Assessing the sensitivity of habitats to fishing: from seabed maps to sensitivity maps.

In the Welsh part of the Irish Sea, a method was developed for assessing the sensitivity of different seabed habitats to existing fishing activities, across a range of potential fishing intensities. The resistance of 31 habitats and their associated biological assemblage to damage by 14 categories of fishing activity were assessed along with the rate at which each habitat would recover following impact (resilience). Sensitivity was scored based on a combination of the resistance of a habitat to damage and its subsequent rate of recovery. The assessments were based, wherever possible, on scientific literature, with expert judgement used to extrapolate results to habitat and gear combinations not directly examined in the published literature. The resulting sensitivity matrices were then subject to further peer review at a series of workshops. Following consensus on the habitat sensitivity, these data were combined with the most resolved sea-floor habitat maps. These habitat sensitivity maps can help inform the development of site-specific management plans, as well as having a place in spatial planning and aiding managers in developing dialogue with other stakeholders. A case study of their application is provided.

The impact of floral resources and omnivory on a four trophic level food web.

Omnivory is common among arthropods, but little is known about how availability of plant resources and prey affects interactions between species operating at the third and fourth trophic level. We used laboratory and field cage experiments to investigate how the provision of flowers affects an omnivorous lacewing, Micromus tasmaniae (Hemerobiidae) and its parasitoid Anacharis zealandica (Figitidae). The adult lacewing is a true omnivore that feeds on both floral resources and aphids, whereas the parasitoid is a life-history omnivore, feeding on lacewing larvae in the larval stage and floral nectar as an adult. We showed that the effect of floral resources (buckwheat) on lacewing oviposition depends on prey (aphid) density, having a positive effect only at low prey density and that buckwheat substantially increases the longevity of the adult parasitoid. In field cages, we tested how provision of flowering buckwheat affects the dynamics of a four trophic level system, comprising parasitoids, lacewings, pea aphids and alfalfa. We found that provision of buckwheat decreased the density of lacewings in the first phase of the experiment when the density of aphids was high. This effect was probably caused by increased rate of parasitism by the parasitoid, which benefits from the presence of buckwheat. Towards the end of the experiment when the aphid populations had declined to low levels, the effect of buckwheat on lacewing density became positive, probably because lacewings were starving in the no-buckwheat treatment. Although presence of buckwheat flowers did not affect aphid populations in the field cages, these findings highlight the need to consider multitrophic interactions when proposing provision of floral resources as a technique for sustainable pest management.

Effect of denervation on the expression of two glucose transporter isoforms in rat hindlimb muscle.

Denervation rapidly (within 24 h) induces insulin resistance of several insulin-responsive pathways in skeletal muscle, including glucose transport; resistance is usually maximal by 3 d. We examined the effect of denervation on the expression of two glucose transporter isoforms (GLUT-1 and GLUT-4) in rat hindlimb muscle; GLUT-4 is the predominant species in muscle. 1 d postdenervation, GLUT-1 and GLUT-4 mRNA and protein concentrations were unchanged. 3 and 7 d postdenervation, GLUT-4 mRNA and protein (per microgram DNA) were decreased by 50%. The minor isoform, GLUT-1 mRNA increased by approximately 500 and approximately 100%, respectively, on days 3 and 7 while GLUT-1 protein increased by approximately 60 and approximately 100%. The data suggest that the insulin resistance of glucose transport early after denervation does not reflect a decrease in total glucose transporter number; however, decreased GLUT-4 expression may contribute to its increased severity after 3 d. Parallel decreases in GLUT-4 mRNA and GLUT-4 protein postdenervation are consistent with pretranslational regulation; GLUT-1 expression may be regulated pre- and posttranslationally. The cell type(s) which overexpress GLUT-1 postdenervation need to be identified. Nervous stimuli and/or contractile activity may modulate the expression of GLUT-1 and GLUT-4 in skeletal muscle tissue.

WITHDRAWN: Anticoagulants or antiplatelet therapy for non-rheumatic atrial fibrillation and flutter.

