Alteration in the redox state of plasma in heart-transplant patients with moderate hyperhomocysteinemia

Hyperhomocysteinemia has recently been suggested to contribute to the progression of the so-called chronic rejection or cardiac allograft vasculopathy (CAV) in heart-transplant patients in which the major determinant of the increase in homocysteine (Hcy) was the progressive decline of renal function. The exact mechanisms of tissue injury by Hcy is unknown, but some aspects of its toxicity have been related to its capacity for altering the redox state of plasma and forming protein adducts by intermediate lactone. To study the relationships between Hcy levels and variations in the redox state governed by thiols, plasma levels of Hcy, cysteine, glutathione, cysteinylglycine, and corresponding disulfides and protein-mixed disulfides were evaluated in subjects with moderate hyperhomocysteinemia represented by heart-transplant patients with (HTRF) and without (HT) renal failure, as well as patients with renal failure of different origin (RF), and compared with those of a control group (C) of normal subjects matched for age and sex. Plasma levels of Hcy and the corresponding protein mixed disulfides increased progressively in HTs, RFs, and HTRFs with respect to control. These changes were correlated with cysteine variations (as cystine and protein-mixed disulfides) but not with glutathione or cysteinylglycine that varied only as disulfides with a similar tendency. Moreover, an alteration in the plasma redox was evidenced by the decrease in thiol/disulfide ratios of cysteine, Hcy, and cysteinylglycine. In all groups, cysteine was directly correlated with Hcy but not with glutathione or cysteinylglycine, which in turn were correlated each other. Therefore levels of plasma cysteine were more linked to Hcy than to metabolism of glutathione. The clinical meaning of cysteine changes remains undefined and requires further study.     

Repeated administration of a Fusarium mycotoxin butenolide to rats induces hepatic lipid peroxidation and antioxidant defense impairment

Butenolide, a mycotoxin elaborated by several toxigenic Fusarium species, frequently contaminates important agricultural products, and has been considered a potential health risk to humans and animals. However, many toxicology issues including toxicity targets and mechanisms of butenolide remain unclear. Previous study indicated that acute butenolide exposure produced hepatic oxidative toxicity, but its chronic toxicity is still unknown. The present study therefore attempted to reveal the adverse effects of repeated butenolide exposure from a viewpoint of oxidative damage focusing on the liver. Intragastic administration of rats with butenolide for seven consecutive weeks resulted in hepatic injury as shown by obvious changes of serum biochemistry parameters indicating liver function. Repeated butenolide exposure also induced oxidative stress as manifested by impairment of antioxidant defenses including depletion of sulfhydryl groups and reduction of glutathione peroxidase activity, and enhancement of lipid peroxidation both in serum and liver. In conclusion, the present study indicated that repeated butenolide exposure induced a significant liver injury, and oxidative damage may serve as a mediator in the toxicity of butenolide. The current findings contribute to the understanding of the toxic profile of butenolide.     

Pancreatic stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction

Background & Aims: Patients with sphincter of Oddi dysfunction are at high risk of developing pancreatitis after endoscopic biliary sphincterotomy. Impaired pancreatic drainage caused by pancreatic sphincter hypertension is the likely explanation for this increased risk. A prospective, randomized controlled trial was conducted to determine if ductal drainage with pancreatic stenting protects against pancreatitis after biliary sphincterotomy in patients with pancreatic sphincter hypertension. Methods: Eligible patients with pancreatic sphincter hypertension were randomized to groups with pancreatic duct stents (n = 41) or no stents (n = 39) after biliary sphincterotomy. The primary measured outcome was pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP). Results: Pancreatic stenting significantly decreased the risk of pancreatitis from 26% to 7% (10 of 39 in the no stent group and 3 of 41 in the stent group; P = 0.03). Only 1 patient in the stent group developed pancreatitis after sphincterotomy, and 2 others developed pancreatitis at the time of stent extraction. Patients in the no stent group were 10 times more likely to develop pancreatitis immediately after sphincterotomy than those in the stent group (relative risk, 10.5; 95% confidence interval, 1.4–78.3). Conclusions: Pancreatic duct stenting protects significantly against post-ERCP pancreatitis in patients with pancreatic sphincter hypertension undergoing biliary sphincterotomy. Stenting of the pancreatic duct should be strongly considered after biliary sphincterotomy for sphincter of Oddi dysfunction; pancreatic sphincter of Oddi manometry identifies which high-risk patients may benefit from pancreatic stenting.GASTROENTEROLOGY 1998;115:1518-1524     

