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C J Mann

Publications and source records attributed to C J Mann.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Animals↗
PubMed1999

Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases.

Although the three-dimensional structure of the dimeric class 3 rat aldehyde dehydrogenase has recently been published (Liu ZJ et al., 1997, Nature Struct Biol 4:317-326), few mechanistic studies have been conducted on this isoenzyme. We have characterized the enzymatic properties of recombinant class 3 human stomach aldehyde dehydrogenase, which is very similar in amino acid sequence to the class 3 rat aldehyde dehydrogenase. We have determined that the rate-limiting step for the human class 3 isozyme is hydride transfer rather than deacylation as observed for the human liver class 2 mitochondrial enzyme. No enhancement of NADH fluorescence was observed upon binding to the class 3 enzyme, while fluorescence enhancement of NADH has been previously observed upon binding to the class 2 isoenzyme. It was also observed that binding of the NAD cofactor inhibited the esterase activity of the class 3 enzyme while activating the esterase activity of the class 2 enzyme. Site-directed mutagenesis of two conserved glutamic acid residues (209 and 333) to glutamine residues indicated that, unlike in the class 2 enzyme, Glu333 served as the general base in the catalytic reaction and E209Q had only marginal effects on enzyme activity, thus confirming the proposed mechanism (Hempel J et al., 1999, Adv Exp Med Biol 436:53-59). Together, these data suggest that even though the subunit structures and active site residues of the isozymes are similar, the enzymes have very distinct properties besides their oligomeric state (dimer vs. tetramer) and substrate specificity.

Aldehyde Dehydrogenase↗
PubMed1998

Alternative dystrophin gene transcripts in golden retriever muscular dystrophy.

Golden retriever muscular dystrophy (GRMD), the canine model of Duchenne muscular dystrophy (DMD), is caused by a splice site mutation in the dystrophin gene. This mutation predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. Western blot analysis of skeletal muscle from GRMD dogs reveals a slightly truncated 390-kD protein that is approximately 91% the size of normal dystrophin. This 390-kD dystrophin suggests that GRMD dogs, like some DMD patients, employ a mechanism to overcome their predicted frameshift. Reverse-transcriptase polymerase chain reaction on GRMD muscle has revealed two in-frame dystrophin transcripts which lack either exons 3-9 or exons 5-12. Both transcripts could be translated into a dystrophin protein of approximately 390 kD. An understanding of how truncated dystrophin is produced in GRMD may allow this mechanism to be manipulated toward a potential therapy for DMD.

Alternative Splicing↗
PubMed1998

Molecular cloning, characterization, and potential roles of cytosolic and mitochondrial aldehyde dehydrogenases in ethanol metabolism in Saccharomyces cerevisiae.

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption delta ald1 and delta ald2 strains on ethanol was marginally slower than that of the parent strain. The delta ald1 delta ald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.

Acetaldehyde↗
PubMed1998

Use of the Ottawa ankle rules by nurse practitioners.

OBJECTIVE: To determine the ability of nurse practitioners to use the Ottawa ankle rules by comparing their requests for radiography with those of senior house officers (SHOs). METHOD: 1365 patients were assessed by a nurse trained in the use of the Ottawa ankle rules. Where indicated the nurse sent the patient for radiography. 700 patients in the same period were seen by SHOs who had not received training in these rules. After three months the SHOs were similarly trained in the use of the rules and a further 700 patients were seen. RESULTS: Nurse practitioners assessed 1365 patients; 72.6% had radiography. 1398 patients were seen only by an SHO. Before their use of the Ottawa ankle rules SHOs requested radiography in 91.0% (p = 0.001); using the rules this fell to 74.2% (p = 0.001). CONCLUSION: Nurse practitioners trained to use the Ottawa ankle rules are as effective as SHOs in the implementation of the rules. Moreover the sensitivity and specificity of the Ottawa ankle rules was similar to that seen in other studies.