BACKGROUND: Atrial fibrillation (AF) carries a high risk of stroke and other thromboembolic events. Appropriate use of drugs to prevent thromboembolism in patients with AF involves comparing the patient's risk of stroke to the risk of hemorrhage from medication use. OBJECTIVES: To quantify risk of stroke, major hemorrhage and death from using medications that have been rigorously evaluated for prevention of thromboembolism in AF. SEARCH STRATEGY: Articles were identified through the Cochrane Collaboration's CENTRAL database and MEDLINE until December 1999. SELECTION CRITERIA: Included Randomized controlled trials of drugs to prevent thromboembolism in adults with non-postoperative AF. Excluded RCTS of patients with rheumatic valvular disease. DATA COLLECTION AND ANALYSIS: Data were abstracted by two reviewers. Odds ratios from all qualitatively similar studies were combined, with weighting by study size, to yield aggregate odds ratios for stroke, major hemorrhage, and death for each drug. MAIN RESULTS: Fourteen articles were included in this review. Warfarin was more efficacious than placebo for primary stroke prevention {aggregate odds ratio (OR) of stroke=0.30 [95% Confidence Interval (C.I.) 0.19,0.48]}, with moderate evidence of more major bleeding { OR= 1.90 [95% C.I. 0.89,4.04].}. Aspirin was inconclusively more efficacious than placebo for stroke prevention {OR=0.68 [95% C.I. 0.29,1.57]}, with inconclusive evidence regarding more major bleeds {OR=0.81[95% C.I. 0.37,1.78]}. For primary prevention, assuming a baseline risk of 45 strokes per 1000 patient-years, warfarin could prevent 30 strokes at the expense of only 6 additional major bleeds. Aspirin could prevent 17 strokes, without increasing major hemorrhage. In direct comparison, there was moderate evidence for fewer strokes among patients on warfarin than on aspirin {aggregate OR=0.64[95% C.I. 0.43,0.96]}, with only suggestive evidence for more major hemorrhage {OR =1.58 [95% C.I. 0.76,3.27]}. However, in younger patients, with a mean age of 65 years, the absolute reduction in stroke rate with warfarin compared to aspirin was low (5.5 per 1000 person-years) compared to an older group (15 per 1000 person-years). Low-dose warfarin or low-dose warfarin with aspirin was less efficacious for stroke prevention than adjusted-dose warfarin. AUTHORS' CONCLUSIONS: The evidence strongly supports warfarin in AF for patients at average or greater risk of stroke, although clearly there is a risk of hemorrhage. Although not definitively supported by the evidence, aspirin may prove to be useful for stroke prevention in sub-groups with a low risk of stroke, with less risk of hemorrhage than with warfarin. Further studies are needed of low- molecular weight heparin and aspirin in lower risk patients.

SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes.

Basic helix-loop-helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates dimerization to create homo- and heterodimers. Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5'-CANNTG-3'. Several bHLH proteins have been identified in the yeast Saccharomyces cerevisiae using traditional genetic methodologies. These proteins regulate diverse biological pathways. The completed sequence of the yeast genome, combined with novel methodologies allowing whole-genome expression studies, now offers a unique opportunity to study the function of these bHLH proteins. It is the purpose of this review to summarize the current knowledge of bHLH protein function in yeast.

A network of yeast basic helix-loop-helix interactions.

The Ino4 protein belongs to the basic helix-loop-helix (bHLH) family of proteins. It is known to form a dimer with Ino2p, which regulates phospholipid biosynthetic genes. Mammalian bHLH proteins have been shown to form multiple dimer combinations. However, this flexibility in dimerization had not been documented for yeast bHLH proteins. Using the yeast two-hybrid assay and a biochemical assay we show that Ino4p dimerizes with the Pho4p, Rtg1p, Rtg3p and Sgc1p bHLH proteins. Screening a yeast cDNA library identified three additional proteins that interact with Ino4p: Bck2p, YLR422W and YNR064C. The interaction with Bck2p prompted us to examine if any of the Bck2p-associated functions affect expression of phospholipid biosynthetic genes. We found that hyperosmotic growth conditions altered the growth phase regulation of a phospholipid biosynthetic gene, CHO1. There are two recent reports of initial whole genome yeast two-hybrid interactions. Interestingly, one of these reports identified five proteins that interact with Ino4p: Ino2p, Hcs1p, Apl2p, YMR317W and YNL279W. Ino2p is the only protein in common with the data presented here. Our finding that Ino4p interacts with five bHLH proteins suggests that Ino4p is likely to be a central player in the coordination of multiple biological processes.

