A new idea of polishing pad called flexible nanobrush pad (FNP) has been proposed for the low down pressure chemical mechanical planarization (CMP) process of Cu/ultra-low-к materials. The FNP was designed with a surface layer of flexible brush-like nanofibers which can ‘actively’ carry nanoscale abrasives in slurry independent of the down pressure. Better planarization performances including high material removal rate, good planarization, good polishing uniformity, and low defectivity are expected in the CMP process under the low down pressure with such kind of pad. The FNP can be made by template-assisted replication or template-based synthesis methods, which will be driven by the development of the preparation technologies for ordered nanostructure arrays. The present work would potentially provide a new solution for the Cu/ultra-low-к CMP process.
Flexible nanobrush pad; Polishing pad; Chemical mechanical planarization; Cu/ultra-low-к integration
The folding and misfolding mechanism of multi-domain proteins remains poorly understood. While thermodynamic instability of the first nucleotide binding domain (NBD1) of ΔF508-CFTR partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508-CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane spanning domain 2) interface stabilization are required for wild-type-like folding, processing and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508-CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multi-domain membrane proteins.
Cyclooxygenase-2 inhibitors have been implicated in adverse cardiac events. We hypothesize that hypercholesterolemia and ischemia may alter the myocardial response to the cyclooxygenase-2 inhibitor celecoxib.
Yorkshire swine fed normal chow (CX, n=6) or high-cholesterol diet (HCX, n=6) underwent left circumflex artery ameroid placement, and were started on celecoxib (200 mg/day). After 7 weeks, ischemic and non-ischemic myocardium were analyzed for thrombogenic ratio (thromboxane content divided by prostacyclin content), total protein oxidative stress, and expression of prostacyclin synthase, thromboxane synthase, myeloperoxidase, and superoxide dismutase. Cardiac function, tissue perfusion, and vessel density were measured.
HCX animals were significantly hypercholesterolemic compared to CX animals. Thrombogenic ratio was significantly higher in the HCX group compared to the CX group, but prostacyclin and thromboxane synthase expression was similar in all tissues. Myocardial perfusion was decreased in the HCX group compared to the CX group. Total oxidative stress, myeloperoxidase, and superoxide dismutase were increased in ischemic tissue compared to non-ischemic tissues, but there was no diet-induced difference between groups. There was no difference in capillary or arteriolar density between groups. LV contractility was greater in the HCX group compared to the CX group, but there was no significant difference in heart rate, mean arterial pressure, or left ventricular pressure.
Hypercholesterolemic patients using celecoxib may be at higher risk for thrombotic events than those with normal cholesterol, but the relationship between dyslipidemia, ischemia, and cyclooxygenase-2 inhibition is likely much more complicated than originally thought.
The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the cardiovascular system remains controversial, especially in patients with cardiovascular comorbidities. We used a swine model of chronic myocardial ischemia to investigate whether hypercholesterolemia alters the cardiovascular effects of the non-selective NSAID naproxen.
Yorkshire swine were fed normal chow (NAP, n=7) or high-fat diet (HF-NAP, n=8). Chronic myocardial ischemia was created in all animals by left circumflex ameroid constrictor placement. All swine were started on oral naproxen (440 mg/day) at the time of ameroid placement. After 7 weeks, myocardial perfusion and microvessel reactivity in the ischemic territory were assessed. Tissue levels of prostanoid metabolites 11-dehydrothromboxane B2 (11-d-TXB2) and 6-keto-prostaglandin F1-α (6-k- PGF1α) were measured. Tissue was analyzed for capillary density and protein expression.
Myocardial perfusion was significantly decreased in the HF-NAP group both at rest and during ventricular pacing. Microvessel relaxation responses to sodium nitroprusside and adenosine 5’-diphosphate were similar between groups. Tissue 11-d-TXB2 levels were similar between groups, but tissue 6-k-PGF1α was significantly decreased in the HF-NAP group (p=0.001). Expression of thromboxane synthase was significantly higher in the HF-NAP group (p=0.02), while prostacyclin synthase expression was significantly decreased in the HF-NAP group (p=0.04). Capillary density was higher in the HF-NAP group (p=0.005). Pro-angiogenic VEGF (p=0.0002) and Akt (p=0.01) were downregulated in the HF-NAP group.
