ABSTRACT
We investigate whether paradigmatic measurements for quantum state tomography, namely mutually unbiased bases and symmetric informationally complete measurements, can be employed to certify quantum correlations. For this purpose, we identify a simple and noise-robust correlation witness for entanglement detection, steering, and nonlocality that can be evaluated based on the outcome statistics obtained in the tomography experiment. This allows us to perform state tomography on entangled qutrits, a test of Einstein-Podolsky-Rosen steering and a Bell inequality test, all within a single experiment. We also investigate the trade-off between quantum correlations and subsets of tomographically complete measurements as well as the quantification of entanglement in the different scenarios. Finally, we perform a photonics experiment in which we demonstrate quantum correlations under these flexible assumptions, namely with both parties trusted, one party untrusted and both parties untrusted.
ABSTRACT
The Heisenberg scaling, which scales as N^{-1} in terms of the number of particles or T^{-1} in terms of the evolution time, serves as a fundamental limit in quantum metrology. Better scalings, dubbed as "super-Heisenberg scaling," however, can also arise when the generator of the parameter involves many-body interactions or when it is time dependent. All these different scalings can actually be seen as manifestations of the Heisenberg uncertainty relations. While there is only one best scaling in the single-parameter quantum metrology, different scalings can coexist for the estimation of multiple parameters, which can be characterized by multiple Heisenberg uncertainty relations. We demonstrate the coexistence of two different scalings via the simultaneous estimation of the magnitude and frequency of a field where the best precisions, characterized by two Heisenberg uncertainty relations, scale as T^{-1} and T^{-2}, respectively (in terms of the standard deviation). We show that the simultaneous saturation of two Heisenberg uncertainty relations can be achieved by the optimal protocol, which prepares the optimal probe state, implements the optimal control, and performs the optimal measurement. The optimal protocol is experimentally implemented on an optical platform that demonstrates the saturation of the two Heisenberg uncertainty relations simultaneously, with up to five controls. As the first demonstration of simultaneously achieving two different Heisenberg scalings, our study deepens the understanding on the connection between the precision limit and the uncertainty relations, which has wide implications in practical applications of multiparameter quantum estimation.
ABSTRACT
An e-waste dismantling industrial park of Taizhou was selected as the sampling center, within a radius of 16 km, and a total of 30 sampling sites were designed in three circles as follows: C (3 km), S (5-10 km) and R (10-16 km). Pollution characteristics and ecological risk of polybrominated diphenyl ethers (PBDEs) in water and sediments were investigated. The concentrations of PBDEs in water ranged from 9.4 to 57.2 ng · L⻹, with a mean value of 25.9 ng · L⻹; and 3.7 to 38,775 ng · g⻹, with an average of 2 779 ng · g⻹ in sediments. BDE-209 was the predominant congener. The spatial distribution patterns of PBDE levels in water and sediment were both in the following order: C > S > R. Furthermore, the concentrations of PBDEs in sediments showed significant negative correlation against the distance from the industrial park (P < 0.01). Compared with other regions around the world, the PBDEs contamination was more serious in the area, which indicated that e-waste dismantling activity was one of the significant sources for PBDEs pollution. It was estimated that a total of 30. 7 t PBDEs (including 28. 9 t BDE- 209) was discharged into surrounding environment as a result of dismantling industrial activities in last 40 years. A preliminary ecological risk assessment for PBDEs in water and sediments was conducted by hazard quotient method. The results demonstrated that the Penta-BDEs in the center of e-waste dismantling area ( a radius of 1.5 km) was at particularly high risk level and could cause serious influence on the ecological safety and human health.
Subject(s)
Electronic Waste , Environmental Monitoring , Halogenated Diphenyl Ethers/analysis , Water Pollutants, Chemical/analysis , China , Ecology , Geologic Sediments/chemistry , Humans , Industrial Waste , Risk Assessment , Water/chemistryABSTRACT
Angiogenesis has emerged as an important therapeutic target in several major diseases, including cancer and age-related macular degeneration. The zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. In this study, we have taken advantage of the transgenic Tg (fli1a:EGFP) zebrafish line to screen the U.S. Drug Collection Library and identified 11 old drugs with antiangiogenic activity, including Closantel, an FDA-approved broad-spectrum salicylanilide antiparasitic drug for a variety of types of animals. Closantel was confirmed to have antiangiogenic activity in zebrafish with a half-inhibitory concentration (IC50) at 1.69 µM on the intersegmental vessels and 1.45 µM on the subintestinal vessels. Closantel also markedly suppressed cancer growth in zebrafish xenotransplanted with human lymphoma, cervical cancer, pancreatic cancer, and liver cancer cells, generally in a dose-dependent manner. These data reveal that Closantel has antiangiogenesis and anticancer effects and could be a potential drug candidate for animal and human cancer treatments. Further study is needed to clarify the mechanisms involved in the antiangiogenesis and anticancer effects of Closantel.
