The receptor for advanced glycation end products (RAGE) enhances autophagy and neutrophil extracellular traps in pancreatic cancer.

Neutrophil extracellular traps (NETs) are formed when neutrophils expel their DNA, histones and intracellular proteins into the extracellular space or circulation. NET formation is dependent on autophagy and is mediated by citrullination of histones to allow for the unwinding and subsequent expulsion of DNA. NETs have an important role in the pathogenesis of several sterile inflammatory diseases, including malignancy, therefore we investigated the role of NETs in the setting of pancreatic ductal adenocarcinoma (PDA). Neutrophils isolated from two distinct animal models of PDA had an increased propensity to form NETs following stimulation with platelet activating factor (PAF). Serum DNA, a marker of circulating NET formation, was elevated in tumor bearing animals as well as in patients with PDA. Citrullinated histone H3 expression, a marker of NET formation, was observed in pancreatic tumors obtained from murine models and patients with PDA. Inhibition of autophagy with chloroquine or genetic ablation of receptor for advanced glycation end products (RAGE) resulted in decreased propensity for NET formation, decreased serum DNA and decreased citrullinated histone H3 expression in the pancreatic tumor microenvironment. We conclude that NETs are upregulated in pancreatic cancer through RAGE-dependent/autophagy mediated pathways.

Acute uterine rupture and its sequelae.

A 41-year-old woman developed hypovolaemic shock following instrumental delivery. This was ultimately discovered to be due to uterine rupture. Management involved massive transfusion of crystalloids, colloids and blood products and subtotal hysterectomy and left salpingo-oopherectomy progressing later to bilateral ligation of internal iliac arteries. Resuscitation was complicated by the development of dilutional coagulopathy, cardiac tamponade and cardiac arrest. The patient survived and was discharged from hospital 28 days after the primary event.

Investigation of potential surrogate organisms and public health risk in UV irradiated secondary effluent.

Watercare's Mangere Wastewater Treatment Plant in Auckland, New Zealand treats sewage from a population equivalent of approximately 1,000,000. The treatment plant is currently undergoing a major upgrade, and as a part of this upgrade the largest UV disinfection plant in the world (at the time of award of the contract) is being constructed. Pilot scale investigations were undertaken at a purpose built facility. The pilot plant employed secondary treatment, and filtration, UV disinfection and a number of low pressure membrane systems. Investigations at the facility focussed on attempting to identify relationships between potential surrogate indicator organisms (including enterococci, faecal coliforms, Clostridium perfringens spores and F-specific bacteriophage) and pathogenic organisms (including culturable human enteric viruses, bacterial pathogens and parasites). The aim of the study was to identify a suitable indicator organism and an associated effluent concentration that would ensure that an acceptable level of public health risk was maintained in the environment. The results showed that no suitable surrogate indicator organism could be found. However the results did indicate that a two tiered operating strategy, based on the concentration of enteroviruses present in raw sewage and an appropriate UV dose, would ensure that an acceptable level of public health risk was maintained in the environment.

Removal of enteric viruses and selected microbial indicators by UV irradiation of secondary effluent.

The Watercare Mangere Wastewater Treatment Facility, which treats wastewater from the greater Auckland New Zealand region, is undergoing a major expansion/upgrading to add advanced treatment and disinfection prior to discharge into a harbor. One important goal of this project is to protect the receiving water from microbial contamination. Since sufficient information on the fate of various microorganisms through wastewater treatment plants in New Zealand was not readily available, extensive pilot- and bench-scale studies were undertaken to develop specific design criteria for the treatment and disinfection systems. The specific objective of this study was to evaluate the removal and inactivation of enteric pathogens and other microbial indicators through treatment processes that employs UV irradiation as a final disinfection process. The removal of indicator organisms through secondary treatment was typically between 2.5-log (99.7% removal) and 2.8-log (99.8% removal) for fecal coliforms and enterococci, respectively. Indigenous F-specific bacteriophage exhibited a mean removal of 1.6-log (i.e. 97.7% removal) and Clostridium perfringens spores showed a mean removal of 1.3-log (i.e. 95% removal). The UV dose required to achieve a one log reduction in the concentration of indigenous F-specific bacteriophage was found to be approximately 20 mWs/cm2 per log removal. The concentration of enterovirus and adenovirus were consistently reduced to the limit of detection (1 TCID50/100L) at UV doses of 35 to 40 mWs/cm2 and 40 to 45 mWs/cm2, respectively. Clostridium perfringens spores were the most resistant indicator organisms, being reduced to less than 200 MPN/100 mL at a UV dose of 75 mWs/cm2.