A high-fat diet impairs tissue perfusion in ischemic myocardium of naproxen-treated swine by shifting the prostanoid balance to favor production of thromboxane over prostacyclin. Thus dietary modification may improve myocardial blood flow and alter the safety profile in chronically ischemic cardiac patients taking naproxen.
We studied the contractile response of human peripheral microvasculature to thromboxane A-2 (TXA-2) before and after cardiopulmonary bypass (CPB), with and without the blockade of TXA-2 receptors, or the inhibition of phospholipase C (PLC), phospholipase A-2 (PLA-2) or protein kinase C-alpha (PKC-α). We also examined the protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC and other TXA-2-related proteins.
Methods and Results
Skeletal muscle arterioles (90–180 μm in diameter) were harvested pre- and post-CPB from patients (n = 28) undergoing cardiac surgery. The post-CPB contractile response of skeletal muscle arterioles to TXA-2 analog U-46619 was significantly impaired compared with pre-CPB (P <0.05). The presence of TXA-2 receptor antagonist SQ-29548 (10−6M) prevented the contractile response to U-46619 (P<0.05). Pretreatment with the PLC inhibitor U-73122 (10−6M) significantly inhibited the U-46619-induced contractile response (P<0.01). Administration of the PLA-2 inhibitor quanacrine (10−6M) or PKC-α inhibitor safingol (2×10−5M), however, failed to affect U-46619-induced contraction. Total protein levels and gene expression of TXA-2 receptors, and TXA-2 synthase of skeletal muscle, were not altered post-CPB. Confocal microscopy showed no significant differences in the expression of PLCβ-3 in the microcirculation. PLCβ-3 was localized to both smooth muscle and endothelium.
CPB decreases the contractile response of human peripheral arterioles to TXA-2 soon after cardiac surgery. This response may be in part responsible for the decrease in vascular tone, and accompanying hypotension sometimes observed after cardiac operations utilizing CPB.
In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2fl/fl mice). In the present study, we used TnIFastCreSod2fl/fl mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2fl/fl mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2fl/fl mice, when compared with control mice. Complex II activity is reduced by 47% in young and by ~90% in old TnIFastCreSod2fl/fl mice, associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2fl/fl mice. Complex II-linked mitochondrial ATP production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2fl/fl mice. Furthermore, in old TnIFastCreSod2fl/fl mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains greater than 2-fold elevated; and oxidative damage (measured as F2 isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2fl/fl mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.
The quality of life of leg amputees can be improved dramatically by using a cyber physical system (CPS) that controls artificial legs based on neural signals representing amputees’ intended movements. The key to the CPS is the neural-machine interface (NMI) that senses electromyographic (EMG) signals to make control decisions. This paper presents a design and implementation of a novel NMI using an embedded computer system to collect neural signals from a physical system - a leg amputee, provide adequate computational capability to interpret such signals, and make decisions to identify user’s intent for prostheses control in real time. A new deciphering algorithm, composed of an EMG pattern classifier and a post-processing scheme, was developed to identify the user’s intended lower limb movements. To deal with environmental uncertainty, a trust management mechanism was designed to handle unexpected sensor failures and signal disturbances. Integrating the neural deciphering algorithm with the trust management mechanism resulted in a highly accurate and reliable software system for neural control of artificial legs. The software was then embedded in a newly designed hardware platform based on an embedded microcontroller and a graphic processing unit (GPU) to form a complete NMI for real time testing. Real time experiments on a leg amputee subject and an able-bodied subject have been carried out to test the control accuracy of the new NMI. Our extensive experiments have shown promising results on both subjects, paving the way for clinical feasibility of neural controlled artificial legs.
Neural-machine interface; prosthetics; high performance computer; trust management
We investigated the contractile response of human coronary microvasculature to thromboxane A-2 (TXA-2), with and without the blockade of TXA-2 receptors or the inhibition of phospholipase-C (PLC) or of protein kinase C-α (PKC-α) in the human coronary microvasculature before and after cardioplegia, followed by reperfusion (CP/Rep). Protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC, and other TXA-2–related proteins was also examined.
