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Biomedical subjects

G J Cooper

Publications and source records attributed to G J Cooper.

At least 19 recordsLinked to original sources
PubMed2002

Expression of a sodium bicarbonate cotransporter in human parotid salivary glands.

The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Preptin derived from proinsulin-like growth factor II (proIGF-II) is secreted from pancreatic islet beta-cells and enhances insulin secretion.

Pancreatic islet beta-cells secrete the hormones insulin, amylin and pancreastatin. To search for further beta-cell hormones, we purified peptides from secretory granules isolated from cultured murine beta TC6-F7 beta-cells. We identified a 34-amino-acid peptide (3948 Da), corresponding to Asp(69)-Leu(102) of the proinsulin-like growth factor II E-peptide, which we have termed 'preptin'. Preptin, is present in islet beta-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated beta TC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.

Amino Acid Sequence↗
PubMed2001

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions.

Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K(+) and HCO(3)(-) out of the cell, tending to lower intracellular pH (pH(i)). With an inwardly directed K/HCO(3) gradient, the cotransporter mediates a net uptake of alkali (i.e., K(+) and HCO(3)(-) influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA(+)) inhibit the inwardly directed cotransporter by interacting at the intracellular K(+) site. We computed the equivalent HCO(3)(-) influx (J(HCO3)) mediated by the cotransporter from the rate of pH(i) increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH(i) 8.0, using a dialysis fluid (DF) free of K(+), Na(+) and Cl(-). Our standard artificial seawater (ASW) also lacked Na(+), K(+) and Cl(-). After halting dialysis, we introduced an ASW containing 437 mm K(+) and 0.5% CO(2)/12 mm HCO(3)(-), which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO(2) influx, followed by a slower plateau-phase pH(i) increase, due to inward cotransport of K(+) and HCO(3)(-). With no QA(+) in the DF, J(HCO3) was approximately 58 pmole cm(-2) sec(-1). With 400 mm tetraethylammonium (TEA(+)) in the DF, J(HCO3) was virtually zero. The apparent K(i) for intracellular TEA(+) was approximately 78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K(+) channels. Introducing 100 mm inhibitor into the DF reduced J(HCO3) to approximately 20 pmole cm(-2) sec(-1) for tetramethylammonium (TMA(+)), approximately 24 for TEA(+), approximately 10 for tetrapropylammonium (TPA(+)), and virtually zero for tetrabutylammonium (TBA(+)). The apparent K(i) value for TBA(+) is approximately 0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA(+)), with an apparent K(i) of approximately 91 microm. Thus, trans-side quaternary ammonium ions inhibit K/HCO(3) influx in the potency sequence PPTEA(+) > TBA(+) > TPA(+) > TEA(+) congruent with TMA(+). The identification of inhibitors of the K/HCO(3) cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.

Animals↗
PubMed2001

Identification and characterization of a bovine myosin light chain-1 fast polymorphism.

This study aimed to identify genes or gene products associated with high lean muscle mass in bovines that may serve as potential markers for selection. An animal with a genetic predisposition to high lean muscle mass, the Belgium Blue, was chosen as a model to compare with the Holstein Friesian, a model that does not. Two-dimensional polyacrylamide gel electrophoresis analysis was utilized to compare the exhibited skeletal muscle proteome between the two animal types at two stages of foetal development. A previously uncharacterized polymorphism of a high expression myofibrillar protein, myosin light chain 1 fast (MLC-1f), was observed. The characterization of this polymorphism revealed a two amino acid insertion in a part of the protein that has been implicated in modulating myosin S1 ATPase activity. Furthermore, this polymorphism was shown to be the product of two alleles that are codominant. Screening studies were carried out on selected herds and showed a very high frequency of one allele. Both isoforms of MLC-1f were produced by recombinant means and purified. The recombinant proteins were exchanged into purified myosin hexamers that were then subject to assays measuring ATP consumption. The sensitivity of the assay utilized could not reveal any significant difference in ATPase activity between hexamers containing one or the other isoform.

