Diversification of mitochondrial genome of Daphnia galeata (Cladocera, Crustacea): Comparison with phylogenetic consideration of the complete sequences of clones isolated from five lakes in Japan.

To characterize genetic diversity and gene flow among Daphnia galeata populations, the complete nucleotide (nt) sequences of the mitochondrial (mt) DNAs of D. galeata clones isolated from five lakes in Japan (Lakes Shirakaba, Suwa, Kizaki, Kasumigaura, and Biwa) were determined. Comparison of non-synonymous (amino acid altering) substitution rates with synonymous substitution rates of D. galeata mt protein-coding genes demonstrated that ATPase8 and COI genes were the most and least susceptible, respectively, to the evolutional forces selecting the aa substitutions. Several non-synonymous substitutions were found in ATPase8 and ATPase6 even in the comparison that no synonymous substitution was found. Comparison of the total number of nt variations among the mt DNAs suggested the phylogenetic relationship ((((Shirakaba/Suwa, Kizaki), Kasumigaura), Biwa), D. pulex). Maximum-likelihood analysis using the total nt sequences of mt protein-coding genes confirmed this relationship with bootstrap values higher than 98%. All the mtDNAs of the analyzed Japanese D. galeata clones contained a control region of essentially the same structure that is distinct from those of the previously reported European Daphnia species of the D. longispina complex. The two control regions of different structures spread among mtDNAs of the Japanese and European Daphnia species, respectively, probably after the divergence of the Japanese D. galeata under different selection pressures associated with their habitats.

Development of an RNA interference method in the cladoceran crustacean Daphnia magna.

Daphnids are small crustaceans ubiquitous in fresh water; they have been a subject of study in ecology, evolution, and environmental sciences for decades. To understand data accumulated in daphnid biology at the molecular level, expressed sequence tags and a genome sequence have been determined. However, these discoveries lead to the problem of how to understand the functions of newly discovered genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a useful tool to achieve specific gene silencing in nontransformable species. Hence, we established a technique to inject exogenous materials into ovulated eggs and developed a dsRNA-based RNAi method for Daphnia magna. Eggs were collected just after ovulation and injected with dsRNA specific to the Distal-less (Dll) gene, which functions in appendage development in invertebrates and vertebrates. We found that the dsRNA successfully triggered the degradation of Dll mRNAs, which induced the truncation of the second antenna in a dose-dependent manner. This effect was sequence specific in that: (1) an unrelated dsRNA did not induce any morphological abnormalities and (2) two non-overlapping Dll dsRNAs generated the same phenotype. This is the first report of an RNAi technique in D. magna and, together with the emerging genome sequences, will be useful for advancing knowledge of the molecular biology of daphnids.

Involvement of Smad3 phosphoisoform-mediated signaling in the development of colonic cancer in IL-10-deficient mice.

Chronic inflammation predisposes to cancer. Transforming growth factor (TGF)-beta, a multifunctional protein, suppresses the growth of normal colonic epithelial cells, whereas it stimulates the proliferation of cancer cells. Interleukin (IL)-10-deficient mice, which develop colitis and colorectal cancer, show an increased level of plasma TGF-beta. Although TGF-beta may be a key molecule in the development of colon cancer arising from chronic colitis in IL-10-deficient mice, the role of TGF-beta still remains unclear. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which converts the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). We studied C57BL/6-IL-10-deficient mice (n=18) at 4 to 32 weeks of age. We investigated histology, and pSmad2/3L, pSmad2/3C, and p53 by immunohistochemistry. pSmad3L staining was detected in the cancer cells in all 10 mice with colonic cancer and in the epithelial cells in 7 of 12 mice with colonic dysplasia, but not in the normal or colitic mice. pSmad3c was detected without any significant difference between stages. p53 was weakly stained in a few cancer cells in 5 out of 10 mice. Smad3L signaling plays an important role in the carcinogenesis of chronic colitis in IL-10-deficient mice.

Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70-75 degrees C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes (udgA and udgB). Expression level of udgA gene was 2-3 times higher than that of udgB at 70, 74, and 78 degrees C when it was monitored by beta-glucosidase reporter assay. We developed hisD(3110), hisD(3113), hisD(3115), and hisD(174) marker allele that can specifically detect G:C-->A:T, C:G-->A:T, T:A-->A:T, and A:T-->G:C base-substitutions, respectively, by His(+) reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene (htk) or pyrE gene insertion in each hisD background, and their spontaneous His(+) reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C-->A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78 degrees C were about 2, 12, and 117 His(+)/10(8)/generation, respectively. At 70 degrees C culture, increased ratio of the mutation rate compared with the udg(+) strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G-->A:T, T:A-->A:T, and A:T-->G:C between udgA,B double mutant and the parent udg(+) strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C-->A:T transition mutations.

