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BACKGROUND: Management of juxta-renal abdominal aortic aneurysms (JRAAA) remains challenging. Both open surgical and endovascular options are feasible, however, there is lack of level 1 evidence to support one modality over the other. Operative interventions frequently necessitate either open repair with a suprarenal clamp positioning (ORSRC) or complex endovascular repair (EVAR) with fenestrated (fEVAR) or parallel stent grafts (chEVAR). AIM: The aim of this study was to compare the renal function deterioration and mortality between ORSRC and fEVAR in patients who were operated on for unruptured JRAAA at a tertiary centre. DESIGN: This was a retrospective observational study of patients. SETTING: Tertiary vascular unit in the United Kingdom. POPULATION: Patients with unruptured juxta-renal AAA. METHODS: A retrospective review of prospectively collected data was performed on patients who underwent repair for unruptured JRAAA between March 2008 and October 2019. Analysis of short and mid-term outcomes after ORSRC (occlusion of the aorta above at least one of the main renal arteries) and fEVAR was conducted. Patients who underwent open repair of JRAAA with an infrarenal clamp were excluded. Procedural data as well as data regarding complications was noted. Acute kidney injury (AKI) and chronic kidney disease were defined using the KDIGO criteria (Kidney Disease: Improving Global Outcomes). Renal impairment data up to 1 year and mortality up to 5 years was recorded. RESULTS: During the study period, 162 patients who underwent JRAAA repair met the inclusion criteria (60.5% of them having ORSRC). Approximately, 85.8% of the population were males (80.6% in the open group compared to 93.7% in the endovascular group; p=0.019). The mean age for patients in the open group was 74.0 (SD=±5.5) years compared to 76.1 (SD=±7.2) years in the fEVAR group (p=0.035). More patients in the ORSRC group were symptomatic (18.4% versus 6.3% in the fEVAR group; p=0.028) and they also had larger aneurysms compared to the fEVAR group (66.9 (±12.7) mm versus 62.6 (±8.6) mm, respectively; p=0.021). In the ORSRC group, all patients required suprarenal clamping, with clamping above only one of the main renal arteries carried out in 43 patients (44.3%) of cases. At baseline, 37.0% and 44.5% of patients had CKD stage 3 or worse in ORSRC and fEVAR groups, respectively (p=0.759). Approximately 47.6% of patients having ORSRC had evidence of acute renal impairment on post-operative day 3 compared to 12.7% of those in the endovascular group (p

Background Cystic artery pseudoaneurysms are rare. It usually occurs as a complication of laparoscopic cholecystectomy, but can arise uncommonly as a complication of acute cholecystitis. Ruptured cystic artery aneurysms present with haemobilia, intraperitoneal or upper gastrointestinal bleeding. We present an unusual case of cystic artery aneurysm presenting as a massive lower gastrointestinal bleed. Case presentation A 47-year-old man was admitted with a thoracic abscess and was noted incidentally on CT to have acute cholecystitis. Subsequently the patient then presented with massive fresh PR bleeding. This was found on CT to be the result of a cystic artery pseudoaneurysm with associated gallbladder fistulation to the hepatic flexure, secondary to cholecystitis. The patient was treated with coil embolisation of the cystic artery made a full recovery and was discharged with a view to performing an elective cholecystectomy. Conclusion Cystic artery pseudoaneurysm is a rare complication of cholecystitis which can present as massive lower gastrointestinal haemorrhage secondary to cholecystocolic fistula. Percutaneous embolization is a safe and effective treatment in the acute phase.

Fluid in the subcutaneous fat is a common finding anterior to the knee on MRI. This may be caused by chronic low-grade shearing injuries in patients who are overweight. The purpose of this study was to determine if there is a difference in the amount of subcutaneous fat around the knee between patients with these appearances and controls.This was a retrospective case-control study. Following a sample size calculation on pilot data, eighteen sequential patients demonstrating hyper-intense subcutaneous signal changes around the knee on fat-saturated T2-weighted MRI were identified from PACS (18 females, mean age 45, range 31-62). Age and gender-matched patients without abnormal T2 MR signal changes were selected. Two observers independently drew regions of interest representing cross-sectional areas of bone and fat. The location of T2 signal hyper-intense lesions was characterized by consensus.Inter and intra-rater intraclass reproducibility was "excellent" (ICC 0.8). The mean cross-sectional area of bone for patients with T2 hyper-intense lesions was 31.79cmSubcutaneous fluid around the knee is associated with an increased amount of subcutaneous fat, anterior to the knee extensor mechanism. This may be caused by shearing injuries in fat with reduced elasticity associated with metabolic syndrome.

