Genome-wide combination profiling of copy number and methylation offers an approach for deciphering misregulation and development in cancer cells

Copy number changes and DNA methylation alterations are crucial to gene regulation in mammals. Recently, a number of microarray studies have been based on copy number and DNA methylation alterations in order to find clinical biomarkers of carcinogenesis. In this study, we attempted to combine profiles of copy number and methylation patterns in four human cancer cell lines using BAC microarray-based approaches and we detected several clinically important genes which showed genetic and epigenetic relationships. Within the clones analyzed, many contained cancer-related genes involved in cell cycle regulation, cell division, signal transduction, tumor necrosis, cell differentiation, and cell proliferation. One clone included the FHIT gene, a well-known tumor suppressor gene involved in various human cancers. Our combined profiling techniques may provide a method by which to find new clinicopathologic cancer biomarkers, and support the idea that systematic characterization of the genetic and epigenetic events in cancers may rapidly become a reality.     

Antibacterial activity of pyrrolidine dithiocarbamate

Pyrrolidine dithiocarbamate (PDTC), an antioxidant with a metal-chelating activity, has been used widely to inhibit the expression of inflammatory genes in vitro and in vivo. This study investigated whether PDTC has an antimicrobial activity against various bacteria. The antibacterial activity of PDTC and other compounds was evaluated in vitro by the broth microdilution method against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Staphylococcus aureus, and Escherichia coli. Bacterial growth was inhibited by PDTC, where a wide range of sensitivity was demonstrated among the tested bacteria. The antibacterial activity of PDTC was reduced by the addition of copper chloride; in contrast, it was enhanced considerably by zinc chloride. Two different zinc chelators, Ca-saturated EDTA (Ca-EDTA) and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, blocked the antibacterial activity of PDTC, whereas Zn-EDTA failed to reduce the activity of PDTC. These results demonstrate for the first time that PDTC possesses an antibacterial activity, for which zinc is required, and suggest that PDTC, possessing a dual anti-inflammatory and antibacterial activity, may be considered for topical use for inflammatory diseases of bacterial origin.     

Clustering orthologous proteins across phylogenetically distant species

The quality of orthologous protein clusters (OPCs) is largely dependent on the results of the reciprocal BLAST (basic local alignment search tool) hits among genomes. The BLAST algorithm is very efficient and fast, but it is very difficult to get optimal solution among phylogenetically distant species because the genomes with large evolutionary distance typically have low similarity in their protein sequences. To reduce the false positives in the OPCs, thresholding is often employed on the BLAST scores. However, the thresholding also eliminates large numbers of true positives as the orthologs from distant species likely have low BLAST scores. To rectify this problem, we introduce a new hybrid method combining the Recursive and the Markov CLuster (MCL) algorithms without using the BLAST thresholding. In the first step, we use InParanoid to produce n(n-1)/2 ortholog tables from n genomes. After combining all the tables into one, our clustering algorithm clusters ortholog pairs recursively in the table. Then, our method employs MCL algorithm to compute the clusters and refines the clusters by adjusting the inflation factor. We tested our method using six different genomes and evaluated the results by comparing against Kegg Orthology (KO) OPCs, which are generated from manually curated pathways. To quantify the accuracy of the results, we introduced a new intuitive similarity measure based on our Least-move algorithm that computes the consistency between two OPCs. We compared the resulting OPCs with the KO OPCs using this measure. We also evaluated the performance of our method using InParanoid as the baseline approach. The experimental results show that, at the inflation factor 1.3, we produced 54% more orthologs than InParanoid sacrificing a little less accuracy (1.7% less) than InParanoid, and at the factor 1.4, produced not only 15% more orthologs than InParanoid but also a higher accuracy (1.4% more) than InParanoid.     

Purification and characterization of branching specificity of a novel extracellular amylolytic enzyme from marine hyperthermophilic Rhodothermus marinus

An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.     

Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties

Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing GTP-Rac translocation to the plasma membrane

Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Inhibitory effects on mushroom tyrosinase by flavones from the stem barks of Morus lhou (S.) Koidz

Five flavones displaying tyrosinase inhibitory activity were isolated from the stem barks of Morus lhou (S.) Koidz., a cultivated edible plant. The isolated compounds were identified as mormin (1), cyclomorusin (2), morusin (3), kuwanon C (4), and norartocarpetin (5). Mormin (1) was characterized as a new flavone possesing a 3-hydroxymethyl-2-butenyl at C-3. The inhibitory potencies of these flavonoids toward monophenolase activity of mushroom tyrosinase were investigated. The IC50 values of compounds 1-5 for monophenolase activity were determined to be 0.088, 0.092, 0.250, 0.135 mM, and 1.2 microM, respectively. Mormin (1), cyclomorusin (2), kuwanon C (4) and norartocarpetin (5) exhibited competitive inhibition characteristics. Interestingly norartocarpetin (5) showed a time-dependent inhibition against oxidation of L-tyrosine: it also operated under the enzyme isomerization model (k5 = 0.8424 min(-1), k6 = 0.0576 min(-1), K(app)(i) = 1.354 microM).