Showing papers by "Simon C. Watkins published in 2001"
TL;DR: Skeletal muscle of trained endurance athletes is markedly insulin sensitive and has a high oxidative capacity, despite having an elevated lipid content, according to quantitative image analysis of Oil Red O staining.
Abstract: We examined the hypothesis that an excess accumulation of intramuscular lipid (IMCL) is associated with insulin resistance and that this may be mediated by the oxidative capacity of muscle. Nine sedentary lean (L) and 11 obese (O) subjects, 8 obese subjects with type 2 diabetes mellitus (D), and 9 lean, exercise-trained (T) subjects volunteered for this study. Insulin sensitivity (M) determined during a hyperinsulinemic (40 mU x m(-2)min(-1)) euglycemic clamp was greater (P < 0.01) in L and T, compared with O and D (9.45 +/- 0.59 and 10.26 +/- 0.78 vs. 5.51 +/- 0.61 and 1.15 +/- 0.83 mg x min(-1)kg fat free mass(-1), respectively). IMCL in percutaneous vastus lateralis biopsy specimens by quantitative image analysis of Oil Red O staining was approximately 2-fold higher in D than in L (3.04 +/- 0.39 vs. 1.40 +/- 0.28% area as lipid; P < 0.01). IMCL was also higher in T (2.36 +/- 0.37), compared with L (P < 0.01). The oxidative capacity of muscle determined with succinate dehydrogenase staining of muscle fibers was higher in T, compared with L, O, and D (50.0 +/- 4.4, 36.1 +/- 4.4, 29.7 +/- 3.8, and 33.4 +/- 4.7 optical density units, respectively; P < 0.01). IMCL was negatively associated with M (r = -0.57, P < 0.05) when endurance-trained subjects were excluded from the analysis, and this association was independent of body mass index. However, the relationship between IMCL and M was not significant when trained individuals were included. There was a positive association between the oxidative capacity and M among nondiabetics (r = 0.37, P < 0.05). In summary, skeletal muscle of trained endurance athletes is markedly insulin sensitive and has a high oxidative capacity, despite having an elevated lipid content. In conclusion, the capacity for lipid oxidation may be an important mediator of the association between excess muscle lipid accumulation and insulin resistance.
1,079 citations
15 May 2001
TL;DR: Current Protocols in Cytometry (CPC), published in affiliation with the International Society for Analytical Cytology, features carefully edited flow and image cytometry methods provided by leading laboratories from around the world.
Abstract: Current Protocols in Cytometry (CPC), published in affiliation with the International Society for Analytical Cytology, features carefully edited flow and image cytometry methods provided by leading laboratories from around the world. All methods included in the one-volume looseleaf manual are rigorously tested and proven before being selected for CPC. Carefully edited, step-by-step protocols replete with material lists, expert commentaries, and safety and troubleshooting tips ensure that you can duplicate the experimental results in your own laboratory. This publication also includes extensive coverage of cytometry instrumentation, safety and quality control, and data processing and analysis. Quarterly updates, which are filed into the looseleaf, keep the set current with the latest developments in cytometry methods. The initial purchase includes one year of updates and then subscribers may renew their annual subscriptions. Current Protocols publishes a family of laboratory manuals for bioscientists, including Molecular Biology, Immunology, Human Genetics, Protein Science, Cell Biology, Neuroscience, Pharmacology, and Toxicology.
644 citations
TL;DR: Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Angeles, CA, and approved August 27, 2001.
Abstract: Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Los Angeles, CA, and approved August 27, 2001 (received for review May 16, 2001)
516 citations
TL;DR: Based on single-fiber analysis, skeletal muscle in obese and type 2 diabetic subjects mani-fests disturbances of oxidative enzyme activity and increased lipid content that are independent of the effect of fiber type are found.
Abstract: In obesity and type 2 diabetes, skeletal muscle has been observed to have a reduced oxidative enzyme activity, increased glycolytic activity, and increased lipid content. These metabolic characteristics are related to insulin resistance of skeletal muscle and are factors potentially related to muscle fiber type. The current study was undertaken to examine the interactions of muscle fiber type in relation to oxidative enzyme activity, glycolytic enzyme activity, and muscle lipid content in obese and type 2 diabetic subjects compared with lean healthy volunteers. The method of single-fiber analysis was used on vastus lateralis muscle obtained by percutaneous biopsy from 22 lean, 20 obese, and 20 type 2 diabetic subjects (ages 35 ± 1, 42 ± 2, and 52 ± 2 years, respectively), with values for BMI that were similar in obese and diabetic subjects (23.7 ± 0.7, 33.2 ± 0.8, and 31.8 ± 0.8 kg/m 2 , respectively). Oxidative enzyme activity followed the order of type I > type IIa > type IIb, but within each fiber type, skeletal muscle from obese and type 2 diabetic subjects had lower oxidative enzyme activity than muscle from lean subjects ( P P
510 citations
TL;DR: It is demonstrated that caspase‐3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury and Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cellDeath occurs.