Background Fluid in the subcutaneous fat is a common finding anterior to the knee on MRI. This may be caused by chronic low-grade shearing injuries in patients who are overweight. The purpose of this study was to determine if there is a difference in the amount of subcutaneous fat around the knee between patients with these appearances and controls. Methods This was a retrospective case-control study. Following a sample size calculation on pilot data, eighteen sequential patients demonstrating hyper-intense subcutaneous signal changes around the knee on fat-saturated T2-weighted MRI were identified from PACS (18 females, mean age 45, range 31–62). Age and gender-matched patients without abnormal T2 MR signal changes were selected. Two observers independently drew regions of interest representing cross-sectional areas of bone and fat. The location of T2 signal hyper-intense lesions was characterized by consensus. Results Inter and intra-rater intraclass reproducibility was “excellent” (ICC > 0.8). The mean cross-sectional area of bone for patients with T2 hyper-intense lesions was 31.79cm2 (SD 2.57) and for controls 30.11cm2 (SD 3.20) which was not significantly different (p = 0.09). The median cross-sectional area of fat for the study group was 62.29cm2 (IQR 57.1–66.5) and for controls was 32.77cm2 (IQR 24.8–32.3) which was significantly different (p

Experiments were conducted at the Lonoke Extension and Applied Research Center greenhouse at Lonoke, AR, to evaluate the effects of urea ammonium nitrate (UAN) on bispyribac and penoxsulam efficacy on barnyardgrass, hemp sesbania, and broadleaf signalgrass. Herbicide treatments included bispyribac at 17.9 or 35.8 g ai/ha or penoxsulam at 24.4 or 48.9 g ai/ha tank mixed with (1) no adjuvant, (2) a nonionic organosilicone (OSL) adjuvant at 0.125% v/v, (3) a methylated seed oil/organosilicone (MSO/OSL) adjuvant at 0.37 L/ha, (4) a proprietary blend of MSO/OSL/UAN at 2% v/v, (5) UAN at 2% v/v, (6) OSL at 0.125% plus UAN at 2% v/v, or (7) MSO/OSL at 0.37 L/ha plus UAN at 2% v/v. In addition to these adjuvants, penoxsulam was also applied with crop oil concentrate (COC) at 2.34 L/ha and with COC at 2.34 L/ha plus UAN at 2% v/v. The addition of UAN to either herbicide plus an adjuvant increased herbicide efficacy on barnyardgrass in the greenhouse, with 95 to 99% biomass reduction of three- to four-leaf barnyardgrass and 88 to 92% biomass reduction of one- to three-tiller barnyardgrass. UAN did not generally increase efficacy on hemp sesbania, as control was 90% or higher with treatments containing either herbicide and a recommended adjuvant. Adding UAN did not increase efficacy on broadleaf signalgrass. Broadleaf signalgrass control was highly variable and no treatment provided more than 65% biomass reduction.

Background and purpose: Studies in cultured hepatocytes demonstrate glycogen synthase (GS) activation with glycogen phosphorylase (GP) inhibitors. The current study investigated whether these phenomena occurred in vivo using a novel GP inhibitor. Experimental approach: An allosteric GP inhibitor, GPi688, was evaluated against both glucagon-mediated hyperglycaemia and oral glucose challenge-mediated hyperglycaemia to determine the relative effects against GP and GS in vivo. Key results: In rat primary hepatocytes, GPi688 inhibited glucagons-mediated glucose output in a concentration dependent manner. Additionally GP activity was reduced and GS activity increased seven-fold. GPi688 inhibited glucagon-mediated hyperglycaemia in both Wistar (65%) & obese Zucker (100%) rats and demonstrated a long duration of action in the Zucker rat. The in vivo efficacy in the glucagon challenge model could be predicted by the equation; % glucagon inhibition=56.9+34.3[log ([free plasma]/rat IC50)], r=0.921). GPi688 also reduced the blood glucose of obese Zucker rats after a 7 h fast by 23%. In an oral glucose tolerance test in Zucker rats, however, GPi688 was less efficacious (7% reduction) than a glycogen synthase kinase-3 (GSK-3) inhibitor (22% reduction), despite also observing activation (by 45%) of GS in vivo. Conclusions and implications: Although GP inhibition can inhibit hyperglycaemia mediated by increased glucose production, the degree of GS activation induced by allosteric GP inhibitors in vivo, although discernible, is insufficient to increase glucose disposal. The data suggests that GP inhibitors might be more effective clinically against fasting rather than prandial hyperglycaemic control. British Journal of Pharmacology (2007) 152, 1239–1247; doi:10.1038/sj.bjp.0707502; published online 15 October 2007

Propanil-resistant barnyardgrass populations, previously verified in Arkansas rice fields and in greenhouse tests, were examined in the laboratory to ascertain if the resistance mechanism in this weed biotype was herbicide metabolism. Propanil-resistant barnyardgrass was controlled >95% in the greenhouse when carbaryl (an aryl acylamidase inhibitor) was applied two days prior to propanil. Laboratory studies with 14 C-radiolabelled propanil indicated that the herbicide was hydrolysed in propanil-resistant barnyardgrass and rice to form 3,4-dichloroaniline, but no detectable hydrolysis occurred in susceptible barnyardgrass. Two additional polar metabolites were detected in propanil-resistant barnyardgrass and rice and tentatively identified by thin layer chromatography. Overall, metabolites in the resistant barnyardgrass had R f values similar to those in rice, indicating similar metabolism for both species. These data, coupled with data from a previous report on the resistant biotype showing no differential absorption/translocation or molecular modification of the herbicide binding site in the resistant biotype, indicate that the resistance mechanism is metabolic degradation of propanil.

