A critical role of the 3' terminus of nascent DNA chains in recognition of stalled replication forks

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3' termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative "3' terminus binding pocket" present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses.     

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.     

Gene cloning and characterization of a Bacillus vietnamensis metalloprotease

A Bacillus vietnamensis metalloprotease (BVMP) with high affinity toward collagen was isolated and purified from the culture supernatant of Bacillus vietnamensis 11-4 occurring in Vietnamese fish sauces. The BVMP gene was cloned and its nucleotide and coded amino acid sequences determined. BVMP consists of 547 amino acid residues, with the zinc-binding sites conserved in common metalloproteases. It shares 57% amino acid identity with thermolysin originating from Bacillus thermoproteolyticus. The three-dimensional structure of BVMP was deduced by computer-aided modeling with the use of the known three-dimensional thermolysin structure as a template. Like thermolysin, BVMP cleaved the oxidized insulin B-chain at the peptide bonds involving the N-terminal sides of hydrophobic and aromatic amino acids. BVMP also showed high hydrolytic activity toward gelatin, collagen, casein, and elastin, especially toward the skeletal proteins at increased NaCl concentration. The high activity was found to be due to enhanced affinity to the substrates. Kinetical data on BVMP indicated that the Km values for the hydrolysis of Cbz-GPGGPA as a collagen model decreased as the concentration of added NaCl increased. Some contribution of this enzyme during the aging of fish sauces at high salt concentrations can thus be expected.     

Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

Histone deacetylases and ASYMMETRIC LEAVES2 are involved in the establishment of polarity in leaves of Arabidopsis

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.     

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Proteinase-activated receptors (PARs; PAR_1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR₄, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR₄ stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.

Methods

EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).

Results

Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR₄ agonist peptide (AYPGKF-NH₂, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR₄ stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR₄-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR₄-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR₄-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR₄ stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.

Conclusion

PAR₄ stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.     

Differential gene expression profiling in blood from patients with digestive system cancers

To develop a non-invasive and sensitive diagnostic test for cancer using peripheral blood, we evaluated gene expression profiling of blood obtained from patients with cancer of the digestive system and normal subjects. The expression profiles of blood-derived total RNA obtained from 39 cancer patients (11 colon cancer, 14 gastric cancer, and 14 pancreatic cancer) was clearly different from those obtained from 15 normal subjects. By comparing the gene expression profiles of cancer patients and normal subjects, 25 cancer-differentiating genes (p < 5.0 × 10⁻⁶ and fold differences >3) were identified and an “expression index” deduced from the expression values of these genes differentiated the validation cohort (11 colon cancer, 8 gastric cancer, 18 pancreatic cancer, and 15 normal subjects) into cancer patients and normal subjects with 100% (37/37) and 87% (13/15) accuracy, respectively. Although, the expression profiles were not clearly different between the cancer patients, some characteristic genes were identified according to the stage and species of the cancer. Interestingly, many immune-related genes such as antigen presenting, cell cycle accelerating, and apoptosis- and stress-inducing genes were up-regulated in cancer patients, reflecting the active turnover of immune regulatory cells in cancer patients. These results showed the potential relevance of peripheral blood gene expression profiling for the development of new diagnostic examination tools for cancer patients.