ABSTRACT
Resolvin D1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) (RvD1), generated from ω-3 fatty docosahexaenoic acids, is believed to exert anti-inflammatory properties including inhibition of neutrophil activation and regulating inflammatory cytokines. In this study, we sought to investigate the effect of RvD1 in modulating alveolar fluid clearance (AFC) on LPS-induced acute lung injury. In vivo, RvD1 was injected i.v. (5 µg/kg) 8 h after LPS (20 mg/kg) administration, which markedly stimulated AFC in LPS-induced lung injury, with the outcome of decreased pulmonary edema. In addition, rat lung tissue protein was isolated after intervention and we found RvD1 improved epithelial sodium channel (ENaC) α, γ, Na,K-adenosine triphosphatase (ATPase) α1, ß1 subunit protein expression and Na,K-ATPase activity. In primary rat alveolar type II epithelial cells stimulated with LPS, RvD1 not only upregulated ENaC α, γ and Na,K-ATPase α1 subunits protein expression, but also increased Na+ currents and Na,K-ATPase activity. Finally, protein kinase A and cGMP were not responsible for RvD1's function because a protein kinase A inhibitor (H89) and cGMP inhibitor (Rp-cGMP) did not reduce RvD1's effects. However, the RvD1 receptor (formyl-peptide receptor type 2 [FPR2], also called ALX [the lipoxin A4 receptor]) inhibitor (BOC-2), cAMP inhibitor (Rp-cAMP), and PI3K inhibitor (LY294002) not only blocked RvD1's effects on the expression of ENaC α in vitro, but also inhibited the AFC in vivo. In summary, RvD1 stimulates AFC through a mechanism partly dependent on alveolar epithelial ENaC and Na,K-ATPase activation via the ALX/cAMP/PI3K signaling pathway.
Subject(s)
Acute Lung Injury/immunology , Acute Lung Injury/metabolism , Alveolar Epithelial Cells/metabolism , Cyclic AMP/metabolism , Docosahexaenoic Acids/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Lipoxin/metabolism , Signal Transduction/drug effects , Sodium-Potassium-Exchanging ATPase/metabolism , Acute Lung Injury/genetics , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/immunology , Animals , Docosahexaenoic Acids/administration & dosage , Enzyme Activation/drug effects , Epithelial Sodium Channels/genetics , Epithelial Sodium Channels/metabolism , Gene Expression Regulation/drug effects , Lipopolysaccharides/immunology , Lung/drug effects , Lung/immunology , Lung/metabolism , Lung/pathology , Male , Rats , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
The zebrafish is increasingly used as a vertebrate animal model for in vivo drug discovery and for assessing chemical toxicity and safety. Numerous studies have confirmed that zebrafish and mammals are similar in their physiology, development, metabolism and pathways, and that zebrafish responses to toxic substances are highly predictive of mammalian responses. Developmental and reproductive toxicity assessments are an important part of new drug safety profiling. A significant number of drug candidates have failed in preclinical tests due to their adverse effect on development and reproductivity. Compared to conventional mammal testing, zebrafish testing for assessing developmental and reproductive toxicity offers several compelling experimental advantages, including transparency of embryo and larva, higher throughput, shorter test period, lower cost, smaller amount of compound required, easier manipulation and direct compound delivery. Toxicity and safety assessments using zebrafish have also been accepted by the FDA and EMEA for investigative new drug (IND) approval.
Subject(s)
Models, Animal , Teratology/methods , Zebrafish/growth & development , Zebrafish/physiology , Animals , Behavior, Animal/drug effects , Drug Evaluation, Preclinical , Larva/drug effects , Larva/growth & development , Reproduction/drug effects , Teratogens/toxicity , Zebrafish/embryologyABSTRACT
Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.
