by
Wilson C. Fok;
Yiqiang Zhang;
Adam B. Salmon;
Arunabh Bhattacharya;
Rakesh Gunda;
Dean Jones;
Walter Ward;
Kathleen Fisher;
Arlan Richardson;
Viviana I. Perez
Because rapamycin, an inhibitor of the nutrient sensor mammalian target of rapamycin, and dietary restriction both increase life span of mice, it has been hypothesized that they act through similar mechanisms. To test this hypothesis, we compared various biological parameters in dietary restriction mice (40% food restriction) and mice fed rapamycin (14 ppm). Both treatments led to a significant reduction in mammalian target of rapamycin signaling and a corresponding increase in autophagy. However, we observed striking differences in fat mass, insulin sensitivity, and expression of cell cycle and sirtuin genes in mice fed rapamycin compared with dietary restriction. Thus, although both treatments lead to significant downregulation of mammalian target of rapamycin signaling, these two manipulations have quite different effects on other physiological functions suggesting that they might increase life span through a common pathway as well as pathways that are altered differently by dietary restriction and rapamycin.
Neisseria meningitidis (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasivemeningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, USNmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of USNmUC isolates was an IS1301 element in the intergenic region separating the capsule ctr-css operons and adjacent deletion of cssA/B/C and a part of csc, encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an ∼20-kb sequence near the cps locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification norB-aniA gene cassette, and strains grow well anaerobically. The cc11 USNmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the Neisseria gonorrheae norB-aniA cassette promoting anaerobic growth.
Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H 2 O 2 ), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H 2 O 2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option.
The oncogenic transcription factor MYC and its binding partner MAX regulate gene expression by binding to DNA at enhancer-box (E-box) elements 5'-CACGTG-3'. In mammalian genomes, the central E-box CpG has the potential to be methylated at the 5-position of cytosine (5mC), or to undergo further oxidation to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), or 5-carboxyl (5caC) forms. We find that MAX exhibits the greatest affinity for a 5caC or unmodified C-containing E-box, and much reduced affinities for the corresponding 5mC, 5hmC or 5fC forms. Crystallization of MAX with a 5caC modified E-box oligonucleotide revealed that MAX Arg36 recognizes 5caC using a 5caC-Arg-Guanine triad, with the next nearest residue to the carboxylate group being Arg60. In an analysis of >800 primary multiple myelomas, MAX alterations occurred at a frequency of ∼3%, more than half of which were single nucleotide substitutions affecting a basic clamp-like interface important for DNA interaction. Among these, arginines 35, 36 and 60 were the most frequently altered. In vitro binding studies showed that whereas mutation of Arg36 (R36W) or Arg35 (R35H/L) completely abolished DNA binding, mutation of Arg60 (R60Q) significantly reduced DNA binding, but retained a preference for the 5caC modified E-box. Interestingly, MAX alterations define a subset of myeloma patients with lower MYC expression and a better overall prognosis. Together these data indicate that MAX can act as a direct epigenetic sensor of E-box cytosine modification states and that local CpG modification and MAX variants converge to modulate the MAX-MYC transcriptional network.
Background: Azelnidipine (AZL), a long-acting dihydropyridine-based calcium antagonist, has been recently approved and used for treating ischemic heart disease and cardiac remodeling after myocardial infarction, however, its effect on hyperglycemia-induced cardiac damage has not been studied.Methods: This study examined the effect of AZL on circulating markers of cardiac damage, altered lipid and cytokines profile and markers of oxidative stress including homocysteine in diabetic rats.Results: STZ induced diabetes caused a significant increase in blood glucose levels. It also resulted in an increase in the levels of homocysteine and cardiac damage markers, like Troponin-1, CK-MB, CK-NAC, uric acid, LDH and alkaline phosphatase. Moreover, there was an increase in the levels of proinflammatory cytokines like TNF-α, IFN-γ, and TGF-β and decrease in the levels of IL-4 and IL-10. Additionally, there was increase in the levels of cholesterol, triglycerides, LDL, VLDL and a decrease in HDL in these animals. There was an altered antioxidant enzyme profile which resulted in a notable increase in the levels of oxidative stress markers like lipid peroxides, nitric oxide and carbonylated proteins. Compared with the untreated diabetic rats, AZL treatment significantly reduced the levels of troponin-1 (P < 0.05), CK-MB (P < 0.05), CK-NAC (P < 0.05), uric acid (P < 0.05), LDH (P < 0.05) and alkaline phosphatase (P < 0.05). It also reduced the levels of the TNF-α (P < 0.05), IFN-γ (P < 0.05), and TGF-β (P < 0.05) and increased the levels of IL-4 (P < 0.05). A significant decrease in the serum cholesterol (P < 0.05), triglycerides (P < 0.05), LDL (P < 0.05), VLDL (P < 0.05) and a significant rise in levels of HDL (P < 0.05) was also observed. Treatment with AZL corrected the distorted antioxidant enzyme profile resulting in a significant decrease in the levels of lipid peroxides, nitric oxide and carbonylated proteins.Conclusion: Our results indicate that AZL treatment can reduce the risk of hyperglycemia induced metabolic disorders and its role can be further extended to explore its therapeutic potential in diabetic patients with cardiac complications.