Actins↗
PubMed2001

Proteomic analysis of the brain in Alzheimer's disease: molecular phenotype of a complex disease process.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder accounting for about 50% of all dementias, yet its pathogenic mechanisms remain poorly understood. In order to provide a more complete picture of pathogenesis in AD, we analysed six human brain regions for alterations in their proteomes. Quantitative proteome analysis was used to compare signals corresponding to individual proteins between post mortem brain tissues from persons with AD, and those from age-matched nondemented control (NC) tissues. In severely injured brain regions, 76 proteins were differentially expressed in AD hippocampus compared with NC, 62 proteins were differentially expressed in temporal cortex, and 39 proteins were differentially expressed in entorhinal cortex. Significant differences were also present in relatively spared regions. Thus, 34 proteins were differentially expressed in AD cerebellum compared with NC, 125 proteins were differentially expressed in cingulate gyrus, and 75 proteins were differentially expressed in sensorimotor cortex. The identity of 37 of these proteins was determined, and the possible relevance of changes in key pathogenic pathways analysed. These studies provide a unique snapshot illustrating the complexity of interrelated disease mechanisms at work in a complex, multifactorial disease, and show that comparative proteome analysis is a method with the power to develop important new insights into pathogenic mechanisms in the dementias.

Alzheimer Disease↗
PubMed2001

Ultrastructural evidence that apoptosis is the mechanism by which human amylin evokes death in RINm5F pancreatic islet beta-cells.

A view is emerging that human amylin (HA) kills pancreatic islet beta-cells by apoptosis. This study strengthens this view by documenting time-dependent morphological and ultrastructural changes in 10 microm HA-treated cultured RINm5F islet beta-cells. Membrane blebbing and microvilli loss were the earliest detectable apoptosis-related phenomena, already evident 1 h after HA exposure. Following 6-12 h of HA-treatment, chromatin margination became evident, consistent with detecting DNA laddering about the same time. Nuclear shrinkage, nuclear membrane convolution and prominent cytoplasmic vacuolization were clearly recognized at 22 h post-treatment. Together, these cellular changes constitute a strong case for HA-induced apoptosis, and further demonstrates that electron microscopy is a more sensitive tool for early apoptosis detection in cultured cells than classical biochemical assays like visualizing DNA laddering. The ultrastructural changes reported here contribute further evidence to be included in the ongoing dissection of molecular mechanisms underlying HA-induced apoptosis, as may occur in type-2 diabetes mellitus.

Amyloid↗
PubMed2001

Membrane cross-talk in the early distal tubule segment of frog kidney: role of calcium stores and chloride.

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

Adenosine Triphosphate↗
PubMed2001

Single aortic cross-clamp technique reduces S-100 release after coronary artery surgery.

BACKGROUND: Neurologic impairment after coronary artery bypass grafting is associated with cerebral embolization. An important cause of embolism is aortic manipulation. Constructing both distal and proximal anastomoses during a single period of aortic cross-clamping avoids this source of embolism and may reduce neurologic injury after coronary artery bypass grafting. METHODS: Fifty consecutive patients undergoing coronary artery bypass grafting were prospectively randomized to group 1, in which a single aortic cross-clamping was used to construct distal and proximal anastomoses, or to group 2, in which the proximal anastomoses were each constructed with a partial occluding aortic clamp. Levels of S-100 and troponin-T release were measured preoperatively and postoperatively. RESULTS: Aortic cross-clamp time was significantly longer in group 1, but other preoperative and intraoperative variables were equally represented in both groups. Control group levels of S-100 and troponin-T were similar. Postoperative S-100 levels were significantly higher in group 2 than in group 1 (p < 0.015). No significant difference was found between the groups in postoperative troponin-T levels. CONCLUSIONS: The results of this trial suggest improved cerebral protection is associated with the single aortic cross-clamp technique for coronary artery bypass grafting with no increase in myocardial damage. The single aortic cross-clamp technique is simple and inexpensive. We recommend its wider use.

Aged↗
PubMed2001

Fatigue testing median sternotomy closures.

