The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.     

In vivo surveillance and elimination of teratoma-forming human embryonic stem cells with monoclonal antibody 2448 targeting annexin A2

This study describes the use of a previously reported chimerised monoclonal antibody (mAb), ch2448, to kill human embryonic stem cells (hESCs) in vivo and prevent or delay the formation of teratomas. ch2448 was raised against hESCs and was previously shown to effectively kill ovarian and breast cancer cells in vitro and in vivo. The antigen target was subsequently found to be Annexin A2, an oncofetal antigen expressed on both embryonic cells and cancer cells. Against cancer cells, ch2448 binds and kills via antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-drug conjugate (ADC) routes. Here, we investigate if the use of ch2448 can be extended to hESC. ch2448 was found to bind specifically to undifferentiated hESC but not differentiated progenitors. Similar to previous study using cancer cells, ch2448 kills hESC in vivo either indirectly by eliciting ADCC or directly as an ADC. The treatment with ch2448 post-transplantation eliminated the in vivo circulating undifferentiated cells and prevented or delayed the formation of teratomas. This surveillance role of ch2448 adds an additional layer of safeguard to enhance the safety and efficacious use of pluripotent stem cell-derived products in regenerative medicine. Thereby, translating the use of ch2448 in the treatment of cancers to a proof of concept study in hESC (or pluripotent stem cell [PSC]), we show that mAbs can also be used to eliminate teratoma forming cells in vivo during PSC-derived cell therapies. We propose to use this strategy to complement existing methods to eliminate teratoma-forming cells in vitro. Residual undifferentiated cells may escape in vitro removal methods and be introduced into patients together with the differentiated cells.     

Acute exposure to 50 Hz magnetic field does not affect hematologic or immunologic functions in healthy young men: A circadian study

Some epidemiological studies report a relationship between magnetic field exposure and such human diseases as leukemia and immune system disturbances. The few published studies on animals do not demonstrate field exposure-related alterations in hematologic and immune systems. The data presented here are part of a broader study designed to investigate the possible effects of acute exposure to a 50 Hz linearly polarized magnetic field (10 μT) on hematologic and immunologic functions. Thirty-two young men (20–30 years old) were divided into two groups (control group, i.e., sham-exposed, 16 subjects; exposed group, 16 subjects). All subjects participated in two 24 h experiments to evaluate the effects of both continuous and intermittent (1 h “off” and 1 h with the field switched “on” and “off” every 15 s) exposure to linearly polarized magnetic fields. The subjects were exposed to the magnetic field (generated by three Helmholtz coils per bed) from 23:00 to 08:00 while lying down. Blood samples were collected during each session at 3 h intervals from 11:00 to 20:00 and hourly from 22:00 to 08:00. No significant differences were observed between sham-exposed (control) and exposed men for hemoglobin concentration, hematocrit, red blood cells, platelets, total leukocytes, monocytes, lymphocytes, eosinophils, or neutrophils. Immunologic variables [CD3, CD4, CD8, natural killer (NK) cells and B cells] were unaltered. To our knowledge, this study is the first to document the effects of a 50 Hz magnetic field on the circadian rhythm of human hematologic and immune functions, and it suggests that acute exposure to either a continuous or an intermittent 50 Hz linearly polarized magnetic field of 10 μT, at least under the conditions of our experiment, does not affect either these functions or their circadian rhythms in healthy young men. © 1996 Wiley-Liss, Inc.     

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K_M, V_max, K_D, and k_obs values for different sugar substrates. K_D and k_obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H⁺ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H⁺ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s⁻¹) and for sugar translocation (2 s⁻¹ − 30 s⁻¹ for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.     

Roles of traditional medicine and traditional healers for rabies prevention and potential impacts on post-exposure prophylaxis: A literature review

IntroductionGlobally, traditional medicine is widely used to treat a variety of injuries and illnesses, including dog bites, and exposures that are risky for rabies. However, efficacy of most traditional remedies used for rabies prevention or treatment has not been demonstrated in controlled trials or proven in community-based surveys.MethodsSix databases were searched including the terms rabies, traditional treatment, traditional remedy, traditional therapy, traditional medicine, and medicinal treatment to review traditional remedies used in the prevention and treatment of rabies. In addition, published literature of rabies transmission dynamics was used to estimate statistical likelihood of dog bite victims developing rabies to provide clarity as to why traditional healers have a high apparent success rate when preventing death from rabies in victims bitten by suspected rabid dogs.ResultsLiterature review yielded 50 articles, including three controlled experiments, that described use of traditional remedies for rabies prevention and treatment. Traditional remedies for rabies ranged from plant- or animal-based products to spiritual rituals; however, only a few controlled mice trials were conducted, and none of these trials demonstrated efficacy in preventing or treating rabies. Risk of dying from rabies after a bite from a dog with unknown rabies status is low, 1.90% (0.05%-29.60%). Therefore, traditional healers had a 98.10% (70.40%-99.95%) apparent success rate in preventing death from suspected rabid dog bites despite inefficaciousness of herbal remedies.ConclusionThere was no universal plant species or route of administration that was consistently used for rabies prevention or treatment across countries. No traditional remedy was efficacious in the prevention or treatment of rabies in randomized controlled experiments. Understanding the cultural context under which traditional remedies are used may facilitate collaboration of traditional healers with the modern medical system to ensure timely and appropriate use of proven therapies for prevention and clinical management of rabies.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

L-Homocysteine and L-homocystine stereospecifically induce endothelial nitric oxide synthase-dependent lipid peroxidation in endothelial cells

Atherothrombotic cardiovascular disease associated with hyperhomocysteinemia has been proposed to result, at least in part, from increased vascular oxidative stress. Here we characterize one mechanism by which homocyteine may induce a vascular cell type-specific oxidative stress. Our results show that L-homocysteine at micromolar levels stereospecifically increases lipid peroxidation in cultured endothelial cells, but not in vascular smooth muscle cells or when medium is incubated in the absence of cells. Consistent with these observations, homocysteine also increases the formation of intracellular reactive oxygen species. The pro-oxidant effect of homocysteine can be fully replicated by an equivalent concentration of homocystine (i.e., an oxidized form of homocysteine), but not with cysteine or glutathione. Homocyst(e)ine-dependent lipid peroxidation is independent of H(2)O(2) and alterations in glutathione peroxidase activity, but dependent on superoxide. Mechanistically, the pro-oxidant effect of homocysteine appears to involve endothelial nitric oxide synthase (eNOS), as it is blocked by the eNOS inhibitor L-N(G)-nitroarginine methyl ester. Thus, homocyst(e)ine actively promotes oxidative stress in endothelial cells via an eNOS-dependent mechanism.     

Epithelial trafficking: new routes to familiar places

Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.