Intracoronary beta-radiation exacerbates long-term neointima formation in balloon-injured pig coronary arteries.

BACKGROUND: Long-term biological effects of ionizing radiation on coronary arteries remain poorly defined. We examined late arterial responses 6 months after balloon angioplasty and beta-radiation in normal pig coronary arteries. METHODS AND RESULTS: Coronary arteries of 25 adult pigs were randomized to receive 20 Gy (n=8) or 30 Gy (n=9) of (186)Re beta-radiation or sham radiation (n=8) immediately after balloon angioplasty. Aspirin was given daily during follow-up. The study vessels were analyzed histopathologically at 6 months. beta-Radiation decreased lumen area (20 Gy, 1.55+/-0.99 mm(2); 30 Gy, 1.03+/-0.82 mm(2); and 0 Gy, 2.05+/-0.80 mm(2); P<0.05) but not overall vessel area. The neointimal area was significantly larger within the injured segment with beta-radiation (20 Gy, 1.92+/-1.23 mm(2); 30 Gy, 1.51+/-0.97 mm(2); and 0 Gy, 0.89+/-0.31 mm(2); 0 Gy versus 20 Gy, P<0.05), and a significant increase of edge stenosis was observed with beta-radiation. Irradiated vessels also had larger thrombus areas within the neointima (30 Gy, 0.24+/-0.61 mm(2); 20 Gy, 0.98+/-1.57 mm2; and 0 Gy, 0.00+/-0.01 mm(2); P<0.05) and larger adventitial areas (20 Gy, 2.25+/-0.75 mm(2); 30 Gy, 2.38+/-0.98 mm(2); and 0 Gy, 1.23+/-0.29 mm(2); 0 Gy versus 20 or 30 Gy, P<0.05) that showed substantial collagen accumulation. CONCLUSIONS: Intracoronary beta-radiation did not inhibit neointima formation in balloon-injured normal pig coronary arteries 6 months after the interventional procedure. Unresorbed thrombus contributed to, but was not the sole component of, augmented neointima formation. Irradiated vessels demonstrated more adventitial thickening and fibrosis. These observations may have relevance for long-term clinical outcomes after intracoronary beta-radiation.

The effect of endovascular irradiation on platelet recruitment at sites of balloon angioplasty in pig coronary arteries.

BACKGROUND: Endovascular irradiation (EI) inhibits balloon-induced neointima formation in animals and is now in clinical trials for restenosis prevention. However, little is known of the effect of EI on vessel thrombogenicity due to delayed arterial healing. We investigated EI effects on platelet recruitment in pig coronary arteries. METHODS AND RESULTS: EI was performed using (90)Sr/Y at 0 Gray (Gy), 15Gy, or 30Gy at 2 mm after balloon overstretch injury. At 1 day, 1 week, and 1 month, platelet recruitment and thrombus formation were assessed using autologous (111)In-oxine-platelet labeling and light and scanning electron microscopy. In balloon-injured nonirradiated vessels, there was complete reendothelialization at 1 month, and platelet recruitment was similar to normal uninjured arteries. In irradiated vessels, scanning electron microscopy showed incomplete reendothelialization at 1 month, and these areas demonstrated attachment of activated platelets. Light microscopy of irradiated coronaries showed adherent partially organized thrombi and incomplete resolution of intramural hemorrhages. There was a significant increase in platelet recruitment at 1 month in arteries receiving EI at 15Gy (5.1+/-2. 8x10(6), P=0.02) or 30Gy (12.5+/-9.9x10(6), P=0.005) compared with nonirradiated controls (2.7+/-1.5x10(6)); 30Gy was also higher than 15Gy (P=0.05). Platelet recruitment was also increased for 30Gy compared with control at 1 day. CONCLUSIONS: Endovascular irradiation at 15Gy or 30Gy after balloon angioplasty results in incomplete endothelial recovery, impaired resolution of intramural hemorrhage, and a dose-dependent increase in platelet recruitment at 1 month.

