Hepatocyte I kappaB kinase beta (IKK beta) inhibits hepatocarcinogenesis by suppressing accumulation of reactive oxygen species (ROS) and liver damage, whereas JNK1 activation promotes ROS accumulation, liver damage, and carcinogenesis. We examined whether hepatocyte p38 alpha, found to inhibit liver carcinogenesis, acts similarly to IKK beta in control of ROS metabolism and cell death. Hepatocyte-specific p38 alpha ablation enhanced ROS accumulation and liver damage, which were prevented upon administration of an antioxidant. In addition to elevated ROS accumulation, hepatocyte death, augmented by loss of either IKK beta or p38 alpha, was associated with release of IL-1 alpha. Inhibition of IL-1 alpha action or ablation of its receptor inhibited carcinogen-induced compensatory proliferation and liver tumorigenesis. IL-1 alpha release by necrotic hepatocytes is therefore an important mediator of liver tumorigenesis.

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-<em>B</em>*1502 and <em>B</em>*1513 which differ only at residues 77-83 which define the <em>B</em>w4 and <em>B</em>w6 serological epitopes, we tested the hypothesis that the presence of the <em>B</em>w4 epitope on class I molecules determines recognition by NK<em>B</em>1+ NK cells. HLA-<em>B</em>*1513 possesses the <em>B</em>w4 epitope, whereas <em>B</em>*1502 has the <em>B</em>w6 epitope. Lysis by NK<em>B</em>1+ NK cell clones of transfected target cells expressing <em>B</em>*1513 as the only HLA-A, -<em>B</em>, or -C molecule was inhibited, whereas killing of transfectants expressing <em>B</em>*1502 was not. Addition of an an anti-NK<em>B</em>1 monoclonal antibody reconstituted lysis of the targets expressing <em>B</em>*1513, but did not affect killing of targets bearing <em>B</em>*1502. The inhibitory effect of <em>B</em>*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the <em>B</em>w4 epitope influences recognition of HLA-<em>B</em> molecules by NK cells that express NK<em>B</em>1, and suggest that the NK<em>B</em>1 molecule may <em>act</em> as a receptor for <em>B</em>w4+ HLA-<em>B</em> alleles. Sequences outside of the <em>B</em>w4 region must also affect recognition by NK<em>B</em>1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the <em>B</em>w4 epitope but are in other ways substantially different from HLA-<em>B</em> molecules, was not increased by addition of the anti-NK<em>B</em>1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the <em>B</em>w4/<em>B</em>w6 region. The glycosylation site of the <em>B</em>w4-positive molecule <em>B</em>*5801 was mutated, and the mutant molecules tested for inhibition of NK<em>B</em>1+ NK cells. Inhibition that could be reversed by addition of the anti-NK<em>B</em>1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NK<em>B</em>1+ NK cell clones.

Elimination of interstitial fluid and solutes plays a role in homeostasis in the brain, but the pathways are unclear. Previous work suggests that interstitial fluid drains along the walls of arteries.

OBJECTIVE

to define the pathways within the walls of capillaries and arteries for drainage of fluid and solutes out of the brain.

METHODS

Fluorescent soluble tracers, dextran (3 kDa) and ovalbumin (40 kDa), and particulate fluospheres (0.02 microm and 1.0 microm in diameter) were injected into the corpus striatum of mice. Brains were examined from 5 min to 7 days by immunocytochemistry and confocal microscopy.

RESULTS

soluble tracers initially spread diffusely through brain parenchyma and then drain out of the brain along basement membranes of capillaries and arteries. Some tracer is takenf up by vascular smooth muscle cells and by perivascular macrophages. No perivascular drainage was observed when dextran was injected into mouse brains following cardiac arrest. Fluospheres expand perivascular spaces between vessel walls and surrounding brain, are ingested by perivascular macrophages but do not appear to leave the brain even following an inflammatory challenge with lipopolysaccharide or kainate.

CONCLUSIONS

capillary and artery basement membranes act as 'lymphatics of the brain' for drainage of fluid and solutes; such drainage appears to require continued cardiac output as it ceases following cardiac arrest. This drainage pathway does not permit migration of cells from brain parenchyma to the periphery. Amyloid-beta is deposited in basement membrane drainage pathways in cerebral amyloid angiopathy, and may impede elimination of amyloid-beta and interstitial fluid from the brain in Alzheimer's disease. Soluble antigens, but not cells, drain from the brain by perivascular pathways. This atypical pattern of drainage may contribute to partial immune privilege of the brain and play a role in neuroimmunological diseases such as multiple sclerosis.

In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.