Subject(s)
Cardiotoxins/toxicity , Heart Diseases/pathology , Toxicity Tests , Abnormalities, Drug-Induced/pathology , Animals , Aspirin/toxicity , Clomipramine/toxicity , Cyclophosphamide/toxicity , Disease Models, Animal , Edema/chemically induced , Edema/pathology , Gentamicins/toxicity , Heart Diseases/chemically induced , Heart Rate/drug effects , Heart Ventricles/drug effects , Heart Ventricles/physiopathology , Larva/drug effects , Microinjections , Nimodipine/toxicity , Pericardium/drug effects , Pericardium/pathology , Quinidine/toxicity , Terfenadine/toxicity , Tetracycline/toxicity , Verapamil/toxicity , Yolk Sac/drug effects , Yolk Sac/pathology , ZebrafishABSTRACT
Edema fluid resorption is critical for gas exchange, and both alveolar epithelial sodium channel (ENaC) and Na,K-ATPase are accredited with key roles in the resolution of pulmonary edema. Alveolar fluid clearance (AFC) was measured in in situ ventilated lungs by instilling isosmolar 5% BSA solution with Evans Blue-labeled albumin tracer (5 ml/kg) and measuring the change in Evans Blue-labeled albumin concentration over time. Treatment with lipoxin A4 and lipoxin receptor agonist (5(S), 6(R)-7-trihydroxymethyl 17 heptanoate) significantly stimulated AFC in oleic acid (OA)-induced lung injury, with the outcome of decreased pulmonary edema. Lipoxin A4 and 5(S), 6(R)-7-trihydroxymethyl 17 heptanoate not only up-regulated the ENaC α and ENaC γ subunits protein expression, but also increased Na,K-ATPase α1 subunit protein expression and Na,K-ATPase activity in lung tissues. There was no significant difference of intracellular cAMP level between the lipoxin A4 treatment and OA group. However, the intracellular cGMP level was significantly decreased after lipoxin A4 treatment. The beneficial effects of lipoxin A4 were abrogated by butoxycarbonyl-Phe-Leu-Phe-Leu-Ph (lipoxin A4 receptor antagonist) in OA-induced lung injury. In primary rat alveolar type II epithelial cells stimulated with LPS, lipoxin A4 increased ENaC α and ENaC γ subunits protein expression and Na,K-ATPase activity. Lipoxin A4 stimulated AFC through activation of alveolar epithelial ENaC and Na,K-ATPase.
Subject(s)
Epithelial Sodium Channel Agonists/administration & dosage , Epithelial Sodium Channels/metabolism , Lipoxins/administration & dosage , Sodium-Potassium-Exchanging ATPase/metabolism , Acute Lung Injury/drug therapy , Acute Lung Injury/immunology , Acute Lung Injury/metabolism , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/immunology , Animals , Cells, Cultured , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Epithelial Sodium Channel Agonists/pharmacology , Epithelial Sodium Channel Blockers/pharmacology , Epithelial Sodium Channels/genetics , Gene Expression/drug effects , Heptanoic Acids/pharmacology , Lipopolysaccharides/pharmacology , Lipoxins/pharmacology , Male , Mucociliary Clearance , Oligopeptides/pharmacology , Peroxidase/metabolism , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/physiopathology , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/metabolismABSTRACT
INTRODUCTION: Numerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish. METHODS: Hepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology. RESULTS: All the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish. DISCUSSION: Larval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.
Subject(s)
Chemical and Drug Induced Liver Injury/diagnosis , Chemical and Drug Induced Liver Injury/genetics , Disease Models, Animal , Phenotype , Animals , Drug-Related Side Effects and Adverse Reactions/diagnosis , Drug-Related Side Effects and Adverse Reactions/genetics , Toxicity Tests/methods , ZebrafishABSTRACT
Quantum dots (QDs) have strong adsorption capacity; therefore, their potential toxicity to aquatic organisms from the facilitated transport of other trace toxic pollutants when they coexist has received increasing interest. However, the impact of cadmium selenium (CdSe) QDs and copper ion (Cu(2+)) joint exposure on zebrafish (Danio rerio) embryo and larvae remains almost unknown. Therefore, the present study was performed to determine the developmental toxicities to zebrafish exposed to combined pollution with CdSe QDs (500 µg/L) and Cu(2+) (0, 0.1, 1, 10, and 100 µg/L CuC1(2)) compared with single exposure. Our findings for the first time revealed that: (1) QDs facilitated the accumulation of Cu(2+) in zebrafish; (2) QDs caused higher mortality, lower hatch rate, and more malformations of the exposed zebrafish; (3) junction, bifurcation, crossing, particles, and aggregation of the exposed FLI-1 transgenic zebrafish larvae can be observed; (4) embryo cell apoptosis appeared in the head and tail region; and (5) synergistic effects played an important role during joint exposure. These observations provide a basic understanding of CdSe QDs and Cu(2+) joint toxicity to aquatic organisms and suggest the need for additional research to identify the toxicological mechanism.
