Biomedical subjects

D Doyle

Publications and source records attributed to D Doyle.

At least 37 records · Page 2Linked to original sources

PubMed1990

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

We have translated RNAs for the two rat asialoglycoprotein receptor polypeptides together in a cell-free system containing dog pancreatic microsomes and immunoprecipitated the products with antibodies that distinguish the two proteins. In this system the proteins oligomerize, as judged by their coprecipitation with either of the subunit-specific antisera. Oligomerization does not occur between subunits synthesized without microsomes or between subunits synthesized on separate microsomes mixed during detergent solubilization. Thus, oligomerization occurs within the microsomal membrane. We calculate that oligomerization proceeds with an efficiency of approximately 85%. The receptor complex appears to represent a specific oligomer because it excludes a third membrane glycoprotein synthesized in the same reaction. Oligomerization of the asialoglycoprotein receptor in vitro should provide a useful system to study the assembly of a membrane-protein complex.

Animals↗

PubMed1990

Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with a type II orientation in the plasma membrane. As shown in a cell-free translation system, the amino-terminal 34 amino acids of rat DPPIV are involved in translocating nascent polypeptide across the membrane of microsomes and in anchoring the translocated polypeptide in the microsomal membrane. The amino-terminal sequence performing this dual function is composed of: a central hydrophobic core of 22 amino acid residues; 6 amino-terminal residues preceding the hydrophobic core (MKTPWK); and 6 residues following the hydrophobic core. The six residues preceding the hydrophobic core are exposed on the outside (cytoplasmic side) of the microsomal membrane. Site-directed mutagenesis studies show that deletion of this cytoplasmic domain, excluding the amino-terminal initiating methionine, does not affect translocation of nascent DPPIV polypeptide, but does affect significantly anchoring of the translocated polypeptide in the microsomal membrane. In contrast, changing the two cytoplasmic Lys to Glu residues or shortening of the hydrophobic core from 22 to 15 residues or converting the last 11e of the shortened hydrophobic core into Ala affects neither translocation across nor anchoring of the DPPIV polypeptide in the microsomal membrane. These and other structural features of the DPPIV amino-terminal signal-anchor sequences are discussed along with other types of sequences for their role in targeting nascent polypeptides to the RER.

Amino Acid Sequence↗

PubMed1990

The role of coronary angioplasty in patients with associated noncardiac medical and surgical conditions.

Percutaneous transluminal coronary angioplasty was performed in 22 patients with associated significant medical or surgical conditions. It was successful in 22 patients. Two had procedure-related complications: one femoral hematoma and one small myocardial infarction. The patients were divided into a 'medical' group (12 patients) and a 'surgical' group (10 patients). In the medical group, mean coronary artery stenosis decreased from 87 +/- 5% to 20 +/- 13% and mean coronary artery stenosis decreased from 57 +/- 2% to 16 +/- 7%. In the surgical group coronary artery stenosis decreased from 83 +/- 9% to 18 +/- 9% and the gradient from 49 +/- 16 to 16 +/- 6 mmHg. Percutaneous transluminal coronary angioplasty allowed the safe management of underlying conditions in all patients so that medical treatment could be continued and noncardiac surgery performed.

Adult↗

PubMed1989

Expression of enzymatically active rat dipeptidyl peptidase IV in Chinese hamster ovary cells after transfection.

Dipeptidyl peptidase IV (DPPIV) is a cell surface membrane glycoprotein expressed in many tissues. We have subcloned the coding region of a full-length cDNA for DPPIV into the inducible eukaryotic expression vector pMSG. The resulting construct was used to transfect Chinese hamster ovary (CHO) cells. Stable transformants were found to express DPPIV, and the expression is enhanced by dexamethasone. Metabolic labeling of the transfected cells with [35S]Met followed by immunoprecipitation revealed the presence of two specific products of apparent Mr 100,000 (100-kDa form) and 110,000 (110-kDa form), respectively. Pulse-chase experiments demonstrated that the 100-kDa form can be chased into the 110-kDa form, suggesting the 100-kDa form is the precursor of the 110-kDa form. Further studies with endo H treatment demonstrated that the carbohydrate structures are of the high-mannose type, and of the complex type for the 100- and 110-kDa forms, respectively. The 110-kDa form is present at the cell surface as shown by its accessibility to cell surface iodination. The DPPIV expressed on the cell surface is resistant to digestion by relatively high concentrations of trypsin. Studies also demonstrated that the surface DPPIV is fairly stable with a half-life for turnover of about 40 h. Furthermore, the DPPIV produced in the transfected cells displays specific dipeptidyl peptidase activity. The stably transfected cells that express enzymatically active DPPIV in an inducible manner will provide an excellent system for further biochemical, functional, and cell biological characterizations of DPPIV.