Right atrial tissue was harvested before and after cold blood cardioplegia, followed by about 10 minutes of reperfusion, from 28 patients undergoing cardiac operations. Coronary arterioles (90 to 170 µm in diameter) were dissected from the harvested tissue.
The post-CP/Rep contractile response of coronary arterioles to TXA-2 analog U-46619 was significantly impaired vs pre-CP/Rep (p < 0.05). The TXA-2 receptor antagonist SQ-29548 (10−6 M) prevented the contractile response to U-46619 (p < 0.05). Pretreatment with the PLC inhibitor U73122 (10−6 M) significantly inhibited the U-46619–induced contractile response (p < 0.05). Administration of the PKC-α inhibitor safingol failed to affect U-46619–induced contraction. Total protein levels and gene expression of TXA-2 receptors, TXA-2 synthase, PLC-β3, phospho–PLC-β3, PLC-γ1, and phospho–PLC-γ1 were not altered after CP/Rep. Confocal microscopy showed no significant differences in the expression of TXA-2 receptors or PLC-β3 in the microcirculation. TXA-2 receptors and PLC-β3 were both present in smooth muscle and endothelium.
Cardioplegia/Rep decreases the contractile response of human coronary arterioles to TXA-2 soon after cardiac operations. The contractile response to the TXA-2 analog U-46619 is through activation of TXA-2 receptors and PLC.
Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2–/– mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear. We report here that Cxcr2 on murine neutrophils inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis. Furthermore, enterocyte-derived Cxcl5 in the gut regulates IL-17/G-CSF levels and contributes to Cxcr2-dependent neutrophil homeostasis. Conversely, G-CSF was required for Cxcl5-dependent regulation of neutrophil homeostasis, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5–/– and Cxcr2–/– mice. Cxcr2–/– mice constitutively expressed IL-17A and showed increased numbers of IL-17A–producing cells in the lung, terminal ileum, and spleen. Most IL-17–producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment in Cxcr2–/– mice markedly decreased IL-17A and G-CSF expression, neutrophilia, and marrow myeloid hyperplasia. These data suggest a critical role for Cxcr2, Cxcl5, and commensal bacteria in regulation of the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites and have implications for the development of treatments for pathologies resulting from either excessive or ineffective neutrophil responses.
We compared the contractile responses to ET-1, with and without the inhibition of ET-A receptors and protein kinase C-alpha (PKC-α) in the human peripheral microvasculature of diabetic and case-matched non-diabetic patients.
Chest wall skeletal muscle was harvested from patients with and without diabetics undergoing cardiac surgery. Peripheral arterioles (90-180 micrometer in diameter) were dissected from the harvested tissue. Microvascular constriction was assessed by videomicroscopy in response to ET-1, with and without an endothelin A (ET-A) receptor antagonist, or an endothelin B (ET-B) antagonist or a PKC-α inhibitor.
ET-1 induced a dose-dependent contractile response of skeletal muscle arterioles from diabetes and non-diabetes. The contractile response of diabetic arterioles from both pre-bypass and post-bypass to ET-1 (10−9mol/L) was significantly decreased compared with those of non-diabetics (P<0.05), respectively. The contractile responses of microvessels of both diabetics and non-diabetics to ET-1 were significantly inhibited in the presence of either ET-A receptor antagonist BQ123 (10−7mol/L, P<0.05, respectively) or the PKC-α inhibitor safingol (2 × 10−5mol/L), respectively. In contrast, the ET-1-induced vasoconstriction was not affected by the administration of the ET-B receptor antagonist BQ788 (10−7mol/L). There were no significant differences in skeletal muscle levels of the ET-A and ET-B receptors between diabetic and non-diabetic groups.
Diabetic patients demonstrated a decreased contractile response to ET-1 in human peripheral microvasculature. The contractile response to ET-1 is via activation of ET-A receptors and PKC-α in diabetics. These results provide novel mechanisms of ET-1-induced contraction in vasomotor dysfunction in patients with diabetes.