Adult↗
PubMed1997

Inhibitory effects of specific apolipoprotein C-III isoforms on the binding of triglyceride-rich lipoproteins to the lipolysis-stimulated receptor.

ApoC-III overexpression in mice results in severe hypertriglyceridemia due primarily to a delay in the clearance of triglyceride-rich lipoproteins. We have, in primary cultures of rat hepatocytes, characterized a lipolysis-stimulated receptor (LSR). The apparent number of LSR that are available on rat liver plasma membranes is negatively correlated with plasma triglyceride concentrations measured in the fed state. We therefore proposed that the primary physiological role of the LSR is to contribute to the cellular uptake of triglyceride-rich lipoproteins. We have now tested the effect of apoC-III on the binding of triglyceride-rich lipoproteins to LSR. Supplementation of 125I-very low density lipoprotein (VLDL) with apoC-III inhibited the LSR-mediated binding, internalization, and degradation of 125I-VLDL in primary cultures of rat hepatocytes. Studies using isolated rat liver plasma membranes showed that enrichment of human VLDL and chylomicrons with synthetic or purified human apoC-III decreased their binding to the LSR by about 40%. Supplementation of triglyceride-rich lipoproteins under the same conditions with human apoC-II had no such inhibitory effect, despite the fact that this apoprotein bound as efficiently as apoC-III to these particles. Preincubation of LDL with apoC-III did not modify its binding to LSR. Partitioning studies using 125I-apoC-III showed that this lack of effect was due to apoC-III's inability to efficiently associate with LDL. Purified human apoC-III1 was as efficient as the synthetic nonsialylated form of apoC-III in inhibiting binding of VLDL to LSR. However, despite a 2-fold greater binding of apoC-III2 to VLDL, this isoform was a less efficient inhibitor of the binding of VLDL to LSR than apoC-III1 or nonsialylated apoC-III. Desialylation of apoC-III2 by treatment with neuraminidase increased the inhibition of VLDL binding to LSR to a level similar to that observed with apoC-III1 and nonsialylated apoC-III. We propose that apoC-III regulates in part the rate of removal of triglyceride-rich particles by inhibiting their binding to the LSR, and that the level of inhibition is determined by the degree of apoC-III sialylation.

Animals↗
PubMed1997

Paramedic interventions increase the rate of return of spontaneous circulation in out of hospital cardiac arrests.

OBJECTIVE: To determine whether paramedic interventions increased the rate of return of spontaneous circulation in the victims of out of hospital cardiac arrest. METHODS: A retrospective analysis of 276 out of hospital cardiac arrests was made. Data analysed included age, sex, presenting rhythm, ambulance response time, presence of a pulse at any point, interventions performed by the ambulance crews, and survival to discharge. RESULTS: 146 patients were treated by paramedics and 130 by technicians. There was no difference in the rate of return of spontaneous circulation or survival to discharge in patients presenting in ventricular fibrillation (VF). In non-VF arrests there was no increase in survival to discharge, but 15% of patients in non-VF arrests achieved a return of spontaneous circulation when treated by paramedics compared to none treated by technicians. There were no other significant differences in any of the variables assessed. CONCLUSIONS: Out of hospital cardiac arrests presenting in VF are managed equally well by paramedics and technicians. However, in non-VF arrests there is a significantly increased rate of return of spontaneous circulation in those patients attended by paramedics.

Adult↗
PubMed1996

Comparison of cardiopulmonary resuscitation techniques using video camera recordings.