Animals↗

PubMed1989

Cloning and analysis of cDNA clones for rat kidney alpha-spectrin.

We have isolated a 3922-base pair (bp) cDNA clone for rat nonerythroid alpha-spectrin from a rat kidney lambda gt11 cDNA library. Sequence analysis revealed that this cDNA contains an open reading frame of 3090 bp encoding for the C-terminal 1030 amino acid sequence of rat kidney alpha-spectrin. The 3'-untranslated region (including a 38-bp poly(A+) tail) contains an 832-bp sequence. A single mRNA of about 8 kilobase pairs was detected in rat liver, kidney, brain, heart, intestine, lung, testis, stomach, spleen, and muscle with varying abundances, which is consistent with and further confirms the presence of spectrins in nonerythroid tissues as demonstrated previously by immunoblot analysis. Southern blot analysis suggested that there is a single gene for nonerythroid alpha-spectrin. The derived amino acid sequence contains sequence from the spectrin 106-residue internal repeat 12 to the C terminus of rat kidney alpha-spectrin. Sequence comparison with human and chicken nonerythroid alpha-spectrin showed that nonerythroid alpha-spectrin is well conserved during evolution. The rat kidney alpha-spectrin sequence, when compared to rat brain alpha-spectrin, contains an extra 76-amino-acid sequence at the C terminus. Sequence comparison of all the internal repeats available revealed that the internal repeat 3, 4, 5, 6, 7, and 8 has highest sequence similarity with internal repeat 12, 13, 14, 15, 16, and 17, respectively. Therefore, internal repeats 3-8 and 12-17 are most likely derived from an ancestral gene through gene duplication, suggesting that the spectrin gene is derived from a half-spectrin gene by gene duplication and divergence during evolution.

Amino Acid Sequence↗

PubMed1989

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels.

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.

Animals↗

PubMed1989

Mineralocorticoid specificity of renal type I receptors: binding and metabolism of corticosterone.

In cytosol preparations renal mineralocorticoid receptors have equivalent affinity for aldosterone and corticosterone; in vivo, such receptors are clearly aldosterone-selective. To explore this difference, we have compared the binding of both steroids in kidney slices, high speed supernatants and true cytosols in vitro, in the presence of RU28362 to exclude steroids from Type II glucocorticoid receptors, and/or in the presence of cortisol 17 beta-COOH to exclude corticosterone from transcortin. In addition, we have explored the effect of tissue preparation and cortisol 17 beta-COOH on the level of activity of the renal enzyme 11 beta-OH steroid dehydrogenase. The results of the present studies support the hypothesis that the predominant aldosterone specific-conferring mechanism for renal Type I receptors in vivo in the enzyme 11 beta-OH steroid dehydrogenase, and underline the potential difficulties in interpreting studies which include multiple potential ligands to multiple potential binding sites.

Aldosterone↗

PubMed1989

Diffuse axonal injury in head injury: definition, diagnosis and grading.

Diffuse axonal injury is one of the most important types of brain damage that can occur as a result of non-missile head injury, and it may be very difficult to diagnose post mortem unless the pathologist knows precisely what he is looking for. Increasing experience with fatal non-missile head injury in man has allowed the identification of three grades of diffuse axonal injury. In grade 1 there is histological evidence of axonal injury in the white matter of the cerebral hemispheres, the corpus callosum, the brain stem and, less commonly, the cerebellum; in grade 2 there is also a focal lesion in the corpus callosum; and in grade 3 there is in addition a focal lesion in the dorsolateral quadrant or quadrants of the rostral brain stem. The focal lesions can often only be identified microscopically. Diffuse axonal injury was identified in 122 of a series of 434 fatal non-missile head injuries--10 grade 1, 29 grade 2 and 83 grade 3. In 24 of these cases the diagnosis could not have been made without microscopical examination, while in a further 31 microscopical examination was required to establish its severity.