Research in the field of molecular biology has helped to provide a better understanding of both the cascade of biochemical events that occurs with Alzheimer disease (AD) and the heterogeneous nature of the disease. One hypothesis that accounts for both the heterogeneous nature of AD and the fact that aging is the most obvious risk factor is that free radicals are involved. The probability of this involvement is supported by the fact that neurons are extremely sensitive to attacks by destructive free radicals. Furthermore, lesions are present in the brains of AD patients that are typically associated with attacks by free radicals (eg, damage to DNA, protein oxidation, lipid peroxidation, and advanced glycosylation end products), and metals (eg, iron, copper, zinc, and aluminum) are present that have catalytic activity that produce free radicals. beta-Amyloid is aggregated and produces more free radicals in the presence of free radicals; beta-amyloid toxicity is eliminated by free radical scavengers. Apolipoprotein E is subject to attacks by free radicals, and apolipoprotein E peroxidation has been correlated with AD. In contrast, apolipoprotein E can act as a free radical scavenger and this behavior is isoform dependent. AD has been linked to mitochondrial anomalies affecting cytochrome-c oxidase, and these anomalies may contribute to the abnormal production of free radicals. Finally, many free radical scavengers (eg, vitamin E, selegeline, and Ginkgo biloba extract EGb 761) have produced promising results in relation to AD, as has desferrioxamine-an iron-chelating agent-and antiinflammatory drugs and estrogens, which also have an antioxidant effect.

The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.

The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.

Arabidopsis COP1 acts as a repressor of photomorphogenesis in darkness, and light stimuli abrogate this suppressive action. COP1, when fused to beta-glucuronidase (GUS), is enriched in the nucleus in darkness, but not in the light, in hypocotyl cells of Arabidopsis seedlings and epidermal cells of onion bulbs. In Arabidopsis hypocotyl cells, the nuclear GUS-COP1 level changes in response to dark-light transitions and quantitatively correlates with the extent of repression of photomorphogenic development. In root cells, GUS-COP1 is constitutively nuclear, consistent with an established role of COP1 in suppressing root chloroplast development in both light and darkness. We conclude that COP1 acts inside the nucleus to suppress photomorphogenesis and that light inactivation of COP1 involves a cell type-specific control of its nucleocytoplasmic partitioning.

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

The transcription factor NF-kappa B is a protein complex which comprises a DNA-binding subunit and an associated transactivation protein (of relative molecular masses 50,000 (50K) and 65K, respectively). Both the 50K and 65K subunits have similarity with the rel oncogene and the Drosophila maternal effect gene dorsal. The 50K DNA-binding subunit was previously thought to be a unique protein, derived from the 105K gene product (p105). We now report the isolation of a complementary DNA that encodes an alternative DNA-binding subunit of NF-kappa B. It is more similar to p105 NF-kappa B than other family members and defines a new subset of rel-related genes. It is synthesized as approximately 100K protein (p100) that is expressed in different cell types, contains cell cycle motifs and, like p105, must be processed to generate a 50K form. A 49K product (p49) can be generated independently from an alternatively spliced transcript; it has specific kappa B DNA-binding activity and can form heterodimers with other rel proteins. In contrast to the approximately 50K protein derived from p105, p49 acts in synergy with p65 to stimulate the human immunodeficiency virus (HIV) enhancer in transiently transfected Jurkat cells. p49/p100 NF-kappa B could therefore be important in the regulation of HIV and other kappa B-containing genes.

Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well-characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T-helper 1 effectors. The effects of nTregs in this model are not antigen-specific but are mediated by activation of the nTregs by ubiquitous self-peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non-specific suppressor effector function by stimulation with interleukin-2 in the absence of engagement of their T-cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B-dependent, partially perforin-independent cytotoxic cells that are capable of specifically killing antigen-presenting B cells. Natural Treg-mediated killing of antigen-presenting cells may represent one pathway by which they can induce long-lasting suppression of autoimmune disease.

Mammals generate a diverse array of antimicrobial proteins, largely represented by defensins or cathelicidins. The direct in vitro microbicidal activity of antimicrobial proteins has long been considered an important innate immune defense, although the in vivo relevance has only very recently been established for certain defensins and cathelicidins. Mammalian defensins and cathelicidins have also been shown to have multiple receptor-mediated effects on immune cells. Beta-defensins interact with CCR6; murine beta-defensin-2 in addition activates TLR4. Cathelicidins act on FPRL1-expressing cells. Furthermore, several defensins have considerable immunoenhancing activity. Thus, it appears that mammalian antimicrobial proteins contribute to both innate and adaptive antimicrobial immunity.

