Spontaneous Mutations Modifying the Activity of Alcohol Dehydrogenase (Adh) in DROSOPHILA MELANOGASTER

In a marked-inversion balanced lethal system of the second chromosome of Drosophila melanogaster, mutations were accumulated under minimum pressure of natural selection in 1000 individual lines that originated essentially from two individuals. After about 300 generations, the specific activities of alcohol dehydrogenase of 69 randomly selected individual lines were measured with replications using four replicated vials (on 2 days—two replications per day) by observing the reduction of NAD⁺ to NADH at 340 nm. Total soluble protein as the basis of standardization of enzyme activity was measured by the Lowry method for each vial. A control experiment was made immediately after the establishment of 20 individual lines from a single genotype. A significant increase in genetic variance was observed among the mutation-accumulating lines but was not detected in the control experiment. The statistical analysis of the data on the basis of the one-band/one-gene hypothesis suggests that many mutations controlling the activity of alcohol dehydrogenase occurred in regions different from the alcohol dehydrogenase locus itself, mainly in the noncoding DNA. Furthermore, it is suggested that transposon-like elements are related to the induction of these changes in alcohol dehydrogenase specific activities. Additional experimental evidence supporting this conclusion is also given.     

Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia

Cancer cells in solid tumors are challenged by various microenvironmental stresses, including hypoxia, and cancer cells in hypoxic regions are resistant to current cancer therapies. To investigate the mechanism of resistance to hypoxia in cancer cells, we examined mouse Lewis lung carcinoma (LLC) cells, which died due to necrosis at high density under hypoxic but not under normoxic conditions. Levels of mammalian target of rapamycin (mTOR), a central regulator of cellular energy, are reported to be suppressed in hypoxia. We found that phosphorylation of two molecules downstream to it, ribosomal p70 S6 kinase (S6K) and ribosomal protein S6, was markedly suppressed by hypoxia. Overexpression of the active form of S6K increased the sensitivity of LLC cells to hypoxia. On the other hand, inhibition of PI3K or mTOR dramatically reduced hypoxia-induced cell death under hypoxic conditions. Under hypoxic conditions, blockade of the PI3K or mTOR pathway increased levels of intracellular ATP and delayed decreases in pH and glucose level in culture medium, without affecting the cell cycle.     

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

The sugary-type isoamylase gene from rice and Aegilops tauschii: characterization and comparison with maize and arabidopsis.

Genes for an isoamylase-like debranching enzyme have been isolated from rice and Aegilops tauschii, the donor of the D genome to wheat. The structures of the genes are very similar to each other and to the maize SU1 isoamylase gene and consist of 18 exons spread over approximately 7.5 kb. Southern analysis and fluorescent in situ hybridization showed the Ae. tauschii gene to be located in the proximal region of the short arm of chromosome 7D, thus showing synteny with the localization of the rice isoamylase gene on rice chromosome 8. Analysis of the expression pattern of wheat sugary isoamylase genes indicates that they are strongly expressed in the developing endosperm 6 days after flowering. Three distinct Sugary-type cDNA sequences were isolated from the wheat endosperm that are likely to correspond to the products of the three genomes. The deduced amino acid sequence of rice and wheat Sugary-type isoamylase is compared with other sequences available in the database and the results demonstrate that there are three types of isoamylase sequences in plants: those containing 18 exons (the Sugary-type isoamylase gene), those containing 21 exons, and those containing only 1 exon. It is possible that different combinations of isoamylase genes are expressed in different tissues.     