The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival.

Activation of the induced receptor for advanced glycation end products (RAGE) leads to initiation of NF-kappaB and MAP kinase signaling pathways, resulting in propagation and perpetuation of inflammation. RAGE-knockout animals are less susceptible to acute inflammation and carcinogen-induced tumor development. We have reported that most forms of tumor cell death result in release of the RAGE ligand, high-mobility group protein 1 (HMGB1). We now report a novel role for RAGE in the tumor cell response to stress. Targeted knockdown of RAGE in the tumor cell, leads to increased apoptosis, diminished autophagy and decreased tumor cell survival . In contrast, overexpression of RAGE is associated with enhanced autophagy, diminished apoptosis and greater tumor cell viability. RAGE limits apoptosis through a p53-dependent mitochondrial pathway. Moreover, RAGE-sustained autophagy is associated with decreased phosphorylation of mammalian target of rapamycin (mTOR) and increased Beclin-1/VPS34 autophagosome formation. These findings show that the inflammatory receptor, RAGE, has a heretofore unrecognized role in the tumor cell response to stress. Furthermore, these studies establish a direct link between inflammatory mediators in the tumor microenvironment and resistance to programmed cell death. Our data suggest that targeted inhibition of RAGE or its ligands may serve as novel targets to enhance current cancer therapies.

Cyclic AMP and cyclic GMP suppress TNFalpha-induced hepatocyte apoptosis by inhibiting FADD up-regulation via a protein kinase A-dependent pathway.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) suppress apoptosis in many cell types, including hepatocytes. We have previously shown that membrane-permeable cAMP and cGMP analogs attenuate tumor necrosis factor alpha plus actinomycin D (TNFalpha/ActD)-induced apoptosis in hepatocytes at a step upstream of caspase activation and cytochrome c release. Recently we have also shown that FADD levels increase 10 folds in response to TNFalpha/ActD. Therefore we hypothesized that cAMP and cGMP would inhibit FADD upregulation. We show here that cyclic nucleotide analogs dibutyryl cAMP (db-cAMP) and 8-bromo-cGMP (Br-cGMP) inhibit cell death and the cleavages of multiple caspases including caspase-10, -9, -8, -3, and -2, as well as suppress FADD protein up-regulation in TNFalpha/ActD-induced apoptosis. The inhibitory effects of cAMP were seen at lower concentrations than cGMP. Both cAMP and cGMP prevented FADD overexpression and cell death in hepatocytes transfected with the FADD gene. A protein kinase A (PKA) inhibitor, KT 5720, reversed the inhibition of FADD protein levels induced by cAMP or cGMP. In conclusion, our findings indicate that cAMP and cGMP prevent TNFalpha/ActD-induced apoptosis in hepatocytes and that this occurs in association with a near complete inhibition of the upregulation of FADD via a PKA-dependent mechanism.

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes.

Hepatocytes are capable of repeated inducible NO synthase (iNOS) expression, which occurs under inflammatory and stress conditions. This iNOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of iNOS in these cells, we characterized the iNOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of iNOS in cytokine-stimulated hepatocytes and that the iNOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released iNOS to the soluble fraction. iNOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted iNOS exhibits a lower specific activity than iNOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric iNOS without a greater relative distribution of iNOS to the peroxisomal pool. Thus, iNOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric iNOS. iNOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme.

Maternal stress response associated with caesarean delivery under general and epidural anaesthesia.

The maternal stress response to caesarean delivery with either general or epidural anaesthesia was investigated. Patients given a general anaesthetic showed statistically significant increases in blood pressure, heart rate, and levels of plasma catecholamines, cortisol and glucose. Epidural anaesthesia, to at least the T6 dermatome, obtunded these responses. The significance of these findings to the choice of method of anaesthesia is discussed.

Combined treatment with ranitidine and saline antacids prior to obstetric anaesthesia.

Ranitidine 150 mg was given to 126 patients requiring elective Caesarean section under general anaesthesia: 43 women had ranitidine alone, 43 had this supplemented by a pre-induction dose of sodium citrate and 40 patients had ranitidine plus sodium bicarbonate. All three sub-groups provided satisfactory gastric pH and volume. Ranitidine 150 mg was given orally every 6 hours to women in labour. Of 221 patients requiring general anaesthesia during labour, 103 women received 30 ml 0.3 M sodium citrate and 118 women, 20 ml of 8.4% sodium bicarbonate 10 minutes before induction of anaesthesia. In the citrate sub-group there was one patient with a gastric pH less than 2.5 (mean pH 6.2, SEM 0.13 range 2.1-8.4). In the bicarbonate sub-group the lowest gastric acidity was 3.8 (mean pH 8.3, SEM 0.11 range 3.8-9.83).

