Showing papers in "The FASEB Journal in 2003"

Oxidative DNA damage: mechanisms, mutation, and disease

Abstract: Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8-oxo-2'deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.

One-thousand-and-one substrates of protein kinase CK2?

Abstract: CK2 (formerly termed "casein kinase 2") is a ubiquitous, highly pleiotropic and constitutively active Ser/Thr protein kinase whose implication in neoplasia, cell survival, and virus infection is supported by an increasing number of arguments. Here an updated inventory of 307 CK2 protein substrates is presented. More than one-third of these are implicated in gene expression and protein synthesis as being either transcriptional factors (60) or effectors of DNA/RNA structure (50) or translational elements. Also numerous are signaling proteins and proteins of viral origin or essential to virus life cycle. In comparison, only a minority of CK2 targets (a dozen or so) are classical metabolic enzymes. An analysis of 308 sites phosphorylated by CK2 highlights the paramount relevance of negatively charged side chains that are (by far) predominant over any other residues at positions n+3 (the most crucial one), n+1, and n+2. Based on this signature, it is predictable that proteins phosphorylated by CK2 are much more numerous than those identified to date, and it is possible that CK2 alone contributes to the generation of the eukaryotic phosphoproteome more so than any other individual protein kinase. The possibility that CK2 phosphosites play some global role, e.g., by destabilizing alpha helices, counteracting caspase cleavage, and generating adhesive motifs, will be discussed.

Exercise and IL-6 infusion inhibit endotoxin-induced TNF-alpha production in humans.

Abstract: During "nondamaging" exercise, skeletal muscle markedly releases interleukin (IL)-6, and it has been suggested that one biological role of this phenomenon is to inhibit the production of tumor necrosis factor (TNF)- alpha, which is known to cause pathogenesis such as insulin resistance and atherosclerosis. To test this hypothesis, we performed three experiments in which eight healthy males either rested (CON), rode a bicycle for 3 h (EX), or were infused with recombinant human IL-6 (rhIL-6) for 3 h while they rested. After 2.5 h, the volunteers received a bolus of Escherichia coli lipopolysaccharide endotoxin (0.06 ng/kg) i.v. to induce low-grade inflammation. In CON, plasma TNF-alpha increased significantly in response to endotoxin. In contrast, during EX, which resulted in elevated IL-6, and rhIL-6, the endotoxin-induced increase in TNF-alpha was totally attenuated. In conclusion, physical exercise and rhIL-6 infusion at physiological concentrations inhibit endotoxin-induced TNF-alpha production in humans. Hence, these data provide the first experimental evidence that physical activity mediates antiinflammatory activity and suggest that the mechanism include IL-6, which is produced by and released from exercising muscles.

Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs.

Abstract: Cellular responses to oxygen are increasingly recognized as critical in normal development and physiology, and are implicated in pathological processes. Many of these responses are mediated by the transcription factors HIF-1 and HIF-2. Their regulation occurs through oxygen-dependent proteolysis of the alpha subunits HIF-1alpha and HIF-2alpha, respectively. Both are stabilized in cell lines exposed to hypoxia, and recently HIF-1alpha was reported to be widely expressed in vivo. In contrast, regulation and sites of HIF-2alpha expression in vivo are unknown, although a specific role in endothelium was suggested. We therefore analyzed HIF-2alpha expression in control and hypoxic rats. Although HIF-2alpha was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, including brain, heart, lung, kidney, liver, pancreas, and intestine. Time course and amplitude of induction varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non-parenchymal in some organs (kidney, pancreas, and brain), predominantly parenchymal in others (liver and intestine) or equally distributed (myocardium). These data indicate that HIF-2 plays an important role in the transcriptional response to hypoxia in vivo, which is not confined to the vasculature and is complementary to rather than redundant with HIF-1.

IL-1beta-mediated up-regulation of HIF-1alpha via an NFkappaB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis.

Abstract: SPECIFIC AIMThere is growing evidence that inflammation is a contributing factor leading to cancer development, yet pathways involved in this progression are not well understood. While HIF-1, a heterodimeric transcription factor composed of inducibly expressed HIF-1α and constitutively expressed HIF-1β, regulates genes that play essential roles in tumor progression and HIF-1 activity is up-regulated in inflammatory conditions, there is no prior evidence suggesting a role for HIF-1 as a link between inflammation and the development of cancer. Therefore, we investigated whether the HIF-1 signaling pathway was stimulated by the proinflammatory cytokine interleukin-1 β (IL-1β) -induced inflammatory signal in A549 cells.PRINCIPAL FINDINGS1. HIF-1α induction by IL-1β is mediated by NFκBTo test whether IL-1β was able to induce HIF-1α protein, we treated the lung epithelial cell line A549 with IL-1β under normoxia. HIF-1α protein was markedly induced by IL-1β. Because the proinflammatory cytokine IL-1β activates ...