Propanil-resistant barnyardgrass was reported in Poinsett County, AR, in 1990. Greenhouse studies were initiated to determine the distribution of propanil-resistant barnyardgrass in the state and to characterize the resistance. Barnyardgrass seeds were obtained in 1991 and 1992 from fields in 19 of the 38 rice producing counties in Arkansas where propanil treatment at recommended rates gave unsatisfactory barnyardgrass control. Barnyardgrass seedlings from the various sources were treated with propanil at 4.5 kg ai/ha and seedling injury response was compared to the response of seedlings collected from known resistant and susceptible barnyardgrass populations. Propanil-resistance of varying levels was confirmed in 115 (16 counties) out of the 138 Arkansas barnyardgrass seed sources. Propanil I 50 values (rate of herbicide required to provide 50% injury/control) were determined to be 14, 20, and 39 kg/ha for slightly, moderately, and highly resistant barnyardgrass, respectively. A resistance factor of 20x was found in the highly resistant barnyardgrass category. Development of resistance was highly correlated with crop rotations where rice was grown one out of two, or two out of three years. Nomenclature: Propanil, N-(3,4-dichlorophenyl)propanamide, barnyardgrass, Echinochloa crus-galli (L.) Beauv. #4 ECHCG; rice, Oryza sativa L.

We report 13C NMR measurements of the flux through aldose reductase in isolated rat sciatic nerve, and its inhibition by an aldose reductase inhibitor of the sulphonylnitromethane class. [1-13C] galactose was used as substrate, and the rate of production of [1-13C] dulcitol was measured. Quantitation required the use both of internal extracellular, and external, standards. The mean net forward flux (+/- SD) was 20 +/- 11 nmol/(mL nerve water)/min (n = 10). In the presence of the inhibitor, flux was reduced significantly (p < 0.001) to 13% of control. Since dulcitol is symmetrical, an estimate of the backward flux, to [6-13C] galactose, is also possible; under our conditions, this was negligible.

Effects of bearded sprangletop interference durations on Lemont and Newbonnet rice cultivars were studied. Interference durations of 63, 70, and 130 d after rice emergence reduced Lemont grain yields 11, 21, and 50%, respectively, and lowered Newbonnet grain yields 11, 13, and 37%, respectively. Interference durations of 21 to 56 d after emergence did not reduce grain yields of either cultivar. Bearded sprangletop grown in Lemont rice produced more biomass than that in Newbonnet. Season-long interference reduced plant height and straw dry weight of Lemont more than that of Newbonnet.

In this study the identity of annexins in human platelets has been determined together with their ability to be released by agents which induce platelet degranulation. The presence of proteins cross-reacting to antibodies against annexins I and V was detected in human platelets. However, the study provided evidence that these annexins are not located on the surface of the plasma membrane in a Ca++ dependent manner. Moreover, activation of platelets with several agents which induced platelet degranulation did not cause release of annexins I or V as determined by both immunoblotting and ELISA.

In the present studies, the mechanisms underlying the inhibitory actions of lipocortin-1 on the pyrogenic and thermogenic properties of cytokines were investigated. Central (icv) injection of corticotropin-releasing factor (CRF, 4.7 micrograms) or the recombinant cytokines interleukin (IL)-1 alpha (50 ng), IL-1 beta (5 ng), IL-6 (20 ng), IL-8 (20 ng), or tumor necrosis factor-alpha (TNF-alpha, 1 microgram) in conscious rats produced significant increases in resting oxygen consumption (VO2, 13-26%) and colonic temperature (0.7-1.6 degrees C) within 2 h postinjection. Administration (icv) of a recombinant fragment (NH2-terminus, 1-188 amino acids) of human lipocortin-1 (1.2 micrograms) produced small increases in VO2 (< 5%) and body temperature (< 0.3 degrees C). Pretreatment (-5 min) with lipocortin-1 significantly attenuated the thermogenic and pyrogenic effects of centrally injected IL-1 beta (80% inhibition), IL-6 (60%), IL-8 (80%), or CRF (60%). However, pretreatment with lipocortin-1 failed to modify the actions of IL-1 alpha or TNF-alpha. We have previously demonstrated that the pyrogenic and thermogenic effects of IL-1 beta, IL-6, and IL-8 are dependent on the central actions of CRF, whereas IL-1 alpha and TNF-alpha act independently of CRF. Fever and thermogenesis induced by all of these cytokines (with the exception of IL-8) can also be prevented by administration of a cyclooxygenase inhibitor. The data presented here suggest that the potent antipyretic effects of lipocortin-1 may result from inhibition of the release or actions of CRF rather than modulation of eicosanoid synthesis.