Humoral and cytokine response during protection of mice against secondary hydatidosis caused by Echinococcus granulosus

Infection of BALB/c mouse with protoscoleces of Echinococcus granulosus constitutes a model for the study of secondary hydatidosis and the associated immune response in immunization and infection trials. The aims of this study were to induce a protective immunity against secondary hydatidosis using conventional vaccination approaches and to analyse the immune responses that accompany this protection. Mice immunized with antigen B (AgB), a component of crude sheep hydatid fluid (CSHF), showed a significant level of protection as indicated by a 98.3% reduction in cyst load. This reduction in cyst development was accompanied by a high concentration of interferon gamma secreted by antigen-stimulated spleen cells, as compared with those secreted by cells of mice immunized with CSHF or protoscoleces homogenate (PSH) antigens. In contrast, interleukin-4 was significantly higher in the supernatants of cells stimulated with CSHF or PSH compared with AgB (191.5, 195.7 and 127.5 pg, respectively). Kinetic analysis of immunoglobulin subclasses showed persistently high levels of IgG1 and IgG2a subclasses in immunized infected animals until 6 months of infection, whereas IgG3 showed a significant decline after 1 month of infection. In infected non-immunized control mice, all IgG subclasses showed a gradual increase after the first month of infection until the experiment termination (8 months after infection).     

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH

Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer’s disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aβ42, Aβ40, and Aβ38. The molar ratio of the cleavage products Aβ42 over Aβ40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aβ42/Aβ40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase.Amyloid precursor protein (APP) is initially cleaved by β-secretase in the extracellular space, producing a membrane-tethered, 99-residue carboxyl-terminal fragment known as APP C99 (1). APP C99 then undergoes sequential cleavages by γ-secretase, first at the ε sites, yielding Aβ49/Aβ48, and eventually at the γ-sites, generating Aβ42/Aβ40/Aβ38 (2–4) (Fig. 1A). The 230-kDa γ-secretase contains four components: presenilin (PS), Pen-2, Aph-1, and nicastrin (NCT), of which PS1 is the target of most mutations derived from early-onset familial Alzheimer’s disease (FAD) patients. Rather than abolishing the protease activity of γ-secretase, these mutations are thought to increase the molar ratio of Aβ42 over Aβ40 (2).Open in a separate windowFig. 1.Cleavage of the amyloid precursor protein (APP) C99 fragment by the archaeal presenilin homolog PSH. (A) A schematic diagram of the APP C99 substrate. The substrate is tagged by a maltose-binding protein (MBP) at the amino terminus and eight histidine residues (8xHis) at the carboxyl terminus. Some of the known γ-secretase cleavage sites are indicated. (B) APP C99 is cleaved by PSH, and the cleavage is inhibited by the γ-secretase–specific inhibitor III-31C. Cleavage of APP C99 produced a 50-kDa amino-terminal fragment and a 10-kDa carboxyl-terminal fragment. Shown here is a SDS-PAGE stained by Coomassie blue. (C) Quantitation of III-31C–mediated inhibition of PSH cleavage reveals an inhibitory constant of ∼10 μM. (D) The carboxyl-terminal cleavage fragment of APP C99 was analyzed by amino-terminal peptide sequencing. The results revealed a major species, LVMLK, and a minor species, VMLKK. Thus, the first cleavages in APP C99 occur either between Thr48 and Leu49, producing Aβ48, or between Leu49 and Val50, generating Aβ49.Among the cleavage products of γ-secretase, Aβ42 is particularly prone to aggregation, leading to formation of β-amyloid plaque in the brain and presumably causing Alzheimer’s disease (3). Other FAD-derived mutations map to APP and PS2, lending support to the causal relationship between formation of β-amyloid plaque and Alzheimer’s disease (5). Therapeutic intervention of Alzheimer’s disease may directly benefit from in vitro investigation of γ-secretase and discovery of its potential modulators (6). Unfortunately, such effort has been hampered by the difficulty in expression, purification, and manipulation of the complex protease. This problem persists despite recent breakthroughs in structural elucidation of γ-secretase and nicastrin (7, 8).The intramembrane aspartate protease PSH from the archaeon Methanoculleus marisnigri JR1 shares 19% sequence identity and 53% sequence similarity with human PS1 (hPS1). The signature motifs for catalysis, ΦYDΦΦ (Φ for a hydrophobic residue) on transmembrane segment 6 (TM6) and ΦGΦGD on TM7, are identical between PSH and hPS1. PSH can be readily overexpressed in Escherichia coli and purified in large quantity (9, 10). Importantly, the crystal structure of PSH is available and offers a valuable guide for the design and improvement of protease modulators (10). Therefore, PSH may represent an attractive surrogate of γ-secretase for modulator screening if PSH cleaves APP C99 and such cleavages recapitulate those by the intact γ-secretase. Notably, due to the presence of two different presenilin variants (hPS1 and hPS2) and two Aph-1 variants (Aph-1A and Aph-1B), there are at least four distinct human γ-secretase complexes, which exhibit different signature Aβ profiles (11). In this study, we compare PSH to the most prevalent γ-secretase complex comprising hPS1 and Aph-1A.     