α-synuclein implicated in Parkinson's disease is present in extracellular biological fluids, including human plasma

Abstract: Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of alpha-synuclein protein (alpha-syn) inside brain cells. It is likely that the formation of alpha-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding alpha-syn have been found in inherited forms of PD. alpha-Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of alpha-syn in conditioned culture media from untransfected and alpha-syn-transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti-alpha-syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, alpha-syn was identified as a single 15 kDa band, which co-migrated with a recombinant form of the protein and reacted with five different antibodies to alpha-syn. Our findings suggest that cells normally secrete alpha-syn into their surrounding media, both in vitro and in vivo. The detection of extracellular alpha-syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.

General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit.

Abstract: General anesthetics are widely used in clinical practice. On the molecular level, these compounds have been shown to modulate the activity of various neuronal ion channels. However, the functional relevance of identified sites in mediating essential components of the general anesthetic state, such as immobility and hypnosis, is still unknown. Using gene-targeting technology, we generated mice harboring a subtle point mutation (N265M) in the second transmembrane region of the beta3 subunit of the GABA(A) receptor. In these mice, the suppression of noxious-evoked movements in response to the intravenous anesthetics etomidate and propofol is completely abolished, while only slightly decreased with the volatile anesthetics enflurane and halothane. beta3(N265M) mice also display a profound reduction in the loss of righting reflex duration in response to intravenous but not volatile anesthetics. In addition, electrophysiological recordings revealed that anesthetic agents were significantly less effective in enhancing GABA(A) receptor-mediated currents, and in decreasing spontaneous action potential firing in cortical brain slices derived from mutant mice. Taken together, our results demonstrate that a single molecular target, and indeed a specific residue (N265) located within the GABA(A) receptor beta3 subunit, is a major determinant of behavioral responses evoked by the intravenous anesthetics etomidate and propofol, whereas volatile anesthetics appear to act via a broader spectrum of molecular targets.

CD105 is important for angiogenesis: evidence and potential applications.

Abstract: Angiogenesis is the propelling force for tumor growth and metastasis, and antiangiogenic therapy represents one of the most promising modalities for cancer treatment. CD105 (endoglin) is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells (EC). It is a receptor for transforming growth factor (TGF) -beta1 and -beta3 and modulates TGF-beta signaling by interacting with TGF-beta receptors I and/or II. Immunohistochemistry studies have revealed that CD105 is strongly expressed in blood vessels of tumor tissues. Intratumoral microvessel density (MVD) determined using antibodies to CD105 has been found to be an independent prognostic indicator, wherein increased MVD correlates with shorter survival. CD105 is able to be shed into the circulation, with elevated levels detected in patients with various types of cancer and positively correlated with tumor metastasis. Tangible evidence of its proangiogenic role comes from knockout studies in which CD105 null mice die in utero as a result of impaired angiogenesis in the yolk sac and heart defects. The potential usefulness of CD105 for tumor imaging has been evaluated in tumor-bearing mice and dogs that have shown the rapid accumulation of radiolabeled anti-CD105 monoclonal antibody in the tumors with a high tumor-to-background ratio. The anti-CD105 antibody conjugated with immunotoxins and immunoradioisotopes efficiently suppressed/abrogated tumor growth in murine models bearing breast and colon carcinoma without any significant systemic side effects. Immunoscintigraphy in patients with renal cell carcinomas has shown specific localization of 99Tcm-labeled CD105 mab in tumor endothelial cells. Thus, CD105 is a promising vascular target that can be used for tumor imaging, prognosis, and bears therapeutic potential in patients with solid tumors and other angiogenic diseases.

Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth

Abstract: Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Resveratrol: from grapevines to mammalian biology

Abstract: With the rapid advances made over the last two decades in biomedical research, there has been an unprecedented interest in unraveling the magical properties of some commonly used natural products. Consequently, a wide variety of natural products are under scrutiny for their clinical potential, both in terms of disease prevention and treatment. Among the compounds under investigation is a family of polymers given the name viniferin. These compounds elicit strong anti-fungal properties and are therefore included under the broad class of plant antibiotics known as phytoalexins (1). One remarkable compound in this list is resveratrol (RSV), a major active ingredient of stilbene phytoalexins, first isolated from the roots of the oriental medicinal plant Polygonum Capsidatum (Kojo-kon in Japanese) (2). Observations that this compound was an active ingredient of a folk plant known for its remedial effects against a host of human afflictions (2, 3) and that it was synthesized by leaf tissue in response to fungal infection of grapevines (Vitis vinifera) (4) provided the impetus for the increase in activity surrounding RSV in the field of biomedical research. The relatively high concentration of RSV in wine (5) and its documented cardioprotective effect (6) form the basis for the so-called “French paradox” (7). Most of the initial work on RSV was centered around its effects on metabolic pathways regulating cardiovascular biology, such as lipid metabolism and platelet function; however, since the reported cancer chemopreventive activity of RSV in animal models of carcinogenesis (8), recent investigations have been directed at understanding the molecular mechanism(s) of its diverse biological effects. As a result, the positive or negative effects of RSV on some important physiological pathways have been proposed as possible mechanisms for its observed cancer chemopreventive, cardioprotective, and neuroprotective activities. These include suppression of cellular proliferation via inhibition of key steps in the signal transduction pathways (9–12) and cyclin-dependent kinases (cdks) (13), promotion of cellular differentiation (14), scavenging/suppression of intracellular reactive oxygen intermediates (ROI) (15), induction of apoptotic cell death through activation of mitochondria-dependent or -independent pathways (16–18), anti-inflammatory activity via down-regulation of proinflammatory cytokines (19, 20), and inhibition of androgen receptor function and estrogenic activity (21, 22). This review is intended to provide the reader with an appreciation of the diverse biological effects of this remarkable compound, which could have tremendous potential as a chemopreventive and/or chemotherapeutic agent in clinical medicine.

Peroxisome proliferator-activated receptor δ controls muscle development and oxydative capability

Abstract: Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors exerting several functions in development and metabolism. The physiological functions of PPARdelta remain elusive. By using a CRE-Lox recombination approach, we generated an animal model for muscle-specific PPARdelta overexpression to investigate the role of PPARdelta in this tissue. Muscle-specific PPARdelta overexpression results in a profound change in fiber composition due to hyperplasia and/or shift to more oxidative fiber and, as a consequence, leads to the increase of both enzymatic activities and genes implicated in oxidative metabolism. These changes in muscle are accompanied by a reduction of body fat mass, mainly due to a large reduction of adipose cell size. Furthermore, we demonstrate that endurance exercise promotes an accumulation of PPARdelta protein in muscle of wild-type animals. Collectively, these results suggest that PPARdelta plays an important role in muscle development and adaptive response to environmental changes, such as training exercise. They strongly support the idea that activation of PPARdelta could be beneficial in prevention of metabolic disorders, such as obesity or type 2 diabetes.

Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense

Abstract: SPECIFIC AIMSLactoperoxidase (LPO) has long been appreciated as a potent antimicrobial enzyme in saliva, milk, and tears, although host sources of hydrogen peroxide supporting this activity have never been described; recently, several homologues of the microbicidal NADPH oxidase of phagocytes were identified in diverse tissues. Our objective was to identify potential sources of reactive oxygen species (ROS) on mucosal surfaces that may support the antimicrobial activity of LPO.1. Duox2, a homologue of the gp91phox component of the phagocyte NADPH oxidase, is detected in major ducts of the salivary glandWe studied Duox expression in salivary glands by Northern blot and in situ hybridization. Figure 1⤻ a shows that Duox2 mRNA is expressed in the human salivary gland; Duox1 was not detected (not shown). Northern blot also detected high Duox2 mRNA levels in rat submandibular salivary gland, using a cDNA probe from the rat Duox2 mRNA 3′-untranslated region (Fig. 1a⤻ ). In situ hybridization on rat submandibula...

Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells

Abstract: Electroporation by using pulsed electric fields with long durations compared with the charging time of the plasma membrane can induce cell fusion or introduce xenomolecules into cells. Nanosecond pulse power technology generates pulses with high-intensity electric fields, but with such short durations that the charging time of the plasma membrane is not reached, but intracellular membranes are affected. To determine more specifically their effects on cell structure and function, human cells were exposed to high intensity (up to 300 kV/cm) nanosecond (10-300 ns) pulsed electric fields (nsPEF) and were analyzed at the cellular and molecular levels. As the pulse duration decreased, plasma membrane electroporation decreased and appearances of apoptosis markers were delayed. NsPEF induced apoptosis within tens of minutes, depending on the pulse duration. Annexin-V binding, caspase activation, decreased forward light scatter, and cytochrome c release into the cytoplasm were coincident. Apoptosis was caspase- and mitochondria-dependent but independent of plasma membrane electroporation and thermal changes. The results suggest that with decreasing pulse durations, nsPEF modulate cell signaling from the plasma membrane to intracellular structures and functions. NsPEF technology provides a unique, high-power, energy-independent tool to recruit plasma membrane and/or intracellular signaling mechanisms that can delete aberrant cells by apoptosis.

Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets

Abstract: Although tissue factor (TF), the principial initiator of physiological coagulation and pathological thrombosis, has recently been proposed to be present in human blood, the functional significance and location of the intravascular TF is unknown. In the plasma portion of blood, we found TF to be mainly associated with circulating microvesicles. By cell sorting with the specific marker CD42b, platelet-derived microvesicles were identified as a major location of the plasma TF. This was confirmed by the presence of full-length TF in microvesicles acutely shedded from the activated platelets. TF was observed to be stored in the α-granules and the open canalicular system of resting platelets and to be exposed on the cell surface after platelet activation. Functional competence of the blood-based TF was enabled when the microvesicles and platelets adhered to neutrophils, as mediated by P-selectin and neutrophil counterreceptor (PSGL-1, CD18 integrins) interactions. Moreover, neutrophil-secreted oxygen radical species supported the intravascular TF activity. The pools of platelet and microvesicle TF contributed additively and to a comparable extent to the overall blood TF activity, indicating a substantial participation of the microvesicle TF. Our results introduce a new concept of TF-mediated coagulation crucially dependent on TF associated with microvesicles and activated platelets, which principally enables the entire coagulation system to proceed on a restricted cell surface.

Impaired insulin secretory capacity in mice lacking a functional vitamin D receptor.

Abstract: It was the aim of this study to further explore the functional role of vitamin D in the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. Pancreatic islets but not exocrine pancreas cells showed strong lacZ reporter gene expression in mutant mice. To rule out possible influences of hypocalcemia on pancreatic endocrine function, a rescue diet enriched with calcium, phosphorus, and lactose was fed to wild-type (WT) and VDR mutant mice. The rescue diet normalized body weight and mineral homeostasis in VDR mutants. In glucose tolerance tests, baseline blood glucose levels were unchanged in fasting VDR mutants. However, blood glucose was elevated after oral or subcutaneous glucose loading, and maximum serum insulin levels were reduced by approximately 60% in VDR mutants vs. WT mice on either diet. In addition, insulin mRNA levels were decreased in VDR mutant mice on both diets, whereas pancreatic beta cell mass, islet architecture, and islet neogenesis were normal. These findings clearly establish a molecular role of the vitamin D-responsive elements in pancreatic insulin synthesis and secretion in vivo.

Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria

Abstract: Several lines of evidence indicate that mitochondria-mediated reactive oxygen species (ROS) generation is a major source of oxidative stress in the cell. Release of cytochrome c from mitochondria is a central event in apoptosis induction and appears to be mediated by ROS. Dissociation of cytochrome c from the IMM, where it is bound to cardiolipin, represents a necessary first step for cytochrome c release. In the present study, the role of ROS and cardiolipin in the release of cytochrome c from rat liver mitochondria was investigated. ROS were produced by mitochondria oxidizing succinate in the nonphosphorylating state. Cytochrome c was quantitated by a new, very sensitive and rapid reverse-phase HPLC method. We found that succinate-supported ROS production resulted in a release of cytochrome c from mitochondria and a parallel loss of cardiolipin content. These effects were directly and significantly correlated and also abolished by ADP, which prevents succinate-mediated ROS production. The ROS-induced cytochrome c release was independent from MPT and appears to involve VDAC. It is suggested that mitochondrial-induced ROS production promotes cytochrome c release from mitochondria by a two-steps process, consisting of the dissociation of this protein from cardiolipin, followed by permeabilization of the outer membrane, probably by interaction with VDAC. The data may help clarify the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol and the role of ROS and cardiolipin in this release.

Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate.