Pre-exposure to glucosamine induces insulin resistance of glucose transport and glycogen synthesis in isolated rat skeletal muscles. Study of mechanisms in muscle and in rat-1 fibroblasts overexpressing the human insulin receptor.

Increased routing of glucose through the hexosamine-biosynthetic pathway has been implicated in the development of glucose-induced insulin resistance of glucose transport in cultured adipocytes. Because both glucosamine and glucose enter this pathway as glucosamine-6-phosphate, we examined the effects of preincubation with glucosamine in isolated rat diaphragms and in fibroblasts overexpressing the human insulin receptor (HIR-cells). In muscles, pre-exposure to glucosamine inhibited subsequent basal and, to a greater extent, insulin-stimulated glucose transport in a time- and dose-dependent manner and abolished the stimulation by insulin of glycogen synthesis. Insulin receptor number, activation of the insulin receptor tyrosine kinase in situ and after solubilization, and the total pool of glucose transporters (GLUT4) were unaffected, and glycogen synthase was activated by glucosamine pretreatment. In HIR-cells, which express GLUT1 and not GLUT4, basal and insulin-stimulated glucose transport were unaffected by glucosamine, but glycogen synthesis was markedly inhibited. Insulin-stimulated activation of protein kinases (MAP and S6) was unaffected, and the fractional velocity and apparent total activity of glycogen synthase was increased in glucosamine-treated HIR-cells. In pulse-labeling studies, addition of glucosamine during the chase prolonged processing of insulin proreceptors to receptors and altered the electrophoretic mobility of proreceptors and processed alpha-subunits, consistent with altered glycosylation. Glucosamine-induced insulin resistance of glucose transport appears to be restricted to GLUT4-expressing cells, i.e., skeletal muscle and adipocytes; it may reflect impaired translocation of GLUT4 to the plasmalemma. The glucosamine-induced imbalance in UDP sugars, i.e., increased UDP-N-acetylhexosamines and decreased UDP-glucose, may alter glycosylation of critical proteins and limit the flux of glucose into glycogen.

A novel variant of glutamine: fructose-6-phosphate amidotransferase-1 (GFAT1) mRNA is selectively expressed in striated muscle.

Glutamine:fructose-6-phosphate amidotransferase(GFAT) is the rate-limiting enzyme of the hexosamine synthesis pathway. Products of this pathway have been implicated in insulin resistance and glucose toxicity. GFAT1 is ubiquitous, whereas GFAT2 is expressed mainly in the central nervous system. In the course of developing a competitive reverse transcriptase-polymerase chain reaction assay, we noted that GFAT1 cDNA from muscle but not from other tissues migrated as a doublet. Subsequent cloning and sequencing revealed two GFAT1 mRNAs in both mouse and human skeletal muscles. The novel GFAT1 mRNA (GFAT1Alt [muscle selective variant of GFAT1]) is likely a splice variant. It is identical to GFAT1 except for a 48 or 54 bp insert in the mouse and human, respectively, at nucleotide position 686 of the coding sequence, resulting in a 16 or 18 amino acid insert at position 229 of the protein. GFAT1Alt is the predominant GFAT1 mRNA in mouse hindlimb muscle, is weakly expressed in the heart, and is undetectable in the brain, liver, kidney, lung, intestine, spleen, and 3T3-L1 adipocytes. In humans, it is strongly expressed in skeletal muscle but not in the brain. GFAT1 and GFAT1Alt expressed by recombinant adenovirus infection in COS-7 cells displayed robust enzyme activity and kinetic differences. The apparent K(m) of GFAT1Alt for fructose-6-phosphate was approximately twofold higher than that of GFAT1, whereas K(i) for UDP-N-acetylglucosamine was approximately fivefold lower. Muscle insulin resistance is a hallmark and predictor of type 2 diabetes. Variations in the expression of GFAT isoforms in muscle may contribute to predisposition to insulin resistance.