Lipocortin-1 (annexin-1), an endogenous phospholipid and calcium binding protein, has been shown to significantly attenuate the damage produced by focal cerebral ischaemia in the rat. In the present study we have therefore investigated its effect on N-methyl-D-aspartate (NMDA) induced neuronal damage. Unilateral intrastriatal infusion of a potent and selective NMDA agonist, cis-2,4-methanoglutamate (MGlu), induced an extensive lesion of the striatum in the rat, which was inhibited (greater than 80%) by prior injection of MK801 (4 mg/kg, i.p.). Infusion of 1.2 micrograms of an active fragment of lipocortin-1 (N-terminal 1-188 aa) immediately after MGlu significantly reduced the extent of damage by 44.2 +/- 8.0%. In contrast, infusion of 3 microliters of neutralizing anti-lipocortin-1 antibody with MGlu increased lesion size by 158.9 +/- 22.0%. These findings indicate that the damage produced by intrastriatal infusion of MGlu is mediated by the NMDA receptor. Lipocortin-1 fragment markedly attenuated, and the neutralizing antibody increased, this NMDA mediated neuronal damage. These observations may explain the neuroprotective action of lipocortin following cerebral ischaemia.

Many of the complications of diabetes seem to be due to aldose reductase (aldehyde reductase 2, ALR2) catalysing the increased conversion of glucose to sorbitol. Therapy with aldose reductase inhibitors (ARIs) could, therefore, decrease the development of diabetic complications. (2,6-Dimethyl-phenylsulphonyl) nitromethane (ICI 215918) is an example from a newly discovered class of ARIs, and we here describe its kinetic properties. Preparations of bovine lens ALR2 exhibit biphasic kinetics with respect to glucose and various inhibitors including ICI 215918. The inhibitor sensitive form (ALR2S) has a higher affinity for glucose than does the inhibitor insensitive form (ALR2I). Only ALR2S was characterized in detail because ALR2I activity is very low at physiological levels of glucose and is difficult to measure with accuracy. Aldehyde reductase (ALR1) is the most closely related enzyme to ALR2. Inhibition of ALR1 was, therefore, investigated in order to assess the specificity of ICI 215918. The values of K i , and K ies (dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for ICI 215918 with bovine kidney ALR1 and bovine lens ALR2S have been determined. When glucose is varied, the compound is an uncompetitive inhibitor of ALR2S ( K ies = 0.10 μ M and K i is ⪢ K ies ), indicating that ICI 215918 associates with an allosteric site on the enzyme. These kinetic characteristics would cause a decrease in the concentration required to give 50% inhibition when glucose levels rise during hyperglycaemia. ICI 215918 is a mixed noncompetitive inhibitor of ALR1 ( K i = 10 μ M and K ies = 1.8 μ M) when glucuronate is varied. Thus, the compound has up to 100-fold specificity in favour of ALR2S relative to ALR1. Therapeutic interest has now centred upon at least three distinct structural types of ARIs: spirohydantoins, acetic acids and sulphonylnitromethanes. Using one representative of each type, we have demonstrated kinetic competition for inhibition of ALR2S. This observation strongly suggests that the different inhibitors use overlapping binding sites.

The presence and localization of endogenous lipocortin-1 (LC-1, a protein which has been proposed to mediate the anti-inflammatory actions of the glucocorticoids) was studied by immunohistochemical techniques in rat brain and pituitary. A polyclonal antiserum specific for a fragment of lipocortin-1 (alpha alpha 1-188) was used to visualize immunoreactive LC-1 (iLC-1) in both neuronal and non-neuronal cell structures. Neuronal staining, which was independent of microtubular axonal transport mechanisms (in that it was not affected by blockade of axonal transport), was found in varicose nerve fibres in various regions of the brain. In addition, iLC-1 was found in the cytoplasm of neuronal cells throughout the brain. Of all brain regions which showed iLC-1, only the hippocampal neurons showed a reduced staining intensity after adrenalectomy. However, iLC-1 was not affected by dexamethasone treatment. Non-neuronal iLC-1 was found in ependymocytes lining the cerebral ventricles and aqueduct. In addition, iLC-1 was found in tanycytes in all circumventricular organs studied and in the ventral walls of the third ventricle, where some of the branching tail processes appeared to envelop local capillaries and neuronal cell bodies. A tancycyte-mediated release of LC-1 from varicose nerve fibres into the portal vasculature is proposed.

was written in ranges that cover the full gamut between soprano and bass registers. Many other compositions from this period, however, are scored in more restricted ranges. There is a long history of music couched in these more constrained textures. Among French polyphonic songs from the first third of the fifteenth century, for exam300 ple, David Fallows has identified a significant repertory of about fifty compositions apparently perceived, at the time of their composition, to be viable works for two voices. Of these, approximately half deploy voices that occupy the same range.' And any student of the sacred music of the late fifteenth century will recall works by such composers as Jacques Barbireau, Gaspar van Weerbecke, Johannes Tinctoris, and Pierre de la Rue, in which both the bassus and superius lie in unusually low registers, 'implying performance by changed voices.2

Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antilipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.

Lipocortins form a group of proteins that have been proposed as mediators of the anti-inflammatory actions of glucocorticoids. Intracerebroventricular injection of a recombinant fragment of lipocortin 1 (NH2-terminal 1-188) caused dose-dependent (0.4-1.2 micrograms) reductions in the acute increases in colonic temperature and oxygen consumption, which occurred in response to central injections of recombinant interleukin 1 beta and gamma-interferon in conscious rats. In contrast the lipocortin fragment did not affect the response to prostaglandin E2, and its activity was prevented by heat treatment or by pretreatment of animals with polyclonal antiserum raised to the fragment. Central injection of antiserum significantly enhanced the thermogenic responses to interleukin 1 beta in rats treated with dexamethasone without affecting the responses in normal animals. These results support a physiological role for lipocortin in the central effects of glucocorticoids.