Abstract: Green tea extract and its main polyphenol constituent (-)-epigallocatechin-3-gallate (EGCG) possess potent neuroprotective activity in cell culture and mice model of Parkinson's disease. The central hypothesis guiding this study is that EGCG may play an important role in amyloid precursor protein (APP) secretion and protection against toxicity induced by beta-amyloid (Abeta). The present study shows that EGCG enhances (approximately 6-fold) the release of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPalpha) into the conditioned media of human SH-SY5Y neuroblastoma and rat pheochromocytoma PC12 cells. sAPPalpha release was blocked by the hydroxamic acid-based metalloprotease inhibitor Ro31-9790, which indicated mediation via alpha-secretase activity. Inhibition of protein kinase C (PKC) with the inhibitor GF109203X, or by down-regulation of PKC, blocked the EGCG-induced sAPPalpha secretion, suggesting the involvement of PKC. Indeed, EGCG induced the phosphorylation of PKC, thus identifying a novel PKC-dependent mechanism of EGCG action by activation of the non-amyloidogenic pathway. EGCG is not only able to protect, but it can rescue PC12 cells against the beta-amyloid (Abeta) toxicity in a dose-dependent manner. In addition, administration of EGCG (2 mg/kg) to mice for 7 or 14 days significantly decreased membrane-bound holoprotein APP levels, with a concomitant increase in sAPPalpha levels in the hippocampus. Consistently, EGCG markedly increased PKCalpha and PKC in the membrane and the cytosolic fractions of mice hippocampus. Thus, EGCG has protective effects against Abeta-induced neurotoxicity and regulates secretory processing of non-amyloidogenic APP via PKC pathway.

Mitochondria-targeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants.

Abstract: Friedreich Ataxia (FRDA), the most common inherited ataxia, arises from defective expression of the mitochondrial protein frataxin, which leads to increased mitochondrial oxidative damage. Therefore, antioxidants targeted to mitochondria should be particularly effective at slowing disease progression. To test this hypothesis, we compared the efficacy of mitochondria-targeted and untargeted antioxidants derived from coenzyme Q10 and from vitamin E at preventing cell death due to endogenous oxidative stress in cultured fibroblasts from FRDA patients in which glutathione synthesis was blocked. The mitochondria-targeted antioxidant MitoQ was several hundredfold more potent than the untargeted analog idebenone. The mitochondria-targeted antioxidant MitoVit E was 350-fold more potent than the water soluble analog Trolox. This is the first demonstration that mitochondria-targeted antioxidants prevent cell death that arises in response to endogenous oxidative damage. Targeted antioxidants may have therapeutic potential in FRDA and in other disorders involving mitochondrial oxidative damage.

The dsRNA binding protein family: critical roles, diverse cellular functions

Abstract: The dsRNA binding proteins (DRBPs) comprise a growing family of eukaryotic, prokaryotic, and viral-encoded products that share a common evolutionarily conserved motif specifically facilitating interaction with dsRNA. Proteins harboring dsRNA binding domains (DRBDs) have been reported to interact with as little as 11 bp of dsRNA, an event that is independent of nucleotide sequence arrangement. More than 20 DRBPs have been identified and reportedly function in a diverse range of critically important roles in the cell. Examples include the dsRNA-dependent protein kinase PKR that functions in dsRNA signaling and host defense against virus infection and DICER, which is implicated in RNA interference (RNAi) -mediated gene silencing. Other DRBPs such as Staufen, adenosine deaminase acting on RNA (ADAR), and spermatid perinuclear RNA binding protein (SPNR) are known to play essential roles in development, translation, RNA editing, and stability. In many cases, homozygous and even heterozygous disruption of DRBPs in animal models results in embryonic lethality. These results implicate the recognition of dsRNA as an evolutionarily conserved mechanism important in the regulation of gene expression and in host defense and underscore the diversity of essential biological tasks performed by dsRNA-related processes in the cell.