High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes.

Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes.

Effects of diabetes and hyperglycemia on the hexosamine synthesis pathway in rat muscle and liver.

In vitro studies suggested that increased flux of glucose through the hexosamine biosynthesis pathway (HexNSP) contributes to glucose-induced insulin resistance. Glutamine:fructose-6- phosphate amidotransferase (GFAT) catalyzes glucose flux via HexSNP; its major products are uridine diphosphate (UDP)-N-acetyl hexosamines (UDP-HexNAc). We examined whether streptozotocin (STZ)-induced diabetes (4-10 days) or sustained hyperglycemia (1-2 h) in normal rats alters absolute or relative concentrations of nucleotide-linked sugars in skeletal muscle and liver in vivo. UDP-HexNAc and UDP-hexoses (UDP-Hex) were increased and decreased, respectively, in muscles of diabetic rats, resulting in an approximately 50% increase in the UDP-HexNAc:UDPHex ratio (P < 0.01). No significant changes in nucleotide sugars were observed in livers of diabetic rats. In muscles of normal rats, UDP-HexNAc concentrations increased (P < 0.01) and UDP-Hex decreased (P < 0.01) during hyperglycemia. The UDP-HexNAc:UDP-Hex ratio increased approximately 40% (P < 0.01) and correlated strongly with plasma glucose concentrations. Changes in liver were similar to muscle but were less marked. GFAT activity in muscle and liver was unaffected by 1-2 h of hyperglycemia. GFAT activity decreased 30-50% in muscle, liver, and epididymal fat of diabetic rats, and this was reversible with insulin therapy. No significant change in GFAT mRNA expression was detected, suggesting post-transcriptional regulation. The data suggest that glucose flux via HexNSP increases in muscle during hyperglycemic hyperinsulinemia and that the relative flux of glucose via HexNSP is increased in muscle in STZ-induced diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle insulin-receptor kinase. Effects of substrate inhibition and diabetes.

Insulin receptor tyrosine kinase activity solubilized from hind limb muscle of control and streptozocin-induced diabetic (STZ-D) rats (2-3 wk) was studied with the substrates histone H2B and poly glutamic acid-tyrosine (glu-tyr) (4:1). Basal and insulin-stimulated kinase activities were inhibited when high concentrations of either substrate were added before initiation of phosphorylation with ATP. Under these conditions, insulin-stimulated activities of diabetic- and control-derived receptor kinase toward H2B were similar at 0.008 mg/ml H2B. However, higher concentrations of H2B (0.04-1 mg/ml) progressively reduced the ratios of diabetic-derived to control-derived receptor kinase activities to approximately 0.5. When inhibition of receptor kinase activities was prevented by allowing maximal autophosphorylation of insulin receptors before addition of H2B, kinase activity of diabetic- and control-derived receptors was similar at all H2B concentrations. Diabetic-derived insulin-receptor tyrosine kinase activity toward poly glu-tyr (4:1) was not significantly different from that of control rats. Under conditions of substrate inhibition (0.4 mg/ml H2B), insulin receptor H2B kinase activity from muscles of rats with severe diabetes (85 mg/kg STZ, 7 days) was significantly decreased, whereas the same activity from rats with moderate diabetes (50 mg/kg STZ, 7 days) was not significantly different from control rats. Insulin receptor alpha,beta dimers were not detectable in muscle preparations from control or diabetic rats. The data suggest that the impairment of muscle-derived insulin-receptor tyrosine kinase activity associated with insulinopenic diabetes reflects, in part, enhanced inhibition by some substrates. If solubilized insulin receptors and the exogenous substrates studied model in vivo events, impaired signaling of the muscle insulin receptor in insulinopenic diabetes may depend on the type and concentration of intracellular tyrosine kinase substrates and the severity of the metabolic derangements.

Effects of left ventricular pressure on sonicated albumin microbubbles: evaluation using an isolated rabbit heart model.

