Validation study to compare effects of processing protocols on measured Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine in blood

Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured N^ε-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, N^ε-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.     

Role of N-(carboxymethyl)lysine in the development of ischemic heart disease in type 2 diabetes mellitus

This study aims to determine the levels of N(epsilon)-(carboxymethyl)lysine (CML) in patients with Type 2 diabetic patients with and without ischemic heart disease (IHD) and to find for a possible association between circulating CML and a number of clinical parameters including lipids, hemoglobin A1c (HbA1c) and malondialdehyde (MDA) in Type 2 diabetic IHD patients. Serum CML levels were measured by enzyme-linked immunosorbent assay using polyclonal anti-CML antibodies. Serum levels of CML and MDA were assessed in 60 IHD patients with Type 2 diabetes, 43 IHD patients without Type 2 diabetes, 64 Type 2 diabetics without IHD, and 80 sex- and age-matched healthy subjects. Correlations studies between CML levels and lipids, HbA1c, and lipid peroxidation were performed in Type 2 diabetes patients with and without IHD. A statistical significance was observed in the levels of serum glucose, lipids (triglyceride, total cholesterol, HDL-cholesterol), MDA, HbA1c, CML and LDL-cholesterol (p<0.05) between the groups of the study. CML levels were significantly increased in diabetic IHD patients compared with Type 2 diabetes patients but without IHD (537.1 +/- 86.1 vs 449.7 +/- 54.9, p<0.001). A positive correlation was observed between serum levels of CML and MDA, r = 0.338 (p = 0.008) in Type 2 diabetes patients with IHD. However, age, HbA1c and lipids had no significant influence on CML levels among diabetics (p>0.05). In conclusion, this study demonstrates the effect of both diabetes and oxidative stress on the higher levels of circulating CML. These results showed that increased serum levels of CML are associated with the development of IHD in Type 2 diabetes mellitus.     

Immune Suppression by Recombinant Interleukin (rIL)-12 Involves Interferon γ Induction of Nitric Oxide Synthase 2 (iNOS) Activity: Inhibitors of NO Generation Reveal the Extent of rIL-12 Vaccine Adjuvant Effect

Recombinant interleukin 12 (IL-12) can profoundly suppress cellular immune responses in mice. To define the underlying mechanism, recombinant murine (rm)IL-12 was given to C57BL/6 mice undergoing alloimmunization and found to transiently but profoundly suppress in vivo and in vitro allogeneic responses and in vitro splenocyte mitogenic responses. Use of neutralizing antibodies and genetically deficient mice showed that IFN-γ (but not TNF-α) mediated rmIL-12–induced immune suppression. Splenocyte fractionation studies revealed that adherent cells from rmIL-12–treated mice suppressed the mitogenic response of normal nonadherent cells to concanavalin A and IL-2. Addition of an inhibitor of nitric oxide synthase (NOS) restored mitogenic responses, and inducible (i)NOS^−/− mice were not immunosuppressed by rmIL-12. These results support the view that suppression of T cell responses is due to NO produced by macrophages responding to the high levels of IFN-γ induced by rmIL-12. When a NOS inhibitor was given with rmIL-12 during vaccination of A/J mice with irradiated SCK tumor cells, immunosuppression was averted and the extent of rmIL-12''s ability to enhance induction of protective antitumor immunity was revealed. This demonstrates that rmIL-12 is an effective vaccine adjuvant whose efficacy may be masked by its transient immunosuppressive effect.     

Genetic research and clinical analysis of β‐globin gene cluster deletions in the Chinese population of Fujian province: A 14‐year single‐center experience