Subject(s)
Cadmium/toxicity , Copper/toxicity , Environmental Pollutants/toxicity , Metal Nanoparticles/toxicity , Quantum Dots , Selenium/toxicity , Zebrafish/embryology , Zebrafish/growth & development , Animals , Animals, Genetically Modified , Apoptosis , Biological Transport , Body Burden , Cadmium/metabolism , Cations, Divalent , Copper/metabolism , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryonic Development , Environmental Pollutants/metabolism , Larva/cytology , Larva/drug effects , Larva/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Selenium/metabolism , Zebrafish/abnormalities , Zebrafish/metabolismABSTRACT
The mammalian heart loses its regenerative potential soon after birth. Adult cardiac myocytes (ACMs) permanently exit the cell cycle, and E2F-dependent genes are stably silenced, although the underlying mechanism is unclear. Heterochromatin, which silences genes in many biological contexts, accumulates with cardiac differentiation. H3K9me3, a histone methylation characteristic of heterochromatin, also increases in ACMs and at E2F-dependent promoters. We hypothesize that genes relevant for cardiac proliferation are targeted to heterochromatin by retinoblastoma (Rb) family members interacting with E2F transcription factors and recruiting heterochromatin protein 1 (HP1) proteins. To test this hypothesis, we created cardiac-specific Rb and p130 inducible double knockout (IDKO) mice. IDKO ACMs showed a decrease in total heterochromatin, and cell cycle genes were derepressed, leading to proliferation of ACMs. Although Rb/p130 deficiency had no effect on total H3K9me3 levels, recruitment of HP1-γ to promoters was lost. Depleting HP1-γ up-regulated proliferation-promoting genes in ACMs. Thus, Rb and p130 have overlapping roles in maintaining the postmitotic state of ACMs through their interaction with HP1-γ to direct heterochromatin formation and silencing of proliferation-promoting genes.
Subject(s)
E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , Myocytes, Cardiac/physiology , Retinoblastoma Protein/metabolism , Retinoblastoma-Like Protein p130/metabolism , Animals , Cell Cycle , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/metabolism , Genes, Retinoblastoma/genetics , Heterochromatin , Jumonji Domain-Containing Histone Demethylases/metabolism , Mice , Mice, Knockout , Myocytes, Cardiac/cytology , Patch-Clamp Techniques , Polymerase Chain Reaction , RNA Interference , RNA, Small Interfering , Retinoblastoma Protein/genetics , Retinoblastoma-Like Protein p130/geneticsABSTRACT
In the title compound, C(10)H(9)FN(2)O(4), the dihedral angle between the benzene ring and the nitro group plane is 11.29â (3)°. The morpholinone ring adopts a twist-chair conformation. In the crystal, mol-ecules are linked by inter-molecular C-Hâ¯O hydrogen bonds into a chain along the a-axis direction.
ABSTRACT
OBJECTIVE: Cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1-like 1 has recently been linked to type 2 diabetes by genome-wide association studies. While CDK5 and its regulatory protein p35 are both expressed and display enzymatic activity in pancreatic ß-cells, their precise role in the ß-cell remains unknown. Because type 2 diabetes is characterized by a deficit in ß-cell mass and increased ß-cell apoptosis, we investigated the role of CDK5 in ß-cell survival. RESEARCH DESIGN AND METHODS: We used INS 832/13 cells, rat islets isolated from wild-type or human islet amyloid polypeptide (h-IAPP) transgenic rats, and pancreatic tissue from rats and humans with and without type 2 diabetes and investigated the effect of CDK5/p35 inhibition (by small interfering RNA or by chemical inhibition) as well as CDK5/p35 overexpression on ß-cell vulnerability to apoptosis. RESULTS: CDK5 inhibition led to increased ß-cell apoptosis. To identify the mechanisms involved, we examined the phosphorylation state of focal adhesion kinase (Fak)(Ser732), a known target of CDK5. Following CDK5 inhibition, the phosphorylation of Fak(Ser732) decreased with resulting attenuation of phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway. Conversely, CDK5 overexpression increased Fak(Ser732) phosphorylation and protected ß-cells against apoptosis induced by the inhibition of the ß-1 integrin signaling pathway. Also, Fak(Ser732) phosphorylation was less abundant in ß-cells in both h-IAPP transgenic rats and humans with type 2 diabetes. CONCLUSIONS: This study shows that by regulating Fak phosphorylation and subsequently PI3K/Akt survival pathway, CDK5 plays a previously unrecognized role in promoting ß-cell survival.