Introduction. Seroma formation is one of the most frequently encountered complications following mastectomy. It may cause significant morbidity, including delayed wound healing, infection and frequent clinic attendance for seroma aspiration. Objective. To evaluate the effect of surgical quilting after mastectomy in the prevention of postoperative seroma and to investigate which factors influence seroma formation. Methods. This was a single-centre prospective cohort study over a 1-year period. All patients who had a mastectomy operation during this period were included in this study. Group 1 patients (quilting) had mastectomy flaps sutured to pectoral muscle using interrupted absorbable sutures. Seroma requiring aspiration, number of aspirations and volume aspirated were recorded postoperatively. Results. During the study period, 168 patients were recruited, with 54 patients in group 1 (quilting) and 114 patients in group 2 (non-quilting). The proportion of patients who developed seroma requiring aspiration was 69% ( n =79) in the non-quilting group and 29% ( n =15) in the quilting group ( p

Glycogen phosphorlyase inhibitors (GPi) act on the glycogenolytic pathway decreasing hepatic glucose output, making them potential candidates for Type 2 diabetes treatment. We established a robust in vivo method to assess GPis efficacy utilising glucagon-stimulated glycogenolysis.Blood glucose was monitored in both male AP Wistar and AP Zucker rats using tail prick samples pre- and post intraperitoneal or subcutaneous glucagon administration. The effect of glycogen phosphorylase inhibitors GPi296 (6-60 mg kg(-1) po) and DAB (5 mg kg(-1) po) upon glucose response to subcutaneous glucagon were examined in both strains.In the Wistar rat glucagon induced dose related increases in blood glucose, with the maximum increase occurring 20 min post dose (4.0+/-0.88 mmol l(-1), intraperitoneal; and 2.8+/-0.72 mmol l(-1), subcutaneous, ns). Intraperitoneal glucagon administration produced shorter duration blood glucose elevation than observed with the subcutaneous route of administration. In the Zucker rat, no differences were observed between the 10 and 13 week old rats in response to glucagon (3-200 microg kg(-1) subcutaneous). The maximum blood glucose increase was lower in the Wistar rat compared to the Zucker rats (2.9+/-0.20 vs 7.7+/-1.22 mmol l(-1), P0.0000018). GPi296 and DAB both produced similar inhibition in each strain.Subcutaneous glucagon administration induced more sustained increases in blood glucose than intraperitoneal administration. Blood glucose response to glucagon was higher in the Zucker rat compared to the Wistar rat; there was no difference in inhibition mediated by either GPi296 or DAB between the two strains. We believe that subcutaneous glucagon administration produces a robust model for the assessment of GPis in either rat strain.

1.Lipocortin-1 and its N-terminal derivatives exert potent inhibitory actions in various models of acute inflammation. The present study examined the ability of lipocortin (LC)-1 to suppress the release of the acute pro-inflammatory mediators, tumour necrosis factor (TNF alpha) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin-1 beta (rhIL-1 beta). 2. LPS (10 micrograms ml-1) stimulated release of TNF alpha and PGE2 from PBMC was significantly inhibited by (4 h) co-incubation of the cells with 10(-6) M dexamethasone (Dex), but not with 10(-9) M to 10(-7) M of a N-terminal fragment (amino acids 1-188) of recombinant human LC-1 (LC-1 fragment). However, Dex suppression of LPS-stimulated TNF alpha and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC-1 fragment (1:10,000 dilution) was included in the medium. rhIL-1 beta (5 x 10(-8) M)-stimulated release of TNF alpha and PGE2 from PBMC (after 18 h) was abolished by co-incubation of the cells with 10(-7) M LC-1 fragment. 3. After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti-LC-1 fragment antibody (which showed to cross-reactivity with human annexins 2 to 6). Dex caused no increase in immunoreactive (ir)LC-1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC-1-like immunoreactivity. This was accompanied by the appearance of irLC-1 in the extracellular medium. 4. The results of the present study implicate endogenous LC-1 in glucocorticoid suppression of TNF alpha and PGE2 release from human PBMC and suggest an extracellular site of action for LC-1. LC-1 may also inhibit rhIL-1 beta-stimulated TNF alpha and PGE2 secretion from PBMC.