BackgroundHeterozygotes of HPFH and δβ thalassemia are clinically asymptomatic or have mild hemoglobin (Hb) values. However, when both HPFH and δβ‐thalassemia are coinherited with heterozygous β‐thalassemia, patients may progress to a clinical phenotype of thalassemia intermedia or thalassemia major. The purpose of this study was to characterize the genotypes and analyze the phenotypes of these disorders in Fujian Province, to offer advice for genetic counseling and accurate prenatal diagnosis in this region. A total of 55 001 subjects were participated in thalassemia screening. 142 subjects with HbF levels ≥10%, before the blood transfusion, were selected for further investigation.MethodsMultiplex ligation‐dependent probe amplification (MLPA) and Gap‐PCR were used to screen for three β‐globin gene cluster deletions: Chinese ^Gγ(^Aγδβ)⁰ thalassemia and Southeast Asia HPFH (SEA‐HPFH) deletion and 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357).ResultsA total of 142 patients with HbF (≥10%) were enrolled to characterize the molecular basis of β‐globin gene cluster deletions in our study; 22 cases 0.04% (22/55 001) were definitively diagnosed with β‐globin gene cluster deletions. Ten cases were heterozygous for the Chinese ^Gγ(^Aγδβ)⁰‐thal mutations, 10 cases were heterozygous for SEA‐HPFH, and one case was compound heterozygous for SEA‐HPFH and the α‐thal mutation. The 1357 bp deletion (NG‐000007.3:g.69997‐71353 del 1357) was detected in one case. Moreover, the hemoglobin A₂ levels in patients who were heterozygous for Chinese ^Gγ(^Aγδβ)⁰‐thal were statistically lower than in cases with SEA‐HPFH deletion(p < 0.05).ConclusionIn Fujian Province, the prevalence of common β‐globin gene cluster deletions was 0.04%. What''s more, the most common β‐globin cluster deletions are the Chinese ^Gγ(^Aγδβ)⁰ and SEA‐HPFH.     

Characterization of Human CD8+ T Cells Reactive with Mycobacterium tuberculosis–infected Antigen-presenting Cells

Previous studies in murine models, including those using the β2 microglobulin knockout mouse, have suggested an important role for CD8⁺ T cells in host defense to Mycobacterium tuberculosis (Mtb). At present, little is understood about these cells in the human immune response to tuberculosis. This report demonstrates the existence of human Mtb-reactive CD8⁺ T cells. These cells are present preferentially in persons infected with Mtb and produce interferon γ in response to stimulation with Mtb-infected target cells. Recognition of Mtb-infected cells by these CD8⁺ T cells is restricted neither by the major histocompatibility complex (MHC) class I A, B, or C alleles nor by CD1, although it is inhibited by anti–MHC class I antibody. The Mtb-specific CD8⁺ T cells recognize an antigen which is generated in the proteasome, but which does not require transport through the Golgi-ER. The data suggest the possible use of nonpolymorphic MHC class Ib antigen presenting structures other than CD1.     

Non-invasive measurement of skin autofluorescence to evaluate diabetic complications

Although the accumulation of advanced glycation end-products (AGEs) of the Maillard reaction in our body is reported to increase with aging and is enhanced by the pathogenesis of lifestyle-related diseases such as diabetes, routine measurement of AGEs is not applied to regular clinical diagnoses due to the lack of conventional and reliable techniques for AGEs analyses. In the present study, a non-invasive AGEs measuring device was developed and the association between skin AGEs and diabetic complications was evaluated. To clarify the association between the duration of hyperglycemia and accumulation of skin fluorophores, diabetes was induced in mice by streptozotocin. As a result, the fluorophore in the auricle of live mice was increased by the induction of diabetes. Subsequent studies revealed that the fingertip of the middle finger in the non-dominant hand is suitable for the measurement of the fluorescence intensity by the standard deviation value. Furthermore, the fluorescence intensity was increased by the presence of diabetic microvascular complications. This study provides the first evidence that the accumulation of fluorophore in the fingertip increases with an increasing number of microvascular complications, demonstrating that the presence of diabetic microvascular complications may be predicted by measuring the fluorophore concentration in the fingertip.     

Biomarkers of oxidative stress and endothelial dysfunction in glucose intolerance and diabetes mellitus

Objectives

To evaluate biomarkers of endothelial dysfunction and oxidative stress in glucose intolerance (GI) compared to overt diabetes (DM2).

Design and methods

140 volunteers including 96 with DM2, 32 with GI and 12 controls (C) were studied. NO metabolites, NO synthase inhibitors, thiols and N-acetyl-β-glucosaminidase (NAGase) activity were analyzed by chemiluminescence, capillary electrophoresis, ELISA and colorimetric assay, respectively.

Results

NO metabolites were higher in GI (NOx: p = 0.03; S-nitrosothiols: p = 0.001) and DM2 (p = 0.006; p = 0.0006) groups in relation to group C, while nitrotyrosine was higher only in the DM2 group in comparison to the other groups. NAGase activity was elevated in GI (p = 0.003) and DM2 (p = 0.0004) groups in relation to group C, as well as, ADMA (p = 0.01; p = 0.003) and GSSG (p = 0.01; p = 0.002).

Conclusions

NO metabolites, NO synthase inhibitors, thiols and NAGase are biomarkers suitable to indicate endothelial dysfunction and oxidative stress in the early stages of impaired response to insulin.     