OBJECTIVES: We used an isolated, crystalloid-perfused rabbit heart model to test the hypothesis that the phasic changes in left ventricular contrast are due to bubble compression and decompression during systole and diastole, respectively. BACKGROUND: Contrast enhancement of the left ventricular cavity has been shown to decrease during ventricular systole. This phenomenon has been attributed to pressure-induced microbubble destruction. Such destruction, if confirmed, would severely confound the quantitative interpretation of contrast echocardiographic data. METHODS: A fixed volume of contrast solution (5% human albumin and Albunex, approximately 400:1 ratio) was introduced into a latex balloon placed within the left ventricular cavity of an isolated paced rabbit heart preparation (n = 12). Instantaneous left ventricular pressure was measured using a high fidelity microtip catheter and digitized on-line. The beating heart was placed in a water tank, and ultrasound images were obtained using a 7.5-MHz transducer and were recorded and digitized off-line at 12 frames/s. Simultaneously, the pacing signal was used for gated on-line acquisition of end-diastolic frames. A simple theoretic model based on surface tension physical principles was used to predict changes in bubble size and, consequently, the reflection intensity in response to the measured changes in left ventricular pressure. RESULTS: We found that under peak left ventricular systolic pressures ranging from 89 to 155 mm Hg, 1) end-diastolic videointensity decreased by 8 +/- 6% (mean +/- SD) over 25 consecutive heart beats; and 2) intracyclic variations in measured videointensity were in close agreement with the theoretic calculations: 80.1 +/- 2.9% versus 80.2 +/- 4.6% of diastolic videointensity at systole. CONCLUSIONS: The major cause of systolic decrease in contrast enhancement is periodic bubble compression (as opposed to bubble destruction) induced by high systolic pressures. The minor progressive decrease in end-diastolic videointensity reflects the degree of instability of Albunex microbubbles under left ventricular pressures. However, the clinical impact of these destructive effects is likely to be only minor because of the rapid transit of microbubbles through the left heart chambers and myocardial microcirculation.

Coronary intimal proliferation after balloon injury and stenting in swine: an animal model of restenosis.

OBJECTIVES: This study was designed to compare the proliferative response in coronary arteries after tantalum stent placement or balloon injury in a normolipemic swine model of restenosis. BACKGROUND: Restenosis remains a significant complication of percutaneous transluminal coronary angioplasty. Efforts to study restenosis have been hampered by the lack of a suitable animal model. METHODS: In an attempt to create lesions resembling those of human restenosis, normolipemic swine underwent injury of either the left anterior descending or the left circumflex coronary artery with either balloon inflation or deployment of a tantalum stent. At 4 weeks, they were killed and the injured vessels processed for histopathologic analysis. Intimal area, lumen area and maximal intimal thickness were measured. The degree of stenosis was expressed as residual lumen area (lumen area/intimal area ratio). RESULTS: Vessels injured by either method demonstrated significant intimal smooth muscle proliferation leading to reduction in lumen area. In the 18 stented vessels residual lumen area measured 0.64 +/- 0.18 and maximal intimal thickness measured 0.6 +/- 0.3 mm; in the 15 balloon-injured vessels these values were 0.75 +/- 0.18 and 0.4 +/- 0.3 mm, respectively (p less than 0.05). In addition, most stented vessels had reactive inflammatory infiltrates surrounding the stent wires composed of lymphocytes, histiocytes and many eosinophils. CONCLUSIONS: These data indicate that coronary artery injury in swine with either balloon inflation or stenting leads to intimal smooth muscle cell proliferation similar to that seen in human restenosis. The degree of intimal proliferation appears to be greater after stenting than after balloon injury. Intracoronary stenting in swine is associated with a marked inflammatory reaction around the stent wires. These models may be helpful in planning systemic and local antirestenosis strategies.

Inhibition of neointima formation by tranilast in pig coronary arteries after balloon angioplasty and stent implantation.