Adolescent↗

PubMed1989

Fatal head injury in children.

A comprehensive neuropathological study was undertaken on 87 children aged between 2 and 15 years with fatal head injuries to identify those features which occurred at the time of head injury (fractured skull, contusions, intracranial haematoma and diffuse axonal injury) and those which were subsequently produced by complicating processes (hypoxic brain damage, raised intracranial pressure, infection and brain swelling). The types of brain brain damage identified were remarkably similar to those seen in adults. The only difference was the prevalence of diffuse brain swelling in children.

Adolescent↗

PubMed1989

Ischaemic brain damage is still common in fatal non-missile head injury.

A detailed neuropathological examination has been undertaken on a consecutive series of head injuries dying in the Institute of Neurological Sciences, Glasgow, between 1968-72 (151 cases) and 1981-82 (112 cases) in order to determine the frequency and distribution of any ischaemic brain damage. Ischaemic damage was found in the brains of 92% of the 1968-72 cases and in 88% of the 1981-82 cases: there was no statistical difference in the amount of moderately severe and severe ischaemic damage in the two groups, 55% and 54% respectively. There was evidence, however, that an increased number of patients with severe ischaemic brain damage was admitted in 1981-82 as a result of a changed admission policy of the Department of Neurosurgery that resulted in an increased detection of intracranial haematomas. It is concluded that ischaemic brain damage is still common after severe head injury, and it seems likely that it remains an important cause of mortality and morbidity.

Adolescent↗

PubMed1989

Brain damage in fatal non-missile head injury in relation to age and type of injury.

Brain damage in a series of 635 fatal non-missile head injuries has been analysed with particular reference to the age of the patient and the type of injury. The differences in the type of brain damage in relation to age were less than we had anticipated, lending further support to the contention that the aged brain has a reduced potential for recovery. The analysis confirms the relationship between road traffic accidents, diffuse axonal injury, gliding contusions and 'basal ganglia' haematomas, and the importance of diffuse brain swelling resulting from a head injury in children.

Adolescent↗

PubMed1989

Acute renal failure due to McArdle's disease.

A 37-year-old previously fit man developed acute renal failure associated with rhabdomyolysis following a brief period of strenuous exercise. Subsequent muscle biopsy and ischaemic arm exercise confirmed the diagnosis of McArdle's disease. This case illustrates that McArdle's disease can present with acute renal failure in the absence of a past history of exercise-induced muscle pain and stiffness.

Acute Kidney Injury↗

PubMed1989

Light and electron microscopic changes in sural nerves in Ethiopian diabetics.

The light and electron microscopic morphological changes in sural nerve biopsies from 32 Ethiopian diabetics and 4 malnourished non-diabetic subjects were studied in order to determine the role of malnutrition in the development of diabetic neuropathy. The most prominent finding in diabetics with clinical neuropathy was reduction in the density of myelinated axons of all diameters especially the large ones. Affected nerves showed abundant intraneural collagen, and evidence of nerve regeneration in some cases. Demyelination, though present, was less marked. Changes in the vasa nervorum were seen in only one case. The morphological changes correlated with the duration and severity of the diabetes and with nerve conduction velocity. No changes were seen in malnourished non-diabetics and diabetics without clinical neuropathy, thus suggesting that malnutrition by itself does not contribute significantly to the development of diabetic neuropathy. Axonal degeneration appears to be the predominant feature in diabetic neuropathy.

Adolescent↗

PubMed1988

Membrane orientation of rat gp110 as studied by in vitro translation.

We have recently isolated a full-length cDNA clone for a membrane glycoprotein with apparent Mr of 110,000, denoted gp110, that is expressed in rat liver, kidney, small intestine, and lung. Analysis of the amino acid sequence derived from cDNA sequencing demonstrated that there is a highly hydrophobic domain at the amino terminus (amino acid residues 1-29) that looks like the amino-terminal signal sequence. Using in vitro transcription and translation systems, we have expressed the full-length gp110 cDNA transcript, as well as transcripts derived from truncated gp110 cDNA that terminate translation at different sites. As expected, the amino-terminal signal sequence can promote the translocation across microsomal membranes of the downstream sequence. Like most translocations, the cotranslational translocation of the downstream sequence initiated by the gp110 amino-terminal signal sequence is mediated by signal recognition particle and docking protein. Unlike most amino-terminal signal sequences of lysosomal, secretory, and membrane proteins, the amino-terminal signal sequence of gp110 is not cleaved and may be involved in anchoring the polypeptide to the membrane. We, therefore, predict that the membrane orientation of gp110 is of type II with an extremely small amino-terminal cytoplasmic domain (6 residues).