Survey of provider perceptions of enhanced recovery after surgery and perioperative surgical home protocols at a tertiary care hospital

Enhanced recovery after surgery (ERAS) and perioperative surgical home (PSH) initiatives are widely utilized to improve quality of patient care. Despite their established benefits, implementation still has significant barriers. We developed a survey for perioperative clinicians to gather information on perception and knowledge of ERAS/PSH programs to guide future expansion of these programs at our institution. The survey included questions about familiarity with ERAS/PSH and perceived value, perceived barriers to protocol implementation, preferred learning methods and prioritization of various ERAS/PSH protocol elements into care delivery and provider education. Faculty surgeons and anesthesiologists, in addition to advanced practice nurses and postgraduate physician trainees in the Departments of Surgery and Anesthesiology were asked to complete the survey. Overall survey participation was 25% (223/888). About half of survey respondents had provided care to a patient on an ERAS/PSH protocol, and a majority felt at least somewhat knowledgeable about ERAS/PSH protocols. Perception of the value of ERAS/PSH was positive. Participants were enthusiastic about on-going learning, with multimodal pain management being the topic of most interest and learning by direct participation in care of protocol patients being the favored educational approach. A significant majority of participants felt that upcoming health providers should receive formal ERAS/PSH education as part of their training. Based on our survey results, we plan to explore teaching methods that successfully engage learners of all levels of clinical expertise and also overcome the major barriers to gaining knowledge about ERAS/PSH identified by study participants, most notably lack of time for busy clinicians.     

Contingent and non-contingent effects of low-dose ethanol on GABA neuron activity in the ventral tegmental area

Ventral tegmental area (VTA) GABA neurons appear to be critical regulators of mesocorticolimbic dopamine (DA) neurotransmission, which has been implicated in alcohol reward. The aim of this study was to evaluate the effects of low-dose “non-contingent” intravenous (IV) ethanol (0.01-0.1 g/kg) on VTA GABA neuron firing rate and synaptic responses, as well as VTA GABA neuron firing rate during low-dose “contingent” IV ethanol self-administration. Intravenous administration of 0.01-0.03 g/kg ethanol significantly increased VTA GABA neuron firing rate and afferent-evoked synaptic responses. In the runway self-administration paradigm, presentation of an olfactory cue (S+; almond extract) or no-cue (S−; no odor) in the Start box was paired with IV administration of low-dose ethanol (0.01 g/kg) or saline in the Target box. Runway excursion times decreased significantly in association during S+, and increased significantly during S− conditions. The firing rate of VTA GABA neurons markedly increased when rats received 0.01 g/kg IV ethanol in the Target box. VTA GABA neuron firing increased in the Start box of the runway in association with S+, but not S−. These findings demonstrate that VTA GABA neurons are activated by low-dose IV ethanol and that their firing rate increases in anticipation of ethanol reward.