Magnesium Carbonate‐Containing Phosphate Binder Prevents Connective Tissue Mineralization in Abcc6 ?/? Mice–Potential for Treatment of Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues primarily in the skin, eyes, and the cardiovascular system. PXE is caused by mutations in the ABCC6 gene. While PXE is associated with considerable morbidity and mortality, there is currently no effective or specific treatment. In this study, we tested oral phosphate binders for treatment of a mouse model of PXE which we have developed by targeted ablation of the corresponding mouse gene (Abcc6 ^−/−). This “knock‐out” (KO) mouse model recapitulates features of PXE and demonstrates mineralization of a number of tissues, including the connective tissue capsule surrounding vibrissae in the muzzle skin which serves as an early biomarker of the mineralization process. Treatment of these mice with a magnesium carbonate‐enriched diet (magnesium concentration being 5‐fold higher than in the control diet) completely prevented mineralization of the vibrissae up to 6 months of age, as demonstrated by computerized morphometric analysis of histopathology as well as by calcium and phosphate chemical assays. The magnesium carbonate‐enriched diet also prevented the progression of mineralization when the mice were placed on that experimental diet at 3 months of age and followed up to 6 months of age. Treatment with magnesium carbonate was associated with a slight increase in the serum concentration of magnesium, with no effect on serum calcium and phosphorus levels. In contrast, concentration of calcium in the urine was increased over 10‐fold while the concentration of phosphorus was markedly decreased, being essentially undetectable after long‐term (>4 month) treatment. No significant changes were noted in the serum parathyroid hormone levels. Computerized axial tomography scan of bones in mice placed on magnesium carbonate‐enriched diet showed no differences in the bone density compared to mice on the control diet, and chemical assays showed a small increase in the calcium and phosphate content of the femurs by chemical assay, in comparison to mice on control diet. Similar experiments with another experimental diet supplemented with lanthanum carbonate did not interfere with the mineralization process in Abcc6 ^−/− mice. These results suggest that magnesium carbonate may offer a potential treatment modality for PXE, a currently intractable disease, as well as for other conditions characterized by ectopic mineralization of connective tissues.     

Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4⁺ T cells. CD4⁺ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4⁺ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1^−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4⁺ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4⁺ T cell activation.     

Translation role of circRNAs in cancers

Circular RNAs (circRNAs) constitute a class of covalently closed RNA molecules. With the continuous advancement of high‐throughput sequencing technology and bioinformatics tools, many circRNAs have been identified in various human tissues and cell lines. Notably, recent studies have indicated that some circRNAs have translational functions. Internal ribosome entry sites and the N6‐methyladenosine modification mediate cap‐independent translation. This review describes these two translation mechanisms and verification methods at the molecular level. Databases (including ORF Finder, Pfam, BLASTp, CircRNADb, CircBase, CircPro, CircCode, IRESite, IRESbase) were used to analyze whether circRNAs have the structural characteristic of translation. CircRNA minigene reporter system containing green fluorescent protein (GFP) confirmed the translation potential of circRNAs. Also, we briefly summarize the roles of proteins/peptides encoded by circRNAs (circFBXW7, circFNDC3B, circLgr4, circPPP1R12A, circMAPK1, circβ‐catenin, circGprc5a, circ‐SHPRH, circPINTexon2, circAKT3) that have been verified thus far in human cancers (triple‐negative breast cancer, colon cancer, gastric cancer, hepatocellular carcinoma, bladder cancer, glioblastoma). Those findings suggest circRNAs have a great implication in translation of the human genome.     

Characterisation of two unusual cases of haemoglobin Bart’s hydrops foetalis caused by –SEA and large novel α-globin gene cluster deletions

BackgroundWe describe 2 unusual haemoglobin (Hb) Bart’s hydrops cases that could not be explained by traditional factors.Case presentation: Two families with a diagnosis or history of foetal hydrops were enrolled. A suspension-array system was used to detect the 23 most frequent mutations in southern China. Multiplex ligation-dependent probe amplification (MLPA) was used to screen for possible deletions. Precise characterisation of the breakpoints of the novel variants and uniparental disomy analysis were performed using a single nucleotide polymorphism (SNP) array. Quantitative fluorescence PCR was used to eliminate maternal cell contamination and nonpaternity. In case 1, the suspension-array system indicated a maternal heterozygous (–^SEA/) deletion, and the paternal sample was negative. The foetal hydrops was caused by the maternal (–^SEA/) deletion and a de novo α-globin gene deletion (–¹⁹³). In case 2, the paternal sample had a heterozygous (–^SEA/) deletion, and MLPA and SNP array analysis revealed a large maternal deletion (–²²⁷) that encompassed the α-globin gene, which explained the history of Hb Bart’s foetal hydrops.ConclusionsOur cases describe 2 new α⁰-thalassaemia deletions and illustrate the importance of using a combination of methods to detect rare types of α-thalassaemia.     

