Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene

A procedure was developed to purify large quantities of PII protein from an Escherichia coli strain which contains a multicopy plasmid harboring the structural gene of PII (the glnB gene). Ultraviolet spectra of uridylylated and unuridylylated PII were obtained using the purified PII and empirical formulas to calculate the concentration of protein and the average number of uridylylated subunits per molecule were derived. A continuous fluorometric assay for the measurement of uridylylated PII (PIID) and adenylyltransferase (ATase) was also established. Rate measurements at various concentrations of PIID and at a fixed concentration of ATase showed that a tetrameric PIID molecule interacts with only one ATase molecule at a time. The complete nucleotide sequence of the glnB gene was determined and parts of the deduced amino acid sequence were confirmed by the results of amino acid sequence analysis of peptides. The PII subunit consists of 103 amino acids (Mr = 11,580). Two tyrosines reside at positions 46 and 51, where Tyr51 is the site of uridylylation. Nucleotide sequence analysis of the upstream region showed no obvious sites for the binding of RNA polymerase, indicating that the glnB gene is a part of an as yet unidentified operon.     

Application of radiotelemetry to the survey of acorn dispersal byApodemus mice

We examined the applicability of radiotelemetry to studies of acorn dispersal byApodemus mice and compared its efficiency with the of this spool-and-line method. Installation of a transmitter (2.2 g) onto acorns did not interfere with the transporting and feeding behavior of the mice. We were able to detect all transmitter-installed acorns and follow the daily changes in the sites in which they were hoarded, while we missed 59% of the spool-tied acorns due to mice breaking the threads. Mice carried transmitter-installed acorns farther than spool-tied ones. The radiotelemetry method is superior to the spool-and-line method and useful for the study of hoarding behavior in rodents.     

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.     

Epigenetic Silencing of CHOP Expression by the Histone Methyltransferase EHMT1 Regulates Apoptosis in Colorectal Cancer Cells

Colorectal cancer (CRC) has a high mortality rate among cancers worldwide. To reduce this mortality rate, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan) or targeted therapy (bevacizumab, cetuximab, and panitumumab) has been used to treat CRC. However, due to various side effects and poor responses to CRC treatment, novel therapeutic targets for drug development are needed. In this study, we identified the overexpression of EHMT1 in CRC using RNA sequencing (RNA-seq) data derived from TCGA, and we observed that knocking down EHMT1 expression suppressed cell growth by inducing cell apoptosis in CRC cell lines. In Gene Ontology (GO) term analysis using RNA-seq data, apoptosis-related terms were enriched after EHMT1 knockdown. Moreover, we identified the CHOP gene as a direct target of EHMT1 using a ChIP (chromatin immunoprecipitation) assay with an anti-histone 3 lysine 9 dimethylation (H3K9me2) antibody. Finally, after cotransfection with siEHMT1 and siCHOP, we again confirmed that CHOP-mediated cell apoptosis was induced by EHMT1 knockdown. Our findings reveal that EHMT1 plays a key role in regulating CRC cell apoptosis, suggesting that EHMT1 may be a therapeutic target for the development of cancer inhibitors.     

Temperature-dependent post-diapause development and prediction of spring emergence of the pine needle gall midge (Dipt., Cecidomyiidae)

Abstract:  We determined the influence of temperature on post-diapause development of overwintered Thecodiplosis japonensis Uchida et Inouye (Dipt., Cecidomyiidae) under various treatments (12, 15, 18, 21, 24, 27 and 30°C) in an effort to predict its spring emergence. Survival and developmental period for the overwintered larvae and pupae were significantly influenced by temperature. Linear and nonlinear regression models quantitatively described temperature-dependent development and survival of T. japonensis . The survival models exhibited right-skewed bell shape patterns for all stages, indicating a more detrimental impact on survival at high temperatures. Theoretical optimum temperatures with highest survival were 22.3, 24.0 and 24.0°C for the overwintered larvae, pupae and total post-diapause development (the larvae to adults) respectively. Pupal mortality was higher at all temperatures than larval mortality and the suitable range of temperature for pupae was narrower than that of larvae. The nonlinear Briere model estimated that optimum temperatures with the fastest development were 29.1°C for larvae, 27.6°C for pupae and 27.0°C for larvae to adults. In a linear model, the lower threshold temperatures were 5.1, 7.1 and 5.9°C for larvae, pupae, and larvae to adults respectively. A predictive degree-day model was developed using trap catches of T. japonensis adult emergence during 1991–1995. The model accounted for 84.6% of year-to-year variation in adult emergence and predicted accurately the median emergence time in 1996.