The influence of age on the onset of anaesthesia with midazolam.

There was a considerable individual variation in response to a standard induction dose of 0.3 mg/kg midazolam in unpremedicated patients. This is shown by variations in time to onset of sleep and the fact that about one quarter of subjects did not lose consciousness in 3 minutes. In 166 fit patients a good negative correlation was demonstrated between the age of the patients and time to loss of consciousness. Patients over 50 years differed from younger patients in a greater reliability of effect and significantly shorter induction time. In elderly patients midazolam is a more reliable induction agent than in the young.

Postanesthetic shivering in primates: inhibition by peripheral heating and by taurine.

There has been little research on the cause(s) of postanesthetic shivering (PAS) and on specific interventions. Therefore, the authors investigated PAS in eight unoperated squirrel monkeys anesthetized with halothane-nitrous oxide mixture. Shivering developed in all monkeys in which body temperature was allowed to decrease (mean +/- SEM, 2.8 +/- 0.6 degrees C) during anesthesia. Shivering occurred in 25% of animals in which body temperature was actively maintained at preanesthetic levels during anesthesia. No shivering occurred in animals warmed both during and after anesthesia. Application of radiant heat to the skin stopped PAS immediately, even though deep body temperature remained low; shivering resumed within seconds after this heating was discontinued. Intracerebroventricular (0.1-2 mg) and intravenous (100 mg/kg) administration of the putative inhibitory neurotransmitter taurine also stopped the shivering in preliminary experiments, but central injection of alpha-melanocyte stimulating hormone (100-300 micrograms), an endogenous antipyretic, did not. The results implicate reduced body temperature and activation of central heat production pathways as major factors in PAS and suggest that halothane-nitrous oxide anesthesia per se, elevation of the thermal set-point, and surgical procedures are not essential to the shivering phenomenon. The results suggest for future study two methods to control PAS: application of radiant heat or administration of taurine.

Diclofenac sodium versus fentanyl for analgesia in laparoscopic sterilization.

The effectiveness of an anaesthetic technique employing diclofenac sodium as an analgesic given preoperatively by intramuscular injection was compared against one employing intravenous fentanyl in patients undergoing laparoscopic sterilization. Postoperative pain was marked and both drugs provided partial relief only. Patients in the diclofenac group had pain scores that were initially higher than those in the fentanyl group and the difference between the groups was statistically significant (P < 0.02). Patients in the diclofenac group who received postoperative supplemental morphine analgesia recorded lower pain scores at 30 min than comparable patients in the fentanyl group (P < 0.03). These findings suggest that neither drug provides sufficient analgesia for laparoscopic sterilization when given as a sole analgesic. Investigation of a combined analgesic technique employing morphine and a non-steroidal anti-inflammatory drug is warranted.

PIP: Pain is a marked feature of laparoscopic tubal ligation. Effective postoperative analgesia would make the procedure more acceptable for day-case anaesthesia. An analgesic suitable for day-case anaesthesia needs to provide analgesia without prolonging recovery or producing other adverse events such as nausea and vomiting. In the attempt to identify such an analgesic, the authors obtained informed oral consent from 80 patients to compare the effectiveness of diclofenac sodium versus fentanyl for analgesia in laparoscopic sterilization. Both drugs were administered preoperatively by intramuscular injection to the gluteal region, diclofenac sodium to 40 women and fentanyl to the remaining 40. Patients were subsequently sterilized by application of Falope rings to the Fallopian tubes. Postoperative pain was marked and both drugs provided only partial relief. Patients in the diclofenac group had pain scores which were initially higher than those in the fentanyl group and the difference between the groups was statistically significant. Patients in the diclofenac group who received postoperative supplemental morphine analgesia recorded lower pain scores at 30 minutes than comparable patients in the fentanyl group. Findings suggest that neither drug provides sufficient analgesia for laparoscopic sterilization when given as a sole analgesic. This warrants the investigation into a combined analgesic technique employing morphine and a nonsteroidal anti-inflammatory drug.

The kinetic and spectral characterization of the E. coli-expressed mammalian CYP4A7: cytochrome b5 effects vary with substrate.

