Anti-platelet effect of aspirin is substantially reduced after administration of heparin during carotid endarterectomy

OBJECTIVES: Aspirin therapy is usually continued throughout the perioperative period to reduce the risk for thromboembolic stroke and myocardial infarction after carotid endarterectomy (CEA). Aspirin irreversibly binds cyclooxygenase-1, thereby reducing platelet aggregation for the lifetime of each platelet. However, recent research from this unit has shown that aggregation in response to arachidonic acid increases significantly, but transiently, during CEA, which suggests that the anti-platelet effect of aspirin is temporarily reversed. The purpose of the current study was to determine when this phenomenon occurs and to identify the possible mechanisms involved. METHODS: Platelet aggregation was measured in platelet-rich plasma from 41 patients undergoing CEA who were stabilized with 150 mg of aspirin daily. Blood was taken at 8 time points: before anesthesia, after anesthesia, before heparinization, 3 minutes after heparinization, 3 minutes after shunt insertion, 10 minutes after flow restoration, 4 hours postoperatively, and 24 hours postoperatively. Platelet aggregation was also measured at similar times in a group of 18 patients undergoing peripheral angioplasty without general anesthesia. RESULTS: All patient platelets were effectively inhibited by aspirin at the start of the operation. There was a significant intraoperative increase in platelet response to arachidonic acid in both groups of patients, which occurred within 3 minutes of administration of unfractionated heparin. In the CEA group this resulted in a greater than 10-fold increase in mean aggregation, to 5 mmol/L of arachidonic acid (5 mmol/L), rising from 3.9% +/- 2.2% preoperatively to 45.1% +/- 29.3% after administration of heparin ( P <.0001). This increased aggregation persisted into the early postoperative period, but by 24 hours post operation aggregation had returned to near preoperative values. Aggregation in response to other platelet agonists (adenosine diphosphate, thrombin receptor agonist peptide) showed only a small increase at the same time, which could be accounted for by a parallel increase in the level of spontaneous aggregation. CONCLUSION: Administration of heparin significantly increases platelet aggregation in response to arachidonic acid, despite adequate inhibition by aspirin administered preoperatively. This apparent reversal in anti-platelet activity persisted into the immediate early postoperative period, and could explain why a small proportion of patients are at increased risk for acute cardiovascular events after major vascular surgery, despite aspirin therapy.

Fate of prions in soil: Detergent extraction of PrP from soils

The transmissible spongiform encephalopathies (TSEs) are caused by infectious agents whose structures have not been fully characterized but include abnormal forms of the host protein PrP, designated PrPSc, which are deposited in infected tissues. The transmission routes of scrapie and chronic wasting disease (CWD) seem to include environmental spread in their epidemiology, yet the fate of TSE agents in the environment is poorly understood. There are concerns that, for example, buried carcasses may remain a potential reservoir of infectivity for many years. Experimental determination of the environmental fate requires methods for assessing binding/elution of TSE infectivity, or its surrogate marker PrPSc, to and from materials with which it might interact. We report a method using Sarkosyl for the extraction of murine PrPSc, and its application to soils containing recombinant ovine PrP (recPrP). Elution properties suggest that PrP binds strongly to one or more soil components. Elution from a clay soil also required proteinase K digestion, suggesting that in the clay soil binding occurs via the N-terminal of PrP to a component that is absent from the sandy soils tested.

Targeting the cell cycle machinery for the treatment of cardiovascular disease

Cardiovascular disease represents a major clinical problem affecting a significant proportion of the world's population and remains the main cause of death in the UK. The majority of therapies currently available for the treatment of cardiovascular disease do not cure the problem but merely treat the symptoms. Furthermore, many cardioactive drugs have serious side effects and have narrow therapeutic windows that can limit their usefulness in the clinic. Thus, the development of more selective and highly effective therapeutic strategies that could cure specific cardiovascular diseases would be of enormous benefit both to the patient and to those countries where healthcare systems are responsible for an increasing number of patients. In this review, we discuss the evidence that suggests that targeting the cell cycle machinery in cardiovascular cells provides a novel strategy for the treatment of certain cardiovascular diseases. Those cell cycle molecules that are important for regulating terminal differentiation of cardiac myocytes and whether they can be targeted to reinitiate cell division and myocardial repair will be discussed as will the molecules that control vascular smooth muscle cell (VSMC) and endothelial cell proliferation in disorders such as atherosclerosis and restenosis. The main approaches currently used to target the cell cycle machinery in cardiovascular disease have employed gene therapy techniques. We will overview the different methods and routes of gene delivery to the cardiovascular system and describe possible future drug therapies for these disorders. Although the majority of the published data comes from animal studies, there are several instances where potential therapies have moved into the clinical setting with promising results.