METHODS: Herein, we examined whether reduced striatal dopamine release in rhesus monkeys chronically treated with interferon-alpha can be restored by administration of the dopamine precursor levodopa via reverse in vivo microdialysis. RESULTS: Levodopa completely reversed interferon-alpha-induced reductions in striatal dopamine release. No changes were found in the 3,4-dihydroxyphenylacetic acid to dopamine ratio, which increases when unpackaged dopamine is metabolized via monoamine oxidase. BACKGROUND: Studies using neuroimaging and in vivo microdialysis in humans and nonhuman primates indicate that inflammatory cytokines such as interferon-alpha reduce dopamine release in the ventral striatum in association with depressive symptoms including anhedonia and psychomotor slowing. CONCLUSIONS: These findings suggest that inflammatory cytokines reduce the availability of dopamine precursors without affecting end-product synthesis or vesicular packaging and/or release and provide the foundation for future studies investigating therapeutic strategies that facilitate availability of dopamine precursors to improve depressive symptoms in patient populations with increased inflammation.
The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.
We developed a robust analytical method for quantification of malondialdehyde (MDA) in exhaled breath condensate (EBC) via derivatization with 2,4-dinitrophenylhydrazine (DNPH). The target MDA-DNPH hydrazone was separated by ultra-performance liquid chromatography using two reversed-phase analytical columns (C18 and phenyl-hexyl) inter-connected via a two-position, six-port switching valve to a single-quadrupole mass spectrometer. The target derivative was analyzed under positive electrospray ionization using single ion monitoring mode (m/z = 235 for the target derivative, and m/z = 237 for its labeled isotopic analog). This pseudo two-dimensional chromatographic separation provided optimum separation conditions for the target derivative resulting in the limit of detection of 0.58 nM in EBC sample (or 36.2 pmol on-column amount), which is comparable to those reported previously using different techniques, including tandem mass spectrometry. Based on the calibration solutions, the method had a linear quantification range of 1.0–200 nM (r2 = 0.998). The method showed good relative recoveries (92.2–102.0%) and acceptable precisions (3.6–12.2% for inter-day precision, and 4.3–12.4% for intra-day precision for two quality control levels, prepared from 5 nM and 25 nM solutions). The derivative was found to be stable at room temperature for 48 h or during analysis. The method was used to analyze 205 exhaled breath condensate samples collected from individuals from a healthy population of student athletes. MDA was detected in approximately 95% of these samples, with concentrations ranging from 1.16 to 149.63 nM. The median concentration was 6.82 nM, (IQR 4.08–9.88). These data demonstrate that our method can be successfully used to measure MDA in population studies.
by
Tom E. Forshaw;
Reetta Holmila;
Kimberly J. Nelson;
Joshua E. Lewis;
Melissa Kemp;
Allen W. Tsang;
Leslie B. Poole;
W. Todd Lowther;
Cristina M. Furdui
Peroxiredoxins have a long-established cellular function as regulators of redox metabolism by catalyzing the reduction of peroxides (e.g., H 2 O 2 , lipid peroxides) with high catalytic efficiency. This activity is also critical to the initiation and relay of both phosphorylation and redox signaling in a broad range of pathophysiological contexts. Under normal physiological conditions, peroxiredoxins protect normal cells from oxidative damage that could promote oncogenesis (e.g., environmental stressors). In cancer, higher expression level of peroxiredoxins has been associated with both tumor growth and resistance to radiation therapies. However, this relationship between the expression of peroxiredoxins and the response to radiation is not evident from an analysis of data in The Cancer Genome Atlas (TCGA) or NCI60 panel of cancer cell lines. The focus of this review is to summarize the current experimental knowledge implicating this class of proteins in cancer, and to provide a perspective on the value of targeting peroxiredoxins in the management of cancer. Potential biases in the analysis of the TCGA data with respect to radiation resistance are also highlighted.