Requirement of Smad3 for mast cell growth

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-beta and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-beta pathway by anti-TGF-beta neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-beta and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-beta-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.     

Acidianus manzaensis sp. nov., a Novel Thermoacidophilic Archaeon Growing Autotrophically by the Oxidation of H2 with the Reduction of Fe3+

A novel thermoacidophilic iron-reducing Archaeon, strain NA−1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H₂ or S⁰ as an electron donor and Fe³⁺ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe³⁺ and O₂ served as electron acceptors for growth. However, S⁰, NO₃ ⁻, NO₂ ⁻, SO₄ ²⁻, Mn⁴⁺, fumarate, and Fe₂O₃ did not serve as electron acceptors. The ranges of growth temperature and pH were 60–90°C (optimum: 80°C) and pH 1.0–5.0 (optimum: pH 1.2–1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1^T = NBRC 100595 = ATCC BAA 1057). Strain NA-1 has been deposited in the culture collections of the National Institute of Technology and Evolution (NBRC 100595) and American Type Culture Collection (ATCC BAA 1057). The 16S rDNA sequence has been deposited at GenBank under accession number AB182498.     

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).     

Rapid and simple screening of transgenic mice: novel extraction-free, filter-based PCR genotyping from blood samples

We evaluated the effectiveness of using Flinders Technology Associates (FTA) filter paper for the polymerase chain reaction (PCR) genotyping of transgenic mice. Tail prick blood sample dried on an FTA filter disc was processed for genomic PCR. It is easy and rapid to prepare DNA templates because the protocol is extraction-free and only requires minimal handling of wash briefly bloodstained FTA filter discs. Progeny of a transgene-positive founder mated with wild-type mice was screened for the presence of the transgene by the filter-based PCR using transgene-specific primers. The resulting amplicons with expected sizes of 3134 bp, 1152 bp, 877 bp and 688 bp were robust and reproducible, allowing a distinction between transgenic (n=44) and wild-type (n=47) mice showing no signal. The filter-based PCR screening took only half a day. The present study confirmed the validity and usefulness of the novel rapid extraction-free genotyping method.     

Clinicopathologic roles of tumor-infiltrating lymphocytes and CD8-positive lymphocytes in lung cancer imprint smears in squamous cell carcinoma and adenocarcinoma

OBJECTIVE: To investigate the presence of tumor-infiltrating lymphocytes (TILs) and CD8-positive lymphocytes (CD8s) in lung cancers and to examine the prognostic significance of their relationship with clinicopathologic parameters. STUDY DESIGN: Primary tumor imprint smears from 83 lung cancers were consecutively obtained at surgery at Tsuchiura Kyodo General Hospital between 1996 and 1998. TILs were observed in Papanicolaou-stained smears, and CD8s were immunocytochemically visualized. RESULTS: TILs were judged positive in 42 cases (51%), and 34 of 83 cases (41%) were positive for CD8s. There was no correlation between the frequency of TILs and CD8s. TILs assessed by Papanicolaou staining in 83 cases showed no correlation with any clinicopathologic factors. CD8s were observed more frequently in squamous cell carcinomas (SCCs) than other adenocarcinomas. A high frequency of CD8s was associated with lymph node metastasis (p = 0.043), stage (p = 0.025) and tumor differentiation (p = 0.03). TILs and CD8s made no significant difference in survival. However, in SCC the patients positive for TILs but negative for CD8s survived and had a significantly better prognosis than did the others (p = 0.037). CONCLUSION: CD8s were associated with tumor progression and development of malignancy in lung cancers. Especially in SCC, the cases had a high frequency of TILs. A low number of CD8s may correlate with a favorable prognosis.     

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.     

Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by atomic force microscopy

A DNA repair-promoting protein, PprA, was isolated from a radiation resistant bacterium, Deinococcus radiodurans [I. Narumi, K. Sato, S. Cui, T. Funayama, S. Kitayama, H. Watanabe, PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation, Mol. Microbiol. 54 (2004) 278-285]. Despite several studies, however, the function of PprA is not still clear. We used atomic force microscopy (AFM) to elucidate the role of this protein in the DNA repair pathway. In the present study, interaction between the linear DNA and PprA protein was imaged and analyzed by AFM without any fixation or staining. Though both end-bound and internally bound PprA was observed, the affinity of the end-bound protein was greater considering the proportion of features of binding analyzed by AFM. In some conditions, looping forms of the DNA-PprA complex were observed. Gel filtration high performance liquid chromatography (HPLC) was also conducted to estimate the molecular weight of this protein. The result of the HPLC analysis suggested that PprA formed multimers in buffer solution without DNA.     

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.