Cyclopropylidenes, bicyclopropylidenes, and vinylcarbenes - some modes of formation and preparative applications

The routes of transformation of the simplest bicyclopropylidene into the derivatives of the second and third generations and synthesis of perspirocyclopropanated [3]rotane and linear spiral [4]- and [5]triangulanes are discussed.

Mechanisms for the formation of organic acids in the gas-phase ozonolysis of 3-carene

This paper describes experimental studies aimed at elucidating mechanisms for the formation of low-volatility organic acids in the gas-phase ozonolysis of 3-carene. Experiments were carried out in a static chamber under 'OH-free' conditions. A range of multifunctional acids-which are analogous to those observed from alpha-pinene ozonolysis-were identified in the condensed phase using gas chromatography coupled to mass spectrometry after derivation. Product yields were determined as a function of different OH radical scavengers and relative humidities to give mechanistic information about their routes of formation. Furthermore, an enone and an enal derived from 3-carene were ozonised in order to probe the early mechanistic steps in the reaction and, in particular, which of the two initially formed Criegee intermediates gives rise to which products. Branching ratios for the formation of the two Criegee Intermediates are determined. Similarities and differences in product formation from 3-carene and alpha-pinene ozonolysis are discussed and possible mechanisms-supported by experimental evidence-are developed for all acids investigated.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions (a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SIDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to similar to 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.

Diacylglycerol-induced protection against injury during ischemia and reperfusion can be achieved without activation of protein kinase C in the rat heart: Comparative studies with ischemic preconditioning

The role of protein kinase C (PKC) activation in ischemic preconditioning remains controversial. Since diacylglycerol is the endogenous activator of PKC and as such might be expected cardioprotective, we have investigated whether: (i) the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) can protect against injury during ischemia and reperfusion; (ii) any effect is mediated via PKC activation; and (iii) the outcome is influenced by the time of administration. Isolated rat hearts were perfused with buffer at 37°C and paced at 400 bpm. In Study 1, hearts (n=6/group) were subjected to one of the following: (1) 36 min aerobic perfusion (controls); (2) 20 min aerobic perfusion plus ischemic preconditioning (3 min ischemia/3 min reperfusion+5 min ischemia/5 min reperfusion); (3) aerobic perfusion with buffer containing DOG (10 μM) given as a substitute for ischemic preconditioning; (4) aerobic perfusion with DOG (10 μM) during the last 2 min of aerobic perfusion. All hearts then were subjected to 35 min of global ischemia and 40 min reperfusion. A further group (5) were perfused with DOG (10 μM) for the first 2 min of reperfusion. Ischemic preconditioning improved postischemic recovery of LVDP from 24±3% in controls to 71±2% (P<0.05). Recovery of LVDP also was enhanced by DOG when given just before ischemia (54±4%), however, DOG had no effect on the recovery of LVDP when used as a substitute for ischemic preconditioning (22±5%) or when given during reperfusion (29±6%). In Study 2, the first four groups of study were repeated (n=4–5/group) without imposing the periods of ischemia and reperfusion, instead hearts were taken for the measurement of PKC activity (pmol/min/mg protein±SEM). PKC activity after 36 min in groups (1), (2), (3) and (4) was: 332±102, 299±63, 521±144, and 340±113 and the membrane:cytosolic PKC activity ratio was: 5.6±1.5, 5.3±1.8, 6.6±2.7, and 3.9±2.1 (P=NS in each instance). In conclusion, DOG is cardioprotective but under the conditions of the present study is less cardioprotective than ischemic preconditioning, furthermore the protection does not appear to necessitate PKC activation prior to ischemia.

Ancient watercourses and biogeography of the Sahara explain the peopling of the desert

Evidence increasingly suggests that sub-Saharan Africa is at the center of human evolution and understanding routes of dispersal “out of Africa” is thus becoming increasingly important. The Sahara Desert is considered by many to be an obstacle to these dispersals and a Nile corridor route has been proposed to cross it. Here we provide evidence that the Sahara was not an effective barrier and indicate how both animals and humans populated it during past humid phases. Analysis of the zoogeography of the Sahara shows that more animals crossed via this route than used the Nile corridor. Furthermore, many of these species are aquatic. This dispersal was possible because during the Holocene humid period the region contained a series of linked lakes, rivers, and inland deltas comprising a large interlinked waterway, channeling water and animals into and across the Sahara, thus facilitating these dispersals. This system was last active in the early Holocene when many species appear to have occupied the entire Sahara. However, species that require deep water did not reach northern regions because of weak hydrological connections. Human dispersals were influenced by this distribution; Nilo-Saharan speakers hunting aquatic fauna with barbed bone points occupied the southern Sahara, while people hunting Savannah fauna with the bow and arrow spread southward. The dating of lacustrine sediments show that the “green Sahara” also existed during the last interglacial (∼125 ka) and provided green corridors that could have formed dispersal routes at a likely time for the migration of modern humans out of Africa.