Abstract: During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.
377 citations
TL;DR: It is shown for the first time that DC readily acquire Ag from other live cells, suggesting that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.
Abstract: Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 microm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.
327 citations
TL;DR: The fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae is studied, indicating that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating thatCFTR is a bona fide ERAD substrate in yeast.
Abstract: Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.
278 citations
TL;DR: It is demonstrated that C1q also binds directly to apoptotic blebs of vascular endothelial cells and PBMC, suggesting that C 1q may participate in the clearance of apoptotic cells from the circulation and from the walls of the vascular lumen.
Abstract: Complement protein C1q is required to maintain immune tolerance. The molecular mechanism responsible for this link has not been determined. We have previously demonstrated that C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes, suggesting that it may participate in clearance of self Ags generated during programmed cell death. Here, we demonstrate that C1q also binds directly to apoptotic blebs of vascular endothelial cells and PBMC. These apoptotic cells are recognized by the globular heads of C1q, which bind specifically to the surface blebs, and deposition increases as the blebs mature on the cell surface. These observations suggest that C1q may participate in the clearance of apoptotic cells from the circulation and from the walls of the vascular lumen. The interaction of surface blebs with the globular heads of C1q suggests that surface blebs may be capable of directly activating the classical pathway of complement under certain circumstances, generating C4- and C3-derived ligands for receptors such as CR1, CR2, CR3, and CR4. Appropriate recognition of apoptotic cells by C1q and targeted clearance of the molecular contents of surface blebs to complement receptors may be critical for the maintenance of immune tolerance.
266 citations
TL;DR: Prostaglandins, through the induction of COX-2, are major participants in rodent postoperative ileus induced by intestinal manipulation, and could be alleviated pharmacologically with selective COx-2 inhibition.
244 citations
TL;DR: In this article, the authors identified and characterized a population of Langerhans cells (LCs) precursors that were resident in human skin and showed that they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage-colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells.
Abstract: Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-β1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage–colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.
225 citations
TL;DR: Assessment of the in vivo function of aspirin-treated DC revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue, and a novel approach to the manipulation of DC for therapeutic application is suggested.
Abstract: Aspirin is the most commonly used analgesic and antiinflammatory agent In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC) CD11c and MHC class I expression were unaffected The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-kappaB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-kappaB p50 Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application
TL;DR: Reduction in the level of Foxf1 caused neonatal pulmonary hemorrhage and abnormalities in alveologenesis, implicating this transcription factor in the regulation of mesenchyme-epithelial interaction critical for lung morphogenesis.
TL;DR: Myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.
Abstract: To better understand the structure and function of Z lines, we used sarcomeric isoforms of α-actinin and γ-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an α-actinin- and γ-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by α-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for α-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.
TL;DR: The data reveal that SEC are dynamic regulators of porosity that respond rapidly and locally to environmental zonal stimuli during liver regeneration.
TL;DR: These findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.
Abstract: Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, α1-syntrophin, and the sarcoglycan complex. The precise role of α-dystrobrevin in skeletal muscle has not yet been determined. To study α-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and α-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.
TL;DR: The data identify novel autocrine and paracrine interactions and indicate that each growth factor acts on a specific set of EC at specific times during revascularization of regenerating liver.
TL;DR: The results suggest that TNF- α by itself is not sufficient to induce apoptosis in cardiac myocytes in vivo, and signaling pathways responsible for T NF-α induced apoptosis should be elucidated.
TL;DR: It is suggested that systemic as well as local sources of iNOS regulate reperfusion, and local iN OS contributes to hepatic injury, while eNOS is protective in warm hepatic ischemia-reperfusion.
Abstract: To determine the contribution of the inducible nitric oxide synthase (iNOS) to hepatic injury following warm ischemia-reperfusion, we developed a model of partial hepatic ischemia-reperfusion in mice and studied the injury response in iNOS knockout (KO) mice. Compared with wild types, iNOS KO animals exhibited lower plasma transaminase levels after 1 and 6 h of reperfusion following 1 h of ischemia. At the 3-h time point, enzyme levels were not different between the two groups, iNOS mRNA was not detectable in the ischemic hepatic lobes of wild-type mice until 3 h of reperfusion; however, perfusion studies identified a significant delay in reperfusion of the ischemic lobe in the iNOS KO mice at the 1-h time point with similar perfusion rates at 3 and 6 h compared with wild type. By way of comparison, mice deficient in the endothelial NOS (eNOS) were also assessed for the degree of hepatic damage 3 h post-reperfusion. Plasma transaminase levels were significantly increased in eNOS KO animals compared with wild-type controls. These data suggest that systemic as well as local sources of iNOS regulate reperfusion, and local iNOS contributes to hepatic injury, while eNOS is protective in warm hepatic ischemia-reperfusion.