Transcriptional readthrough of Hox genes Ubx and Antp and their divergent post-transcriptional control during crustacean evolution.

Hox genes are in principle tandemly arranged in an order colinear with their order of expression along the anterior-posterior axis. Combinations of Hox proteins encode information that specifies the unique characteristics of axial regions in the metazoan body plan. The independent regulation of Hox genes achieved by differential promoter activity is essential for the expression of Hox proteins in distinct territories and thereby creating a full repertoire of Hox codes. Here we report the abundant expression of transcriptional readthrough products of two adjacent Hox genes, Ubx, and Antp, in five crustacean species of Branchiopoda and Malacostraca. Bicistronic mRNA places Antp under the control of the Ubx promoter, which is active in the posterior segments of two branchiopodans Daphnia and Artemia, and would normally reduce the complexity of Hox codes if translated. This does not occur, however, as the translational capability of the bicistronic mRNA is limited. In Daphnia, bicistronic Ubx/Antp mRNA produced no significant level of either UBX or ANTP. In Artemia, on the other hand, the bicistronic mRNA produced only UBX, and replaced the role of monocistronic Ubx mRNA. In this way, multiple post-transcriptional control mechanisms in two extant branchiopodans can be seen as preventing the potentially deleterious consequences of Hox gene fusion.

Tissue-specific expression of a bHLH-PAS protein homologous to ARNT during the development of crustacean Daphnia magna.

cDNAs encoding a Daphnia magna homolog of aryl hydrocarbon receptor nuclear translocator (ARNT) were isolated and the structural and functional features as well as the expression pattern of their product, DmagARNT, were analyzed. Among the known bHLH-PAS proteins, the deduced amino acid sequences of DmagARNT showed the highest degree of identity to that of Drosophila ARNT (TGO). Expression of DmagARNT in ARNT-lacking mouse Hepa-c4 cells resulted in the compensation for the loss of hypoxia response, suggesting the formation of a dimer with mouse HIF-1alpha and that the resulting heterodimer binds to the hypoxia-responsive elements (HRE), leading to transcription of the downstream luciferase gene. Expression of D. magna ARNT was evident at the middle to late stages of embryonic development (about 25 h to 48 h after ovulation) in several tissues, including a pair of the 1st antenna, 2nd antenna, 2nd maxilla, five pairs of the thoracic limbs, the central nerve system, anus, dorsal organ, maxillary gland, and carapace. As observed in other species, the D. magna ARNT is likely to function broadly as an expressed dimerization partner in developmental processes. In contrast, expression of ARNT in adult D. magna was limited to the epipodites of thoracic limbs, suggesting that ARNT plays a role solely in hypoxia response in adult Daphnia.

beta-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes.

Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75 degrees C. Because the frequency of DNA damage, such as deamination, depurination and single-strand breaks, increases as the temperature rises, the regulation of expression as well as the specificities and activities of T.thermophilus DNA repair systems are of particular interest. To study those systems, we developed a gene expression vector using the T.thermophilus beta-glucosidase gene (bgl) with host strain JOS9 (Deltabgl) derived from the T.thermophilus wild-type strain HB27. Since HB27 has two putative beta-galactosidase genes, the use of a single bgl gene as a reporter in combination with a Deltabgl host strain permits the study of gene expression against a low background level. We assayed Bgl activity with 2-nitrophenyl-beta-d-glucopyranoside as the substrate at 80 degrees C. We measured the expression of seven genes involved in DNA repair--three nucleotide excision repair genes (uvrA, uvrB and uvrC) and four recombinational repair genes (recA, ruvA, ruvB and ruvC). Expression levels of uvrA and uvrB were about three times those of uvrC, while those of ruvA, ruvB and ruvC were almost equal. Both ruvA and ruvC formed an operon with their adjacent 5'-upstream gene paaG and ftsQAZ, respectively. recA was transcribed as an operon of four genes, amt-cinA-ligT-recA. All seven DNA repair genes were expressed constitutively, and the DNA damaging agent mitomycin C did not increase their expression.

Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna.

Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH(2)-terminus [Kato et al., Gene 334 (2004) 157-165]. Both genes had a 17-exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH(2)-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids.

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

Exploring embryonic germ line development in the water flea, Daphnia magna, by zinc-finger-containing VASA as a marker.

