Regulation of microfilament organization by Kaposi sarcoma-associated herpes virus-cyclin.CDK6 phosphorylation of caldesmon

Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here we report the identification of the actin- and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin.CDK substrate, which is not phosphorylated by D.CDK. CALD1 plays a central role in the regulation of microfilament organization, consequently controlling cell shape, adhesion, cytokinesis and motility. K-cyclin.CDK6 specifically phosphorylates four Ser/Thr sites in the human CALD1 carboxyl terminus, abolishing CALD1 binding to its effector protein, actin, and its regulator protein, calmodulin. CALD1 is hyperphosphorylated in cells following K-cyclin expression and in KSHV-transformed lymphoma cells. Moreover, expression of exogenous K-cyclin results in microfilament loss and changes in cell morphology; both effects are reliant on CDK catalysis and can be reversed by the expression of a phosphorylation defective CALD1. Together, these data strongly suggest that K-cyclin expression modulates the activity of caldesmon and through this the microfilament functions in cells. These results establish a novel link between KSHV infection and the regulation of the actin cytoskeleton.     

Suppression of hypothalamic deiodinase type II activity blunts TRH mRNA decline during fasting

Fasting is characterized by disrupted thyroid feedback, with suppressed levels of thyroid hormones and paraventricular thyrotropin releasing hormone (TRH). We found that third ventricle administration of the deiodinase inhibitor, iopanoic acid, dose-dependently reduced deiodinase type II (DII) activity selectively in the hypothalamus. This suppression of DII by iopanoic acid during fasting prevented elevated DII activity and blunted the decline in hypothalamic TRH mRNA levels. Because fasting-induced elevation in hypothalamic DII activity is paralleled by increased hypothalamic T3 concentration, our study suggests that T3 formation by DII in the hypothalamus is the cause of disrupted thyroid feedback during fasting.     

NF1 gene mutations represent the major molecular event underlying neurofibromatosis-Noonan syndrome

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.     

Identification of the mitochondrial GTP/GDP transporter in Saccharomyces cerevisiae

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K(m) of 1 microm for GTP and 5 microm for GDP. It was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H(+)-compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.     

beta-Arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor beta-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with beta-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from beta-arrestin activate any G-protein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.     

A leucine-rich repeat motif of Leishmania parasite surface antigen 2 binds to macrophages through the complement receptor 3

Membrane glycoconjugates on the Leishmania parasites, notably leishmanolysin and lipophosphoglycan, have been implicated in attachment and invasion of host macrophages. However, the function of parasite surface Ag 2 (PSA-2) and membrane proteophosphoglycan (PPG) has not been elucidated. In this study we demonstrate that native and recombinant Leishmania infantum PSA-2, which consists predominantly of 15 leucine-rich repeats (LRR) and a recombinant LRR domain derived from L. major PPG, bind to macrophages. The interaction is restricted to macrophages and appears to be calcium independent. We have investigated the PSA-2-macrophage interaction to identify the host receptor involved in binding and we show that binding of PSA-2 to macrophages can be blocked by Abs to the complement receptor 3 (CR3, Mac-1). Data derived from mouse macrophage studies were further confirmed using cell lines expressing human CR3, and showed that PSA-2 also binds to the human receptor. This is the first demonstration of a functional role for PSA-2. Our data indicate that in addition to leishmanolysin and lipophosphoglycan, parasite attachment and invasion of macrophages involve a third ligand comprising the LRRs shared by PSA-2 and PPG and that these interactions occur via the CR3.     

A protective effect of the synthetic coumarine derivative Cloricromene against DNB-colitis in the rat

Biologic therapies, namely antibodies against tumor necrosis factor-alpha (TNF- alpha) or its receptors, have been recently introduced for the treatment of patients with inflammatory bowel disease (IBD). In the present study the effects of cloricromene, an agent with known antithrombotic actions and with demonstrated anti-TNF- alpha activity were investigated in a rat model of experimental colitis induced with dinitrobenzenesulphonic acid (DNB)/ethanol. We investigated three experimental groups: (i) sham-colitis with vehicle-treatment (controls, n = 6), (ii) colitis with vehicle-treatment (saline, 0.1 ml s.c., daily) (DNB-V, n = 7), (iii) colitis with cloricromene-treatment (10 mg/kg/day s.c.; DNB-C, n = 8). After 7 days, the weight gain, colon wet weight, macroscopic damage score, coagulation parameters, colon mucosal myeloperoxidase activity (MPO), and tissue concentrations of TNF- alpha and of macrophage inhibitory peptide-2 (MIP-2) were assessed. The macroscopic damage scores, colon wet weights, and tissue MIP-2 levels were significantly increased in untreated and in cloricromene-treated rats compared with controls. Cloricromene treatment was associated with a minor body weight loss (p < 0.025) and significantly reduced tissue concentrations of MPO and TNF-alpha (p < 0.02, both). Blood coagulation parameters were not affected by treatment. In the DNB-model treatment with cloricromene effectively reduces tissue levels of TNF- alpha and of myeloperoxidase, whereas MIP-2 concentrations were not influenced. Blood coagulation parameters remained unchanged indicating safety of treatment. Since biological therapies frequently fail to improve disease course of IBD, other therapies with similar targets should be further investigated.     

