Amyloids are a class of noncrystalline, yet ordered, protein aggregates. A new approach was used to provide the initial structural data on an amyloid fibril--comprising a peptide (beta 34-42) from the C-terminus of the beta-amyloid protein--based on measurement of intramolecular 13C-13C distances and 13C chemical shifts by solid-state 13C NMR and individual amide absorption frequencies by isotope-edited infrared spectroscopy. Intermolecular orientation and alignment within the amyloid sheet was determined by fitting models to observed intermolecular 13C-13C couplings. Although the structural model we present is defined to relatively low resolution, it nevertheless shows a pleated antiparallel beta-sheet characterized by a specific intermolecular alignment.

SummaryA randomised double-blind controlled study was performed to examine the effect of diclofenac on skin bleeding time and in vitro whole blood platelet aggregation. Twenty thoracotomy patients were studied; I0 were given diclofenac 75 mg intramuscularly at induction of anaesthesia, and I0 formed a control group. Skin bleeding times and platelet aggregation tests were performed the day before and repeated one hour after induction of anaesthesia. Diclofenac prolonged skin bleeding time and reduced platelet aggregation. There were no significant changes in the control group. Key wordsAnalgesics; diclofenac. Blood; coagulation.The use of nonsteroidal anti-inflammatory drugs (NSAIDs) to provide postoperative analgesia is becoming accepted practice. Intramuscular diclofenac was shown to be an effective analgesic,' but there is concern over the perioperative use of the NSAIDs since they have potential side effects related to their mechanism of action, inhibition of the enzyme cyclo-oxygenase.2 This enzyme is essential in platelets for the production of thromboxane A, which is an important mediator of platelet aggregation and vasoconstriction. These are processes that constitute the primary haemostatic response to vessel injury. Peri-operative administration of a NSAID could affect platelet function and inhibit haemostasis.It is clear that NSAIDs and aspirin, which has a similar inhibitory effect on cyclo-oxygenase, inhibit aggregation and prolong bleeding time in v~lunteers,~,~ but there is little information on the peri-operative situation where the haemostatic response may be altered by the stress of surgery. It has recently become possible to study platelet aggregation in whole b l~o d .~.~ This may be more physiological than traditional turbidometric methods using platelet-rich plasma, since the platelets are left in their natural milieu surrounded by red and white cells that can influence the aggregatory respon~e.~.~ To our knowledge there is no previous study which uses the whole blood aggregation technique to investigate the effect of NSAIDs on platelet aggregation in the peri-operative situation. The aim of this study was to investigate the effect of intramuscular diclofenac on skin bleeding time and whole blood platelet aggregation in a randomised, double-blind, controlled, peri-operative study. MethodsTwenty ASA 1 and 2 patients aged 27 to 79 years undergoing thoracotomy were studied. The investigation was approved by the regional ethics committee and each subject provided written informed consent before enrolment. Exclusion criteria consisted of a history of peptic ulcer disease, bleeding tendency, asthma, allergies, recent aspirin or NSAID ingestion, and alcohol or narcotic abuse. The general anaesthetic technique was standardised (premedication, papaveretum and hyoscine; induction, thiopentone, suxamethonium; maintenance, nitrous oxide, oxygen, enflurane, fentanyl, alcuronium). Subcutaneous heparin (2500 IU subcutaneously, one hour before operation) was given as prophylaxis against venous thrombosis. A...

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (, , and), which contain consensus sites for phosphory-lation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphor-ylation of-and-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opi-oid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala 2 ,N-Me-Phe 4 ,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to 12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor cheleryth-rine, and the L-type calcium channel antagonist nimodip-ine attenuated opioid-mediated effects on PKC and-receptor down-regulation, suggesting that this is a process partially regulated by Ca 2-dependent PKC iso-forms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca 2-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-) and novel (PKC-) isoforms, SH-SY5Y cells also contain a DAG-and Ca 2-independent, atypical PKC isozyme (PKC-), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-is similarly sensitive to activation by-opioids. PKC-translocates from the cytosol to the membrane with kinetics similar to those of PKC-and in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by-opioid agonists is involved in the processes that result in-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved. Key Words: Protein kinase-Receptor-Down-regulation-Second messenger-Isoenzyme. J. Neurochem. 72, 594-604 (1999). The recent cloning of the major opioid receptor types (, , and) has provided essential information about their plasticity (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993; Wang et al., 1993; Yasuda et al., 1993). Repeated or prolonged exposure to opioid ago-nists reduces the responsiveness of opioid receptors to its agonists over time. This loss of receptor function is hypothesized to contribute to the cellular and biochemical changes that lead to opiate tolerance, dependence, and, possibly, addiction in humans (Nestler, ...