241 research outputs found
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Nitric Oxide Activates Intradomain Disulfide Bond Formation in the Kinase Loop of Akt1/PKBĪ± after Burn Injury
Severe burn injury is an acute inflammatory state with massive alterations in gene expression and levels of growth factors, cytokines and free radicals. During the catabolic processes, changes in insulin sensitivity and skeletal muscle wasting (unintended loss of 5ā15% of lean body mass) are observed clinically. Here, we reveal a novel molecular mechanism of Akt1/protein kinase BĪ± (Akt1/PKBĪ±) regulated via cross-talking between dephosphorylation of Thr308 and S-nitrosylation of Cys296 post severe burn injury, which were characterized using nano-LC interfaced with tandem quadrupole time-of-fight mass spectrometry (Q-TOF)micro tandem mass spectrometry in both in vitro and in vivo studies. For the in vitro studies, Akt1/PKBĪ± was S-nitrosylated with S-nitrosoglutathione and derivatized by three methods. The derivatives were isolated by SDS-PAGE, trypsinized and analyzed by the tandem MS. For the in vivo studies, Akt1/PKBĪ± in muscle lysates from burned rats was immuno-precipitated, derivatized with HPDP-Biotin and analyzed as above. The studies demonstrated that the NO free radical reacts with the free thiol of Cys296 to produce a Cys296-SNO intermediate which accelerates interaction with Cys310 to form Cys296-Cys310 in the kinase loop. MS/MS sequence analysis indicated that the dipeptide, linked via Cys296-Cys310, underwent dephosphorylation at Thr308. These effects were not observed in lysates from sham animals. As a result of this dual effect of burn injury, the loose conformation that is slightly stabilized by the Lys297-Thr308 salt bridge may be replaced by a more rigid structure which may block substrate access. Together with the findings of our previous report concerning mild IRS-1 integrity changes post burn, it is reasonable to conclude that the impaired Akt1/PKBĪ± has a major impact on FOXO3 subcellular distribution and activities
Recent Decisions
Comments on recent decisions by George N. Tompkins, James M. Corcoran, John L. Rosshirt, Ronald P. Mealey, Patrick J. Foley, Lawrence J. Dolan, Edward J. Griffin, John W. Thornton, and Thomas R. King
Significance Analysis of Time Course Microarray Experiments
Characterizing the genome-wide dynamic regulation of gene expression is important and will be of much interest in the future. However, there is currently no established method for identifying differentially expressed genes in a time course study. Here we propose a significance method for analyzing time course microarray studies that can be applied to the typical types of comparisons and sampling schemes. This method is applied to two studies on humans. In one study, genes are identified that show differential expression over time in response to in vivo endotoxin administration. Using our method 7409 genes are called significant at a 1% FDR level, whereas several existing approaches fail to identify any genes. In another study, 417 genes are identified at a 10% FDR level that show expression changing with age in the kidney cortex. Here it is also shown that as many as 47% of the genes change with age in a manner more complex than simple exponential growth or decay. The methodology proposed here has been implemented in the freely distributed and open-source EDGE software package
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The Quorum Sensing Volatile Molecule 2-Amino Acetophenon Modulates Host Immune Responses in a Manner that Promotes Life with Unwanted Guests
Increasing evidence indicates that bacterial quorum sensing (QS) signals are important mediators of immunomodulation. However, whether microbes utilize these immunomodulatory signals to maintain infection remain unclear. Here, we show that the Pseudomonas aeruginosa QS-regulated molecule 2-amino acetophenone (2-AA) modulates host immune responses in a manner that increases host ability to cope with this pathogen. Mice treated with 2-AA prior to infection had a 90% survival compared to 10% survival rate observed in the non-pretreated infected mice. Whilst 2-AA stimulation activates key innate immune response pathways involving mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-, and pro-inflammatory cytokines, it attenuates immune response activation upon pretreatment, most likely by upregulating anti-inflammatory cytokines. 2-AA host pretreatment is characterized by a transcriptionally regulated block of c-JUN N-terminal kinase (JNK) and NF- activation, with relatively preserved activation of extracellular regulated kinase (ERK) 1/2. These kinase changes lead to CCAAT/enhancer-binding protein- activation and formation of the complex that prevents NF- activation. 2-AA's aptitude for dampening the inflammatory processes while increasing host survival and pathogen persistence concurs with its ability to signal bacteria to switch to a chronic infection mode. Our results reveal a QS immunomodulatory signal that promotes original aspects of interkingdom communication. We propose that this communication facilitates pathogen persistence, while enabling host tolerance to infection
Developing Effective Alzheimer's Disease Therapies: Clinical Experience and Future Directions
Alzheimer's disease (AD) clinical trials, focused on disease modifying drugs and conducted in patients with mild to moderate AD, as well as prodromal (early) AD, have failed to reach efficacy endpoints in improving cognitive function in most cases to date or have been terminated due to adverse events. Drugs that have reached clinical stage were reviewed using web resources (such as clinicaltrials.gov, alzforum.org, company press releases, and peer reviewed literature) to identify late stage (Phase II and Phase III) efficacy clinical trials and summarize reasons for their failure. For each drug, only the latest clinical trials and ongoing trials that aimed at improving cognitive function were included in the analysis. Here we highlight the potential reasons that have hindered clinical success, including clinical trial design and choice of outcome measures, heterogeneity of patient populations, difficulties in diagnosing and staging the disease, drug design, mechanism of action, and toxicity related to the long-term use. We review and suggest approaches for AD clinical trial design aimed at improving our ability to identify novel therapies for this devastating disease
Evaluation of Human Skin Reconstituted from Composite Grafts of Cultured Keratinocytes and Human Acellular Dermis Transplanted to Athymic Mice
This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections, Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds
Gram-Negative Bacterial Infection in Thigh Abscess Can Migrate to Distant Burn Depending on Burn Depth
Sepsis remains the major cause of death in patients with major burn injuries. In the present investigation we evaluated the interaction between burn injuries of varying severity and preexisting distant infection. We used Gram-negative bacteria (Pseudomonas aeruginosa and Proteus mirabilis) that were genetically engineered to be bioluminescent, which allowed for noninvasive, sequential optical imaging of the extent and severity of the infection. The bioluminescent bacteria migrated from subcutaneous abscesses in the leg to distant burn wounds on the back depending on the severity of the burn injury, and this migration led to increased mortality of the mice. Treatment with ciprofloxacin, injected either in the leg with the bacterial infection or into the burn eschar, prevented this colonization of the wound and decreased mortality. The present data suggest that burn wounds can readily become colonized by infections distant from the wound itself
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Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle
Objective: Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. Methods: A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. Results: Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3Ī² was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. Conclusions: Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients
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A Small Volatile Bacterial Molecule Triggers Mitochondrial Dysfunction in Murine Skeletal Muscle
Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) metabolomics, in vivo 31P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by 1H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-Ī³, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo 31P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections
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