A 188-amino acid NH2-terminal fragment of recombinant human lipocortin-1 (rhLC-1) (LC-1 fragment) mimics glucocorticoid (and rhLC-1) inhibition of corticotrophin-releasing hormone (CRH)-stimulated release of adrenocorticotrophin (ACTH) from rat anterior pituitary and cytokine-stimulated CRH release from rat hypothalamus in vitro. The present in vivo study examined the effect of LC-1 fragment on CRH stimulation of rat plasma ACTH and release of rat hypothalamic CRH. Coinjection of LC-1 fragment inhibited the increase in plasma ACTH concentration stimulated by either central (76% inhibition) or peripheral (72% inhibition) injection of CRH and abolished the (62%) depletion of hypothalamic immunoreactive (ir)CRH stimulated by central injection of interleukin-1 beta. Central injection of the CRH functional analogue sauvagine led to a 46% reduction (P > 0.05, 2-way analysis of variance) in rat hypothalamic irCRH content, which was reversed by coinjection of LC-1 fragment. These results indicate that LC-1 can suppress the activity of the hypothalamic-pituitary axis in the rat, possibly by inhibiting a positive feedback mechanism controlling release of hypothalamic CRH.

Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522 follows uncompetitive kinetics with respect to glucuronate (K(ii) = 39 nM), indicating a selectivity greater than 5-fold for bovine aldose reductase relative to aldehyde reductase.

The mechanisms underlying age-related impairments in febrile responses were investigated in female C57Bl/lcrf-a(t) mice. Injection of norepinephrine, to assess total thermogenic capacity, significantly increased oxygen consumption (VO2) in all age groups, although the responses of the aged mice were significantly reduced. Injection of lipopolysaccharide or murine interleukin-1 beta (mIL-1 beta) significantly increased body temperature and VO2 in the young and adult mice but not in the aged mice. The impaired responses to mIL-1 beta in the aged mice were normalized by either injection of the glucocorticoid receptor antagonist RU-38486 or by injection of an antiserum to lipocortin-1 or its purified immunoglobulin G fraction. Injection of prostaglandin E2 significantly increased VO2 and body temperature in all age groups. Resting plasma corticosterone concentrations were significantly elevated in the aged and adult mice, whereas injection of mIL-1 beta significantly raised plasma corticosterone concentrations in all animals. These findings indicate that the impaired febrile response of aged female C57Bl/lcrf-a(t) mice may be caused by increased concentrations and/or sensitivity to endogenous glucocorticoids. The impaired febrile responses of aged mice appear to be mediated by endogenous lipocortin-1.

Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2,6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E. NADPH → E*. NADPH). Reduction of aldehyde appears rate-limited by the step E. NADP+ → E. NADP. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*. NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*. NADPH complex. (3) Acceleration of the E*. NADP+ → E. NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.

A model is described for determining the pharmacodynamics of inhibitors of arachidonate metabolism in mice. Bioavailability and selectivity were assessed by ex vivo RIA of TXB2, LTB4, and 12-HETE from ionophore-challenged blood. Inhibition of LTB4 and 12-HETE was measured using a single LTB4 RIA, following extraction and separation of these eicosanoids from plasma. Separation on cyanopropyl mini-columns yielded hexane/ether and methanol fractions, which contained 12-HETE and LTB4, respectively. Analgesic efficacy was measured by inhibition of phenylbenzoquinone-induced abdominal constriction. The NSAIDs, indomethacin ibuprofen, flurbiprofen, and benoxaprofen, were analgesic and selective cyclo-oxygenase inhibitors. BW775C was also analgesic, but inhibited cyclo-oxygenase, 5-lipoxygenase and 12-HETE formation. Other in vitro 5-lipoxygenase inhibitors, NDGA, quercetin, and nafazatrom, were inactive in vivo, although NDGA reduced abdominal constrictions. The results indicate that this model has utility in determining the mechanism/selectivity of action and analgesic potential of 5-lipoxygenase inhibitors.

Two radioimmunoassays, the first specific for 6-oxo-PGF1 alpha and the second for 6-oxo-PGF1 alpha, 13, 14-dihydro-6-oxo-PGF1 alpha, 13,14-dihydro-6, 15-dioxo-PGF1 alpha and 6-oxo-PGE1 are described. These radioimmunoassays were used to measure levels of immunoreactive 6-oxo-PGF1 alpha and alleged metabolites of prostacyclin in human venous plasma. Procedures for the direct measurement and extraction of plasma 6-oxo-PGF1 alpha are described and the limitations to which radioimmunoassay of plasma 6-oxo-PGF1 alpha is exposed are discussed. Direct measurement of plasma immunoreactive 6-oxo-PGF1 alpha gave maximal levels of 6.1 pg ml-1 and 4 pg ml-1 in the first and second radioimmunoassays respectively. The latter value reflected combined levels of less than 2 pg ml-1 of 6-oxo-PGF1 alpha and cross-reacting metabolites in venous blood. Extraction of human plasma gave 88.7 +/- 2.2% recovery (mean +/- S.E.M. n = 5 experiments) of [3H]-6-oxo-PGF1 alpha and 80 - 86% recovery of exogenous 6-oxo-PGF1 alpha as immunoreactive 6-oxo-PGF1 alpha. Basal plasma levels of extractable immunoreactive 6-oxo-PGF1 alpha were less than 2.5 pg ml-1. Prostacyclin incubated in vitro with blood was recovered in plasma as immunoreactive 6-oxo-PGF1 alpha and confirmed that conversion to metabolites that cross-reacted with the second antibody, in particular 6-oxo-PGE1, did not occur under experimental conditions. Extraction of [3H]-6-oxo-PGF1 alpha from acidified plasma with methanol resulted in formation of a prostanoid that had properties consistent with the methylated hemiketal isomer of 6-oxo-PGF1 alpha. Under radioimmunoassay conditions this prostanoid was less immunogenic than native [3H]-6-oxo-PGF1 alpha and [3H]-6-oxo-PGF1 alpha extracted from plasma using methyl formate. The low levels of plasma 6-oxo-PGF1 alpha which we report questions the validity of clinical studies that previously described altered levels of plasma 6-oxo-PGF1 alpha in pathophysiological conditions. These studies which were based upon measurement of plasma 6-oxo-PGF1 alpha by radioimmunoassay and GC/MS should now be re-evaluated. Preliminary results from this study have been reported elsewhere (1, 2).