Comprehensive analysis of N6‐methyladenosine‐related long non‐coding RNAs for prognosis prediction in liver hepatocellular carcinoma

BackgroundLiver hepatocellular carcinoma (LIHC) is a lethal cancer. This study aimed to identify the N⁶‐methyladenosine (m⁶A)‐targeted long non‐coding RNA (lncRNA) related to LIHC prognosis and to develop an m⁶A‐targeted lncRNA model for prognosis prediction in LIHC.MethodsThe expression matrix of mRNA and lncRNA was obtained, and differentially expressed (DE) mRNAs and lncRNAs between tumor and normal samples were identified. Univariate Cox and pathway enrichment analyses were performed on the m⁶A‐targeted lncRNAs and the LIHC prognosis‐related m⁶A‐targeted lncRNAs. Prognostic analysis, immune infiltration, and gene DE analyses were performed on LIHC subgroups, which were obtained from unsupervised clustering analysis. Additionally, a multi‐factor Cox analysis was used to construct a prognostic risk model based on the lncRNAs from the LASSO Cox model. Univariate and multivariate Cox analyses were used to assess prognostic independence.ResultsA total of 5031 significant DEmRNAs and 292 significant DElncRNAs were screened, and 72 LIHC‐specific m⁶A‐targeted binding lncRNAs were screened. Moreover, a total of 29 LIHC prognosis‐related m⁶A‐targeted lncRNAs were obtained and enriched in cytoskeletal, spliceosome, and cell cycle pathways. An 11‐m⁶A‐lncRNA prognostic model was constructed and verified; the top 10 lncRNAs included LINC00152, RP6‐65G23.3, RP11‐620J15.3, RP11‐290F5.1, RP11‐147L13.13, RP11‐923I11.6, {"type":"entrez-nucleotide","attrs":{"text":"AC092171.4","term_id":"19526159"}}AC092171.4, KB‐1460A1.5, LINC00339, and RP11‐119D9.1. Additionally, the two LIHC subgroups, Cluster 1 and Cluster 2, showed significant differences in the immune microenvironment, m⁶A enzyme genes, and prognosis of LIHC.ConclusionThe m⁶A‐lncRNA prognostic model accurately and effectively predicted the prognostic survival of LIHC. Immune cells, immune checkpoints (ICs), and m⁶A enzyme genes could act as novel therapeutic targets for LIHC.     

Contribution of ?Virus-specific CD8+ Cytotoxic T Cells to Virus Clearance or Pathologic Manifestations of Influenza Virus Infection in a T Cell Receptor Transgenic Mouse Model

The ability of influenza virus to evade immune surveillance by neutralizing antibodies (Abs) directed against its variable surface antigens provides a challenge to the development of effective vaccines. CD8⁺ cytotoxic T lymphocytes (CTLs) restricted by class I major histocompatibility complex molecules are important in establishing immunity to influenza virus because they recognize internal viral proteins which are conserved between multiple viral strains. In contrast, protective Abs are strain-specific. However, the precise role of effector CD8⁺ CTLs in protection from influenza virus infection, critical for understanding disease pathogenesis, has not been well defined. In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8⁺ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose. The data suggest a dual role for CD8⁺ CTLs against influenza, which may present a challenge to the development of effective CTL vaccines. Effector mechanisms used by CD8⁺ CTLs in orchestrating clearance of virus and recovery from experimental influenza infection, or potentiation of lethal pathology, are discussed.     

