Recent data suggests that the presenilins may be aspartyl proteinases with the specificity of gamma-secretase. Although APP and the presenilins are present in detergent-insoluble, cholesterol- and glycosphingolipid-rich lipid rafts, they do not behave as typical lipid raft proteins, and thus it is unclear whether these membrane domains are the sites for proteolytic processing of APP.

ADAM Proteins↗
PubMed2000

A prospective study of a quantitative PCR ELISA assay for the diagnosis of CMV pneumonia in lung and heart-transplant recipients.

BACKGROUND: Qualitative polymerase chain reaction (PCR) for the identification of cytomegalovirus (CMV) infection has a low predictive value for the identification of CMV pneumonia. This study prospectively evaluated the application of a quantitative PCR Enzyme-Linked Immuno-Sorbent Assay (ELISA) assay in 9 lung- and 18 heart-transplant recipients who did not receive ganciclovir prophylaxis. METHODS: DNA was collected from peripheral blood polymorphonuclear leucocytes (PMNL) posttransplantation. Oligonucleotide primers for the glycoprotein B gene (149 bp) were used in a PCR ELISA assay using an internal standard for quantitation. CMV disease was defined as histological evidence of end organ damage. RESULTS: The median level CMV genome equivalents in patients with CMV disease was 2665/2 x 10(5) PMNL (range 1,200 to 61,606) compared to 100 x 10(5) PMNL (range 20 to 855) with infection but no CMV disease (p = 0.036). All patients with CMV disease had genome equivalents levels of >1200/2 x 10(5) PMNL. A cut-off level of 1,200 PMNL had a positive predictive value for CMV disease of 100% and a negative predictive value of 100%. The first detection of levels of CMV genome equivalents above a level of 1200/2 x 10(5) PMNL was at a median of 58 days (range 47 to 147) posttransplant. CONCLUSIONS: Quantitative PCR assays for the diagnosis of CMV infection may predict patients at risk of CMV disease and thereby direct preemptive treatment to high-risk patients.

Antigens, Viral↗
PubMed2000

Respiratory viral infections in primary immune deficiencies: significance and relevance to clinical outcome in a single BMT unit.

Respiratory viral infections are major causes of morbidity and mortality in children with SCID and other primary immunodeficiencies who require BMT. Twenty-two of 73 (30%) such children were admitted with respiratory viral infections, of whom 13/22 (59%) died. All viruses were detected in nasopharyngeal aspirate (NPA). Virus was only found in BAL in those with LRTI. Eleven of 22 (50%) had paramyxovirus infections, all with severe viral pneumonitis which worsened post BMT. Five of 11 (45.5%) survived overall. All 11 received aerosolised ribavirin; five of 11 received additional inhaled immunoglobulin and corticosteroid. Three of 5 (60%) survived compared with two of six (33.3%) not thus treated. Three of 22 (13.6%) had adenoviruses; one died of disseminated disease, including pneumonia despite intravenous ribavirin. Eleven patients had rhinovirus detected; nine of 11 (82%) were asymptomatic or coryzal and survived. Two patients with additional severe lung pathologies had LRT rhinovirus and died. All patients received intravenous immunoglobulin. No treatments resulted in viral clearance without successful T cell engraftment. Respiratory viruses, particularly paramyxoviruses and adenoviruses are common, significant pathogens in these patients, significantly worsening outcome of BMT. NPA is an ideal specimen for diagnosis and monitoring of infection. Aggressive treatments may reduce viral replication and damage. Nebulised immunoglobulin and corticosteroid in LRTI may improve respiratory function and outcome.

Adenoviridae Infections↗
PubMed2000

What does it mean to be a donor offspring? The identity experiences of adults conceived by donor insemination and the implications for counselling and therapy.

In the absence of research with adult donor offspring, this study begins to bridge that gap by asking individuals about their experiences as donor offspring and considering the implications for psychotherapeutic and counselling practice. Sixteen participants (13 male, three female, age range 26-55 years) recruited through donor insemination support networks in the UK, USA, Canada and Australia, were sent semi-structured questionnaires by E-mail and post. Using identity process theory as a framework for understanding participants' accounts, the data were qualitatively analysed using interpretative phenomenological analysis. Participants consistently reported mistrust within the family, negative distinctiveness, lack of genetic continuity, frustration in being thwarted in the search for their biological fathers and a need to talk to a significant other (i.e. someone who would understand). These experiences could be postulated as being indicative of a struggle to assimilate, accommodate and evaluate information about their new identities as donor offspring. Psychotherapists and counsellors need to be aware of these identity issues if they are to meet the needs of donor offspring within therapeutic practice.

Adult↗
PubMed2000

Functional significance of the isoforms of endothelin-converting enzyme-1.

