Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

The role of CD98 in astrocytic neoplasms

The high expression of CD98 was reported in some normal tissues, including blood brain barrier, activated lymphocytes, the basal layer of skin, proximal tubles of kidney, placenta, testis and a wide variety of tumors. The CD98 complex consists of an 80-85kD heavy chain (4F2hc/FRP-1) and a 40-45kD light chain. CD98hc, 4F2hc, and FRP-1 are the same glycosylated protein each other and define antigenicity of CD98. LAT1, the sodium-independent L-type amino acid transporter 1, has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells such as glioma cells. Increased LAT1 expression was found in various malignancies including human gliomas. Several studies implicated the important role of LAT1 and 4F2hc in malignant transformation and carcinogenesis. The LAT1-CD98 pathway may represent a unique therapeutic target for cancer intervention.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Some Characteristics of Red Light-induced Substance(s) against Botrytis cinerea Produced in Broad Bean Leaflets

The red light-induced antifungal substance(s) produced in broad bean was of relatively high molecular weight, water soluble, heat stable and fungi specific. Cellulose thin layer chromatography (TLC) of infection droplets of Botrytis cinerea or water droplets without spores of B. cinerea, recovered from inoculated broad bean leaflets kept under red light for 48 h, displayed inhibition zones at approximate Rf values of 0.0 and 0.6. Inhibition zones observed in cellulose TLC of water droplets were relatively faint compared to those of infection droplets. In a time-course study of accumulation of the antifungal substance(s), antifungal activity in both water and infection droplets recovered from red light irradiated broad bean leaflets occurred after 24 h irradiation. However, the antifungal activity in infection droplets was significantly higher than in water droplets. The antifungal substance(s) was less active against Botrytis fabae than B. cinerea.     

Phylogenetic relationships of Australian skinks of the Mabuya group (Reptilia: Scincidae) inferred from mitochondrial DNA sequences

Portions of two mitochondrial genes (12S and 16S ribosomal RNAs) were sequenced to analyze the phylogenetic relationships of the Mabuya group from the Australian region (Corucia, Egernia and Tiliqua). Results indicated the monophyly of these genera and their divergence from Asian and African members of this group. This suggests that the diversity of the Mabuya group in the Australian region has increased through an endemic radiation, not through multiple colonizations from outside. Among the genera from this region, Corucia and Tiliqua were closest to each other. This result contradicts with those of the previous hypotheses on the basis of morphological and immunological data that, respectively, suggested closest affinities between Corucia and Egernia, and Egernia and Tiliqua. We suppose that the morphological characters exclusively joining Corucia and Egernia are actually in plesiomorphic state.     

Characterization of peroxidase in buckwheat seed

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.     

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.     

Induction of efficient antitumor immunity using dendritic cells activated by recombinant Sendai virus and its modulation by exogenous IFN-beta gene

Dendritic cell (DC)-based cancer immunotherapy has been paid much attention as a new and cancer cell-specific therapeutic in the last decade; however, little clinical outcome has been reported. Current limitations of DC-based cancer immunotherapy include sparse information about which DC phenotype should be administered. We here report a unique, representative, and powerful method to activate DCs, namely recombinant Sendai virus-modified DCs (SeV/DC), for cancer immunotherapy. In vitro treatment of SeV without any bioactive gene solely led DCs to a mature phenotype. Even though the expression of surface markers for DC activation ex vivo did not always reach the level attained by an optimized amount of LPS, superior antitumor effects to B16F1 melanoma, namely tumor elimination and survival, were obtained with use of SeV-GFP/DC as compared with those seen with LPS/DC in vivo, and the effect was enhanced by SeV/DC-expressing IFN-beta (SeV-murine IFN-beta (mIFN-beta)/DC). In case of the treatment of an established tumor of B16F10 (7-9 mm in diameter), a highly malignant subline of B16 melanoma, SeV-modified DCs (both SeV-GFP/DC and SeV-mIFN-beta/DC), but not immature DC and LPS/DC, dramatically improved the survival of animals. Furthermore, SeV-mIFN-beta/DC but not other DCs could lead B16F10 tumor to the dormancy, associated with strongly enhanced CD8+ CTL responses. These results indicate that rSeV is a new and powerful tool as an immune booster for DC-based cancer immunotherapy that can be significantly modified by IFN-beta, and SeV/DC, therefore, warrants further investigation as a promising alternative for cancer immunotherapy.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.