Identification of transfer RNA suppressors in Escherichia coli. II. Duplicate genes for tRNA2Gln.

The number of gene copies for tRNA₂^Gln in λpsu⁺2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su⁺2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNA_m. When the su⁺2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA₂^Gln, instead of the amber-suppressing tRNA₂^Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA₂^Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA₂^Gln genes, one su⁺2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNA_m^Met gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA₂^Gln at 15 minutes on the bacterial chromosome. su⁺2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA₂^Gln genes could also become ochre suppressors. A tRNA_m^Met gene is located near, but not between, these two tRNA₂^Gln genes.     

Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.     

Micromechanical evaluation of mineralized multilayers

The biomechanical stability of osseointegrated implants is of particular importance, especially the stability which is achieved from structural manipulation at the interface between the implant surface and the bone tissues. Nanoscale β-tricalcium phosphate-immobilized titanium was prepared by discharge into a physiological buffered saline solution. Compared with hydroxyapatite, it has been shown to be effective in generating a bone-like chemical structure on the surface by cooperative interaction between osteoblastic cells and the β-tricalcium phosphate. The present study, after cell cultivation, investigates the nanostructures and biomechanical property differences of a mineralized layer formed on two samples of nano-calcium phosphate-immobilized titanium. A scanning probe microscope study revealed that the mineralized tissue formed on the β-tricalcium phosphate samples after 1 week of cell culture showed significantly higher roughness, compared with hydroxyapatite samples. Nanoindentation micromechanical evaluation of the in vitro generated multilayered structures exhibited thicker bone-like mineralized layers on the β-tricalcium phosphate samples. A successful modification of titanium implants through the cooperative interaction between osteoblastic cells and nano β-tricalcium phosphate is anticipated.     

Development of EST-SSR markers from an inner bark cDNA library of Fagus crenata (Fagaceae)

Fagus crenata Blume is widely distributed throughout Japanese cool-temperate deciduous broad-leaved forests, but there are two divergent groups of populations in areas with contrasting winter climates separated by Japan’s Central Mountain Range. To facilitate investigations of adaptive genetic differentiation of the species using potentially functional genes, we have collected Expressed Sequence Tags and developed Simple Sequence Repeat markers using a cDNA library constructed from cambium and surrounding tissues. In total, 270 primer pairs were designed, and 87 of the corresponding loci showed polymorphism in 16 individuals, with 2–21 alleles per locus and expected heterozygosities ranging from 0.06 to 0.97. EST-SSR markers developed in the present study will be useful for genomic analyses of F. crenata populations.     

Changes in the glycolipid composition and characteristic activation of GM3 synthase in the thymus of mouse after administration of dexamethasone

Glycolipids in the thymus of mice after administration of dexamethasone were compared with those in control mice. In parallel with a decrease in the tissue weight due to the disappearance of immature thymocytes in the cortex, the amounts of GlcCer, Gg₄Cer and GM1 decreased from 18 h after intraperitoneal administration of dexamethasone, but those of Gb₄Cer and Forssman glycolipid did not change, indicating the differential distribution of ganglio- and globo-series glycolipids in the thymus, GlcCer, Gg₄Cer and GM1 being on dexamethasone-sensitive cortical thymocytes, and Gb₄Cer and Forssman glycolipid on dexamethasone-resistant cells including thymic stromal cells, respectively. At the same time, a characteristic increase in GM3, whose amount per thymus and concentration per mg of thymus were increased 4-fold and 13-fold compared to those in the control mice, respectively, was observed at the onset of the decrease in tissue weight and was due to the increased activity of LacCer sialyltransferase with the enhanced expression of its gene and the concomitant decrease in cytosolic sialidase activity. One can suggest that endogenous accumulation of GM3 is involved in the dexamethasone-induced apoptosis of cortical thymocytes. On radiolabeling of the thymus with CMP-[¹⁴C]-NeuAc, the incorporation of radioactivity into GM3 was preferentially observed in the thymuses of dexamethasone-administered mice, but not in those of control mice, suggesting the possible involvement of plasma membrane-associated sialytransferase in GM3 synthesis in the thymuses of dexamethasone-administered mice. Published in 2005.     

Frequent detection of anti-recoverin cytotoxic T-lymphocyte precursors in peripheral blood of cancer patients by using an HLA-A24-recoverin tetramer

Recoverin is a photoreceptor-specific calcium binding protein that is only expressed in the retina in normal tissues. Aberrant expression of recoverin, however, has been observed in several cancer tissues and may cause a very rare autoimmune disease, cancer-associated retinopathy (CAR). It has been suggested that CAR-positive cancer patients have a more favorable prognosis than CAR-negative cancer patients. To estimate the status of recoverin-specific T cells in such cancer patients, we generated an HLA-A24-recoverin peptide tetramer. By use of the tetramer, we could directly assess the frequency of CTL precursors (CTLp) of 20 HLA-A24(+) cancer patients with ten colon, six stomach and four breast cancers, and seven healthy individuals. Four cancer patients showed a CTLp frequency higher than 0.9%. Seven cancer patients including the former four patients and two healthy individuals showed specific anti-recoverin cytotoxic responses in an HLA-A24-restricted manner after in vitro stimulation with the recoverin peptide. Moreover, five cancer patients analyzed in an independent experiment using different peripheral blood mononuclear cells (PBMC) samples showed similar CTLp and cytotoxic T lymphocyte (CTL) frequencies and cytotoxic responses, suggesting that the CTLp frequency analyzed by the tetramer and the cytotoxic response may have a good correlation. Thus, we hypothesize that anti-recoverin CTLp may exist in some cancer patients, and that anti-recoverin CTL may be readily induced.