OBJECTIVE: Sternal dehiscence is commonly due to wire cutting through bone. With a biological model, we measured the rate of cutting through bone, of standard steel wire closure, peristernal steel wire, figure-of-eight closure, polyester and sternal bands sternotomy closure techniques. METHODS: Polyester, figure-of-eight, peristernal and sternal band closures were tested against standard closure eight times using adjacent paired samples, to eliminate biological variables. Fatigue testing was performed by a computerized materials-testing machine, cycling between loads of 1 and 10 kg. The displacements at maximum and minimum loads were measured during each cycle. Cutting through, manifested by the displacement at the maximum load between the 1st and 150th cycles was measured. The percentage cut-through of each closure method versus standard closure was calculated. RESULTS: The differences in the displacement between each of the polyester (1.01 mm), figure-of-eight (0.52 mm), peristernal (0.72 mm) and sternal band (0.66 mm) groups versus standard closure (0.22, 0.22, 2.1, 3.2 mm) in the paired samples were statistically significant (Student's paired t-test; P<0.01). There were statistically significant differences in the percentage cut-through of polyester, figure-of-eight, peristernal and sternal bands (ANOVA, P<0.001), versus standard closure. CONCLUSIONS: In our sheep sternum model, we have quantified the differing rate of cutting through bone of five types of median sternotomy closure techniques. We have controlled for bone variables by testing each closure versus standard closure using paired adjacent bone samples. Peristernal and sternal band closure techniques are significantly superior to standard closure. The use of polyester and figure-of-eight closures requires caution.

Analysis of Variance↗
PubMed2001

Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development.

Amylin and calcitonin gene-related peptide (CGRP) are homologous 37 amino acid peptides that are found in the circulation. Both peptides belong to the calcitonin family. Similar to calcitonin, amylin and CGRP inhibit osteoclast activity, although they are much less potent than calcitonin. Calcitonin is known to act on the latter stages of osteoclast development, inhibiting the fusion of committed preosteoclasts to form mature multinucleated cells; however, whether or not calcitonin acts earlier in the formation of the precursor osteoclasts is controversial. The question of osteoclast development has never been examined with respect to amylin and CGRP. These issues are addressed in the present study. We studied the effects of calcitonin (salmon and rat), amylin (human and rat), and CGRP (human and rat) in mouse bone marrow cultures stimulated to generate osteoclasts using 1alpha,25-dihydroxyvitamin D3. Calcitonin dose-dependently decreased the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as TRAP-positive mono-/binucleated cells at concentrations >10(-13) mol/L. Amylin and CGRP showed similar effects at concentrations >10(-9) mol/L. In addition, calcitonin substantially reduced the ratio of TRAP-positive multinucleated to mono-binucleated cells, indicating an effect on fusion of osteoclast precursors. The present data establish that this family of peptides not only acts on mature osteoclasts but also inhibits their development in bone marrow cultures. This activity is shared by amylin and CGRP. The much greater potency of calcitonin than amylin and CGRP is consistent with the action of these peptides being mediated by calcitonin receptors.

Amyloid↗
PubMed2001

Plasma amylin concentration is related to the severity of intestinal ischemic injury in rats.

BACKGROUND: Previous work has demonstrated that intestinal ischemia increases plasma amylin concentration. This study examined the relationship between the degree of intestinal ischemia injury and plasma amylin in an experimental rat model. METHODS: Wistar rats were divided into a control group (n = 6); a sham-operated group (n = 9); and 3 intestinal ischemia-reperfusion groups (n = 8 in each), which underwent clamping of the superior mesenteric artery for either 15, 30, or 45 minutes followed by 15 minutes of reperfusion. Samples were then collected for intestinal histology and measurement of amylin, insulin, and glucose. RESULTS: There was a positive correlation between the histologic score of the intestinal injury and the measured plasma amylin concentration (R = 0.48, P =.007). The median plasma concentration of amylin was 62 pmol/L (range, 42-97 pmol/L) in the 30-minute intestinal ischemia group and 58 pmol/L (42-86 pmol/L) in the 45-minute intestinal ischemia group. Both these groups were increased compared with the sham-operated group (29 pmol/L; range, 22-57 pmol/L; P <.001 and P <.005, respectively) and the control group (28 pmol/L; range, 26-42 pmol/L; P <.001 and P <.0005, respectively). The median plasma concentration of insulin in the 30-minute intestinal ischemia group was 4230 pmol/L (range, 1360-5770 pmol/L), which was increased compared with both the control group (950 pmol/L; range, 550-1510 pmol/L; P <.005) and the sham-operated group (720 pmol/L; range, 280-4180 pmol/L; P<.005). There were no differences between any of the other groups either for glucose, insulin, or amylin. CONCLUSIONS: Plasma amylin concentration is related to the severity of intestinal ischemic injury.

Amyloid↗
PubMed2001

Evidence that amylin stimulates lipolysis in vivo: a possible mediator of induced insulin resistance.