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH2 metabolism in favour of PGI2, a potent vasodilator and anti-platelet agent. While redirection has been observed ex vivo there are conflicting reports of its occurrence in vivo . We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH2 for the study of PGH2 metabolism. Following challenge, plasma concentrations of TXB2, 6-oxo-PGF1α, alleged metabolites of PGI2 (PGI2m) and PGE2 were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB2 while AA and PGH2, in addition, elevated 6-oxo-PGF1α and PGI2m. Injection of PGH2 elevated 6-oxo-PGF1α, PGI2m, TXB2 and PGE2 levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg−1), collagen (100 kg−1), AA (1mg kg−1) and PGH2 (5μg kg−1) and measurement of eicosanoids 0.5min following challenge (5μg kg−1) and measurement of eicosanoids 0.5min following challenge were optimal for detection of redirection of PGH2 metabolism in vivo . The identity of immunoreactive TXB2 and 6-oxo-PGF1α was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [3H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect in vivo biosynthesis.

In clinical and pre-clinical research the pharmacodynamics of selective 5-lipoxygenase and dual 5-lipoxygenase/cyclo-oxygenase inhibitors may be studied by direct RIA of plasma LTB4. Although immunoreactive LTB4 in plasma from A23187 stimulated human blood has the characteristics of authentic LTB4 our results show, particularly in mice and rats, that exposure to A23187 produces large quantities of 12-HETE. Since in different species the levels of 12-HETE increase with platelet concentration we suggest that the 12(S)-HETE is produced by platelet lipoxygenase. However, we do not rule out the possibility that a proportion of 12-HETE may exist as the (R)-stereoisomer. The latter has greater potential for interference in the direct RIA of LTB4. Biosynthesis of 12-HETE may be measured either by RPHPLC/U.V. abs. (8) or by RIA (9) and LTB4 by a more specific antibody described in this report. We conclude that the combined ex vivo RIA of plasma TXB2, LTB4 and 12-HETE has utility in determining the selectivity of inhibitors of arachidonate metabolism and in distinguishing between selective 5-lipoxygenase inhibitors which interact directly with the enzyme and anti-oxidant or free radical scavenging types which may be less specific.

The plasma concentrations of immunoreactive thromboxane B2 and 6-keto-prostaglandin-F1 alpha were determined in coronary venous blood sampled from open-chested, anaesthetised beagle dogs by local vein catheterisation. Thromboxane levels were high immediately following catheterisation (2.08 +/- 0.75 ng X ml-1, mean +/- SEM, n = 6) and fell over 25 min to 0.48 +/- 0.07 ng X ml-1. Initial trauma of the coronary veins (agitation of the catheter) produced a large but variable increase to 5.13 +/- 2.27 ng X ml-1 (0.1 greater than P greater than 0.05) within 2 min, but arterial trauma (repeated momentary occlusions) produced 0.4 +/- 0.03 ng X ml-1, a value not significantly different from control. After 7 min ischaemia an increase to 0.98 +/- 0.26 ng X ml-1 was detected (P less than 0.05). Reperfusion after 15 min ischaemia caused a fall to 0.43 +/- 0.09 ng X ml-1 at 2 min. In contrast, 6-keto-prostaglandin-F1 alpha levels varied little with trauma but increased to 0.64 +/- 0.14 ng X ml-1 (P less than 0.01) within 2 min ischaemia, remaining elevated. On reperfusion, levels fell to 0.27 +/- 0.06 ng X ml-1 (P less than 0.05) at 2 min, progressively increasing to 0.35 +/- 0.05 ng X ml-1 at 30 min. We conclude that intimal trauma of coronary veins induced thromboxane release and that the physical effects of venous sampling may contribute to apparent thromboxane release from ischaemic muscle. Although transient arterial and venous trauma had no significant effect on 6-keto-prostaglandin-F1 alpha, it is still possible that its release during ischaemia may follow prolonged arterial clamping.

