Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling

The Notch pathway is an evolutionarily conserved signaling mechanism that is essential for cell-cell interactions. The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, we have analyzed the functional involvement of these Deltex domains in Notch signaling. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain was indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaved as a dominant-negative form. This dominant-negative Deltex inhibited Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting the dominant-negative Deltex might prevent the activation of the Notch receptor. We found that Deltex formed a homo-multimer, and mutations in the RING-H2 finger domain abolished this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupted the function of Deltex in vivo. However, when the same mutant was fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein had normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex.     

The primary structure of the Chloroflexus aurantiacus reaction-center polypeptides

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.     

An experimental device for studying seed responses to naturally fluctuating temperature of surface soil under a constant water table

1. A simple experimental device was designed, in which seeds can be exposed to natural fluctuation of surface soil temperature under a constant soil moisture condition maintained by an automatic water supply system based on the principle of a Mariotte siphon.
2. Except for the period during summer drought, surface soil temperature at a depth of 5 cm and its fluctuation within the device were largely similar to the temperature of the surface soil subjected to natural fluctuation of moisture.
3. The seeds of Persicaria lapathifolia placed at the depth of 0·5 cm in the soil within the device germinated during the natural germination season of the species, while the seeds placed at the depth of 5 cm or beneath the 10 cm-thick layer of litter failed to germinate.
4. The ungerminated seeds retrieved in August did not germinate at the favourable alternating temperature in the laboratory test unless exposed to previous moist chilling, suggesting the induction of secondary dormancy. Therefore, higher summer temperatures but not summer drought or moisture fluctuation seem to have been responsible for the dormancy induction, because the dormancy was induced in the fully hydrated seeds.     

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.     

Establishment and characterization of the NCC–SS1–C1 synovial sarcoma cell line

Synovial sarcoma is an aggressive mesenchymal malignancy characterized by unique gene fusions. Tissue culture cells are essential tools for further understanding tumorigenesis and anti-cancer drug development; however, only a limited number of well-characterized synovial sarcoma cell lines exist. Thus, the objective of this study was to establish a patient-derived synovial sarcoma cell line. We established a synovial sarcoma cell line from tumor tissue isolated from a 72-year-old female patient. Prepared cells were analyzed for the presence of gene fusions by fluorescence in situ hybridization, RT-PCR, and karyotyping. In addition, the resulting cell line was characterized by viability, short tandem repeat, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell line were examined by mass spectrometric protein expression profiling and KEGG pathway analysis. Our analyses revealed that the primary tumor and NCC–SS1–C1 cell line harbored the SS18–SSX1 fusion gene typical of synovial sarcoma and similar proteomics profiles. In vitro analyses also confirmed that the established cell line harbored invasive, colony-forming, and spheroid-forming potentials. Moreover, drug screening with chemotherapeutic agents and tyrosine kinase inhibitors revealed that doxorubicin, a subset of tyrosine kinase inhibitors, and several molecular targeting drugs markedly decreased NCC–SS1–C1 cell viability. Results from the present study support that the NCC–SS1–C1 cell line will be an effective tool for sarcoma research.     

Mutagenic activity of 2-phenylbenzotriazole derivatives related to a mutagen, PBTA-1, in river water.

A mutagen, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]5-ami no-7-bromo-4-chloro-2H-benzotriiazole (PBTA-1), isolated from water of the Nishitakase River in Kyoto exhibits potent mutagenic activity in Salmonella typhimurium TA98 with S9 mix and has characteristic moieties, including bromo, chloro, acetylamino, bis(2-methoxyethyl)amino and primary amino groups on a 2-phenylbenzotriazole skeleton. The mutagenicities of PBTA-1, its congeners and five related 2-phenylbenzotriazoles were examined in S. typhimurium TA98 with S9 mix in order to elucidate the structure-activity relationships. The data obtained suggest that a primary amino group plays an essential role in the mutagenic activity as do aromatic amines including heterocyclic amines in cooked foods. The effect of planarity of the 2-phenylbenzotriazole ring was significant, and in addition, halogen groups of PBTA-1 influenced the enhancement of the mutagenic activity.     

Mice-lacking LMP2, immuno-proteasome subunit,as an animal model of spontaneous uterine leiomyosarcoma

Uterine tumors are the most common type of gynecologic neoplasm. Uterine leiomyosarcoma (LMS) is rare, accounting for 2% to 5% of tumors of the uterine body. Uterine LMS develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Radiographic evaluation combined with PET/CT can be useless in the diagnosis and surveillance of uterine LMS. Importantly, a diagnostic biomarker, which distinguishes malignant LMS and benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS in order to establish a method of treatment. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40% by 14 months of age. It is therefore of interest whether human uterine LMS shows a loss of LMP2 expression. We found LMP2 expression is absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is potentially a diagnostic biomarker for uterine LMS, and gene therapy with LMP2-encording DNA may be a new therapeutic approach.     

Breast cancer resistance protein directly confers SN-38 resistance of lung cancer cells

Breast cancer resistance protein (BCRP), an ABC half-transporter, is overexpressed in cancer cell lines selected with doxorubicin/verapamil, topotecan, or mitoxantrone. BCRP-overexpressing cells show cross-resistance to camptothecin derivatives such as irinotecan, SN-38 (the active metabolite of irinotecan), and topotecan. To test whether BCRP confers SN-38 resistance, we selected two SN-38 resistant sublines from PC-6 human small-cell lung cancer cells by SN-38, and then characterized these cells. Compared to PC-6 cells, the resistant sublines PC-6/SN2-5 and PC-6/SN2-5H were approximately 18- and 34-fold resistant, respectively. The intracellular SN-38 accumulation was reduced in the sublines, and BCRP mRNA was overexpressed in proportion to the degree of SN-38 resistance. These findings suggest that BCRP confers SN-38 resistance in the sublines. To confirm this hypothesis, PC-6/SN2-5 cells were transfected with antisense oligonucleotides complementary to portions of BCRP mRNA. The antisense oligonucleotides significantly suppressed BCRP mRNA expression, and enhanced SN-38 sensitivity in the subline. These data indicate that BCRP is directly involved with SN-38 resistance, by efflux transport of SN-38.     

Efficient sequential gene regulation via FLP-and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells

Site-specific recombinase is widely applied for the regulation of gene expression because its regulatory action is strict and efficient. However, each system can mediate regulation of only one gene at a time. Here, we demonstrate efficient "sequential" gene regulation using Cre-and FLP-expressing recombinant adenovirus (rAd) in two different monitor cell lines, for regulation of one gene (OFF-ON-OFF) and for two genes (ON-OFF and OFF-ON, independently). Generally, serial use of Cre-and FLP-expressing rAd tends to cause significant cytotoxicity, but we here described optimum dose of the rAds for serial regulation. We also established an efficient method of rAd infection to mouse ES cell lines after removing feeder cells, showing that this system is useful for removal of FRT-flanked drug-resistance gene cassette from recombinant ES cells prior to introduction of ES cells into blastocytes for chimeric mice production. Because our sequential gene-regulation system offers efficient purpose-gene regulation and strict OFF-regulation, it is potentially valuable for elucidating not only novel gene functions using cDNA microarray analysis but also for "gene switching" in development and regeneration research.