c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFbeta-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation.     

Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging

Taking advantage of the phenomenon of bioluminescence resonance energy transfer (BRET), we developed a bioluminescent probe composed of EYFP and Renilla reniformis luciferase (RLuc)--BRET-based autoilluminated fluorescent protein on EYFP (BAF-Y)--for near-real-time single-cell imaging. We show that BAF-Y exhibits enhanced RLuc luminescence intensity and appropriate subcellular distribution when it was fused to targeting-signal peptides or histone H2AX, thus allowing high spatial and temporal resolution microscopy of living cells.     

Lake-wide assessment of microplastics in the surface waters of Lake Baikal,Siberia

Limnology - Small microplastic particles < 330 µm, sometimes called mini-microplastics (MMP), are far more abundant than those larger than 330 µm....     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Immunohistochemical analysis for cell proliferation-related protein expression in small cell carcinoma of the esophagus; a comparative study with small cell carcinoma of the lung and squamous cell carcinoma of the esophagus

Small cell carcinoma is a rare neoplasm in the esophagus. To evaluate cell proliferation activity and its underlying mechanisms in this tumor, we examined immunohistochemically 5 cases of small cell carcinoma of the esophagus (SCCE) for expressions of tumor suppressor proteins, oncoproteins and cell proliferation markers including p53, p21WAF1/CIP1, retinoblastoma (Rb) protein, bcl-2, Ki-67 and PCNA, and compared the results with those of 5 cases of small cell carcinoma of the lung (SCCL) and 10 cases of squamous cell carcinoma of the esophagus (SQCE). The prevalence and labeling index of p53-immunoreactivity tended to be higher in SCCE (4/5; 56.6%) and SCCL (4/5; 79.9%) than in SQCE (6/10; 48.8%). Expression of p21WAF1/CIP1 was observed in 2 of 10 cases of SQCE. In contrast, its expression could not be detected in any cases of SCCE and SCCL examined. Expression of Rb protein was observed in 9 out of 10 cases of SQCE, but not in any cases of SCCE and SCCL. SCCE and SCCL showed more frequent and intense immunoreactivity for bcl-2 than SQCE. In expression of cell proliferation markers (Ki-67 and PCNA), no remarkable difference was observed among SCCE, SCCL and SQCE. These results suggest that SCCE and SCCL could share some genetic alternations including mutation of p53, loss of Rb gene and overexpression of bcl-2, and these may be related to the similar biological potentials between the two. Furthermore, SCCE was different from SQCE in expression of Rb protein and bcl-2, and these two types of esophageal carcinoma could arise through different molecular mechanisms.     

Comparison of Japanese isolates of Coxiella burnetii by PCR-RFLP and sequence analysis

The genetic variation of Japanese isolates of Coxiella burnetii, the agent of Q fever, was found for the first time. Forty-nine out of 72 isolates had the chronic pattern of the isocitrate hydrogenase gene. Sequence analysis revealed that the isolates have a specific nucleotide sequence. The putative amino acid sequence was the same as that of chronic reference strains. These results suggest the variation of C. burnetii isolates in Japan.     

In vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of Coxiella burnetii

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.     

Essential structural features of acetogenins: role of hydroxy groups adjacent to the bis-THF rings

The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins. To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues which lack either or both of the hydroxy groups, and investigated their inhibitory activities with bovine heart mitochondrial complex I. Our results indicate that the presence of either of the two hydroxy groups sufficiently sustains a potent inhibitory effect.