Subject(s)
Cyclin-Dependent Kinase 5/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western , Cell Line , Cell Survival/drug effects , Cell Survival/genetics , Cyclin-Dependent Kinase 5/genetics , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/metabolism , Focal Adhesion Protein-Tyrosine Kinases/genetics , Humans , In Vitro Techniques , Insulin-Secreting Cells/drug effects , Islet Amyloid Polypeptide/genetics , Islet Amyloid Polypeptide/metabolism , Proto-Oncogene Proteins c-akt/genetics , RNA, Small Interfering , Rats , Rats, Transgenic , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
OBJECTIVE: The islet in type 2 diabetes is characterized by ß-cell apoptosis, ß-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in ß-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum-associated degradation/ubiquitin/proteasome system. RESEARCH DESIGN AND METHODS: We used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated. RESULTS: We report accumulation of polyubiquinated proteins and UCH-L1 deficiency in ß-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in ß-cells induced endoplasmic reticulum stress leading to apoptosis. CONCLUSIONS: Our results indicate that defective protein degradation in ß-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises ß-cell viability.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Insulin-Secreting Cells/physiology , Islet Amyloid Polypeptide/pharmacology , Proteasome Endopeptidase Complex/metabolism , Ubiquitin Thiolesterase/deficiency , Ubiquitin/metabolism , Animals , Autopsy , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Humans , Hypoglycemic Agents/therapeutic use , Insulin/therapeutic use , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/pathology , Islet Amyloid Polypeptide/genetics , Nuclear Proteins/drug effects , Nuclear Proteins/metabolism , Obesity/complications , Obesity/pathology , Rats , Rats, Transgenic , Ubiquitin/drug effects , Ubiquitin Thiolesterase/drug effectsABSTRACT
Type 2 diabetes involves aberrant misfolding of human islet amyloid polypeptide (h-IAPP) and resultant pancreatic amyloid deposits. Curcumin, a biphenolic small molecule, has offered potential benefits in other protein misfolding diseases, such as Alzheimer's disease. Our aim was to investigate whether curcumin alters h-IAPP misfolding and protects from cellular toxicity at physiologically relevant concentrations. The effect of curcumin on h-IAPP misfolding in vitro was investigated by electron paramagnetic resonance spectroscopy, ThT fluorescence and electron microscopy. Our in vitro studies revealed that curcumin significantly reduces h-IAPP fibril formation and aggregates formed in the presence of curcumin display alternative morphology and structure. We then tested a potential protective effect of curcumin against h-IAPP toxicity on ß-cells. Micromolar concentrations of curcumin partially protect INS cells from exogenous IAPP toxicity. This protective effect, however, is limited to a narrow concentration range, as curcumin becomes cytotoxic at micromolar concentrations. In different models of endogenous over-expression of h-IAPP (INS cells and h-IAPP transgenic rat islets), curcumin failed to protect ß-cells from h-IAPP-induced apoptosis. While curcumin has the ability to inhibit amyloid formation, the present data suggest that, without further modification, it is unlikely to be therapeutically useful in protection of ß-cells in type 2 diabetes.
Subject(s)
Curcumin/pharmacology , Islet Amyloid Polypeptide/metabolism , Islets of Langerhans/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Electron Spin Resonance Spectroscopy , Humans , In Vitro Techniques , Islet Amyloid Polypeptide/toxicity , Islets of Langerhans/drug effects , Islets of Langerhans/ultrastructure , Microscopy, Electron , Rats , Rats, TransgenicABSTRACT
The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by beta cells. It is increasingly appreciated that the toxic form of amyloidogenic proteins is not amyloid but smaller membrane-permeant oligomers. Using an antibody specific for toxic oligomers and cryo-immunogold labeling in human IAPP transgenic mice, human insulinoma and pancreas from humans with and without T2DM, we sought to establish the abundance and sites of formation of IAPP toxic oligomers. We conclude that IAPP toxic oligomers are formed intracellularly within the secretory pathway in T2DM. Most striking, IAPP toxic oligomers appear to disrupt membranes of the secretory pathway, and then when adjacent to mitochondria, disrupt mitochondrial membranes. Toxic oligomer-induced secretory pathway and mitochondrial membrane disruption is a novel mechanism to account for cellular dysfunction and apoptosis in T2DM.