Inversion Clines in Populations of DROSOPHILA MELANOGASTER

Twenty different natural populations of Drosophila melanogaster were sampled to determine the frequencies of inversions. Based on their frequencies and geographical distributions, the inversions could be classified as follows: (1) Common cosmopolitan inversions that are present in many populations in frequencies exceeding five percent and that may exhibit frequency clines over large geographical regions; (2) Rare cosmopolitan inversions that occur throughout the species range but usually at frequencies below five percent and that may be absent in many populations; (3) Recurrent endemic inversions that are found in several adjacent populations in frequencies usually not exceeding one or two percent; and (4) Unique endemic inversions that are found only among the progeny of a single individual and that may represent one aspect of the syndrome termed "hybrid dysgenesis". Four common cosmopolitan inversions that exhibit highly significant clines in populations in the eastern United States are In(2L)t, In(2R)NS, In(3L)P and In(3R)P.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Photosynthetic acclimation and photoinhibition on exposure to high light in shade-developed leaves of Fagus crenata seedlings

The physiological response of leaves developed in low light (L) on Fagus crenata seedlings exposed to different levels of high light (H: high light, M: medium light) was studied. Measurements were conducted on potted seedlings in the F. crenata forest understory. The seedlings with leaves developed in L were transferred to H (L–H) and M (L–M) in summer. On exposure to high light, the photochemical efficiency of dark-adapted PSII (F_v/F_m) immediately decreased and was followed by a subsequent recovery in both L–H and L–M leaves. The mean value of F_v/F_m in L–H leaves was lower than that in L–M leaves through experiments, indicating that the degree of photoinhibition in L–H leaves was greater than that in L–M leaves. About 1 month after transfer, 37% and 5% of leaves had fallen in L–H and L–M seedlings, respectively. This result also indicated the greater photoinhibition in L–H leaves. Moreover, the photosynthetic capacity (P_Nmax) of L–H leaves decreased. In contrast, the P_Nmax of L–M leaves increased, although the P_Nmax was lower than that of M control leaves. An increase in the xanthophyll cycle pool (VAZ), indicating an increase of the photoprotective function, was found in both L–H and L–M leaves. Especially, the VAZ pool in L–M leaves was higher than that in M leaves by the end of experiments. L–M leaves may avoid photoinhibition effectively by the decrease in excess light with the increase of the P_Nmax or VAZ pool, compared to L–H leaves. Thus, the physiological acclimation on exposure to high light depended on the degree of high light. To achieve successful photosynthetic acclimation with slight photoinhibition, the variation of light intensity before and after exposure to high light would be an important factor because of the difference in excess light.     

Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation

Whole genome duplication leads to autopolyploidy and brings about an increase in cell size, concentration of secondary metabolites and enhanced cytosine methylation. The increased cell size offers a positive advantage to polyploids for cell-surface-related activities, but there is a differential response to change in body size across species and taxonomic groups. Although polyploidy has been very extensively studied, having genetic, ecological and evolutionary implications, there is no report that underscores the significance of native secondary metabolites vis-à-vis body size with ploidy change. To address this problem we targeted unique diploid-autotetraploid paired sets of eight diverse clones of six species of Cymbopogon- a species complex of aromatic grasses that accumulate qualitatively different monoterpene essential oils (secondary metabolite) in their vegetative biomass. Based on the qualitative composition of essential oils and the plant body size relationship between the diploid versus autotetraploid paired sets, we show that polyploidy brings about enhanced accumulation of secondary metabolites in all cases, but exerts differential effects on body size in various species. It is observed that the accumulation of alcohol-type metabolites (e.g. geraniol) does not inhibit increase in body size with ploidy change from 2× to 4× (r = 0.854, P < 0.01), but aldehyde-type metabolites (e.g. citral) appear to drastically impede body development (r = -0.895). Such a differential response may be correlated to the metabolic steps involved in the synthesis of essential oil components. When changed to tetraploidy, the progenitor diploids requiring longer metabolic steps in production of their secondary metabolites are stressed, and those having shorter metabolite routes better utilize their resources for growth and vigour. In situ immunodetection of 5-methylcytosine sites reveals enhanced DNA methylation in autopolyploids. It is underpinned that the qualitative composition of secondary metabolites found in the vegetative biomass of the progenitor diploid has a decisive bearing on the body size of the derived autotetraploids and brings about an enhancement in genome-wide cytosine methylation.