Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.

Mitochondrial DNA Damage Following Isolated Muscle Exercise: A Preliminary Investigation Into HO-1 Gene Expression

PURPOSE: This preliminary investigation was designed to test the hypothesis that high intensity single-leg exercise can cause extensive cell DNA damage, which subsequently may affect the expression of the HO-1 gene. METHODS: Six (n=6) apparently healthy male participants (age 27 + 7 yrs, stature 174 + 12 cm, body mass 79 + 4 kg and BMI 24 + 4 kg/m2) completed 100 isolated and continuous maximal concentric contractions (minimum force = 200 N, speed of contraction = 60°/sec) of the rectus femoris muscle. Using a spring-loaded and reusable Magnum biopsy gun with a 16-gauge core disposable biopsy needle, skeletal muscle micro biopsy tissue samples were extracted at rest and following exercise. mRNA gene expression was determined via two-step quantitative real-time PCR using GAPDH as a reference gene. RESULTS: The average muscle force production was 379 + 179 N. High intensity exercise increased mitochondrial 8-OHdG concentration (P < 0.05 vs. rest) with a concomitant decrease in total antioxidant capacity (P < 0.05 vs. rest). Exercise also increased protein oxidation as quantified by protein carbonyl concentration (P < 0.05 vs. rest). HO-1 expression increased (> 2-fold change vs. rest) following exercise, and it is postulated that this change was not significant due to low subject numbers (P > 0.05). CONCLUSION: These preliminary findings tentatively suggest that maximal concentric muscle contractions can cause intracellular DNA damage with no apparent disruption to the expression of the antioxidant stress protein HO-1. Moreover, it is likely that cell oxidant stress is required to activate the signal transduction cascade related to the expression of HO-1. A large-scale study incorporating a greater subject number is warranted to fully elucidate this relationship.

EpsinR an AP1/clathrin interacting protein involved in vesicle trafficking

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

AINT/ERIC/TACC: an expanding family of proteins with C-terminal coiled coil domains:an expanding family of proteins with C-terminal coiled coil domains

The AINT/ERIC/TACC genes encode novel proteins with a coiled coil domain at their C-terminus. The founding member of this expanding family of genes, transforming acidic coiled coil 1 (TACC1), was isolated from a BAC contig spanning the breast cancer amplicon-1 on 8p11. Transfection of cells in vitro with TACC1 resulted in anchorage-independent growth consistent with a more "neoplastic" phenotype. Database searches employing the human TACC1 sequence revealed other novel genes, TACC2 and TACC3, with substantial sequence homology particularly in the C-terminal regions encoding the coiled coil domains. TACC2, located at 10q26, is similar to anti-zuai-1 (AZU-1), a candidate breast tumour suppressor gene, and ECTACC, an endothelial cell TACC which is upregulated by erythropoietin (Epo). The murine homologue of TACC3, murine erythropoietin-induced cDNA (mERIC-1) was also found to be upregulated by Epo in the Friend virus anaemia (FVA) model by differential display-PCR. Human ERIC-1, located at 4p16.3, has been cloned and encodes an 838-amino acid protein whose N- and C-terminal regions are highly homologous to the shorter 558-amino acid murine protein, mERIC-1. In contrast, the central portions of these proteins differ markedly. The murine protein contains four 24 amino acid imperfect repeats. ARNT interacting protein (AINT), a protein expressed during embryonic development in the mouse, binds through its coiled coil region to the aryl hydrocarbon nuclear translocator protein (ARNT) and has a central portion that contains seven of the 24 amino acid repeats found in mERIC-1. Thus mERIC-1 and AINT appear to be developmentally regulated alternative transcripts of the gene. Most members of the TACC family discovered so far contain a novel nine amino acid putative phosphorylation site with the pattern [R/K]-X(3)-[E]-X(3)-Y. Genes with sequence homology to the AINT/ERIC/TACC family in other species include maskin in Xenopus, D-TACC in Drosophila and TACC4 in the rabbit. Maskin contains a peptide sequence conserved among eIF-4E binding proteins that is involved in oocyte development. D-TACC cooperates with another conserved microtubule-associated protein Msps to stabilise spindle poles during cell division. The diversity of function already attributed to this protein family, including both transforming and tumour suppressor properties, should ensure that a new and interesting narrative is about to unfold.

Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene

Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1.