The subcellular localization of endothelin-converting enzyme-1 (ECE-1) is a matter of some controversy, further complicated by the discovery of its multiple isoforms. ECE-1 is a critical enzyme in the biosynthesis of the potent vasoconstrictor peptide endothelin (ET), and, as such, represents a potential target for drug therapy in the control of disease states involving the ET system. Knowledge of the precise locations of the isoforms and their regulation would aid in the design of drugs to target specifically ECE-1. In this study, the subcellular localization and potential targeting pathways of the ECE-1 isoforms were investigated. Antipeptide antibodies were raised to the unique N-terminal sequence of ECE-1b and were then used in the investigation of its subcellular distribution. Mutagenesis of proposed targeting sequences within the cytoplasmic tails of the isoforms was carried out to determine their significance in subcellular localization.

Animals↗
PubMed2000

Protein processing mechanisms: from angiotensin-converting enzyme to Alzheimer's disease.

Angiotensin-converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein are two examples of membrane-bound proteins that are released in a soluble form by a post-translational proteolytic cleavage event involving a secretase. Site-specific antibodies and matrix-assisted laser desorption ionization-time-of-flight ('MALDI-TOF') MS have been used to map the secretase cleavage site in somatic ACE to Arg-1203/Ser-1204, 24 residues proximal to the membrane-anchoring domain. Trypsin, which can solubilize ACE from the membrane, cleaves the protein at the same site. The use of structurally related hydroxamic acid-based zinc metalloproteinase inhibitors indicate that tumour necrosis factor-alpha convertase, a member of the ADAMs ('a disintegrin and metalloproteinase') family of proteins, is not involved in the proteolytic release of ACE, or in the constitutive or regulated alpha-secretase release of the amyloid precursor protein from a human neuronal cell line.

ADAM Proteins↗
PubMed2000

Serological diagnosis of syphilis. PHLS Syphilis Serology Working Group.

The availability of an increasing number of enzyme immunoassays (EIAs) for detecting syphilis antibodies makes it appropriate to review approaches to syphilis serology and to assess the role of syphilis EIAs in routine diagnostic microbiology laboratories. This paper summarises the principles and practice of syphilis serology and provides recommendations on the use of laboratory tests for syphilis in UK diagnostic microbiology laboratories. The main recommendations are summarised in a testing algorithm. Treponemal EIAs are an appropriate alternative to the use of combined Venereal Disease Research Laboratories/rapid plasma reagin and Treponema pallidum haemagglutination assay (TPHA) tests for screening for syphilis. If a treponemal EIA is used for screening an alternative treponemal test, such as TPHA, should be used for confirmatory testing. The fluorescent treponemal antibody-absorbed test is probably best reserved for specimens giving discrepant results. Such specimens may be referred to the PHLS laboratories that provide confirmatory treponemal testing for reference testing and to facilitate collection of surveillance data on what remains an important public health problem.

Algorithms↗
PubMed2000

Ultrasound measurement of biparietal diameter and umbilical artery blood flow in the normal fetal guinea pig.

BACKGROUND AND PURPOSE: Measurement of the biparietal diameter (BPD) by use of B-mode ultrasound provides a useful means for assessment of gestational age and brain growth in humans during pregnancy. Recording of flow velocity waveforms from the umbilical artery, using Doppler ultrasound, is used to assess development of the fetal placental circulation. We sought to measure these ultrasound parameters during normal pregnancy in the guinea pig and develop normative data. METHODS: Measurements of BPD were made on 205 fetuses of various gestational ages; 114 fetuses had 2 or more serial studies performed (total n = 474). RESULTS: BPD increased from 0.806cm at 22 to 26 days, to 1.922cm at term (69 days), (y = -0.00043x2 + 0.06881x - 0.75941, with an r value of 0.995, where x = days' gestation, y = biparietal diameter [cm]). Umbilical artery flow velocity waveform resistance index (RI) decreased as gestation advanced (y = -0.012x + 1.294 with an r value of 0.887, where x = days gestation, y = RI) reflecting expansion of the placental vascular bed. CONCLUSIONS: It is possible to use ultrasound to study pregnancy in the guinea pig. The BPD may be used to estimate gestational age. Resistance to blood flow in the placenta may be assessed using the RI derived from the umbilical artery flow velocity waveform.

Animals↗
PubMed1999

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein.

Lipid rafts are regions of the plasma membrane that are enriched in cholesterol, glycosphingolipids and acylated proteins, and which have been proposed as sites for the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Lipid rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C, with the resulting low-density, detergent-insoluble glycolipid-enriched fraction (DIG) being isolated by flotation through a sucrose density gradient. The detergent-insolubility of APP in mouse cerebral cortex relative to a variety of DIG marker proteins (alkaline phosphatase, flotillin, F3 protein and prion protein) and non-DIG proteins (alkaline phosphodiesterase I, aminopeptidase A and clathrin) has been examined. Alkaline phosphatase, flotillin, F3 protein and the prion protein were present exclusively in the DIG region of the sucrose gradient over a range of protein/detergent ratios used to solubilize the membranes and displayed a characteristic enrichment in the low-density fraction as the protein/detergent ratio was decreased. In contrast, most of the APP, alkaline phosphodiesterase I, aminopeptidase A and clathrin was effectively solubilized at all of the protein/detergent ratios examined. However, a minor proportion of these latter proteins was detected in DIGs at levels which remained constant irrespective of the protein/detergent ratio. When DIGs were isolated from the sucrose gradients and treated with excess Triton X-100, both the DIG marker proteins and APP, alkaline phosphodiesterase I and clathrin were predominantly resistant to detergent extraction at 37 degrees C. These results show that, although a minor proportion of APP is present in DIGs, where it is detergent-insoluble even at 37 degrees C, it behaves as an atypical lipid raft protein and raises questions as to whether lipid rafts are a site for its proteolytic processing.