Endothelin; Diabetics; Cardiopulmonary bypass; Microcirculation; Receptors; Skeletal Muscle; Protein Kinase C
We investigated the effects of cardiopulmonary bypass (CPB) on the contractile response of human peripheral microvasculature to endothelin-1 (ET-1), examined the role of specific ET receptors and protein kinase C-alpha (PKC-α), and analyzed ET-1-related gene/protein expression in this response.
Methods and Results
Human skeletal muscle arterioles (90 to 180 μm in diameter) were dissected from tissue harvested pre- and post-CPB from 30 patients undergoing cardiac surgery. In vitro contractile response to ET-1 was assessed by videomicroscopy, with and without an endothelin-A (ET-A) receptor antagonist, an endothelin-B (ET-B) antagonist, or a PKC-α inhibitor. The post-CPB contractile response of peripheral arterioles to ET-1 was significantly decreased compared with pre-CPB. The response to ET-1 was significantly inhibited in the presence of the ET-A antagonist BQ123, but unchanged in the presence of the ET-B receptor antagonist BQ788. Pretreatment with PKC-α inhibitor safingol reversed ET-1-induced response from contraction to relaxation. The total protein levels of ET-A and ET-B receptors were not altered post-CPB. Microarray analysis showed no significant changes in the gene expression of ET- receptors, ET-1 related proteins and protein kinases after CPB.
CPB decreases myogenic contractile function of human peripheral arterioles in response to ET-1. The contractile response to ET-1 is via activation of ET-A receptors and PKC-α. CPB has no effects on ET-1 related gene/protein expression. These results provide novel mechanisms of ET-1-induced contraction in the setting of vasomotor dysfunction after cardiac surgery.
cardiopulmonary bypass; endothelin; genes; microcirculation; vasoconstriction
We investigated the contractile function in responses to endothelin-1 (ET-1) in the human coronary microvasculature and the roles of endothelin receptors and protein kinase C-α (PKC-α) in these responses.
Human atrial tissue was harvested from patients undergoing cardiac surgery pre- and post-cardioplegia/cardiopulmanory bypass (CP/CPB). Microvascular constriction was assessed in pre- and post-CP/CPB samples in responses to ET-1, in the presence and absence of an endothelin-A (ET-A), or an endothelin-B (ET-B) receptor antagonist, or a PKC-α inhibitor, respectively. The expression and localization of ET-A and ET-B receptors were also examined using immunoblot and immunofluorescence photomicroscopy.
The post-CP/CPB contractile response of coronary arterioles to ET-1 was significantly decreased compared with the pre-CP/CPB responses. The response to ET-1 was significantly inhibited in the presence of the ET-A antagonist BQ123 (10–7M), but unchanged with the ET-B receptor antagonist BQ788 (10–7M). Pretreatment with PKC-α inhibitor safingol (2.5 × 10-5M) reversed the ET-1 responses from contraction into relaxation. The total polypeptide levels of ET-A and ET-B receptors were not altered post-CP/CPB. Immunoblot and immunofluorescent staining displayed strong signals for ET-A receptors and relatively weak signals for ET-B receptors localized on coronary microvasculature.
CP/CPB decreases the contractile function of human coronary microvessels in responses to ET-1. ET-A receptors are predominantly localized in the human coronary microcirculation, whereas ET-B receptors appear to be less abundant. The contractile response to ET-1 is in part through activation of ET-A receptors and PKC-α. These results suggest a role of ET-1-induced contraction in the vasomotor dysfunction after cardiac surgery.
Endothelins; Receptors; protein kinase C; Cardioplegia; Cardiopulmonary bypass; vasomotor dysfunction; Microcirculation
Impaired endothelium-independent vasodilation is a known consequence of type-1 and 2 diabetes, and the mechanism of impaired vasodilation is not well understood. The following study investigated the effects of type-1 and 2 diabetes in endothelial-independent vasodilation associated with coronary vascular smooth muscle (VSM) relaxation and contractile signaling mechanisms.