VASA is an ATP-dependent RNA helicase belonging to the DEAD-box family that, in many organisms, is specifically expressed in germ line cells throughout the life cycle, making it a powerful molecular marker to study germ line development. To obtain further information on germ line development in crustaceans, we cloned VASA cDNAs from three branchiopod species: water fleas Daphnia magna and Moina macrocopa, and brine shrimp Artemia franciscana. RNA helicase domains in branchiopod VASA were highly conserved among arthropod classes. However, N-terminal RNA-binding domains in branchiopod VASA were highly diverged and, unlike other arthropod VASA reported so far, possessed repeats of retroviral-type zinc finger (CCHC) motifs. Raising specific antibodies against Daphnia VASA revealed that the primordial germ cells (PGCs) in this organism segregate at a very early cleavage stage of embryogenesis in parthenogenetic and sexual eggs. Clusters of PGCs then start to migrate inside the embryo and finally settle at both sides of the intestine, the site of future gonad development. RNA analyses suggested that maternally supplied vasa mRNA was responsible for early VASA expression, while zygotic expression started during blastodermal stage of development.

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH(2)-terminal kinase during human colorectal carcinogenesis.

Conversion of normal epithelial cells to tumors is associated with a shift in transforming growth factor-beta (TGF-beta) function: reduction of tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal carcinogenesis remain to be elucidated. TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal adenocarcinomas. In particular, late-stage invasive and metastatic cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11 adenomas was intermediate between those in normal epithelial cells and adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive adenocarcinomas. In contrast, pSmad3C gradually decreased as the tumor stage progressed. Activated c-Jun NH(2)-terminal kinase in cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at codon 361 in one adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the type II TGF-beta receptor and Smad2 genes were observed in the tumors. In conclusion, pSmad3C, which favors tumor suppressor activity of TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal kinase tended to induce the phosphorylation of Smad2/3L in human colorectal adenoma-carcinoma sequence.

Development of agmatine sensor using the combination of putrescine oxidase and agmatinase for squid freshness.

Agmatine (Agm) is an indicator of squid freshness. The Agm sensor was developed using flow injection analysis (FIA) that consisted of the putrescine oxidase (PuOx) reactor, the agmatinase (AUH)-PuOx reactor and two oxygen electrodes. In the proposed sensor, the first step is that coexisting cadaverine (Cad) and putrescine (Put) are removed by passing through the PuOx reactor and the initial decomposition is determined by the amount of oxygen consumed, simultaneously. The second step is that the amount of Agm is determined by the amount of oxygen consumed in the AUH-PuOx reactor. The optimum conditions for the use of the Agm sensor were as follows: 50 mM HEPES containing MnSO4 at a final concentration of 5 mM, pH 8.0, flow rate of 0.6 mL min(-1) and injection volume of 50 microL. A single assay could be completed in approximately 3 min. A linear relationship was obtained between the output and the Agm concentration in the range of 0.01-1 mM Agm with a correlation coefficient of 0.999. The detection limit was 0.005 mM. The relative standard deviations (RSDs) were 3.14 and 1.19% (n = 20) for 0.1 and 0.3 mM Agm, respectively. The extracts of squid were injected into the proposed sensor and the results were compared with those obtained using the conventional high-performance liquid chromatography (HPLC) method. A correlation was observed between the results obtained by the proposed sensor and those obtained by the conventional method. The determination of squid freshness is one of the good uses of the proposed Agm sensor.

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

A vitellogenin chain containing a superoxide dismutase-like domain is the major component of yolk proteins in cladoceran crustacean Daphnia magna.

A cDNA encoding vitellogenin (VTG), a precursor of a major yolk protein, vitellin (VTN), was isolated from cladoceran crustacean Daphnia magna. The deduced amino acid sequence of DmagVTG1, the polypeptide encoded by the cDNA, contained a possible signal peptide sequence of 16 amino acid (aa) residues. The possible mature form of DmagVTG1 consists of 1985 aa residues with a calculated molecular mass of 223,070 Da. The large lipid transfer (LLT) module and a part of the von Willebrand factor D (VWD) module found in the aa sequences of VTGs of many other organisms are well conserved in DmagVTG1. Phylogenetic analysis suggested that the LLT module of DmagVTG1 is more closely related to those of insect VTGs than those of decapodan crustaceans. A unique feature of DmagVTG1 is that it has a superoxide dismutase (SOD)-like domain at its NH(2)-terminus. Antisera against the SOD-like domain, the NH(2)-terminal part of the VTG domain and the COOH-terminal part of the VTG domain, respectively, were prepared and used for analysis of D. magna yolk proteins. Six species (I to VI) of major protein complexes were found in D. magna parthenogenetic eggs isolated immediately after ovulation. Complexes IV and V were the most abundant. DmagVTG1 was a component of Complexes III, IV and V, and the most abundant polypeptide in D. magna eggs. The protein complexes underwent gradual proteolysis during development. One of the primary sites of cleavage was between the two successive Arg residues located at the 1454th and 1455th positions of DmagVTG1.

Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent.

Of the four known globin genes that exist in the fresh-water crustacean Daphnia magna, several are individually induced by hypoxia, lending pale normoxic animals a visible red color when challenged by oxygen deprivation. The promoter regions of the Daphnia globin genes each contain numerous hypoxia response elements (HREs) as potential binding sites for hypoxia-inducible factors (HIFs). Daphnia HIF, bound to human HRE sequences, was detected in extracts from hypoxic (red), but not normoxic (pale), animals. Taking advantage of the phylogenetically conserved HIF/HRE recognition, we employed heterologous transfections of HIF-expressing human and Drosophila cells to model HIF signaling in Daphnia. These experiments revealed that three functional HREs within the promoter of the D. magna globin-2 gene cooperate for maximal hypoxic induction of a downstream luciferase reporter gene. Two of these three cis-elements, at promoter positions -258 and -107, are able to specifically bind human, Drosophila, or Daphnia HIF complexes in vitro. The same two sites are also necessary for maximal induction of reporter transcription under low oxygen tension in the presence of either endogenous human or overexpressed Drosophila HIF proteins. The third motif of the globin-2 gene promoter, a CACGTG palindrome at position -146, functions as a docking site for an unknown constitutive transcription factor. In human cells, this -146 complex interferes with HIF occupancy at the adjacent -107 HRE and thus controls the extent of HIF-mediated hypoxic activation of the downstream target.

p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo.

Distortion of autocrine transforming growth factor beta signal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells.

Resistance to growth inhibitory effects of transforming growth factor (TGF)-beta is a frequent consequence of malignant transformation. On the other hand, serum concentrations of TGF-beta, plasminogen activator inhibitor type 1 (PAI-1), and vascular endothelial growth factor (VEGF) are elevated as tumor progresses. The molecular mechanism of autocrine TGF-beta signaling and its effects on PAI-1 and VEGF production in human hepatocellular carcinoma (HCC) is unknown. TGF-beta signaling involves TGF-beta type I receptor-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. To investigate the involvement of autocrine TGF-beta signal in cell growth, PAI-1 and VEGF production of HCC, we made stable transfectants of human HCC line (HuH-7 cells) to express a mutant Smad2(3S-A), in which serine residues of SSXS motif were changed to alanine. The transfectants demonstrated an impaired Smad2 signaling. Along with the resistance to growth inhibition by TGF-beta, forced expression of Smad2(3S-A) induced endogenous TGF-beta secretion. Moreover, this increased TGF-beta enhanced ligand-dependent signaling through the activated Smad3 and Smad4 complex, and transcriptional activities of PAI-1 and VEGF genes. In conclusion, distortion of autocrine TGF-beta signals in human HCC accelerates their malignant potential by enhancing cell growth as well as PAI-1 and VEGF production.

Photomutagenicity of thiabendazole, a postharvest fungicide, in bacterial assays.

We investigated the photomutagenicity of thiabendazole (TBZ), a postharvest fungicide commonly used on imported citrus fruits. Using UVA light (320-400 nm), we irradiated bacterial cultures with or without TBZ in a 24-well multiplate. UVA-irradiation without TBZ was not mutagenic to the tester strains, nor was unirradiated TBZ. TBZ was strongly photomutagenic in Escherichia coli WP2uvrA and WP2uvrA/pKM101 strains, weakly photomutagenic in Salmonella typhimurium TA100 and TA98, and not photomutagenic in S. typhimurium TA1535 and TA1538. The photomutagenicity of TBZ was more evident in WP2uvrA/pKM101, which carries the trpE65 ochre mutation (TAA), than in TA100, which carries the hisG46 missense mutation (CCC). In E. coli WP3101-WP3106 and the corresponding pKM101-containing strains, photoactivated TBZ induced predominantly G:C-->A:T transitions and A:T-->T:A transversions. In the plasmid-containing strains only, TBZ induced a moderate number of A:T-->G:C transitions and a few A:T-->C:G and G:C-->T:A transversions. The observation that UVA-irradiated TBZ mutated both G:C and A:T basepairs may explain why WP2uvrA/pKM101 was more sensitive to its mutagenicity than TA100. TBZ that was irradiated before it was added to the WP2uvrA/pKM101 cells was not photomutagenic, which suggests that the photomutagenic products of TBZ were unstable or rapidly reacted with other molecules before being incorporated into cells.