GHMP Kinases - Structures, Mechanisms and Potential for Therapeutically Relevant Inhibition

The GHMP kinases are a structurally related family of small molecule kinases named after four of its members - galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase. The group also includes the enzymes N-acetylgalactosamine kinase, arabinose kinase, mevalonate 5-diphosphate decarboxylase, archeal shikimate kinase and 4-(cytidine 5'-diphospho)-2-c-methyl-D-erythritol kinase. In addition the group includes two members not known to be catalytically active, the Caenorhabditis elegans sex-fate determining protein XOL-1 and the Saccharomyces cerevisiae transcriptional activator Gal3p. Two catalytic mechanisms have been proposed for GHMP kinases. The structure of mevalonate kinase suggests that an aspartate residue acts as an active site base, removing a proton from the substrate to facilitate attack on the ? phosphate of MgATP. In contrast, in homoserine kinase there is no potential catalytic base and it is proposed that catalysis is driven by transition state stabilisation. Potential chemotherapeutic interventions against GHMP kinases fall into three main categories: inhibition of galactokinase to assist suffers of galactosemia, inhibition of mevalonate kinase or mevalonate 5-diphosphate decarboxylase to reduce flux through the cholesterol biosynthesis pathway and inhibition of bacterial GHMP kinases for novel anti-microbial therapies. These are in the early stages of development, but the accumulation of structural and mechanistic data will assist future progress.

Respiratory syncytial virus infections enhance cigarette smoke induced COPD in mice

Respiratory syncytial viral (RSV) infections are a frequent cause of chronic obstructive pulmonary disease (COPD) exacerbations, which are a major factor in disease progression and mortality. RSV is able to evade antiviral defenses to persist in the lungs of COPD patients. Though RSV infection has been identified in COPD, its contribution to cigarette smoke-induced airway inflammation and lung tissue destruction has not been established. Here we examine the long-term effects of cigarette smoke exposure, in combination with monthly RSV infections, on pulmonary inflammation, protease production and remodeling in mice. RSV exposures enhanced the influx of macrophages, neutrophils and lymphocytes to the airways of cigarette smoke exposed C57BL/6J mice. This infiltration of cells was most pronounced around the vasculature and bronchial airways. By itself, RSV caused significant airspace enlargement and fibrosis in mice and these effects were accentuated with concomitant smoke exposure. Combined stimulation with both smoke and RSV synergistically induced cytokine (IL-1a, IL-17, IFN-c, KC, IL-13, CXCL9, RANTES, MIF and GM-CSF) and protease (MMP-2, -8, -12, -13, -16 and cathepsins E, S, W and Z) expression. In addition, RSV exposure caused marked apoptosis within the airways of infected mice, which was augmented by cigarette smoke exposure. RSV and smoke exposure also reduced protein phosphatase 2A (PP2A) and protein tyrosine phosphates (PTP1B) expression and activity. This is significant as these phosphatases counter smoke-induced inflammation and protease expression. Together, these findings show for the first time that recurrent RSV infection markedly enhances inflammation, apoptosis and tissue destruction in smoke-exposed mice. Indeed, these results indicate that preventing RSV transmission and infection has the potential to significantly impact on COPD severity and progression.

A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD)

Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined P ADJ = 1.15 × 10(-6), OR 1.332 (1.187-1.496)]. This association was characterized by a highly significant sex difference (P diff = 0.0032) in that it was clearly confined to females with genome-wide significance [P ADJ = 2.62 × 10(-8), OR 1.541 (1.324-1.796); males: P ADJ = 0.382, OR 1.084 (0.905-1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMSaveD pathogenesis.

The photocatalytic destruction of the cyanotoxin, nodularin using TiO 2

Titanium dioxide (TiO₂) photocatalysis has been used to initiate the destruction of nodularin, a natural hepatotoxin produced by cyanobacteria. The destruction process was monitored using liquid chromatography-mass spectrometry analysis which has also enabled the identification of a number of the photocatalytic decomposition products. The reduction in toxicity following photocatalytic treatment was evaluated using protein phosphatase inhibition assay, which demonstrated that the destruction of nodularin was paralleled by an elimination of toxicity. 

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.

Curvature of clathrin-coated pits driven by epsin

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P(2) in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic alpha-helix in epsin is coupled to PtdIns(4,5)P(2) binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50 %, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70 %). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (n = 10) and HF (n = 6) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.

Identification of protease and ADP-ribose 1''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3.

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1''-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.