The addition of concanavalin A-stimulated supernatants of the helper T cell clone, D9.1, to cultures of lipopolysaccharide (LPS)-stimulated T-depleted mouse spleen cells caused more than a 100-fold increase in immunoglobulin (Ig) E production. These supernatants cause a 10-fold to 15-fold increase in IgG1, a fivefold to 10-fold increase in IgA, and a fivefold to 10-fold decrease in IgG3. These effects are optimal when the supernatants are added 1 to 2 days after stimulation with LPS. Cells from mouse strains that normally give little or no IgE response in vivo give normal IgE levels in response to LPS plus the supernatant of Concanavalin A-stimulated D9.1 cells in vitro. The enhancement of both IgE and IgG1 can be completely inhibited by relatively low concentrations of interferon-gamma (IFN-gamma). Both the IgE-enhancing activity and IFN-gamma act directly upon purified B cells.

Follicle-stimulating hormone (FSH) is central to reproduction in mammals. It acts through a G-protein-coupled receptor on the surface of target cells to stimulate testicular and ovarian functions. We present here the 2.9-A-resolution structure of a partially deglycosylated complex of human FSH bound to the extracellular hormone-binding domain of its receptor (FSHR(HB)). The hormone is bound in a hand-clasp fashion to an elongated, curved receptor. The buried interface of the complex is large (2,600 A2) and has a high charge density. Our analysis suggests that all glycoprotein hormones bind to their receptors in this mode and that binding specificity is mediated by key interaction sites involving both the common alpha- and hormone-specific beta-subunits. On binding, FSH undergoes a concerted conformational change that affects protruding loops implicated in receptor activation. The FSH-FSHR(HB) complexes form dimers in the crystal and at high concentrations in solution. Such dimers may participate in transmembrane signal transduction.

We have investigated the cell surface recognition mechanisms used by human monocyte-derived macrophages (M phi) in phagocytosis of intact aging human neutrophils (PMNs) undergoing apoptosis. This study shows that the adhesive protein thrombospondin (TSP) was present in the interaction, both associated with the M phi surface and in solution at a mean concentration of 0.59 micrograms/ml. The interaction was inhibited by treatment of M phi (but not aged PMN) with cycloheximide, but could be "rescued" by replenishment with exogenous TSP. Under control conditions, M phi recognition of aged PMNs was specifically potentiated by purified platelet TSP at 5 micrograms/ml, present either in the interaction or if preincubated with either cell type, suggesting that TSP might act as a "molecular bridge" between the two cell types. In support, both aged PMN and M phi were found to adhere to TSP, and phagocytosis of aged PMN was specifically inhibited by (a) excess soluble TSP; (b) antibodies to TSP that also inhibit TSP-mediated adhesion to aged PMN; and (c) down-regulation of M phi receptors for TSP by plating M phi on TSP-coated surfaces. Furthermore, inhibition with mAbs/Arg-Gly-Asp-Ser peptide of the candidate M phi receptors for TSP, CD36, and alpha v beta 3 exerted synergistic effects on both M phi recognition of aged PMN and M phi adhesion to TSP, indicating that "two point" adhesion of TSP to these M phi structures is involved in phagocytosis of aged PMN. Our findings indicate newly defined roles for TSP and CD36 in phagocytic clearance of senescent neutrophils, which may limit inflammatory tissue injury and promote resolution.

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to beta-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (+/- dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca(2+)-ATPase for Ca2+ in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the beta-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the heart's contractile responses to beta-adrenergic agonists.

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31-mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.

Evidence implicates hyperglycemia-derived oxygen free radicals as mediators of diabetic complications. However, intervention studies with classic antioxidants, such as vitamin E, failed to demonstrate any beneficial effect. Recent studies demonstrate that a single hyperglycemia-induced process of overproduction of superoxide by the mitochondrial electron-transport chain seems to be the first and key event in the activation of all other pathways involved in the pathogenesis of diabetic complications. These include increased polyol pathway flux, increased advanced glycosylation end product formation, activation of protein kinase C, and increased hexosamine pathway flux. Superoxide overproduction is accompanied by increased nitric oxide generation, due to an endothelial NOS and inducible NOS uncoupled state, a phenomenon favoring the formation of the strong oxidant peroxynitrite, which in turn damages DNA. DNA damage is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP-ribose) polymerase. Poly(ADP-ribose) polymerase activation in turn depletes the intracellular concentration of its substrate NAD(+), slowing the rate of glycolysis, electron transport, and ATP formation, and produces an ADP-ribosylation of the GAPDH. These processes result in acute endothelial dysfunction in diabetic blood vessels that, convincingly, also contributes to the development of diabetic complications. These new findings may explain why classic antioxidants, such as vitamin E, which work by scavenging already-formed toxic oxidation products, have failed to show beneficial effects on diabetic complications and may suggest new and attractive "causal" antioxidant therapy. New low-molecular mass compounds that act as SOD or catalase mimetics or L-propionyl-carnitine and lipoic acid, which work as intracellular superoxide scavengers, improving mitochondrial function and reducing DNA damage, may be good candidates for such a strategy, and preliminary studies support this hypothesis. This "causal" therapy would also be associated with other promising tools such as LY 333531, PJ34, and FP15, which block the protein kinase beta isoform, poly(ADP-ribose) polymerase, and peroxynitrite, respectively. While waiting for these focused tools, we may have other options: thiazolinediones, statins, ACE inhibitors, and angiotensin 1 inhibitors can reduce intracellular oxidative stress generation, and it has been suggested that many of their beneficial effects, even in diabetic patients, are due to this property.