Alkaline Phosphatase↗
PubMed1999

Sequencing, expression and biochemical characterization of the Porphyromonas gingivalis pepO gene encoding a protein homologous to human endothelin-converting enzyme.

We have determined the nucleotide sequence of the clone pAL2 obtained from Porphyromonas gingivalis 381 in the previous study [Ansai et al. (1995) Microbiology 141, 2047-20521. The DNA sequence analysis of this fragment revealed one complete ORF and one incomplete ORF. The ORF encoded a protein (PgPepO) of 690 amino acids with a calculated molecular weight of 78796. The deduced amino acid sequence exhibited a significant homology with human endothelin-converting enzyme (ECE)-1. Recombinant PgPepO was purified to homogeneity and characterized. The purified enzyme was strongly inhibited by phosphoramidon, and converted big endothelin-1 to endothelin-1. Furthermore, the purified PgPepO strongly cross-reacted with a monoclonal antibody against rat ECE-1. These results indicate that PgPepO has striking similarity to mammalian ECE in structure and function.

Amino Acid Sequence↗
PubMed1999

Cleavage of Alzheimer's amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells.

The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.

Alzheimer Disease↗
PubMed1999

Endothelin converting enzyme is located on alpha-actin filaments in smooth muscle cells.

OBJECTIVE: Endothelin converting enzyme is the key enzyme in the generation of endothelin-1 from big-endothelin-1. The mature endothelin-1 is a potent vasoconstrictor which also promotes mitogenesis and proliferation of smooth muscle cells. The objectives were to demonstrate in smooth muscle cells the presence of a phosphoramidon-sensitive endothelin converting enzyme activity, reveal the subcellular localization of the enzyme protein and determine the effects of the metalloproteinase inhibitor, phosphoramidon, the lysosomotrophic drug, chloroquine, and colchicine on the cycling pathway of the enzyme. METHODS: Subcellular localization of endothelin converting enzyme on human smooth muscle cells and the rat cell line, A7r5, was by immunofluorescence and confocal microscopy or by biotinylation of cell cultures and immunoblotting, after treatment of cell cultures with cytochalasin D, colchicine, chloroquine and phosphoramidon. Converting enzyme activity was determined by high performance liquid chromatographic assay. RESULTS: We detected phosphoramidon-sensitive endothelin converting enzyme activity in smooth muscle cells. In addition to its plasma membrane location, for the first time we revealed a striking co-localization of endothelin converting enzyme and alpha-actin filaments in smooth muscle cells. Colchicine treatment results in a perinuclear accumulation of endothelin converting enzyme. An increased level of endothelin converting enzyme protein was shown to be present in smooth muscle cells which had been grown in the presence of phosphoramidon or chloroquine. CONCLUSION: The 120 kDa endothelin converting enzyme co-localizes with alpha-actin in smooth muscle cells and resembles that found in endothelial cells in that it is present on both the plasma membrane and intracellularly.

Actins↗
PubMed1999

The development of a quantitative PCR ELISA to determine HCMV DNAaemia levels in heart, heart/lung and lung transplant recipients.

The adaptation of the polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) using a co-amplified DNA standard to quantitate the human cytomegalovirus (HCMV) glycoprotein B (gB) gene in clinical samples is described. The PCR ELISA is a solution hybridisation system with colorimetric end stage detection of amplicons. A DNA internal standard (IS) was designed by replacing a probe sequence used currently within the gB region with a heterogeneous sequence, allowing co-amplification with the same oligonucleotide primer sets and differentiation by probe hybridisation. Two DNA fragments homologous to the gB and IS sequences were generated and used for co-amplification studies to construct a standard curve. From this the copy number of the gB gene present in clinical samples could be interpolated. Co-amplification with 1000 IS copies allowed quantitation of 10-1000000 gB DNA copies in a single PCR. This assay was validated subsequently using blood samples tested by the HCMV antigenaemia assay and showed a trend of increasing HCMV DNAaemia with increasing antigenaemia levels. This rapid assay avoids using gel electrophoresis and cumbersome quantitative systems. It has the potential for early identification of patients at high risk of developing HCMV disease, and for therapeutic monitoring.

Base Sequence↗