Type-1 diabetes was induced in Yucatan mini-swine via alloxan injection and treated with or without insulin(DM and IDM). Non-diabetic swine served as controls(ND). Expression and/or phosphorylation of determinants of VSM relaxation and contraction signaling were examined in coronary arteries and microvessels. Coronary microvessel relaxation was assessed using sodium nitroprusside(SNP). In addition, SNP-induced vasodilation and myosin light chain (MLC) phosphorylation was determined in coronary microvessels isolated from ND and type-2 diabetic human atrial appendage.
Diabetic impairment in SNP-induced relaxation was completely normalized by insulin. Soluble guanylate cyclase (sGC) VSM expression decreased in both DM and IDM groups and did not correlate with vasorelaxation. Phosphorylation of MLC and myosin phosphatase increased in the DM group and MLC phosphorylation strongly correlated with impaired VSM relaxation(r=.670, p<0.01). Coronary microvessels from type-2 diabetic human patients exhibited similarly impaired vasodilation and enhanced VSM MLC phosphorylation.
Impaired vasodilation in type-1 diabetes correlates with enhanced VSM MLC phosphorylation. In addition, enhanced VSM MLC phosphorylation is associated with impaired vasodilation in type 2 diabetes in humans.
Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4–6 months) and old (26–28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.
Oxidative damage; Mn superoxide dismutase; Pathology; Aging
Hydrogen sulfide (H2S) is produced endogenously in response to myocardial ischemia and thought to be cardioprotective. The mechanism underlying this protection has yet to be fully elucidated, but may be related sulfide’s ability to limit inflammation. This study investigates the cardioprotection provided by exogenous H2S, and its potential anti-inflammatory mechanism of action.
The mid-LAD coronary artery in 14 Yorkshire swine was acutely occluded for 60 minutes, followed by reperfusion for 120 minutes. Controls(7) received placebo, and treatment animals(7) received sulfide 10 minutes prior to and throughout reperfusion. Hemodynamic and functional measurements were obtained. Evans blue and TTC staining identified the area-at-risk and infarction. Coronary microvascular reactivity was assessed. Tissue was assayed for myeloperoxidase activity and pro-inflammatory cytokines.
Pre-I/R hemodynamics were similar between groups, whereas post-I/R mean arterial pressure (mmHg) was reduced by 28.7±5.0 in controls vs. 6.7±6.2 in treatment animals (p=0.03). +LV dP/dt (mmHg/sec) was reduced by 1325±455 in controls vs. 416±207 in treatment animals (p=0.002). Segmental shortening in the area-at-risk was better in treatment animals. Infarct size (% of area-at-risk) in controls was 41.0±7.8% vs. 21.2±2.5% in the treated group (p=0.036). Tissue levels of IL-6, IL-8, and TNFα and MPO activity decreased in the treatment group. Treated animals demonstrated improved microvascular reactivity.
Therapeutic sulfide provides protection in response to I/R injury, improving myocardial function, reducing infarct size, and improving coronary microvascular reactivity, potentially through its anti-inflammatory properties. Exogenous sulfide may have therapeutic utility in clinical settings in which I/R injury is encountered.
Hypercholesterolemia is prevalent in patients who experience myocardial ischemia-reperfusion injury (IR). We investigate the impact of dietary induced hypercholesterolemia on the myocardium in the setting of acute IR.
Methods and Results
In normocholesterolemic (NC,n=7) and hypercholesterolemic (HC,n=7) Yucatan male pigs, the left anterior descending coronary artery was occluded for 60 min, followed by reperfusion for 120 min. Hemodynamic values were recorded and TTC staining was used to assess necrosis. Oxidative stress was measured. Specific cell death and survival signaling pathways were assessed by Western blot and TUNEL staining. Infarct size was 45% greater in HC vs. NC (42% vs. 61%,p<.05), whereas the area at risk (AAR) was similar in both groups (p=0.61). While global LV function (+dP/dt,p<.05) was higher during entire period of IR in HC vs. NC, regional function deteriorated more following reperfusion in HC (p<.05). Ischemia increased indices of myocardial oxidative stress such as protein oxidation (p<.05), lipid peroxidation (p<.05), and nitrotyrosylation in HC vs. NC, as well as the expression of phospho-eNOS (p<.05). The expression of myeloperoxidase, p38 MAPK, and phospho-p38 MAPK was higher in HC vs. NC (all p<05). Ischemia caused higher expression of the pro-apoptotic protein PARP (p<.05), and lower expression of the pro-survival proteins Bcl2 (p<.05), phospho-Akt, (p<.05), and phospho-PKCε (p<.05) in the HC vs. NC. TUNEL positive cell count was 3.8 fold (p<.05) higher in the AAR of HC vs. NC.