In Vitro Synergistic Activities of Essential Oils and Surfactants in Combination with Cosmetic Preservatives Against Pseudomonas aeruginosa and Staphylococcus aureus

The aim of this study is to evaluate possible synergistic antimicrobial interactions between common cosmetic preservatives and selected essential oils or surfactants. The antimicrobial efficacy of six essential oils, three surfactants and five preservatives against Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 43387 was assessed by a broth micro-dilution assay. MICs for individual and combined antimicrobials were determined and then transformed to fractional inhibitory concentration (FIC) indexes. All essential oils exhibited antibacterial activity; among surfactants, bacteria resulted most susceptible to the cationic agent. Synergy was observed when essential oils of eucalyptus and mint were combined with methylparaben against P. aeruginosa, while essential oils of mint, oregano and sage combined with propylparaben and imidazolidinyl urea acted against S. aureus. Many binary mixtures of preservatives and surfactants produced synergistic activity with the most effective interactions involving the cationic and amphoteric compounds under study. FIC indexes demonstrated synergistic effects when preservatives were combined with either essential oils or surfactants against both bacterial strains. These results highlight the potential usefulness of essential oils and surfactants to enhance the activities of conventional biocides. This kind of study should contribute to the selection and optimization of preservative systems for cosmetic preparations.     

DNA vaccination strategies for anti-tumour effective gene therapy protocols

After more than 15 years of experimentation, DNA vaccines have become a promising perspective for tumour diseases, and animal models are widely used to study the biological features of human cancer progression and to test the efficacy of vaccination protocols. In recent years, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has revealed a number of advantages: antigen-specific DNA vaccination stimulates both cellular and humoral immune responses; multiple or multi-gene vectors encoding several antigens/determinants and immune-modulatory molecules can be delivered as single administration; DNA vaccination does not induce autoimmune disease in normal animals; DNA vaccines based on plasmid vectors can be produced and tested rapidly and economically. However, DNA vaccines have shown low immunogenicity when tested in human clinical trials, and compared with traditional vaccines, they induce weak immune responses. Therefore, the improvement of vaccine efficacy has become a critical goal in the development of effective DNA vaccination protocols for anti-tumour therapy. Several strategies are taken into account for improving the DNA vaccination efficacy, such as antigen optimisation, use of adjuvants and delivery systems like electroporation, co-expression of cytokines and co-stimulatory molecules in the same vector, different vaccination protocols. In this review we discuss how the combination of these approaches may contribute to the development of more effective DNA vaccination protocols for the therapy of lymphoma in a mouse model.     

Bronchial malondialdehyde DNA adducts, tobacco smoking, and lung cancer

Tobacco smoking is a major risk factor for lung cancer causing, among other effects, oxidative stress and lipid peroxidation. Malondialdehyde (MDA)-DNA adducts can be induced by direct DNA oxidation and by lipid peroxidation. We measured the relationship between bronchial MDA-DNA adducts and tobacco smoking, cancer status, and selected polymorphisms in 43 subjects undergoing a bronchoscopic examination for diagnostic purposes. MDA-DNA adducts were higher in current smokers than in never smokers (frequency ratio (FR) = 1.51, 95% confidence interval (CI) 1.01-2.26). MDA-DNA adducts were also increased in lung cancer cases with respect to controls, but only in smokers (FR = 1.70, 95% CI 1.16-2.51). Subjects with GA and AA cyclin D1 (CCND1) genotypes showed higher levels of MDA-DNA adducts than those with the wild-type genotype (FR = 1.51 (1.04-2.20) and 1.45 (1.02-2.07)). Lung cancer cases with levels of MDA-DNA adducts over the median showed a worse, but not statistically significant, survival, after adjusting for age, gender, and packyears (hazard ratio = 2.48, 95% CI 0.65-9.44). Our findings reinforce the role of smoking in lung carcinogenesis through oxidative stress. Subjects who carry at least one variant allele of the CCND1 gene could accumulate DNA damage for altered cell-cycle control and reduced DNA repair proficiency.