The present study investigated the role of amylin in lipid metabolism and its possible implications for insulin resistance. In 5- to 7-h-fasted conscious rats, infusion of rat amylin (5 nmol/h for 4 h) elevated plasma glucose, lactate, and insulin (P <0.05 vs. control, repeated-measures ANOVA) with peak values occurring within 60 min. Despite the insulin rise, plasma nonesterified fatty acids (NEFA) and glycerol were also elevated (P < 0.001 vs. control), and these elevations (80% above basal) were sustained over the 4-h infusion period. Although unaltered in plasma, triglyceride content in liver was increased by 28% (P < 0.001) with a similar tendency in muscle (18%, P = 0.1). Infusion of the rat amylin antagonist amylin-(8-37) (125 nmol/h) induced opposite basal plasma changes to amylin, i.e., lowered plasma NEFA, glycerol, glucose, and insulin levels (all P < 0.05 vs. control); additionally, amylin-(8-37) blocked amylin-induced elevations of these parameters (P < 0.01). Treatment with acipimox (10 mg/kg), an anti-lipolytic agent, before or after amylin infusion blocked amylin's effects on plasma NEFA, glycerol, and insulin but not on glucose and lactate. We conclude that amylin could exert a lipolytic-like action in vivo that is blocked by and is opposite to effects of its antagonist amylin-(8-37). Further studies are warranted to examine the physiological implications of lipid mobilization for amylin-induced insulin resistance.

Amyloid↗
PubMed2001

Cloning and functional expression of an MIP (AQP0) homolog from killifish (Fundulus heteroclitus) lens.

The major intrinsic protein (MIP) of lens fiber cells is a member of the aquaporin (AQP) water channel family. The protein is expressed at very high levels in lens fiber cells, but its physiological function is unclear. By homology to known AQPs, we have cloned a full-length cDNA encoding an MIP from the lens of killifish (Fundulus heteroclitus). The predicted protein (263 amino acids; GenBank accession no. AF191906) shows 77% identity to amphibian MIPs, 70% identity to mammalian MIPs, and 46% identity to mammalian AQP1. Expression of MIPfun in Xenopus laevis oocytes causes an approximately 40-fold increase in oocyte water permeability. This stimulation is comparable to that seen with AQP1 and substantially larger than that seen with other MIPs. The mercurials HgCl(2) and p-chloromercuribenzenesulfonate inhibit the water permeability of MIPfun by approximately 25%. MIPfun is not permeable to glycerol, urea, or formic acid but is weakly permeable to CO(2).

4-Chloromercuribenzenesulfonate↗
PubMed2001

A potential role for adrenomedullin as a local regulator of bone growth.

Bone remodeling is a complex process of coordinated resorption and formation of bone, which is regulated by systemic hormones and by local factors. We have previously shown that the peptide hormone adrenomedullin is mitogenic to osteoblastic cells in vitro and that it promotes bone growth in vivo. The aim of the present study was to characterize the expression of molecules that may mediate adrenomedullin signaling in osteoblasts and to investigate the expression of adrenomedullin itself in these cells. The first adrenomedullin receptor that was cloned is the seven-transmembrane G protein-coupled receptor, L1. Two additional receptors for adrenomedullin, which arise from interactions between calcitonin receptor-like receptor and receptor activity modifying proteins 2 or 3, have now been described. In the current study, we used RT-PCR and Northern blot analysis to demonstrate that messenger RNA for the three adrenomedullin receptors, as well as for adrenomedullin itself, is expressed in primary rat osteoblasts. Treating primary osteoblasts with transforming growth factor-beta and insulin-like growth factor-I moderately reduced adrenomedullin RNA levels, whereas PTH had no effect. We have shown by immunocytochemistry that adrenomedullin peptide is present in osteoblasts, and by competitive binding assays that (125)I-adrenomedullin binds with high affinity to intact osteoblasts and to osteoblast cell membranes. Coexpression of adrenomedullin and adrenomedullin receptors in osteoblasts, taken together with our previous finding that adrenomedullin is mitogenic to these cells, raises the possibility that this peptide is a local regulator of bone growth.

Adrenomedullin↗
PubMed2001

Systemic administration of adrenomedullin(27-52) increases bone volume and strength in male mice.