This study demonstrates that experimental hypercholesterolemia is associated with increased myocardial oxidative stress and inflammation, attenuation of cell survival pathways and induction of apoptosis in the ischemic territory, which together may account for the expansion of myocardial necrosis in the setting of acute IR.
apoptosis; hypercholesterolemia; ischemia; myocardial infarction; risk factors
Aging is a risk factor for the development of adult-onset neuro-degenerative diseases. While some of the molecular pathways regulating longevity and stress resistance in lower organisms are defined (i.e., those activating the transcriptional regulators DAF-16 and HSF-1 in C. elegans), their relevance to mammals and disease susceptibility are unknown. We studied the signaling controlled by the mammalian homolog of DAF-16, FOXO3a, in model systems of motor neuron disease. Neuron death elicited in vitro by excitotoxic insult or the expression of mutant SOD1, mutant p150glued or polyQ expanded androgen receptor was abrogated by expression of nuclear-targeted FOXO3a. We identify a compound (Psammaplysene A, PA) that increases nuclear localization of FOXO3a in vitro and in vivo and show that PA also protects against these insults in vitro. Administration of PA to invertebrate model systems of neurodegeneration similarly blocked neuron death in a DAF-16/FOXO3a-dependent manner. These results indicate that activation of the DAF-16/FOXO3a pathway, genetically or pharmacologically, confers protection against the known causes of motor neuron diseases.
excitotoxicity; proteotoxicity; Amyotrophic Lateral Sclerosis; motor neuron; psammaplysene A; superoxide dismutase; p150glued; androgen receptor; polyQ expansion
The nucleus is the primary site of protein aggregation in many polyglutamine diseases, suggesting a central role in pathogenesis. In SBMA, the nucleus is further implicated by the critical role for disease of androgens, which promote the nuclear translocation of the mutant androgen receptor (AR). To clarify the importance of the nucleus in SBMA, we genetically manipulated the nuclear localization signal of the polyglutamine-expanded AR. Transgenic mice expressing this mutant AR displayed inefficient nuclear translocation and substantially improved motor function compared with SBMA mice. While we found that nuclear localization of polyglutamine-expanded AR is required for SBMA, we also discovered, using cell models of SBMA, that it is insufficient for both aggregation and toxicity and requires androgens for these disease features. Through our studies of cultured motor neurons, we further found that the autophagic pathway was able to degrade cytoplasmically retained expanded AR and represents an endogenous neuroprotective mechanism. Moreover, pharmacologic induction of autophagy rescued motor neurons from the toxic effects of even nuclear-residing mutant AR, suggesting a therapeutic role for autophagy in this nucleus-centric disease. Thus, our studies firmly establish that polyglutamine-expanded AR must reside within nuclei in the presence of its ligand to cause SBMA. They also highlight a mechanistic basis for the requirement for nuclear localization in SBMA neurotoxicity, namely the lack of mutant AR removal by the autophagic protein degradation pathway.
Regulation of the androgen receptor (AR) is critical to prostate cancer (PCa) development; therefore, AR is the first line therapeutic target for disseminated tumors. Cell cycle dependent accumulation of cyclin D1 negatively modulates the transcriptional regulation of the AR through discrete, CDK4-independent mechanisms. The transcriptional co-repressor function of cyclin D1 resides within a defined motif termed ther repressor domain (RD), and it was hypothesized that this motif could be utilized as a platform to develop new strategies for blocking AR function. Here, we demonstrate that expression of the RD peptide is sufficient to disrupt AR transcriptional activation of multiple, prostate-specific AR target genes. Importantly, these actions are sufficient to specifically inhibit S-phase progression in AR-positive PCa cells, but not in AR-negative cells or tested AR-positive cells of other lineages. As expected, impaired cell cycle progression resulted in a suppression of cell doubling. Additionally, cell death was observed in AR-positive cells that maintain androgen dependence and in a subset of castrate-resistant PCa cells, dependent on Akt activation status. Lastly, the ability of RD to cooperate with existing hormone therapies was examined, which revealed that RD enhanced the cellular response to an AR antagonist. Together, these data demonstrate that RD is sufficient to disrupt AR-dependent transcriptional and proliferative responses in PCa, and can enhance efficacy of AR antagonists, thus establishing the impetus for development of RD-based mimetics.