OBJECTIVE: To use video recordings to compare the performance of resuscitation teams in relation to their previous training in cardiac resuscitation. METHODS: Over a 10 month period all cardiopulmonary resuscitations carried out in an accident and emergency (A&E) resuscitation room were videotaped. The following variables were monitored: (1) time to perform three defibrillatory shocks; (2) time to give intravenous adrenaline (centrally or peripherally); (3) the numbers and grade of medical and nursing staff involved in the resuscitation; (4) the experience and training of these personnel. RESULTS: Of 101 resuscitations recorded, 69 were carried out by the A&E team alone and 32 by the hospital cardiac arrest team. Resuscitation procedures were carried out significantly more rapidly by the former. Skills and protocols were most effectively used when the resuscitation team was led by an experienced doctor who had received specific training in cardiopulmonary resuscitation, that is, Advanced Life Support course (ALS) or Advanced Cardiac Life Support course (ACLS). Such an individual was always present at A&E team resuscitations but in only 6% of cardiac arrest team resuscitations. CONCLUSIONS: ALS course completion should be regarded as a vital part of the training of any doctor involved in cardiopulmonary resuscitation.

Cardiopulmonary Resuscitation↗
PubMed1996

Use of an accident and emergency department by hospital staff.

OBJECTIVE: To assess the number of attendances by hospital staff at an accident and emergency (A&E) department, and reasons for their attendance. METHODS: A&E attendances by hospital staff were studied for a 12 month period. Comparison was made with attendances by non-hospital staff in full or part time employment. Differences between the observed and expected numbers of attendances were analysed using chi 2 analysis. RESULTS: 560 staff attendances were recorded out of 78,103 total attendances. There was an observed excess number of attendances by nursing staff for work related incidents when compared to the control group (P = 0.01). However, there were fewer attendances by nursing staff for non-work related incidents (P = 0.01). Staff other than doctors and nurses attended the A&E department more frequently than the control group for non-work related incidents (P = 0.01), but their attendance for incidents occurring at work were the same as the control group (P = NS). CONCLUSIONS: The increased use of A&E resources by staff other than doctors and nurses may be inappropriate and further research into their reasons for attendance is warranted.

Confidentiality↗
PubMed1995

Characterization of the slow folding reactions of trp aporepressor from Escherichia coli by mutational analysis of prolines and catalysis by a peptidyl-prolyl isomerase.

Escherichia coli trp aporepressor (TR) is a highly helical, dimeric protein whose folding has been shown to involve three phases whose relaxation times range from 200 ms to 50 s at 25 degrees C and pH 7.6 [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. All three phases are urea and protein concentration independent below 3 M urea, suggesting that cis/trans proline isomerization might limit the folding of TR under these conditions. This hypothesis was tested by measuring the sensitivity of the folding reaction to site-directed mutagenesis and to cyclophilin, a peptidyl-prolyl isomerase. Each of the four proline residues in TR was replaced singly as well as simultaneously, and the effects on the folding mechanism were assessed. All of these mutants, including the version lacking prolines (des-Pro TR), retain three slow, denaturant-independent folding phases similar to those observed for wild-type TR. However, the pattern of catalysis of the two slower folding phases in wild-type and mutant TRs by cyclophilin shows that cis/trans isomerization of the Thr44/Pro45 peptide bond can limit folding in proteins containing Pro45. The observation of three urea-independent, slow folding phases in des-Pro TR demonstrates that proline isomerization is not solely responsible for this complex folding behavior. Other types of isomerization or conformational rearrangement reactions appear to limit the folding of this dimeric protein under strongly folding conditions.

Amino Acid Isomerases↗
PubMed1995

Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant.

The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L.E., & Maki, A.H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor.

Bacterial Proteins↗
PubMed1995

Mechanism of activation and functional significance of the lipolysis-stimulated receptor. Evidence for a role as chylomicron remnant receptor.

