VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

5'-O-masked 2'-deoxyadenosine analogues as lead compounds for hepatitis C virus (HCV) therapeutic agents

On the basis of our previous study on antiviral agents against the severe acute respiratory syndrome (SARS) coronavirus, a series of nucleoside analogues whose 5'-hydroxyl groups are masked by various protective groups such as carboxylate, sulfonate, and ether were synthesized and evaluated to develop novel anti-hepatitis C virus (HCV) agents. Among these, several 5'-O-masked analogues of 6-chloropurine-2'-deoxyriboside (e.g., 5'-O-benzoyl, 5'-O-p-methoxybenzoyl, and 5'-O-benzyl analogues) were found to exhibit effective anti-HCV activity. In particular, the 5'-O-benzoyl analogue exhibited the highest potency with an EC(50) of 6.1 microM in a cell-based HCV replicon assay. Since the 5'-O-unmasked analogue (i.e., 6-chloropurine-2'-deoxyriboside) was not sufficiently potent (EC(50)=47.2 microM), masking of the 5'-hydroxyl group seems to be an effective method for the development of anti-HCV agents. Presently, we hypothesize two roles for the 5'-O-masked analogues: One is the role as an anti-HCV agent by itself, and the other is as a prodrug of its 5'-O-demasked (deprotected) derivative.     

High-throughput SNP detection using nano-scale engineered biomagnetite

A semi-automated system for the large-scale detection of single nucleotide polymorphisms (SNPs) has been developed based on allele-specific oligonucleotide hybridization and thermal dissociation curve analysis using nano-scale engineered biomagnetite (bacterial magnetic particles; BacMPs). For reliable detection in large numbers of samples, several conditions for the capture of target DNA on nano-sized BacMPs and the denaturation of double-stranded DNA were optimized. The most efficient target DNA capture was observed using short PCR amplicons (69 bp). Captured DNAs were denatured using 50 mM NaOH. With these optimizations, large-scale SNP detection was performed on 822 samples of the transforming growth factor (TGF)-beta1 gene, which is rich in both GC content and repetitive sequences. High reliability for the semi-automated BacMP-based SNP detection system was confirmed following comparison to traditional sequencing-based methods.     

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Centromere dynamics in the primitive red alga Cyanidioschyzon merolae

Centromeres are universally conserved functional units in eukaryotic linear chromosomes, but little is known about the structure and dynamics of the centromere in lower photosynthetic eukaryotes. Here we report the identification of a centromere marker protein CENH3 and visualization of centromere dynamics in the ultra-small primitive red alga Cyanidioschyzon merolae. Immunoblotting and immunofluorescence microscopy showed that CENH3 increased rapidly during S phase, followed by a drastic reconstitution into two discrete foci adjacent to the spindle poles at metaphase, suggesting the cell-cycle-regulated expression of CENH3. Immunoelectron microscopy revealed that the CENH3 signals were associated with the nuclear envelope, implying interplay between the kinetochore complex and the nuclear envelope. These results demonstrate dynamic centromere reconstitution during the cell cycle in an organism in which the chromosomes do not condense at metaphase.     

CP5484, a novel quaternary carbapenem with potent anti-MRSA activity and reduced toxicity

A new series of 1beta-methyl carbapenems possessing a 6,7-disubstituted imidazo[5,1-b]thiazol-2-yl group directly attached to the C-2 position of the carbapenem nucleus was prepared, and the activities of these compounds against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. To study the effect of basic moieties on anti-MRSA activity, we introduced an amino, or imino, or amidino group at the 6-position of imidazo[5,1-b]thiazole in place of the carbamoylmethyl moiety of CP5068. Anti-MRSA activities of almost all basic group-substituted carbapenems were improved, though some of the compounds showed stronger acute toxicity in mice than IPM. In order to decrease the toxicity without decreasing the activity, we introduced various additional functionalities around the basic moiety. Finally, we obtained CP5484, which has excellent anti-MRSA activity and low acute toxicity.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Soluble and particulate methane monooxygenase gene clusters in the marine methanotroph Methylomicrobium sp. strain NI

Soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO) gene clusters in the marine methanotroph Methylomicrobium sp. strain NI were completely sequenced and analysed. Degenerated primers were newly designed and used to amplify the gene fragments containing intergenic mmoX-Y and mmoD-C regions and a partial pmoC region. Phylogenetic analysis of amino acid sequences deduced from mmoX and pmoA, as well as of 16S rRNA gene sequences, indicated that this strain was most closely related to the halotolerant methanotroph Methylomicrobium buryatense. There were putative sigma(54)- and sigma(70)-dependent promoter sequences upstream of the sMMO and pMMO genes, respectively, and mmoG, which is known to be related to the expression and assembly of sMMO, existed downstream of the sMMO genes. These findings suggest that the major components and regulation of MMOs in this marine methanotroph are quite similar to those in freshwater methane oxidizers, despite the difference in their habitats.     

Differential distribution of the endoplasmic reticulum network in filamentous fungi

Filamentous fungi are composed of hyphal compartments divided by septa, which communicate via septal pores. Apical compartments can elongate to over 100 microm without septum formation and possess a polarized distribution of organelles. In Aspergillus, subapical compartments are arrested in interphase but can reinitiate mitosis and growth by branching. Recent reports using green fluorescent protein (GFP) technology have demonstrated the highly differentiated localization of the endoplasmic reticulum (ER) network in various regions of the hyphae: the gradient distribution from the apical region, the localization along the septum, differential distributions in adjacent compartments, and the dynamic morphological change during septum formation. In this review the spatial regulation of the ER network in multicellular filamentous fungi is discussed.