Androgen Receptor; Testosterone; Prostatic Adenocarcinoma; Cell Cycle
Ischemia-reperfusion (I/R) injury, often encountered clinically, results in myocardial apoptosis and necrosis. Hydrogen sulfide (H2S) is produced endogenously in response to ischemia and thought to be cardioprotective, although its mechanism of action is not fully known. This study investigates cardioprotection provided by exogenous H2S, generated as sodium sulfide on apoptosis following myocardial I/R injury.
The mid-LAD coronary artery in Yorkshire swine (n=12) was occluded for 60 minutes, followed by reperfusion for 120 minutes. Controls (n=6) received placebo, and treatment animals (n=6) received sulfide 10 minutes prior to and throughout reperfusion. Hemodynamic, global, and regional functional measurements were obtained. Evans blue/TTC staining identified the area-at-risk (AAR) and infarction. Serum CK-MB, troponin I, and FABP were assayed. Tissue expression of bcl-2, bad, apoptosis-inducing-factor (AIF), total & cleaved caspase-3, and total & cleaved PARP were assessed. PAR and TUNEL staining were performed to assess apoptotic cell counts, and poly-ADP ribosylation, respectively.
Pre-I/R hemodynamics were similar between groups. Post-I/R, mean arterial pressure (mmHg) was reduced by 30.2±4.3 in controls vs. 8.2±6.9 in treatment animals (p=0.01). +LV dP/dt (mmHg/sec) was reduced by 1308±435 in controls vs. 403±283 in treatment animals (p=0.001). Infarct size (% of AAR) in controls was 47.4±6.2% vs. 20.1±3.3% in the treated group (p=0.003). In treated animals, CK-MB and FABP were lower by 47.0% (p=0.10) and 45.1% (p=0.01), respectively. AIF, caspase-3, and PARP expression was similar between groups, whereas cleaved caspase-3 and cleaved PARP was lower in treated animals (p=0.04). PAR staining was significantly reduced in sulfide treated groups (p=0.04). TUNEL staining demonstrated significantly fewer apoptotic cells in sulfide treated animals (p=0.02).
Sodium sulfide is efficacious in reducing apoptosis in response to I/R injury. Along with its known effects on reducing necrosis, sulfide’s effects on apoptosis may partially contribute to providing myocardial protection. Exogenous sulfide may have therapeutic utility in clinical settings in which I/R injury is encountered.
Ischemia; Reperfusion; Apoptosis; Cardiac; Sulfide; Protection
We investigated the role of calcium-activated potassium (KCa) channel activity in human skeletal muscle microvascular function in the setting of cardiopulmonary bypass (CPB).
Methods and Results
Human skeletal muscle arterioles (80- to 180 μm in diameter) were dissected from tissue harvested before and after CPB. In vitro relaxation responses of precontracted arterioles in a pressurized no-flow state were examined in the presence of KCa channel activators/blockers and several other vasodilators. Post-CPB responses to the activator of intermediate (IKCa) and small conductance (SKCa) KCa channels, NS309, to the endothelium-dependent vasodilator adenosine 5′-diphosphate (ADP), and to substance P were reduced compared with pre-CPB responses (P < .05), respectively, whereas responses to the activator of large conductance (BKCa) KCa channels, NS1619, and to the endothelium-independent vasodilator, sodium nitroprusside (SNP) were unchanged. Endothelial denudation decreased NS309-induced relaxation and abolished that induced by ADP or substance P (P< .05), but had no effect on relaxation induced by either NS1619 or SNP. Polypeptide levels of BKCa, IKCa, and SK3Ca were not altered post-CPB.