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

Abstract: Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

Increased cardiac BNP expression associated with myocardial ischemia

Abstract: Congestive heart failure is accompanied by increased cardiac brain natriuretic peptide (BNP) gene expression with elevated plasma concentrations of BNP and its precursor, proBNP. We investigated if myocardial ischemia in the absence of overt heart failure may be another mechanism for increased myocardial BNP expression. The BNP expression was examined in hypoxic myocardium of patients undergoing coronary bypass grafting surgery, in patients with coronary artery disease and normal left ventricular function undergoing percutaneous transluminal intervention therapy, and in heart failure patients without coronary artery disease. BNP mRNA was quantified by real-time PCR, and plasma BNP and proBNP concentrations were measured with radioimmunoassays. Quantitative analysis of BNP mRNA in atrial and ventricular biopsies from coronary bypass grafting patients revealed close associations of plasma BNP and proBNP concentrations to ventricular, but not atrial, BNP mRNA levels. Plasma BNP and proBNP concentrations were markedly increased in patients with coronary artery disease but without concomitant left ventricular dysfunction. These results are compatible with the notion that myocardial ischemia, even in the absence of left ventricular dysfunction, augments cardiac BNP gene expression and increases plasma BNP and proBNP concentrations. Thus, elevated BNP and proBNP concentrations do not necessarily reflect heart failure but may also result from cardiac ischemia.

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

Abstract: SPECIFIC AIMSThe 37 kDa protein annexin 1 (Anx-1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and postulated to be a mediator of glucocorticoid action in inflammation. To test this hypothesis, we have generated for the first time an Anx-1 null mouse line and compared its sensitivity to inflammatory stimuli and its response to glucocorticoids with wild-type controls. A dual-purpose targeting vector designed to simultaneously inactivate the gene and report on the activity of the Anx-1 promoter allowed us to measure gene expression.PRINCIPAL FINDINGS1. Generation of Anx-1−/− miceAnx-1−/− animals were viable and appeared healthy. There was no obvious difference between any of the (sex-matched) groups Anx-1−/− Anx-1+/− and Anx-1+/+ littermate control mice in terms of gross physical appearance, behavior, or weight. Anx-1−/− animals of both sexes bred normally.2. Anx-1 and related protein expression in Anx-1−/− miceThere was strong Anx-1 gene expression ...

UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin

Abstract: DNA damage induced by UV radiation is a critical event in skin photocarcinogenesis. However, the role of racial/ethnic origin in determining individual UV sensitivity remains unclear. In this study, we examined the relationships between melanin content and DNA damage induced by UV exposure in situ in normal human skin of different racial/ethnic groups, phototypes, and UV sensitivities. The minimal erythema dose (MED) was established for each subject exposed to UVA/UVB radiation, and skin was biopsied before as well as 7 min, 1 day, and 1 wk after UV exposure. There was great variation among individuals in the amount of DNA damage incurred and rates of its removal. The results show that after exposure to 1 MED of UV, the skin of subjects from all groups suffered significant DNA damage, and that increasing content of constitutive melanin inversely correlated with the amount of DNA damage. It is clear from these results that measured erythemal UV sensitivity of the skin (MED) is a more useful predictor of DNA photodamage than is racial/ethnic origin or skin phototype and that rates of DNA damage removal following UV radiation may be the critical determinant of the UV sensitivity (including predisposition to cancer) of the skin.

Syndecans in inflammation

Abstract: Cell surface heparan sulfate (HS) influences a multitude of molecules, cell types, and processes relevant to inflammation. HS binds to cell surface and matrix proteins, cytokines, and chemokines. These interactions modulate inflammatory cell maturation and activation, leukocyte rolling, and tight adhesion to endothelium, as well as extravasation and chemotaxis. The syndecan family of transmembrane proteoglycans is the major source of cell surface HS on all cell types. Recent in vitro and in vivo data suggest the involvement of syndecans in the modulation of leukocyte-endothelial interactions and extravasation, the formation of chemokine and kininogen gradients, participation in chemokine and growth factor signaling, as well as repair processes. Thus, the complex role of HS in inflammation is reflected by multiple functions of its physiological carriers, the syndecans. Individual and common functions of the four mammalian syndecan family members can be distinguished. Recently generated transgenic and knockout mouse models will facilitate analysis of the individual processes that each syndecan is involved in.