In cultured human and rat cells, the lipolysis-stimulated receptor (LSR), when activated by free fatty acids (FFA), mediates the binding of apoprotein B- and apoprotein E-containing lipoproteins and their subsequent internalization and degradation. To better understand the physiological role of LSR, we developed a biochemical assay that optimizes both the activation and binding steps and, thus, allows the estimation of the number of LSR binding sites expressed in the livers of living animals. With this technique, a strong inverse correlation was found in rats between the apparent number of LSR binding sites in liver and the postprandial plasma triglyceride concentration (r = -0.828, p < 0.001, n = 12). No correlation existed between the number of LSR and plasma triglycerides measured in the same animals after 24 h of fasting. The same membrane binding assay was used to elucidate the mechanism by which FFA induce lipoprotein binding to LSR. The LSR activation step was mediated by direct interaction of FFA with LSR candidate proteins of apparent molecular masses of 115 and 90 kDa and occurred independently of the membrane lipid environment. The FFA-induced conformational shift that revealed the lipoprotein binding site remained fully reversible upon removal of the FFA. However, occupancy of the site by the apoprotein ligand stabilized the active form of LSR. Comparison of the effect of different FFA alone or in combination indicated that the same binding site is revealed by different FFA and that the length and saturation of the FFA monomeric carbon chain are critical in determining the potency of the FFA activating effect. We propose that the LSR pathway represents a limiting step for the cellular uptake of intestinally derived triglyceride-rich lipoproteins and speculate that FFA liberated by lipolysis initiate this process by altering the conformation of LSR to reveal the lipoprotein binding site.

Animals↗
PubMed1995

Inhibitory effect on the lipolysis-stimulated receptor of the 39-kDa receptor-associated protein.

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.

Animals↗
PubMed1995

Lipolysis-stimulated receptor: a newcomer on the lipoprotein research scene.

It has been widely accepted that the remnants of the intestinally-derived lipoprotein chylomicrons, i.e., chylomicron remnants (CMR), are cleared from the circulation by a receptor genetically distinct from the well-known LDL-receptor. This second receptor was initially considered as a receptor specific for apo E, in contrast to the LDL-receptor, which binds both apo B and apoE. This article critically examines the current dogma of the putative CMR receptor, as well as both supporting and conflicting evidence for the recently-proposed identity of this receptor with the LDL-receptor related protein (LRP). Next, we introduce the lipolysis-stimulated receptor, LSR, which bears all the biochemical characteristics of the CMR receptor. In addition, the apparent number of LSR expressed in the liver is inversely correlated with nonfasting levels of plasma triglycerides. A change in LSR expression and parallel inverse change in plasma triglycerides is observed in rats treated with hyperlipidemic (retinoic acid) or hypolipidemic (fish oil in MaxEPA) agents, indicating that LSR represents a definite target for pharmacological management of hyperlipidemia. In support of this notion is the observation that MaxEPA, which causes an increase in LSR expression, also reduces both plasma triglyceride and cholesterol levels in the thus far intractable homozygous Watanabe heritable hyperlipidemic rabbit.

Animals↗
PubMed1995

[Liver mechanisms for the elimination of lipoproteins of intestinal origin].

This article critically examines the concept of the putative chylomicron remnant receptor (CMR). The molecular nature of this second lipoprotein receptor remains disputed. Indeed, two proteins, the low density lipoprotein receptor-related protein (LRP) and the lipolysis stimulated receptor (LSR) have been proposed as candidates for this function. The LRP bears significant structural homology with the LDL receptor and mediates the internalisation of beta-VLDL enriched with apo E. In addition, LRP binds several ligands not related to the lipoprotein system. Thus, LRP's contribution to the clearance of CMR has been questioned. The precise biochemical structure of LSR remains unclear. However, a series of observations support the hypothesis that LSR is the CMR receptor. LSR, which is activated by free fatty acis (FFA), the products of lipolysis, is present in primary cultures of rat hepatocytes. It displays the highest affinity for triglyceride-rich lipoproteins and is inhibited by lactoferrin. The existence of a strong inverse correlation in rats between the apparent number of hepatic LSR and the plasma triglyceride concentration measured in the post-prandial state, indicate that LSR represents a rate-limiting step for the removal of triglyceride-rich lipoproteins. Moreover, the ability of MAXEPA to enhance the expression of LSR in parallel with its well documented hypotriglyceridemic effect indicates that, contrary to popular belief, the putative CMR receptor represents a target for pharmacological management of hyperlipidemia.

Chylomicrons↗