Amino Acid Sequence↗

PubMed1988

Identification of a complex of the three forms of the rat liver asialoglycoprotein receptor.

We have generated antibodies against synthetic peptides which represent the carboxyl terminus of either the major, or the two minor, forms of the rat hepatic lectin which recognizes galactose-terminated glycoproteins (asialoglycoproteins). The antibodies were shown to be specific for the form of the lectin containing the immunizing peptide sequence by the following: reaction with purified lectin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation of sodium dodecyl sulfate-denatured lectin, immunoprecipitation of lectin synthesized in vitro. These antibodies, however, precipitated all three rat hepatic lectin forms from nonionic detergent extracts of hepatocytes labeled with 125I via the lactoperoxidase catalyzed technique. A similar result was obtained if antibody was bound to intact cells prior to solubilization with detergent and collection of the immune complexes. We conclude that at least the plasma membrane-associated fraction of the rat hepatic lectin forms exists as a heterotypic complex.

Animals↗

PubMed1988

Differential phosphorylation of murine class I major histocompatibility antigens.

Recent studies have shown that the H-2K and H-2D transplantation antigens are expressed differentially in different tissues of mouse. Our previous investigations also established that in thioglycolate-stimulated peritoneal macrophages the H-2Dk antigen exists in distinct cell surface and intracellular forms. These two forms are glycosylated differently. In this report, we have found that (1) H-2Dk antigen is phosphorylated whereas H-2Kk antigen is not, and (2) only the cell surface form of H-2Dk antigen is phosphorylated in thioglycolate-stimulated macrophages derived from C3H/Heha mice. This differential phosphorylation of H-2 antigens will provide a model system for further studies on the molecular mechanism and function of phosphorylation of H-2 antigens.

Animals↗

PubMed1988

Analysis of renal and hippocampal type I and type II receptors by fast protein liquid chromatography.

Type I and Type II adrenal steroid receptors from rat renal and hippocampal cytosols were studied by the technique of Fast Protein Liquid Chromatography. Type I receptors were labelled with [3H]aldosterone plus excess RU26988, and Type II receptors with [3H]dexamethasone. On a Mono Q anion exchange column the molybdate-stabilized renal and hippocampal Type I receptors both eluted as single symmetrical peaks at 0.27 M NaCl, with a recovery of approximately 90% and 60-fold purification (renal) and 10-15-fold (hippocampal). Molybdate-stabilized Type II binding sites from both hippocampal and renal cytosols co-eluted with the Type I sites. On Superose gel filtration renal Type I receptor-steroid complexes consistently eluted two fractions later than hippocampal Type I complexes, suggesting that the renal complexes are smaller; Type II receptor-steroid complexes from both cytosols co-eluted, consistently one fraction behind hippocampal Type I sites. Sequential gel filtration and anion exchange chromatography achieved a 1000-fold purification of renal Type I binding sites, with an overall recovery of 10%.

Androstanols↗

PubMed1988

Asialoglycoprotein receptor genes are linked on chromosome 11 in the mouse.

The asialoglycoprotein receptor on the hepatocyte plasma membrane recognizes galactose-terminated glycoproteins and internalizes them for subsequent degradation in lysosomes. The rat receptor, also known as rat hepatic lectin (RHL), is comprised of three protein subunits called RHL-1, RHL-2, and RHL-3; two genes code for RHL-1 and RHL-2/3, respectively. We have cloned and sequenced the gene for RHL-2/3, and demonstrated that homologous asialoglycoprotein receptor genes exist in the mouse genome. Biochemical studies have demonstrated that receptor subunits exhibit the same temporal expression during development and function in a coordinate manner. This study asks if mouse receptor genes are linked and thus could possibly respond to shared cis-acting regulatory elements. Using restriction fragment length polymorphisms (RFLPs) and recombinant inbred lines, we mapped two closely linked mouse hepatic lectin (MHL) genes to chromosome 11. Asgr is designated to name asialoglycoprotein gene loci. Coordinate regulation of this linked gene family is discussed.

Animals↗