Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

We have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of [32P]Pi-labeled and [3H]leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fractionation studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.     

Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

Summary Rice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO₂). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO₂ resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO₂ (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO₂ (twice).     

Mineral nutrition, of cultured chlorophyllous cells of tobacco VII. Effects of the NH4/NO3 ratio and of Cl in the media on the growth and ionic balance of cells

NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (C—A) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO₃/liter as the sole N source. (C—A) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH₄ 10+NO₃ 42 in meq/liter,had lower growth rates and lower (C—A) values as comparedwith NG on media containing NO₃ as the sole N source. NG, cultured on media containing NH₄ 30+NO₃ 42 in meq/liter,had high growth rates and (A—C) values of 22 to 53 meq/100gof dry cells. In this case, the (A—C) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|C—A| contents [|C—A| being the absolute valuesof (C—A)]. (Received May 15, 1980; )     

Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

Mineral nutrition of cultured chlorophyllous cells of tobacco IV. Kinetic studies of K and Mg uptake by the cells

1. When applying the adsorption theory to the selectivity coefficient,K and Mg uptakes by cells in a K-Mg replacement series of mediaare not regulated only by a common mechanism, under the assumptionthat b values, which indicate the affinities between the ionand the adsorptive surface, do not change.
2. Regulation of Kand Mg uptakes by a common multiphasic mechanismin the cellsis possible when the selectivity coefficient b_K/b_Mgvaries inverselywith the M_K/M_Mg ratio in the external medium.
3. K and Mg uptakesare not regulated only by their respectivesingle specific mechanisms.
4. Another possibility is regulation of K and Mg uptakes bothbycommon and specific mechanisms. The common mechanism maybemultiphasic.

(Received December 2, 1975; )     

Isolation and characterization of different-sized nucleoli from Ehrlich ascites tumor cells : Evidence of different nucleolar ribosomal cistrons

A procedure was developed for isolation of variously sized nucleoli in order to study the mechanism of nucleolar formation from multiple nucleolar organizers and to compare the compositions of different-sized nucleoli from Ehrlich ascites tumor cells. Relatively small nucleoli and large nucleoli from Ehrlich ascites tumor cells were separated by centrifugation at 400 g for 5 min in a layer of 0.34 M sucrose over 0.88 M sucrose. Small nucleoli remained in the 0.34 M sucrose layer while the large nucleoli accumulated in the 0.88 M sucrose.Three fractions, provisionally named small, intermediate and large nucleoli, containing 0.33, 0.41 and 0.84 pg DNA/nucleolus, respectively, were separated. Unfractionated nucleoli contained 0.59 pg DNA/nucleolus. The RNA content also increased with the size of the nucleolus and no significant difference was observed in the RNA/DNA ratios in the three fractions. Large nucleoli incorporated more [³H]uridine and [³²P]orthophosphate into RNA than did small nucleoli, but the base compositions of the RNAs extracted from the different-sized nucleoli were similar. No significant fragmentation occurred on sonication of large nucleoli for 3 min, so the observed difference in the DNA contents was not due to mechanical damage of the nucleoli.The DNAs of these different-sized nucleoli were analysed on CsCl gradients. The nucleoli contained similar percentages of satellite DNA (20–22%) which were also similar to those of total, unfractionated nucleoli. Approx. 10% of the extranucleolar DNA is satellite DNA—thus the nucleolar fractions were probably not appreciably contaminated with extranucleolar DNA. The DNA of small nucleoli contained a slightly lower percentage (0.058%) of ribosomal cistrons than large nucleoli (0.081%). This means that the higher content of DNA in the large nucleoli is not merely due to longer sized chromatin with extra regions of the vicinity of nucleolar organizers. Thus these results suggest that the total content of ribosomal cistrons/nucleolus is roughly proportional to the DNA content of the nucleoli, at least in Ehrlich ascites tumor cells. Namely, the number of ribosomal cistrons per nucleolus for small, intermediate and large nucleoli is 40, 60 and 130, respectively.     

Sulfonamide-based non-alkyne LpxC inhibitors as Gram-negative antibacterial agents

We attempted to optimize sulfonamide-based non-alkyne LpxC inhibitors by focusing on improvements in enzyme inhibitory and antibacterial activity. It was discovered that inhibitors possessing 2-aryl benzofuran as a hydrophobe exhibited good activity. In particular, compound 21 displayed impressive antibacterial activity (E. coli MIC = 0.063 μg/mL, K. pneumoniae MIC = 0.5 μg/mL, and P. aeruginosa MIC = 0.5 μg/mL), and is a promising lead for further exploration as an antibacterial agent.     

Plant stem cell research is uncovering the secrets of longevity and persistent growth

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project ‘Principles of pluripotent stem cells underlying plant vitality’ was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.