OBJECTIVES: We evaluated the effect of orally administered tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, on histologic and histomorphometric changes after angioplasty or stent implantation in pig coronary arteries. BACKGROUND: Tranilast, which has antikeloid and antiallergic properties and therefore may modulate the fibrotic and inflammatory tissue responses to angioplasty and stenting, has been shown to inhibit angiographic restenosis in small clinical trials. However, its effect on histomorphometric changes in coronary arteries after angioplasty and stenting is unknown. METHODS: Following initial pharmacokinetic studies in two pigs to determine desirable plasma levels of orally administered tranilast, 36 crossbred juvenile pigs were randomized to placebo or tranilast before undergoing balloon angioplasty in both the left anterior descending and left circumflex plus stent implantation in the right coronary artery. Oral tranilast was administered at 3 g/day starting 3 days before coronary injury and continued for 28 days until euthanasia. Injured vessels were harvested and sections analyzed by computer-assisted microscopic planimetry. RESULTS: In balloon-injured vessels, tranilast was associated with a 37% reduction in neointimal area normalized to fracture length (0.47 +/- 0.01 vs. 0.74 +/- 0.03 mm; p < 0.001) and a 23% reduction in adventitial area normalized to vessel size (0.43 +/- 0.02 vs. 0.56 +/- 0.03; p = 0.003). In stented arteries, neointimal area normalized to injury score was 32% lower in the tranilast-treated group compared to control (1.94 +/- 0.17 vs. 2.86 +/- 0.29; p = 0.01). CONCLUSIONS: In pig coronary arteries, tranilast was associated with a reduction in neointima formation and adventitial reaction after balloon injury. In stented vessels, tranilast was associated with a reduction in neointima formation normalized to injury score.

Expression and structural analysis of 14-3-3 proteins.

The 14-3-3 family of proteins plays a role in a wide variety of cellular functions including regulation of protein kinase C and exocytosis. Using antisera specific for the N termini of 14-3-3 isoforms described previously and an additional antiserum specific for the C terminus of epsilon isoform, protease digestion of intact 14-3-3 showed that the N-terminal half of 14-3-3 (a 16 kDa fragment) was an intact, dimeric domain of the protein. Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerization. Intact 14-3-3 is a potent inhibitor of protein kinase C, but the N-terminal domain does not inhibit PKC activity. Site-specific mutagenesis of several regions in the tau isoform of 14-3-3, including the mutation of a putative pseudosubstrate site to a potential substrate sequence, did not alter its inhibitory activity. Intact 14-3-3 proteins are phosphorylated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorylated much more efficiently by this kinase. This may imply that the proteins may adopt a different structural conformation, possibly upon binding to the membrane, which could modulate their activity. 14-3-3 proteins are found at high concentration on synaptic plasma membranes and this binding is mediated through the N-terminal 12 kDa of 14-3-3.

Binding of GM-CSF to adherent neutrophils activates phospholipase D.

When the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor was incubated with neutrophils adherent to plastic tissue culture plates or plates coated with extracellular matrix proteins, a rapid (3 min) but transient formation of phosphatidic acid was observed. This stimulation was dependent on the dose of GM-CSF, with an EC50 of 140 pM, and was further enhanced (up to 350%) with the PA phosphatase inhibitor propranolol in a dose-dependent manner. Conversely, GM-CSF was unable to trigger any PA formation in neutrophils maintained in suspension, even in the presence of soluble fibronectin. However, GM-CSF did prime the cells for enhanced PA formation in the presence of a secondary stimulus (fMet-Leu-Phe or PAF). GM-CSF also caused a time-dependent stimulation of diacylglycerol formation in adherent, but not suspended, cells and elicited a time-dependent stimulation of phosphatidylethanol formation, with a concomitant decrease in the formation of PA only at early (< 7 min) times. These observations were consistent with a rapid activation of the enzyme phospholipase D in adherent cells stimulated with GM-CSF. Additional data indicated that the source of DAG was PLD coexisting with PLC, especially at later times ( > 7 min) of stimulation with GM-CSF. Finally, the formation of PA and PEt, and to a minor extent, DAG, were inhibited by the protein tyrosine kinase inhibitor erbstatin in conditions in which tyrosine phosphorylation occurred. Taken together the data indicate that GM-CSF rapidly activates PLD in adherent cells, which is responsible for the generation of PA. Thus, PLD activation is an early event in neutrophil signal transduction following exposure of adherent cells to GM-CSF.

