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D J Clarke

Publications and source records attributed to D J Clarke.

At least 19 recordsLinked to original sources
PubMed1992

Characterisation of a human bilirubin UDP-glucuronosyltransferase stably expressed in hamster lung fibroblast cell cultures.

A cDNA encoding a human bilirubin UDP-glucuronosyltransferase has been isolated and stably expressed in Chinese hamster V79 lung fibroblast cell line. Western blotting of cell homogenates with anti-UGT antibody revealed a highly expressed protein of approx. 55.5 kDa in size. The expressed enzyme specifically catalysed the formation of bilirubin mono- and diglucuronides, and also catalysed the glucuronidation of two phenolic compounds, which are good substrates for other human UGT isoenzymes, at low rates.

Animals↗
PubMed1992

Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa.

The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated 1-naphthol, but not bilirubin or steroids, and displayed a pI of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.

Animals↗
PubMed1992

Isolation and characterisation of a new hepatic bilirubin UDP-glucuronosyltransferase. Absence from Gunn rat liver.

A novel UDP-glucuronosyltransferase that conjugates bilirubin IX alpha, bilirubin monoglucuronide and an arylalkanoic acid was purified to homogeneity from clofibrate treated Wistar rats. The enzyme displayed a subunit molecular mass of 54 kDa, a pI of 7.6 and was demonstrated to be N-glycosylated. Sequence analysis of peptides derived by endoproteinase Glu-C cleavage of the purified enzyme indicated that it was a new member of the recently identified UGT1 subfamily. Immunoblot analysis demonstrated that this enzyme was absent from Gunn rat liver. The molecular derivation of this enzyme and the lack of it in Gunn rats is discussed.

Amino Acid Sequence↗
PubMed1992

The properties of cultured fetal human and rat brain tissue and its use as grafts for the relief of the parkinsonian syndrome.

Primary cultures were derived from human fetal ventral mesencephalon and cerebral cortex at 7-11 weeks gestation, and from fetal rat mesencephalon and cortex at embryonic day 14-15. Immunohistochemical analysis of the mesencephalic cultures using antibodies to tyrosine hydroxylase (TH) showed between 0.1-0.5% of human cells to be TH positive and 0.1-1% of rat cells to be TH positive. HPLC analysis of extracts from the cultures showed that they had the ability to synthesise and store dopamine. Implantation of the cultured human and rat mesencephalic tissue into a 6-hydroxydopamine rat model of Parkinson's disease produced marked recovery from amphetamine induced rotational asymmetry in the recipient rats, but no such recovery was observed following implantation of cortical cultures. Histological examination demonstrated the presence of surviving human mesencephalic and cortical grafts at least 6 months after implantation. Implants of cultured fetal rat tissue were less obviously but still significantly effective in these experiments. These rat tissue grafts were detectable for periods of at least 6-8 weeks by histological staining.

Animals↗
PubMed1992

Differential expression and induction of UDP-glucuronosyltransferase isoforms in hepatic and extrahepatic tissues of a fish, Pleuronectes platessa: immunochemical and functional characterization.

Glucuronidation of three substrates prototypical for different UDP-glucuronosyltransferase (UDPGT) isoforms in hepatic, renal, intestinal, and branchial microsomes of corn oil, 3-methylcholanthrene, Aroclor 1254, and clofibrate-pretreated plaice was investigated. The differential expression of UDPGT in the four tissues clearly demonstrated for the first time that multiple isoforms with differing substrate specificities were present in fish. The liver was quantitatively the most important site for the glucuronidation of all three compounds studied. Phenol UDPGT activity was ubiquitous to all tissues and was induced by 3-methylcholanthrene and Aroclor 1254 in hepatic tissue and by Aroclor 1254 in renal tissue. The glucuronidation of testosterone was restricted to liver and intestinal tissue, while conjugation of bilirubin was expressed solely in hepatic tissue. The biotransformation of the endogenous compounds was not induced in the xenobiotic-treated animals. The presence of immunoreactive UDPGTs in the four tissues was demonstrated by immunoblot analysis using sheep anti-plaice UDPGT antibodies. Hepatic tissue displayed a range of immunoreactive polypeptides of 52 to 57 kDa, while a 55-kDa polypeptide was detected in extrahepatic tissues. An increased intensity of the latter polypeptide species was demonstrated in liver and kidney microsomes in which there was a concomitant induction of phenol UDPGT activity in xenobiotic-treated fish. The results indicate that the 55-kDa polypeptide was the major polyaromatic hydrocarbon-inducible UDPGT isoenzyme in hepatic and renal microsomes.

Animals↗
PubMed1992

ESCA investigation of low-temperature ammonia plasma-treated polyethylene substrate for immobilization of protein.