The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins. CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E. coli by adding the first 10 codons of CYP17alpha producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies. CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min(-1) and corresponding Km values of 74.5, 27, and 16.7 microM, respectively, in the presence of cytochrome b5. In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min(-1) and corresponding Km values of 3.9 and 33 microM, respectively. Arachidonate was not metabolized in the absence of cytochrome b5. Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min(-1) and Km values of 74 and 25 microM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role. Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (KS) in the absence and presence of cytochrome b5 (13 and 43 microM, respectively). In stopped-flow kinetics experiments, the flavin reduction (approximately 90 s(-1)) and heme reduction (approximately 9 s(-1)) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5. Estimations of the rate of CPR (0.3 s(-1)) or cytochrome b5 (9.1 s(-1)) binding with CYP4A7 were also determined.

Influence of plasma proteins on the onset of hypnotic action of intravenous midazolam.

The action of midazolam is influenced by serum protein binding as seen in the relationship between the time of onset of action of a fixed dose of the drug and the plasma albumin. Pretreatment with intravenous aspirin produces a decrease in the in vitro binding of midazolam. In vivo this is manifest as an increase in the rapidity of action of the benzodiazepines. Probenecid pretreatment will also cause a decrease in the onset time of midazolam. However, this is not due to altered plasma protein binding of the sedative.

Expressed CYP4A4 metabolism of prostaglandin E(1) and arachidonic acid.

Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their omega-hydroxylated products. Although the physiological function of this enzyme is unknown, prostaglandins play an important role in the regulation of reproductive, vascular, intestinal, and inflammatory systems and 20-hydroxyeicosatetraenoic acid, the omega-hydroxylated product of arachidonate, is a potent vasoconstrictor. Therefore, it is important to obtain sufficient quantities of the protein for kinetic and biophysical characterization. A CYP4A4 construct was prepared and expressed in Escherichia coli. The enzyme was purified, and its activity with substrates prostaglandin E(1) (PGE(1)) and AA was examined in the presence and absence of cytochrome b(5) (cyt b(5)) and with a heme-depleted form of cyt b(5) (apo b(5)). The stimulatory role played by cyt b(5) in this system is not dependent on electron transfer from cyt b(5) to the CYP4A4 as similar stimulation was observed with apo b(5). Rapid kinetic measurement of CYP4A4 electron transfer rates confirmed this result. Both flavin and heme reduction rates were constant in the absence and presence of cyt b(5) or apo b(5). CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b(5) or apo b(5). Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydroxy-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-UDYA) were used to probe the substrate-binding pocket of CYP4A4. The short-chain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE(1) or AA metabolism. All but 10-UDYA were effective inhibitors of CYP4A4.

Identification of unique amino acids that modulate CYP4A7 activity.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

Nitric oxide from the inducible nitric oxide synthase (iNOS) increases the expression of cytochrome P450 2E1 in iNOS-null hepatocytes in the absence of inflammatory stimuli.

Nitric oxide (NO) can modulate numerous genes through several pathways, yet some genes may be modulated only in the presence of the inflammatory stimuli that upregulate the inducible nitric oxide synthase (iNOS) rather than by NO alone. Furthermore, the role of prior expression of iNOS in the modulation of genes by NO is unknown. We addressed these issues in hepatocytes harvested from iNOS-null (iNOS(-/-)) mice exposed to NO by treatment with NO donors or by infection with an adenovirus-expressing human iNOS (Ad-iNOS), rather than by stimulation with inflammatory cytokines. Differential display and gene array analyses performed on mRNA derived from iNOS(-/-) hepatocytes demonstrated that infection with Ad-iNOS, but not infection with a control adenovirus expressing the beta-galactosidase gene (Ad-LacZ), induced a gene fragment identical to cytochrome P450 2E1 (CYP2E1). Northern analysis performed with this fragment demonstrated that treatment of iNOS(-/-) hepatocytes with Ad-iNOS or with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not control treatment or infection with Ad-LacZ, resulted in increased expression of CYP2E1. Inhibition of soluble guanylyl cyclase partially blocked the induction of CYP2E1 mRNA by Ad-iNOS. Rat hepatocytes treated with SNAP also exhibited increased expression of CYP2E1 mRNA. Preliminary studies, however, suggest that the induction of CYP2E1 in the rat hepatocytes treated with cytokines was not reduced in the presence of a NOS inhibitor. Our results suggest that CYP2E1 can be induced solely by NO derived from iNOS, at least partly in a cyclic GMP-dependent manner and independently of inflammatory stimuli or of prior exposure to NO.