Insulin-stimulated phosphorylation of a 195K protein from muscle and liver in the presence of poly-L-lysine.

Substrates of the insulin receptor tyrosine kinase have not been identified in skeletal muscle, a major target organ of insulin action. We observed the insulin-stimulated phosphorylation of a 195K protein (pp195) in extracts prepared from rat skeletal muscle and liver. pp195 copurifies with the insulin receptor on wheat germ agglutinin affinity chromatography. pp195 is not related to the insulin receptor, as assessed by lack of recognition by antinsulin receptor antibodies and by phosphopeptide mapping. Reduction of sulfhydryl bonds does not affect its apparent mol wt. Phosphorylation of pp195 has an absolute requirement in vitro for Mn2+ or Mg2+ and for certain basic poly-amino acids, i.e. poly-L-lysine or poly-L-ornithine. In the presence of 1 microM poly-L-lysine insulin stimulates pp195 phosphorylation in a dose-dependent manner (k0.5, approximately 5 x 10(-10) M; maximum approximately 10(-8) M insulin); pp195 phosphorylation by insulin-like growth factor-I requires about 100-fold higher doses. By phosphoamino acid analysis, pp195 is predominantly phosphorylated on tyrosine, and it is recognized by antiphosphotyrosine antibodies. Insulin receptors isolated from rat muscles 5 min after insulin injection induce about 2-fold greater phosphorylation of pp195 in vitro than receptors isolated from saline-injected controls. Streptozotocin-induced diabetes results in marked diminution of insulin-stimulated pp195 phosphorylation in extracts of muscle and liver (approximately 50% when normalized to protein content of wheat germ agglutinin eluates or approximately 80% reduction when normalized to equal receptor number). The defect is reversible by insulin therapy in vivo.

Phosphorylation of the rodent negative acute-phase protein alpha 1-inhibitor-III by the insulin receptor tyrosine kinase.

alpha 1-Inhibitor-III (alpha 1I3), a broad range proteinase inhibitor, member of the alpha-macroglobulin family, is abundant in normal rat plasma. Insulin-dependent tyrosine phosphorylation of a monomeric 195K glycoprotein (pp195) was observed in wheatgerm agglutinin (WGA)-Sepharose-purified insulin receptor preparations from rat liver and muscle. Phosphorylation of pp195 in vitro required a basic poly-amino acid, i.e. poly-L-lysine. We present evidence identifying pp195 as alpha 1I3. In situ perfusion with saline essentially removed pp195 from rat livers. Addition of normal rat plasma to liver homogenates or to WGA eluates restored insulin-stimulated phosphorylation of pp195; plasma from streptozotocin-diabetic rats was much less effective. Liver-derived pp195 copurified with an abundant plasma protein, with the characteristics of alpha 1I3, on size exclusion and ion-exchange chromatography. An approximately 195K protein, comigrating with alpha 1I3, was markedly diminished in plasma from diabetic rats, and alpha 1I3 concentration was decreased by approximately 70% upon immunoblot analysis. Highly purified alpha 1I3 was phosphorylated by muscle- or liver-derived insulin receptors in the presence of 1 microM poly-L-lysine and comigrated with pp195 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 1I3 phosphorylation was half-maximal at approximately 70 nM and was stimulated by insulin 7-fold. Hindlimb perfusion removed more than 90% plasma albumin but only approximately 20% pp195 from muscles. alpha 1I3 messenger RNA was identified in liver but not in muscle. A specific antibody against alpha 1I3 immunoprecipitated phosphorylated pp195 in WGA-purified insulin receptor preparations from nonperfused liver and from saline perfused and nonperfused muscle. alpha 1I3 is bound and internalized by alpha-macroglobulin receptors; whether it is phosphorylated in vivo is unknown. Hepatic alpha 1I3 synthesis may diminish in diabetic rats.