A sensitive RIA for LTB 4 (which cross-reacts 60% with 6-traps LTB 4 ) has been developed with a minimal detectable mass of 7.4 × 10 −15 mole. The properties of a second RIA more specific for 6-trans LTB 4 are also described. The latter has utility in the measurement of the enzymatic and non-enzymatic transformations of LTA 4 and in the characterisation of inhibitors of LTA 4 epoxide hydrolase. Conditions for the direct RIA of immunoreactive LTB 4 in human plasma are described. Addition of calcium ionophore, A23187, to human blood increased basal immmunoreactive LTB 4 levels from −1 to 259 ± 23ng ml −1 (mean ± SEM, n=16). Extraction and RPHPLC confirmed that >90% of immunoreactive LTB 4 co-eluted with synthetic and [ 3 H]LTB 4 . Pharmacological characterisation of immunoreactive eicosanoids revealed that in vitro the NSAIDs: aspirin, indomethacin, flurbiprofen and benoxaprofen did not inhibit LTB 4 but inhibited TXB 2 , consistent with cyclo-oxygenase inhibition whereas the prototype mixed inhibitor BW755c inhibited both LTB 4 and TXB 2 . This experimental paradigm may be used in the clinical measurement of the bio-availability of novel agents that inhibit eicosanoid biosynthesis.

In the preceding paper we described the characterisation of an acute intravenous challenge model for the evaluation of the effects of thromboxane synthase inhibition (TXSI) on eicosanoid metabolism. Herein we describe the biochemical pharmacology of two TXSI and aspirin in this model. Both TXSI caused significant inhibition of plasma TXB2 in vivo without elevation of 6-oxo-PGF1 alpha levels. Similar results were obtained when combined levels of 6-oxo-PGF1 alpha,13,14 dihydro 6-oxo-PGF1 alpha,13,14 dihydro 6,15-dioxo-PGF1 alpha and 6-oxo-PGE1 were measured as an index of PGI2 biosynthesis (PGI2m). Thus no evidence of in vivo redirection of PGH2 to PGI2 was found. Ex vivo experiments performed in serum gave an apparent stimulation of immunoreactive 6-oxo-PGF1 alpha following TXSI but RPHPLC analysis of extracted serum showed that this stimulation was accounted for by increase in a product co-eluting with [3H]PGF2 alpha. The implications of these findings in relation to TXSI and receptor antagonists are discussed.

Membrane-bound avachidonic acid is a precursor for PG and LT biosynthesis*. Members of this family of autocoids have been implicated in pathophysiology including regulation of microvascular blood flow and permeability, leukocyte chemotaxis, hyperalgesia and anaphylaxis. Consequently, in recent years considerable effort has been expended in the search for agents that block the synthesis and antagonize the actions of these autocoids. Most of the biologically stable arachidonic metabolites can now be measured by RIA, and this technique finds widespread use in the search for AI and analgesic drugs. Within a mechanistic approach to new drug development, RIA of eicosanoids can be used at the pre-clinical and clinical stages of drug development and now plays an integral part in hypothesis testing and in the progression of pharmacological curiosities to effective medicines.

1. Levels of immunoreactive 6-oxo-prostaglandin F1α (6-oxo-PGF1α) and thromboxane B2 (TXB2) were measured in peripheral venous plasma in a group of volunteers and non-insulin dependent diabetic patients (NIDDS). Levels of these eicosanoids were close to the limit of sensitivity of the radioimmunoassays and consequently data are reported as maximal values. Basal plasma levels of 6-oxo-PGF1α did not exceed 5 pg/ml in either group and maximal levels of immunoreactive TXB2 were 125 ± 14 and 128 ± 8 pg/ml for volunteers and NIDDS respectively. 2. Attempts to elicit peripheral vascular prostacyclin biosynthesis in volunteers by using forearm ischaemia produced no increase in plasma 6-oxo-PGF1α levels. Measurement of the combined plasma levels of 6-oxo-PGF1α, 13,14-dihydro-6-oxo-PGF1α, 13,14-dihydro-6,15-dioxo-PGF1α and 6-oxo-PGE1 indicated that these were also low (less than 5 pg/ml) and that failure to demonstrate increased 6-oxo-PGF1α levels was unlikely to have arisen from metabolism of prostacyclin to one or more of these metabolites. 3. Measurement of 6-oxo-PGF1α and TXB2 in peripheral venous plasma before and during chloropropamide alcohol flushing (CPAF) did not provide evidence for a role for these eicosanoids in the etiology of this phenomenon. 4. These findings point to the need for a reappraisal of studies that have described altered plasma levels of 6-oxo-PGF1α and TXB2 in CPAF and other pathophysiological conditions in man.

The metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells was studied. LiCl, BeCl2 and NaF inhibited the hydrolysis of both monophosphates with half maximal inhibition occurring at 1.2 mM, 0.3 microM, 0.25 mM (Ins 1P) and 0.14 mM, 0.56 microM, 0.28 mM (Ins 4P) respectively. Lithium was an uncompetitive inhibitor with respect to both substrates. Ins 4P inhibited the hydrolysis of Ins 1P in a concentration dependent manner, suggesting that it acts as a competing substrate for the same enzyme. Half maximal inhibition occurred at 120 microM Ins 4P. The lithium sensitive activity responsible for the metabolism of both monophosphates was present in a soluble fraction made from the cells. Taken together these data suggest that Ins 1P and Ins 4P are hydrolysed by a single soluble enzyme activity which is sensitive to inhibition by lithium, beryllium and fluoride.