IFN-α antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3

Abstract: Eighty percent of patients newly infected with the hepatitis C virus (HCV) develop chronic infection, suggesting that HCV can develop effective strategies to escape the unspecific and specific immune response of the host. Because SOCS molecules have been recognized to be powerful inhibitors of cytokine signaling via the Jak/STAT pathway, virus-induced expression of these molecules should be an efficient instrument to counteract the cellular response toward interferons (IFNs), an essential part of first line antiviral immune response. This study shows that overexpression of HCV core protein inhibits IFN-alpha-induced tyrosine phosphorylation and activation of STAT1 in hepatic cells. With the use of a STAT1-YFP fusion protein, further evidence is given that HCV core is capable to inhibit nuclear translocation of STAT1. Inhibition of STAT1-tyrosine phosphorylation was accompanied by the induction of SOCS3-mRNA expression, suggesting that the HCV core protein impairs IFN-alpha-induced signal transduction via induction of SOCS3 expression. HCV core protein was competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist. These IFN-antagonistic properties of the HCV core protein may be part of the molecular basis of IFN-alpha unresponsiveness in about one-half of chronically infected HCV-patients.

γ-Tocopherol, but not α-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats

Abstract: γ-Tocopherol (γT), the major form of vitamin E in U.S. diets, and its physiological metabolite 2, 7, 8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (γ-CEHC), in contrast to α-tocopherol (αT), the primary vitamin E in supplements, inhibit cyclooxygenase-catalyzed synthesis of prostaglandin E2 (PGE2) in activated macrophages and epithelial cells. Here we report that in carrageenan-induced inflammation in male Wistar rats, administration of γT (33 or 100 mg/kg) and γ-CEHC (2 mg/pouch), but not αT (33 mg/kg), significantly reduced PGE2 synthesis at the site of inflammation. γT, but not αT, significantly inhibited the formation of leukotriene B4, a potent chemotactic agent synthesized by the 5-lipoxygenase of neutrophils. Although γT had no effect on neutrophil infiltration, it significantly attenuated the partial loss of food consumption caused by inflammation-associated discomfort. Administration of γT led consistently to a significant reduction of inflammation-mediated increase in 8-isoprostane, a biomarke...

The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-α

Abstract: In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-α (TNF) induced prostaglandin E2 (PGE2) production by ...

Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson's disease.

Abstract: SPECIFIC AIMSThe specific aim of this study was to determine the nature of glial involvement in the pathogenesis of Parkinson’s disease (PD). Exploiting the advantages of cell culture systems, we e...

Activation of microglia by human neuromelanin is NF-κB dependent and involves p38 mitogen-activated protein kinase: implications for Parkinson’s disease

Abstract: It has been suggested that microglial inflammation augments the progression of Parkinson's disease (PD). However, endogenous factors initiating microglial activation are largely unknown. We therefore investigated the effects of human neuromelanin (NM) on the release of neurotoxic mediators and the underlying signaling pathways from rat microglia in vitro. The addition of NM to microglial cultures induced positive chemotactic effects, activated the proinflammatory transcription factor nuclear factor kappaB (NF-kappaB) via phosphorylation and degradation of the inhibitor protein kappaB (IkappaB), and led to an up-regulation of tumor necrosis factor alpha, interleukin-6, and nitric oxide. The impairment of NF-kappaB function by the IkappaB kinase inhibitor sulfasalazine was paralleled by a decline in neurotoxic mediators. NM also activated p38 mitogen-activated protein kinase (MAPK), the inhibition of this pathway by SB203580 diminished phosphorylation of the transactivation domain of the p65 subunit of NF-kappaB. These findings demonstrate a crucial role of NM in the pathogenesis of PD by augmentation of microglial activation, leading to a vicious cycle of neuronal death, exposure of additional neuromelanin, and chronification of inflammation. The antagonization of microglial activation by a pharmacological intervention targeting microglial NF-kappaB or p38 MAPK could point to additional venues in the treatment of PD.

Glial neuronal signaling in the central nervous system

Abstract: Glial cells are known to interact extensively with neuronal elements in the brain, influencing their activity. Astrocytes associated with synapses integrate neuronal inputs and release transmitters that modulate synaptic sensitivity. Glial cells participate in formation and rebuilding of synapses and play a prominent role in protection and repair of nervous tissue after damage. For glial cells to take an active part in plastic alterations under physiological conditions and pathological disturbances, extensive specific signaling, both within single cells and between cells, is required. In recent years, intensive research has led to our first insight into this signaling. We know there are active connections between astrocytes in the form of networks promoting Ca2+ and ATP signaling; we also know there is intense signaling between astrocytes, microglia, oligodendrocytes, and neurons, with an array of molecules acting as signaling substances. The cells must be functionally integrated to facilitate the enormous dynamics of and capacity for reconstruction within the nervous system. In this paper, we summarize some basic data on glial neuronal signaling to provide insight into synaptic modulation and reconstruction in physiology and protection and repair after damage.