TL;DR: In the rat, 60 minutes of transient ischemia to the middle cerebral artery results in infarction of the caudate putamen, and bcl-2 appears to be a major determinant in this model of induced tolerance to focal ischemies.
Abstract: In the rat, 60 minutes of transient ischemia to the middle cerebral artery results in infarction of the caudate putamen. Ischemic preconditioning with 20 minutes of transient focal ischemia produced tolerance (attenuated infarction volume) to 60 minutes of subsequent focal ischemia administered three days, five days, or seven days later. Western blots from tolerant caudate putamen demonstrated increased bcl-2 expression, maximum at 3 days and persisting through 7 days. Immunocytochemical examination found that bcl-2 was expressed in cells with both neuronal and nonneuronal morphology in striatum after preconditioning ischemia. bcl-2 antisense oligodeoxynucleotides (ODNs), bcl-2 sense ODNs, or artificial cerebrospinal fluid (CSF, vehicle) was infused into the lateral ventricle for the 72 hours between the 20-minute ischemic preconditioning and the 60-minute period of ischemia. Antisense ODN treatment reduced expression of bcl-2 in the striatum and blocked the induction of tolerance by preconditioning ischemia. Sense and CSF treatments had no effect on either bcl-2 expression or tolerance. In this model of induced tolerance to focal ischemia, bcl-2 appears to be a major determinant.
TL;DR: In vivo administration of liver DEC205+B220+CD19− cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.
Abstract: Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.
TL;DR: Data demonstrate that AdCREBm1 causes apoptosis and elimination of germ cells and suggest that CREB is required to produce a Sertoli cell-derived factor that is critical for germ cell survival.
Abstract: FSH binding to Sertoli cells is required for optimal production of sperm in mammals. The cAMP response element-binding protein (CREB) is a major mediator of FSH-induced changes in gene expression. To determine whether CREB is required for spermatogenesis, an adenovirus encoding a phosphorylation-defective CREB mutant (AdCREBm1) was used to inhibit CREB activity in Sertoli cells. Addition of AdCREBm1 to primary rat Sertoli cell cultures completely abolished induction of the CREB-regulated c-fos gene. Injection of an adenovirus encoding ss-galactosidase into the rat testis seminiferous tubules in vivo demonstrated that predominately Sertoli cells were infected by adenovirus. AdCREBm1-directed expression of CREBm1 in seminiferous tubules did not affect Sertoli cell viability, but resulted in the apoptosis of meiotic spermatocyte germ cells within 4 days of adenovirus injection into seminiferous tubules. Disrupted spermatogenesis, defined by at least a 75% reduction of round spermatids, was observed in 42 +/- 5.8% of seminiferous tubules 14 days after AdCREBm1 infection, whereas using this criteria, testes injected with a control adenovirus did not display disrupted spermatogenesis. These data demonstrate that AdCREBm1 causes apoptosis and elimination of germ cells and suggest that CREB is required to produce a Sertoli cell-derived factor that is critical for germ cell survival.
TL;DR: Adenoviral inducible nitric oxide synthase (iNOS) gene transfer could inhibit intimal hyperplasia (IH) in porcine internal jugular veins interposed into the carotid artery circulation with potential for iNOS-based genetic modification of vein grafts to prolong graft patency.
TL;DR: It is demonstrated that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells.
Abstract: Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.
TL;DR: Local upregulation of IL-11 may represent an adaptive response designed to limit the extent of intestinal damage in NEC, and decreased IL-12 levels may contribute to the pathogenesis of NEC by allowing bacteria to escape host defenses.
TL;DR: It is reported that Ras proteins are associated with lipid rafts in resting HIRcB fibroblasts and mobilization of active Raf-1 through the endocytic compartment is required for completion of the MAPK cascade.
TL;DR: It is demonstrated that TBI results in extensive DNA damage, which includes both single- and double-strand breaks in injured cortex and hippocampus, and the presence of multiple types of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.