IK/SK-mediated relaxation is predominantly endothelium dependent, whereas BK-mediated relaxation seems to be largely independent of endothelial function in human skeletal muscle microvasculature. CPB-associated microvascular dysfunction likely arises in part from impaired function of endothelial SK and IK channels in the peripheral microvasculature.
Cardioplegic arrest (CP) followed by reperfusion after cardiopulmonary bypass induces coronary microvascular dysfunction. We investigated the role of calcium-activated potassium (KCa) channels in this dysfunction in the human coronary microvasculature.
Methods and Results
Human atrial tissue was harvested before CP from a nonischemic segment and after CP from an atrial segment exposed to hyperkalemic cold blood CP (mean CP time, 58 minutes) followed by 10-minute reperfusion. In vitro relaxation responses of precontracted arterioles (80 to 180 μm in diameter) in a pressurized no-flow state were examined in the presence of KCa channel activators/blockers and several other vasodilators. We also examined expression and localization of KCa channel gene products in the coronary microvasculature using reverse transcriptase-polymerase chain reaction, immunoblot, and immunofluorescence photomicroscopy. Post-CP reperfusion relaxation responses to the activator of intermediate and small conductance KCa channels (IKCa/SKCa), NS309 (10-5 M), and to the endothelium-dependent vasodilators, substance P (10-8 M) and adenosine 5′diphosphate (10-5 M), were significantly reduced compared with pre-CP responses (P<0.05, n=8/group). In contrast, relaxation responses to the activator of large conductance KCa channels (BKCa), NS1619 (10-5 M), and to the endothelium-independent vasodilator, sodium nitroprusside (10-4 M), were unchanged pre- and post-CP reperfusion (n=8/group). Endothelial denudation significantly diminished NS309-induced vasodilatation and abolished substance P- or adenosine 5′ diphosphate-induced relaxation (P<0.05), but had no effect on relaxation induced by either NS1619 or sodium nitroprusside. The total polypeptide levels of BKCa, IKCa, and SKCa and the expression of IKCa mRNA were not altered post-CP reperfusion.
Cardioplegic arrest followed by reperfusion after cardiopulmonary bypass causes microvascular dysfunction associated with and likely in part due to impaired function of SKCa and IKCa channels in the coronary microcirculation. These results suggest novel mechanisms of endothelial and smooth muscle microvascular dysfunction after cardiac surgery.
calcium-activated potassium channels; cardioplegia; ischemia and reperfusion; microcirculation
To identify genes involved in phenotypes that increase one's risk for developing asthma, a complex disease that is likely genetically heterogeneous. Unlike other approaches to locus discovery in the presence of heterogeneity, this method seeks loci that segregate in all or most ascertained families while recognizing that other genes and environmental factors that modify the action of the common gene may vary across families.
The method is based on seeking groups of families that differ, between groups, in the way affected idndividuals express the genotype. Then we use the distance of each individual to the cluster center for his family to define a quantitative trait. This quantitative trait is then subjected to a genome scan using variance components methods.
The method is applied to a data set of 27 multigenerational families with asthma, and a novel locus at 2q33 (at 210 cM) is identified.
The proposed method has the potential to identify loci near genes that increase risk for asthma related phenotypes. The method could be used for other complex disorders that exhibit locus heterogeneity.
Asthma; Linkage analysis; Locus heterogeneity; Quantitative trait locus
Adeno-associated viral (AAV) vectors have been shown to direct stable gene transfer and expression in hepatocytes, which makes them attractive tools for treatment of inherited disorders such as hemophilia B. While substantial levels of coagulation factor IX (F.IX) have been achieved using AAV serotype 2 vectors, use of a serotype 5 vector further improves transduction efficiency and levels of F.IX transgene expression by 3- to 10-fold. In addition, the AAV-5 vector transduces a higher proportion of hepatocytes (∼15%). The subpopulations of hepatocytes transduced with either vector widely overlap, with the AAV-5 vector transducing additional hepatocytes and showing a wider area of transgene expression throughout the liver parenchyma.
Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.