The phytohormone abscisic acid (ABA) acts in seed dormancy, plant development, drought tolerance, and adaptive responses to environmental stresses. Structural mechanisms mediating ABA receptor recognition and signaling remain unknown but are essential for understanding and manipulating abiotic stress resistance. Here, we report structures of pyrabactin resistance 1 (PYR1), a prototypical PYR/PYR1-like (PYL)/regulatory component of ABA receptor (RCAR) protein that functions in early ABA signaling. The crystallographic structure reveals an alpha/beta helix-grip fold and homodimeric assembly, verified in vivo by coimmunoprecipitation. ABA binding within a large internal cavity switches structural motifs distinguishing ABA-free "open-lid" from ABA-bound "closed-lid" conformations. Small-angle x-ray scattering suggests that ABA signals by converting PYR1 to a more compact, symmetric closed-lid dimer. Site-directed PYR1 mutants designed to disrupt hormone binding lose ABA-triggered interactions with type 2C protein phosphatase partners in planta.

Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl(-/-) haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl(+/+) HSPCs, and transduction of C-CBL mutants into c-Cbl(-/-) HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl(+/+) background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.

Mammalian telomeres are formed by tandem repeats of the TTAGGG sequence, which are progressively lost with each round of cell division. Telomere protection requires a minimal length of TTAGGG repeats to allow the binding of shelterin, which prevents the activation of a DNA damage response (DDR) at chromosome ends. Telomere elongation is carried out by telomerase. Telomerase can also act as a transcriptional modulator of the Wnt-β-catenin signalling pathway and has RNA-dependent RNA polymerase activity. Dysfunctional telomeres can lead to either cancer or ageing pathologies depending on the integrity of the DDR. This Review discusses the role of telomeric proteins in cancer and ageing through modulating telomere length and protection, as well as regulating gene expression by binding to non-telomeric sites.

Lipid peroxidation (LPO) has been shown to induce disturbance of membrane organization and functional loss and modification of proteins and DNA bases, and it has been implicated in the pathogenesis of various diseases. At the same time, LPO products have been shown to act as redox signaling mediators. Free and ester forms of both polyunsaturated fatty acids and cholesterol are important substrates for LPO in vivo and they are oxidized by both enzymatic and nonenzymatic mechanisms to give a variety of products. The results of numerous studies reported in the literatures show that the levels of LPO products in plasma of healthy human subjects are below 1 muM and that the molar ratios of LPO products to the respective parent lipids are below 1/1000, that is, below 0.1%. The levels of LPO products in human erythrocytes were found to be higher than those in plasma. Considerable levels of cholesterol oxidation products were observed. Although many LPO products exert cytotoxicity, sublethal concentrations of LPO products induce cellular adaptive responses and enhance tolerance against subsequent oxidative stress through upregulation of antioxidant compounds and enzymes. This adaptive response is observed not only for chemically reactive alpha,beta-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal and 15-deoxy-delta-12,14-prostaglandin J(2) but also for chemically stable compounds such as hydroxyoctadecadienoic acid, hydroxylcholesterol, and lysophosphatidylcholine. Such opposite dual functions of LPO products imply that LPO, and probably oxidative stress in general, may exert both deleterious and beneficial effects in vivo. LPO as well as reactive oxygen and nitrogen species has been shown to play an important role as a regulator of gene expression and cellular signaling messenger. In order to exert physiologically important functions as a regulator of gene expression and mediator of cellular signaling, the formation of LPO products must be strictly controlled and programmed. In contrast to LPO products by enzymatic oxidation, it appears difficult to regulate the formation of free radical-mediated LPO products. Even such unregulated LPO products may exert beneficial effects at low levels, but excessive unregulated LPO may lead to pathological disorders and diseases.