Modelling of Johne's disease control options in beef cattle: a decision support approach

Johne's disease in cattle is a contagious wasting disease caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johne's infection is characterised by a long subclinical phase and can therefore go undetected for long periods of time during which substantial production losses can occur. The protracted nature of Johne's infection therefore presents a challenge for both veterinarians and farmers when discussing control options due to a paucity of information and limited test performance when screening for the disease. The objectives were to model Johne's control decisions in suckler beef cattle using a decision support approach, thus implying equal focus on ‘end user’ (veterinarian) participation whilst still focusing on the technical disease modelling aspects during the decision support model development. The model shows how Johne's disease is likely to affect a herd over time both in terms of physical and financial impacts. In addition, the model simulates the effect on production from two different Johne's control strategies; herd management measures and test and cull measures. The article also provides and discusses results from a sensitivity analysis to assess the effects on production from improving the currently available test performance. Output from running the model shows that a combination of management improvements to reduce routes of infection and testing and culling to remove infected and infectious animals is likely to be the least-cost control strategy.

Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (d,l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

Restriction fragment length polymorphism of polymerase chain reaction products applied to the differentiation of poultry campylobacters for epidemiological investigations

A technique for subtyping Camplobacter jejuni isolates has been developed by using the restriction fragment length polymorphism (Rnp) of polymerase chain reaction (PCR) products of the fluA and flaB genes. The technique was validated by using strains representing 28 serotypes of C jejuni and it may also be applied to C coli. From these strains 12 distinct RFLP profiles were observed but there was no direct relationship between the RFLP profile and the serotype. One hundred and thirty-five campylobacter isolates from 15 geographically distinct broiler flocks were investigated. All the isolates could be subtyped by using the RFLP method. Isolates from most of the flocks had a single RFLP profile despite data indicating that several serotypes were involved. Although it is possible that further restriction analysis may have demonstrated profile variations in these strains, it is more likely that antigenic variation can occur within genotypically related campylobacters. As a result, serotyping may give conflicting information for veterinary epidemiological purposes. This RFLP typing scheme appears to provide a suitable tool for the investigation of the sources and routes of transmission of campylobacters in chickens.

Mutant prevention concentrations of ciprofloxacin and enrofloxacin for Salmonella enterica

Objectives: To determine the mutant prevention concentrations (MPCs) of ciprofloxacin and enrofloxacin against four strains of Salmonella enterica serovar Enteritidis and four strains of S. Typhimurium including one fully susceptible, one multiply resistant (MAR), one GyrA mutant and one GyrA/MAR mutant. Further, to examine mutants arising after exposure to sub-MPC concentrations of the antibiotics for susceptibility to ciprofloxacin and enrofloxacin, and cyclohexane tolerance. Methods: MICs were determined using the agar dilution method of the BSAC. The MPC was recorded as the lowest concentration of antibiotic to inhibit growth from an inoculum of 10(10) cfu. Results: The MPCs and resulting MPC/MIC ratios of enrofloxacin were generally two- to four-fold higher than for ciprofloxacin. At 24 h for both antibiotics, MPCs were lowest for the fully susceptible strains (0.25-0.5 mg/L), similar for the MAR (1-4 mg/L) and GyrA (2-4 mg/L) mutants and highest for the GyrA/MAR mutants (1-8 mg/L). MPC/MIC ratios at 24 h were 2-16 for all strains except those for the MAR strains without mutation in gyrA where the ratios were 8-64. Conclusions: The ability to eradicate Salmonella in vivo depends on many factors such as antibiotic susceptibility of the strain, dose and route of administration. It is suggested that these MPC values will be useful when considering dosing strategies. In view of the high MPC/MIC ratio, MAR strains with wild-type gyrA, although susceptible to ciprofloxacin (MICs 0.06-0.13 mg/L), may give rise to treatment failures.

Trafficking of storage proteins in developing grain of wheat

The processing properties of the wheat flour are largely determined by the structures and interactions of the grain storage proteins (also called gluten proteins) which form a continuous visco-elastic network in dough. Wheat gluten proteins are classically divided into two groups, the monomeric gliadins and the polymeric glutenins, with the latter being further classified into low molecular weight (LMW) and high molecular weight (HMW) subunits. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. However, determination of the precise routes of trafficking and deposition of individual gluten proteins in developing wheat grain has been limited in the past by the difficulty of developing monospecific antibodies. To overcome this limitation, a single gluten protein (a LMW subunit) was expressed in transgenic wheat with a C-terminal epitope tag, allowing the protein to be located in the cells of the developing grain using highly specific antibodies. This approach was also combined with the use of wider specificity antibodies to compare the trafficking and deposition of different gluten protein groups within the same endosperm cells. These studies are in agreement with previous suggestions that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or accumulating directly within the lumen of the ER. They also suggest that the same individual protein could be trafficked by either pathway, possibly depending on the stage of development, and that segregation of gluten proteins both between and within protein bodies may occur.