A low-temperature radiofrequency plasma excited in anhydrous ammonia was used to modify polyethylene substrate surfaces for covalent immobilization of proteins. Electron spectroscopy for chemical application (ESCA) was used for surface characterization of polyethylene to a depth scale of 7 nm. The data revealed that surface modification is extensive and occurs in seconds at low discharge power. Primary amino functionalities were detected on the polyethylene surface and the level is dependent on plasma parameters. 125I-labelled antibodies covalently attached to amino groups via glutaraldehyde allowed the conditions for optimum level of primary amine to be established. Both ESCA data and protein loadings are in excellent agreement.

Ammonia↗
PubMed1992

Functional and immunochemical comparison of hepatic UDP-glucuronosyltransferases in a piscine and a mammalian species.

1. The aglycone specificity of hepatic microsomal glucuronidation was compared under uniform conditions in a fish, Pleuronectes platessa and a mammal, Rattus norvegicus, representative of the most primitive and advanced vertebrate classes. 2. Both species exhibited comparable UDP-glucuronosyltransferase (UDPGT) activity towards planar phenolic substrates (1-naphthol, 4-nitrophenol); however, plaice activity towards bulky non-planar substrates such as (-)-morphine was either 200-fold lower, or for an arylacetic acid (RS-2-phenylpropionic acid) and an aryloxyacetic acid (clofibric acid) non-detectable. 3. Conjugation of the endogenous substrates, bilirubin and steroids were 4- to 40-fold lower in the plaice than in the rat. Whilst both species formed diglucuronides of the asymmetrical bilirubin IX alpha, they displayed a reciprocal preference for the initial esterification, conjugation of the C-8 side chain predominating in the rat and of C-12 in the fish. 4. Immunoblot analysis using two polyclonal antisera preparations raised against rat UDPGTs demonstrated the presence of multiple weakly cross-reacting polypeptides in fish microsomes indicative of multiple isoforms and conservation of common structural motifs over more than 350 million years since evolutionary divergence of the mammals.

Animals↗
PubMed1992

Mental impairment in the West Midlands.

The case records of 55 people from the West Midlands (UK) fulfilling the Mental Health Act 1983 criteria for mental impairment or severe mental impairment, were studied. Most were young men with mild mental retardation. 73 per cent were resident in (or on leave from) mental handicap hospitals, and 27 per cent resident in special hospitals. 29 per cent were subject to a Restriction Order. Most had lived in hospital for more than six years. The commonest problem behaviours were aggression, property offences and inappropriate or offending sexual behaviour. 31 per cent were mentally ill or had a past history of mental illness. A diverse range of services appears necessary to meet the needs of this group of people.

Adolescent↗
PubMed1991

Analysis of pH-induced population oscillations of Saccharomyces cerevisiae and Escherichia coli using photon correlation spectroscopy.

Following a recent successful application of p.c.s. to liquid chromatography in the biotechnology industry, its usefulness as a contamination monitor in the fermentation industry was assessed. It was found that: (i) the intensity bias of the technique limits its uses to the detection of contaminants when they are larger than the host; (ii) the inherent heterogeneity of microbial cultures prevents the use of multiangle studies, and (iii) the large size of bacteria make the use of p.c.s. in flowing, on-line systems impractical.

Absorptiometry, Photon↗
PubMed1991

Phenylketonuria and anorexia nervosa.

A girl with phenylketonuria and mild mental retardation developed a behaviour disorder and anorexia nervosa at the age of 14. The anorexic symptoms disappeared at the age of 20, but the behaviour disorder worsened, leading to compulsory detention in a psychiatric hospital. Serum phenylalanine levels may have influenced the course of the disorder.

Adult↗
PubMed1990

Electroacoustic production of murine hybridomas.

The optimal conditions for the production of murine hybridomas by electroacoustic fusion of cells in sugar solutions and of cells in ionic strength media (up to 115 mM NaCl) have been investigated. In the electroacoustic fusion technique cells were brought into contact in a 3 MHz ultrasonic standing wave field and were fused by application of an electric pulse. Hybridomas of murine splenocytes and X63-Ag8.653 mouse myelomas were successfully produced in low ionic strength mannitol solutions and in a range of salt concentrations up to 115 mM. The yield of hybridomas by electroacoustic fusion of cells in mannitol was at least as good as that obtained when cells were fused following conventional dielectrophoresis induced contact. The electroacoustic fusion yield was also comparable to conventional yields when cells were exposed to a pulse in higher ionic strength media where dielectrophoresis induces heating effects. Hybridomas were produced at similar electric field strengths when cells were suspended in high ionic strength media (up to 115 mM NaCl) or in mannitol. The effective electric field strength for hybridoma production was close to that at which trypan blue tests indicated membrane damage.