Adrenomedullin is a 52-amino acid peptide first described in a human phaeochromocytoma but since been found to be present in many tissues, including the vascular system and bone. Because of its structural similarity to amylin and calcitonin gene-related peptide, both of which have actions on bone cells, we have previously assessed the effects of adrenomedullin on the skeleton, and found that it increases osteoblast proliferation in vitro and bone formation following local injection in vivo. The present study carries this work forward by assessing the effects on bone of the systemic administration of a fragment of this peptide lacking the structural requirements for vasodilator activity. Two groups of 20 adult male mice received 20 injections of human adrenomedullin(27-52) 8.1 microg or vehicle over a 4-week period and bone histomorphometry and strength were assessed. In the tibia, adrenomedullin(27-52) produced increases in the indices of osteoblast activity, osteoid perimeter and osteoblast perimeter (P<0.05 for both using Student's t-test). Osteoclast perimeter was not affected. There was a 21% increase in cortical width and a 45% increase in trabecular bone volume in animals treated with adrenomedullin(27-52) (P<0.002 for both). Assessment of bone strength by three-point bending of the humerus showed both the maximal force and the displacement to the point of failure were increased in the animals treated with adrenomedullin(27-52) (P<0.03 for both). There was also a significant increase in the thickness of the epiphyseal growth plate. No adverse effects of the treatment were noted. It is concluded that adrenomedullin(27-52) acts as an anabolic agent on bone. These findings may be relevant to the normal regulation of bone mass and to the design of agents for the treatment of osteoporosis.

Adrenomedullin↗
PubMed2001

Alteration in phosphorylation of P20 is associated with insulin resistance.

We have recently identified a small phosphoprotein, P20, as a common intracellular target for insulin and several of its antagonists, including amylin, epinephrine, and calcitonin gene-related peptide. These hormones elicit phosphorylation of P20 at its different sites, producing three phosphorylated isoforms: S1 with an isoelectric point (pI) value of 6.0, S2 with a pI value of 5.9, and S3 with a pI value of 5.6 (FEBS Letters 457:149-152 and 462:25-30, 1999). In the current study, we showed that P20 is one of the most abundant phosphoproteins in rat extensor digitorum longus (EDL) muscle. Insulin and amylin antagonize each other's actions in the phosphorylation of this protein in rat EDL muscle. Insulin inhibits amylin-evoked phosphorylation of S2 and S3, whereas amylin decreases insulin-induced phosphorylation of S1. In rats made insulin resistant by dexamethasone treatment, levels of the phosphoisoforms S2 and S3, which were barely detectable in healthy rats in the absence of hormone stimulation, were significantly increased. Moreover, the ability of insulin to inhibit amylin-evoked phosphorylation of these two isoforms was greatly attenuated. These results suggested that alterations in the phosphorylation of P20 might be associated with insulin resistance and that P20 could serve as a useful marker to dissect the cellular mechanisms of this disease.

Adipose Tissue↗
PubMed2001

Association of atypical chest pain presentations by African Americans and the lack of utilization of reperfusion therapy.

OBJECTIVE: One possible factor resulting in delays in using reperfusion therapy in patients with acute myocardial infarction (AMI) is the failure to recognize cardiac symptomatology early in certain subgroups of patients. These patients may undergo extensive evaluation for gastrointestinal or musculoskeletal complaints before it is recognized that they are suffering from an AMI. METHODS: The records of patients (52% Black and 48% White; 49% male and 51% female) presenting to an urban teaching hospital with enzyme documented myocardial infarctions were retrospectively examined for traditional elements of atypia in their initial chest pain descriptions to the emergency department (ED). The rate of reperfusion therapy utilization was also determined for this group. RESULTS: Of the patients meeting the study criteria (166 total), 43% were found to have atypical elements in the character of their pain description. This high prevalence of atypia also coincided with a low reperfusion intervention rate of 38%. In examining the subgroups, it appears that African Americans and women had the highest rates of atypical pain (56% and 46%, respectively) while also having the lowest utilization rates for reperfusion therapies (31% and 33%, respectively). This compares to rates of 48% for Whites and 60% for White males who had more typical chest pain. CONCLUSIONS: While no direct correlation can be drawn from the data, it has been suggested that atypical presentations may result in early failure to recognize myocardial infarction and cause delay in or prevent appropriate therapy. It is thought that chest pain should be de-emphasized as a part of the indication criteria for thrombolytics and emergent angioplasty.

Adult↗