Effects of hypoxia and nitric oxide on ferritin content of alveolar cells

Concentrations of ferritin in alveolar cells and on the alveolar surface are increased in patients with a variety of respiratory disorders. Ferritin synthesis by cells is modulated by iron content but is also influenced by stimuli other than iron. In this study we sought to determine whether in vitro exposure to hypoxia- or nitric oxide (NO)-induced ferritin accumulation or release by human alveolar macrophages (AMs) or a lung cancer-derived epithelial cell line (A549). Changes in cell content of iron and ferritin (L- and H-types), as well as ferritin content of cell supernatants, were determined after in vitro exposure to hypoxia (1% or 10% O(2), 18 hours) or the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.01-1.0 mmol/L, 18 hours). Exposure to 1% O(2) increased ferritin content in both cell types (>fourfold increase; P <.005) without changing iron content. Treatment with SNAP increased ferritin content of A549 cells in a dose-dependent manner, whereas treatment of AMs decreased cellular iron and ferritin content and increased supernate ferritin content. Pretreatment of cells with N-acetylcysteine (500 micromol/L) reduced hypoxia-induced ferritin accumulation in alveolar cells and completely inhibited NO-induced ferritin accumulation in A549 cells. These findings indicate that exposure to 1% O(2)can increase ferritin content in alveolar cells, whereas NO can increase ferritin content (A549 cells) or decrease ferritin content (AMs).     

Age-related changes in endothelial function and blood flow regulation

Vascular endothelial dysfunction is regarded as a primary phenotypic expression of normal human aging. This senescence-induced disorder is the likely culprit underlying the increased cardiovascular and metabolic disease risks associated with aging. The rate of this age-dependent deterioration is largely influenced by the poor-quality lifestyle choice, such as smoking, sedentary daily life, chronic alcohol ingestion, high salt intake, unbalanced diet, and mental stress; and it is accelerated by cardiovascular and metabolic diseases. Although minimizing these detrimental factors is the best course of action, nonetheless chronological age steadily impairs endothelial function through reduced endothelial nitric oxide synthase (eNOS) expression/action, accelerated nitric oxide (NO) degradation, increased phosphodiesterase activity, inhibition of NOS activity by endogenous NOS inhibitors, increased production of reactive oxygen species, inflammatory reactions, decreased endothelial progenitor cell number and function, and impaired telomerase activity or telomere shortening. Endothelial dysfunction in regional vasculatures results in cerebral hypoperfusion triggering cognitive dysfunction and Alzheimer's disease, coronary artery insufficiency, penile erectile dysfunction, and circulatory failures in other organs and tissues. Possible prophylactic measures to minimize age-related endothelial dysfunction are also summarized in this review.     

Improved prediction of early-onset coronary artery disease using APOE epsilon4, BChE-K, PPARgamma2 Pro12 and ENOS T-786C in a polygenic model

OBJECTIVES: Coronary artery disease (CAD) is often polygenic due to multiple mutations that contribute small effects to susceptibility. Since most prior studies only evaluated the contribution of single candidate genes, we therefore looked at a combination of genes in predicting early-onset CAD [apolipoprotein E (APOE) epsilon4, butyrylcholinesterase (BChE) K, peroxisome proliferator-activated receptor gamma2 (PPARgamma2) Pro12Ala and endothelial nitric oxide synthase (ENOS) T-786C]. DESIGN AND METHODS: We examined the frequencies, individually and in combination, of all four alleles among patients with early-onset CAD (n = 150; <50 years), late-onset CAD (n = 150; >65 years) and healthy controls (n = 150, age range 47-93 years). Differences in the proportion of subjects in each group with the given gene combination were assessed and likelihood ratios (LR) were calculated using logistic regression to combine the results of multiple genes. RESULTS: Early-onset CAD patients had increased, but non-significant, frequencies of PPARgamma2 Pro12/Pro12 (P = 0.39) and ENOS T-786C (P = 0.72), while BChE-K was only significantly higher in early-onset CAD patients compared to controls (P = 0.03). There were significantly more APOE epsilon4 alleles alone (P = 0.02) or in combination with BChE-K (P = 0.02) among early-onset CAD patients compared to late-onset CAD ones or controls. When combined, there was a higher prevalence of all four alleles in early-onset CAD (early-onset CAD patients: 10.7%, late-onset CAD patients: 3.3% and controls: 2.7%, P = 0.01). LR for early-onset CAD for a single allele was relatively small (1.08 for PPARgamma2 to 1.70 for APOE epsilon4). This increased to 2.78 (1.44-5.37) when combining all four alleles, therefore increasing the pre-test probability of CAD from 5% to a post-test probability of 12.7%. CONCLUSIONS: While any single mutation causes only a mildly increased LR (none > 1.7), in combination, the likelihood of early-onset CAD increased to 2.78 with four mutations. The genetics of early-onset CAD appear to be multifactorial, requiring polygenic models to elucidate risk.     