Subject(s)
Amyloid/toxicity , Diabetes Mellitus, Type 2/pathology , Insulin-Secreting Cells/pathology , Amyloid/metabolism , Amyloid/pharmacology , Animals , Diabetes Mellitus, Type 2/metabolism , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/ultrastructure , Insulinoma/metabolism , Insulinoma/pathology , Intracellular Space/drug effects , Intracellular Space/metabolism , Islet Amyloid Polypeptide , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Islets of Langerhans/ultrastructure , Mice , Mice, Transgenic , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Protein Multimerization/physiology , Rats , Secretory Pathway/drug effects , Secretory Vesicles/drug effects , Secretory Vesicles/metabolism , Secretory Vesicles/pathologyABSTRACT
In the title compound, C(11)H(19)NO(5), the five-membered pyrrolidine ring adopts an envelope conformation. The dihedral angles between the carboxyl group plane, the pyrrolidine ring and the meth-oxy group are 59.50â (3) and 62.02â (1)°, respectively. In the crystal, inter-molecular O-Hâ¯O hydrogen bonds link the mol-ecules into chains along [100]. The absolute configuration is assigned in accord with that of (2R,4R)-1-(tert-but-oxy-carbon-yl)-4-hy-droxy-pyrrolidine-2-carb-oxy-lic acid, which was the starting material in the synthesis.
ABSTRACT
The islet in type 2 diabetes (T2DM) and the brain in neurodegenerative diseases share progressive cell dysfunction, increased apoptosis, and accumulation of locally expressed amyloidogenic proteins (islet amyloid polypeptide (IAPP) in T2DM). Excessive activation of the Ca(2+)-sensitive protease calpain-2 has been implicated as a mediator of oligomer-induced cell death and dysfunction in neurodegenerative diseases. To establish if human IAPP toxicity is mediated by a comparable mechanism, we overexpressed human IAPP in rat insulinoma cells and freshly isolated human islets. Pancreas was also obtained at autopsy from humans with T2DM and nondiabetic controls. We report that overexpression of human IAPP leads to the formation of toxic oligomers and increases beta cell apoptosis mediated by increased cytosolic Ca(2+) and hyperactivation of calpain-2. Cleavage of alpha-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca(2+)-calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM.
Subject(s)
Apoptosis , Calcium/metabolism , Calpain/metabolism , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/physiopathology , Insulin-Secreting Cells/enzymology , Insulin-Secreting Cells/pathology , Adult , Aged , Aged, 80 and over , Amyloid/toxicity , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cytosol/drug effects , Cytosol/metabolism , Diabetes Mellitus, Type 2/pathology , Dipeptides/pharmacology , Enzyme Activation/drug effects , Female , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/ultrastructure , Islet Amyloid Polypeptide , Male , Middle Aged , Protein Transport/drug effects , Rats , Recombinant Fusion Proteins/metabolism , Spectrin/metabolismABSTRACT
An obstacle to development of methods to quantify beta-cell turnover from pancreas tissue is the lack of conversion factors for the frequency of beta-cell replication or apoptosis detected by immunohistochemistry to rates of replication or apoptosis. We addressed this obstacle in islets from 1-mo-old rats by quantifying the relationship between the rate of beta-cell replication observed directly by time-lapse video microscopy (TLVM) and the frequency of beta-cell replication in the same islets detected by immunohistochemistry using antibodies against Ki67 and insulin in the same islets fixed immediately after TLVM. Similarly, we quantified the rate of beta-cell apoptosis by TLVM and then the frequency of apoptosis in the same islets using TdT-mediated dUTP nick-end labeling and insulin. Conversion factors were developed by regression analysis. The conversion factor from Ki67 labeling frequency (%) to actual replication rate (%events/h) is 0.025 +/- 0.003 h(-1). The conversion factor from TdT-mediated dUTP nick-end labeling frequency (%) to actual apoptosis rate (%events/h) is 0.41 +/- 0.05 h(-1). These conversion factors will permit development of models to evaluate beta-cell turnover in fixed pancreas tissue.