Animals↗
PubMed1990

Synaptic connections formed by grafts of different types of cholinergic neurons in the host hippocampus.

The present experiment was performed to determine whether different types of grafted central cholinergic neurons are able to form synaptic contacts with host hippocampal neurons. Grafts from the septal-diagonal band area, which contain the neurons that normally innervate the hippocampal formation, were compared to those from the nucleus basalis magnocellularis region (NBM), the striatum, the pontomesencephalic tegmentum of the brain stem, and the spinal cord. The regions were dissected from 14- to 16-day-old rat fetuses, and the same number of viable cells (35 x 10(4] from each of the different regions was stereotaxically injected as a cell suspension into the hippocampus of rats subjected to a complete fimbria-fornix lesion, transecting the intrinsic septohippocampal pathways. At 14 to 17 weeks after transplantation, the brains were processed for choline acetyltransferase (ChAT) immunocytochemistry at the light and electron microscopic levels and acetylcholinesterase (AChE) histochemistry at the light microscopic level. There was a great variation in the number of surviving ChAT-positive cells among the different graft types. The septal grafts contained the highest number of ChAT-positive cells, and the striatal grafts showed the lowest numbers. The NBM, brain stem, and spinal cord grafts were in between. The differences in the number of ChAT-positive neurons between the groups matched, in general, the differences found in the magnitude of graft-derived AChE-positive fiber growth into the host hippocampal formation. At the electron microscopical level, all types of grafts were capable of forming synaptic contacts with host elements, however, with vast differences in the number of synapses found. The septal grafts produced the highest number of contacts, whereas the striatal and spinal cord grafts produced very few contacts. The ultrastructure of the cholinergic fibers from grafts obtained from the forebrain areas, i.e., septum, NBM, and striatum all appeared normal, whereas brain stem and spinal cord grafts produced different types of anomalies. The results show that grafted cholinergic neurons, that normally do not innervate the hippocampus, can send axons and form synaptic contacts in the host hippocampus. The ability to reinnervate the denervated hippocampal target appears to be shared by the embryologically closely related forebrain cholinergic neuron types, i.e., the septal, NBM, and striatal neurons. The marked differences in overall fiber ingrowth and number of synapses observed between these different types of grafts could be explained largely on the basis of differences in survivability of each grafted neuron type. By contrast, the reinnervation obtained from the grafted brain stem and spinal cord neurons were both quantitatively and qualitatively abnormal.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗
PubMed1990

Microbial content of aerosols produced from suspensions exposed to megahertz frequency ultrasound.

A piezo-electric bowl transducer was used to generate aerosols by focusing ultrasound in the frequency range 1-7 MHz at a liquid/air interface. Atomization at the liquid surface and the production of a fountain contributed to aerosol formation. When the liquid consisted of suspensions of representatives from the viral, bacterial, and yeast groups of micro-organisms (covering a 0.2-11.5 microns size range) living organisms were isolated from the aerosols at all frequencies. The fountains were implicated as a major source of air-borne micro-organisms because significant numbers of isolates were obtained in the presence of fountains but in the absence of obvious atomization, and theoretical predictions make the sizes of droplets arising from atomization at the higher frequencies too small to have carried some of the larger organisms.

Aerosols↗
PubMed1990

Fetal striatal neurons grafted into the ibotenate lesioned adult striatum: efferent projections and synaptic contacts in the host globus pallidus.

The efferent projections to the host brain from intrastriatal grafts have been examined at the ultrastructural level. Cell suspensions of E14 rat fetal striatal tissue were implanted into the ibotenic acid lesioned caudate-putamen of adult rats. After survival times of at least five months, the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin was injected into the grafts. Consistent with our previous light microscopical analysis, anterogradely labelled fibres could be followed from the grafts into the host globus pallidus along the normal trajectory of the striatopallidal pathway in the internal capsule, and a few fibres also reached the entopeduncular nucleus and the substantia nigra. In the electron microscope, the graft-derived efferents were seen to be myelinated as they projected caudally along the fibre bundles of the internal capsule, and they were thus similar to the neostriatal projection in normal control animals. In the host globus pallidus, the graft-derived fibres ramified into a terminal network forming morphologically normal synaptic contacts with neuronal elements in the host globus pallidus. A total of 118 synaptic contacts were identified, all of them formed symmetric membrane specializations. The major postsynaptic targets were dendritic shafts (90%), with only a few contacts with spines or small shafts (8%) and perikarya (2%). The morphology of the synaptic contacts and their distribution on different postsynaptic targets were similar to that which was found in the globus pallidus after tracer-injections into the caudate-putamen of normal control animals. The results show that grafted fetal striatal neurons can grow along the myelinated territory of the internal capsule to reinstate a seemingly normal synaptic input to the previously denervated neurons in the host globus pallidus.

Animals↗