New biosynthetic step in the melanin pathway of Wangiella (Exophiala) dermatitidis: evidence for 2-acetyl-1,3,6,8-Tetrahydroxynaphthalene as a novel precursor

The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.     

Imaging of molecular dynamics regulated by electrical activities in neural circuits and in synapses

One of the major challenges in brain research is to unravel a network of molecules, neurons, circuits and systems that are responsible for dynamic and hierarchical brain functions. To understand molecular events that occur in synapses could be an important key to exploring the mechanism of information processing. A spatiotemporal recording method is required to observe neuronal activities in a particular local circuit and to resolve single synaptic potential with high resolution. As alternative methods, real-time imaging using fluorescent probes and optical recording methods are also a powerful approach for investigating the molecular dynamics of biological events in neurons in vitro and in vivo. Recently, optical imaging techniques have become of great importance to visualize the molecular dynamics in a micron-sized compartment of a single neuron such as neuronal synapse. In general, the presynaptic axon forms synapses at the postsynaptic site on the dendritic spines in the mammalian central nervous system. Subsets of the synapses undergo a series of enduring changes in spine shape and density as well as alterations in electrophysiological functions. Here we describe recent optical imaging studies conducted by elaborate methods and techniques that provide evidence for the link between neural activity and molecular dynamics.     

Biochemical properties of CikA,an unusual phytochrome-like histidine protein kinase that resets the circadian clock in Synechococcus elongatus PCC 7942

We recently described the cikA (circadian input kinase A) gene, whose product supplies environmental information to the circadian oscillator in the cyanobacterium Synechococcus elongatus PCC 7942. CikA possesses three distinct domains: a GAF, a histidine protein kinase (HPK), and a receiver domain similar to those of the response regulator family. To determine how CikA functions in providing circadian input, we constructed modified alleles to tag and truncate the protein, allowing analysis of each domain individually. CikA covalently bound bilin chromophores in vitro, even though it lacks the expected ligand residues, and the GAF domain influenced but did not entirely account for this function. Full-length CikA and truncated variants that carry the HPK domain showed autophosphorylation activity. Deletion of the GAF domain or the N-terminal region adjacent to GAF dramatically reduced autophosphorylation, whereas elimination of the receiver domain increased activity 10-fold. Assays to test phosphorelay from the HPK to the cryptic receiver domain, which lacks the conserved aspartyl residue that serves as a phosphoryl acceptor in response regulators, were negative. We propose that the cryptic receiver is a regulatory domain that interacts with an unknown protein partner to modulate the autokinase activity of CikA but does not work as bona fide receiver domain in a phosphorelay.     

Adenovirus-mediated gene transfer of interferon alpha inhibits hepatitis C virus replication in hepatocytes

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases.     

Kinetic analysis of C-terminally truncated RNA-dependent RNA polymerase of hepatitis C virus.

The biochemical properties of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) truncated with C-terminal 21 amino acids and expressed in insect cells were analyzed. The enzyme carried copy-back and de novo RNA synthesis activity but not terminal nucleotidyl transferase activity. k(pol) and K(m) for de novo RNA synthesis were calculated as 10.0 pmol/microg/h and 2.5 microM under 0.5 mM GTP and 2.0 pmol/microg/h and 3.5 microM under 50 microM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol), 1.8 pmol/microg/h; K(m), 3.0 microM). De novo RNA synthesis was activated by 0.5 mM GTP. However, the ratio of GTP to three other NTPs was important for activation. Our HCV RdRp showed high activity for the complementary sequence of the HCV internal ribosomal entry site and a synergistic effect of Mg(2+) to Mn(2+).     

Alternative splicing variants of the human DBL (MCF-2) proto-oncogene

The DBL (MCF-2) proto-oncogene is a prototype guanine nucleotide exchange factor (GEF) that modulates the Rho family of GTPases. In this communication we describe the isolation of three novel splicing variants of Dbl. The prototype Dbl gene (designated var.1 here) contains 25 exons, while splicing variant 2 (var.2) lacks exons 23 and 24. Var.3 contains additional 3 exons from 5(')-UTR in place of exon 1, while var.4, var.2, and var.3 contain a 48bp insertion between exons 10 and 11, resulting in the insertion of 16 amino acids. We found that var.1 was expressed only in brain, whereas var.3 was expressed in heart, kidney, spleen, liver, and testis, and var.4 in brain, heart, kidney, testis, placenta, stomach, and peripheral blood. The Dbl protein was detectable in brain, heart, kidney, intestine, muscle, lung, and testis. An assay for GEF activity revealed that the var.2 exhibits decreased GEF activity towards Cdc42, var.3 exhibits a weak but significant activity toward Rac1 and Cdc42, var.4 exhibits significant activity toward RhoA and Cdc42, while var.1 exhibits no activity toward RhoA, Rac1, or Cdc42. In summary, we describe 4 splicing variants of the human DBL proto-oncogene that show different tissue distributions and GEF specificities.     

The carboxyl-terminal half region of ADAMTS-1 suppresses both tumorigenicity and experimental tumor metastatic potential

ADAMTS-1 is an ECM-anchored metalloproteinase with proteoglycan-degrading activity as well as an angiogenesis inhibiting activity. Here, we examined the effects of ADAMTS-1 overexpression on in vivo tumor growth and tumor metastasis. Overexpression of only the C-terminal half region of ADAMTS-1, consisting of TSP type I motifs and the spacer region, suppressed Chinese hamster ovary (CHO) tumor growth in mice. In addition, a significant reduction in tumor metastatic potential was observed in ADAMTS-1-transfected CHO cells in an experimental metastasis assay. Furthermore, deletional analyses revealed that the C-terminal half region of ADAMTS-1 is responsible for its experimental metastasis-inhibitory activity. Our data suggest that the C-terminal half region of ADAMTS-1 has therapeutic potential as an inhibitor of tumor growth and metastasis.     

Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.