Abstract: DNA damage is a common sequela of traumatic brain injury (TBI). Available techniques for the in situ identification of DNA damage include DNA polymerase I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of DNA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). While TUNEL has been widely utilized to detect primarily double-strand DNA breaks, the use of PANT to detect primarily single-strand DNA breaks and Klenow to detect both single- and double-strand DNA breaks has not been reported after TBI. Accordingly, coronal brain sections from naive rats and rats at 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical impact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsilateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (cortex = 66.3 ± 15.8, dentate gyrus 58.6 ± 12....
TL;DR: Cardiac mtDNA repair mechanisms, mtDNA damage, and mitochondrial structure and function in mice with heart failure secondary to overexpression of TNF-&agr; (TNF1.6 mice) are studied to suggest that mtDNA mutations and mitochondrial structural and functional alterations in TNF–induced heart failure may be associated with reduced mt DNA repair activity.
Abstract: Background Recent studies suggest that mutations in cardiac mitochondrial DNA (mtDNA) may contribute to the development of dilated cardiomyopathy. The mechanisms that regulate those mutations, however, remain undefined. Thus, we studied cardiac mtDNA repair mechanisms, mtDNA damage, and mitochondrial structure and function in mice with heart failure secondary to overexpression of TNF-α (TNF1.6 mice). Methods and Results We studied mtDNA repair by measuring the uracil DNA glycosylase (mtUDG) and base excision repair activities. mtDNA damage was assessed by Southern blot of Fpg protein–digested mtDNA. Mitochondrial ultrastructural changes were examined by electron microscopy, and function by cytochrome c oxidase and succinate dehydrogenase activity assays. The results showed that both mtUDG and base excision repair activities were significantly reduced in TNF1.6 mouse heart. Fpg-sensitive sites were markedly increased in TNF1.6 mouse cardiac mtDNA, suggesting increased mtDNA damage. Mitochondrial function a...
TL;DR: The persistence of a poly-L-lactic acid (PLLA) interference screw 2.5 years after anterior cruciate ligament (ACL) reconstruction with Achilles tendon allograft shows that biodegradation of PLLA material in the knee joint causes no irritation and can take several years, even if the material is in contact with the synovial fluid.
Abstract: We report the persistence of a poly-L-lactic acid (PLLA) interference screw 2.5 years after anterior cruciate ligament (ACL) reconstruction with Achilles tendon allograft. The arthroscopy was performed because the patient sustained a reinjury of the ACL graft, making ACL revision surgery necessary. At the time of arthroscopy, both PLLA screws were macroscopically still intact but could not be removed in 1 piece. No inflammation could be observed either macroscopically or in the histologic analysis. The biopsy specimen from the femoral insertion of the graft showed parts of the PLLA material surrounded by scar tissue. This case shows that biodegradation of PLLA material in the knee joint causes no irritation and can take several years, even if the material is in contact with the synovial fluid.
TL;DR: It is concluded that NOX reduces hepatocellular injury after endotoxemia, and may be useful in the management of hepatic dysfunction secondary to sepsis or other diseases associated with excessive NO production.
Abstract: Sustained upregulation of inducible nitric oxide (NO) synthase in the liver after endotoxin [lipopolysaccharide (LPS)] challenge may result in hepatocellular injury. We hypothesized that administra...
TL;DR: Evidence indicating that the interaction of S1A with CFTR and ENaC reduces channel currents in a syntaxin-isoform-specific manner is summarized, and it is suggested that phosphorylation-dependent interactions of these channels with SNARE proteins may acutely regulate this process.
Abstract: The apical membrane ion channels, CFTR and ENaC, undergo regulated trafficking as a means of controlling their plasma membrane density. This provides a mechanism for regulating the Cl and Na conductance properties of epithelial apical membranes, and thus the transepithelial ion transport rates. Physical and functional interactions between these channels and SNARE proteins, in particular syntaxin 1A (S1A), provide a mechanism for linking the known vesicle fusion machinery with this process. In this paper we summarize evidence indicating that the interaction of S1A with CFTR and ENaC reduces channel currents in a syntaxin-isoform-specific manner. The acute cAMP-regulated CFTR trafficking event, which is reported by an increase in membrane capacitance in response to cAMP, is also inhibited by exogenous S1A expression. We tagged both channels with flag epitopes on their extracellular surfaces to monitor their plasma membrane expression as a function of S1A co-expression. The data indicate that the reduction in current caused by S1A is associated with a marked decrease in the amount of CFTR or ENaC detected at the cell surface. These findings suggest that S1A inhibits ion channel insertion into the plasma membrane, either by disrupting the stoichiometry of SNARE protein associations that mediate channel trafficking, or by physically associating with the channels to prevent their insertion. These data link the SNARE machinery to the regulation of apical membrane ion channel density, and suggest that phosphorylation-dependent interactions of these channels with SNARE proteins may acutely regulate this process.