Oxycodone and morphine have distinctly different pharmacological profiles: radioligand binding and behavioural studies in two rat models of neuropathic pain

Previously, we reported that oxycodone is a putative kappa-opioid agonist based on studies where intracerebroventricular (i.c.v.) pre-treatment of rats with the kappa-selective opioid antagonist, nor-binaltorphimine (nor-BNI), abolished i.c.v. oxycodone but not morphine antinociception, whereas pretreatment with i.c.v. naloxonazine (mu-selective antagonist) produced the opposite effects. In the present study, we used behavioural experiments in rat models of mechanical and biochemical nerve injury together with radioligand binding to further examine the pharmacology of oxycodone. Following chronic constriction injury (CCI) of the sciatic nerve in rats, the antinociceptive effects of intrathecal (i.t.) oxycodone, but not i.t. morphine, were abolished by nor-BNI. Marked differences were found in the antinociceptive properties of oxycodone and morphine in streptozotocin (STZ)-diabetic rats. While the antinociceptive efficacy of morphine was abolished at 12 and 24 weeks post-STZ administration, the antinociceptive efficacy of s.c. oxycodone was maintained over 24 weeks, albeit with an approximately 3- to 4-fold decrease in potency. In rat brain membranes irreversibly depleted of mu- and delta-opioid binding sites, oxycodone displaced [(3)H]bremazocine (kappa(2)-selective in depleted membranes) binding with relatively high affinity whereas the selective mu- and delta-opioid ligands, CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)) and DPDPE ([D-Pen(2,5)]-enkephalin), respectively, did not. In depleted brain membranes, the kappa(2b)-ligand, leu-enkephalin, prevented oxycodone's displacement of high-affinity [(3)H]bremazocine binding, suggesting the notion that oxycodone is a kappa(2b)-opioid ligand. Collectively, the present findings provide further support for the notion that oxycodone and morphine produce antinociception through distinctly different opioid receptor populations. Oxycodone appears to act as a kappa(2b)-opioid agonist with a relatively low affinity for mu-opioid receptors.     

The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion

Myocardial ischemia-reperfusion injury is an important cause of impaired heart function in the early postoperative period subsequent to cardiac surgery. Reactive oxygen species (ROS) generation increases during both ischemia and reperfusion and it plays a central role in the pathophysiology of intraoperative myocardial injury. Unfortunately, the cellular source of these ROS during ischemia and reperfusion is often poorly defined. Similarly, individual ROS members tend to be grouped together as free radicals with a uniform reactivity towards biomolecules and with deleterious effects collectively ascribed under the vague umbrella of oxidative stress. This review aims to clarify the identity, origin, and progression of ROS during myocardial ischemia and reperfusion. Additionally, this review aims to describe the biochemical reactions and cellular processes that are initiated by specific ROS that work in concert to ultimately yield the clinical manifestations of myocardial ischemia-reperfusion. Lastly, this review provides an overview of several key cardioprotective strategies that target myocardial ischemia-reperfusion injury from the perspective of ROS generation. This overview is illustrated with example clinical studies that have attempted to translate these strategies to reduce the severity of ischemia-reperfusion injury during coronary artery bypass grafting surgery.     

Apoptosis and oxidants in the heart

Cardiomyocyte (CM) apoptosis has been reported in a variety of cardiovascular diseases, including myocardial infarction, ischemia/reperfusion, end-stage heart failure, arrhythmogenic right ventricular dysplasia, and adriamycin-induced cardiomyopathy. The role of CM apoptosis in the development and progression of cardiac diseases merits further investigation. Cumulative evidence suggests that reactive oxygen species (ROS), which have been implicated in cardiac pathophysiology, can trigger myocyte apoptosis by up-regulating proapoptotic proteins, such as Bax and caspases, and the mitochondria-dependent pathway. These apoptotic proteins and pathways are inhibited by various antioxidants, as well as by overexpression of the antiapoptotic protein Bcl-2 by way of the antioxidant pathway. Detection of CM apoptosis with the terminal transferase-mediated DNA nick-end labeling assay alone has recently been questioned because of technical concerns regarding its sensitivity and specificity. Because CMs are mononuclear or binuclear, if only one nucleus or a certain percentage of fragmented nuclei is stained with TUNEL assay at the early stage of apoptotic cell death, it remains unknown whether this particular early apoptotic CM is still functionally active. The issue of TUNEL specificity further questions reports of high percentages of apoptotic CM nuclei (0.02%-35%) in the heart. Nevertheless, oxidative stress is a major apoptotic stimulus in many cardiovascular diseases and the process can be inhibited by antioxidants both in vitro and in vivo.