A comprehensive software suite for the analysis of cDNAs

We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initia- tion and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction. The software package is freely available under the GNU General Public License at http： / /www.g-language.org/ data/cdna/.     

Hybrid dynamic/static method for large-scale simulation of metabolism

Background  

Many computer studies have employed either dynamic simulation or metabolic flux analysis (MFA) to predict the behaviour of biochemical pathways. Dynamic simulation determines the time evolution of pathway properties in response to environmental changes, whereas MFA provides only a snapshot of pathway properties within a particular set of environmental conditions. However, owing to the large amount of kinetic data required for dynamic simulation, MFA, which requires less information, has been used to manipulate large-scale pathways to determine metabolic outcomes.     

Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement

We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.     

Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus <Emphasis Type="Italic">Tricyrtis</Emphasis>

Summary The liliaceous perennial plants, Tricyrtis spp., are cultivated as ornamental plants in Japan. Natural populations of several Japanese Tricyrtis spp. are severely threatened by indiscriminate collection and habitat destruction. In this study, a plant regeneration system based on somatic embryogenesis has been developed for efficient clonal propagation of T. hirta, T. hirta var. albescens, T. formosana, T. formosana cv. Fujimusume, T. flava ssp. ohsumiensis, and T. macrantha ssp. macranthopsis. Flower tepal explants of these genotypes were cultured on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) alone or in combination with N-(1,2,3-thiadiazol-5-yl)-N′-phenylurea (thidiazuron, TDZ). Calluses induced on media containing 2,4-D produced somatic embryos following their transfer to a plant growth regulator-free medium, indicating that these calluses were embryogenic. A combination of 4.5μM2,4-D and 0.45 μM TDZ was most effective for inducing embryogenic calluses from tepal explants. Among various explant sources, filaments were most suitable for inducing embryogenic calluses on a medium containing 4.5μM 2,4-D and 0.45 μM TDZ. Embryogenic calluses were only obtained from filament explants for T. macrantha ssp. macranthopsis. Embryogenic calluses could be maintained by subculturing monthly onto the same medium, and a 1.5–3.5-fold increase in fresh weight was obtained after 1 mo. of subculture. Depending on the plant genotype, 50–500 somatic embryos per 0.5g fresh weight of embryogenic callus was obtained 1 mo. after transfer to a plant growth regulator-free medium. Most of the embryos developed into plantlets, and they were successfully acclimatized to greenhouse conditions. Regenerated plants showed no alteration in the ploidy level as indicated by chromosome observation and flow cytometric analysis.     

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.     

Accumulation of menaquinones with incompletely reduced side chains and loss of alpha-tocopherol in rice mutants with alternations in the chlorophyll moiety

The rice mutants M249 and M134 accumulate chlorophyllides a and b which are esterified with incompletely reduced alcohols such as geranylgeraniol, dihydrogeranylgeraniol, and tetrahydrogeranylgeraniol. Quantities of alpha-tocopherol, phylloquinone, and menaquinones in leaves of these mutants were determined by high performance liquid chromatography (HPLC) with a fluorescence detector after post-column chemical reduction to convert quinones to fluorescent quinols. Methylnaphthoquinones, varying in the reduction state of the side chain (menaquinones), were detected in leaf segments of the rice mutants on HPLC analyses with both high selectivity and sensitivity to plant quinones. Mutant M249 preferentially accumulated menaquinone, which contains tetrahydrogeranylgeraniol as its side chain. However, mutant M134 exhibited preferential accumulation of menaquinone with a geranylgeraniol side chain. In both mutants, the accumulation patterns of menaquinones with different prenyl side chains were similar to those of chlorophyll with the corresponding prenyl side chains. The content of P700, the photosystem I primary electron donor, in the wild type was greater than that of either mutant, on both a chlorophyll and a fresh weight basis. However, the ratios of total methylnaphthoquinones to P700 were similar in both the wild type and the mutants. Since no comparative large differences in photosynthetic activity exist between the wild type and the mutants, these results suggest that the hydrogenation of the methylnaphthoquinone side chain to phytol is not an essential requirement for it to function as an electron acceptor in photosystem I. On the other hand, alpha-tocopherol was detected in fully developed leaves of the wild type, but not in those of the mutants. Accumulation of menaquinones and the loss of alpha-tocopherol in mutant leaves suggest that the reduction of chlorophyll-geranylgeraniol to phytol and that of geranylgeranyl pyrophosphate to phytyl pyrophosphate are catalysed by the same enzyme.     

Boron nutrition of cultured tobacco BY-2 cells. IV. Genes induced under low boron supply

Genes whose expression was up-regulated in low boron (B)-acclimated tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells, which had been selected under a low supply of B, were screened by the cDNA differential subtraction method. Thirteen genes were identified, including early salicylate-inducible glucosyltransferase, glutamine synthetase, glutathione S-transferase, and a pathogenesis-related protein, which might constitute a rescue system for oxidative damage. This indicates that B deficiency might impose cellular redox imbalance on the cells. Two of the 13 genes were induced within 30 min of B removal in the parent cells, indicating fast signal transfer from the cell walls to the cytoplasm.     

Effects of acute bouts of running and swimming exercise on PGC-1alpha protein expression in rat epitrochlearis and soleus muscle

The purpose of this study was to elucidate the mechanisms underlying low-intensity exercise-induced peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein expression in rat skeletal muscles. Rats (5-6 wk old) swam without a load and ran on the treadmill at a speed of 13 m/min, respectively, in two 3-h sessions separated by 45 min of rest. PGC-1alpha content in epitrochlearis muscle (EPI) was increased by 75 and 95%, immediately and 6 h after swimming, respectively, with no increase in PGC-1alpha content in the soleus (SOL). After running, PGC-1alpha content in EPI was unchanged, whereas a 107% increase in PGC-1alpha content was observed in SOL 6 h after running. Furthermore, in EPI and SOL as well as other muscles (triceps, plantaris, red and white gastrocnemius), PGC-1alpha expression was enhanced concomitant with reduced glycogen postexercise, suggesting that expression of PGC-1alpha occurs in skeletal muscle recruited during exercise. PGC-1alpha content in EPI was increased after 18-h in vitro incubation with 0.5 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and 4 mM caffeine. However, AICAR incubation did not affect PGC-1alpha content in the SOL, whereas caffeine incubation increased it. These results suggest that exercise-induced PGC-1alpha expression in skeletal muscle may be mediated by at least two exercise-induced signaling factors: AMPK activation and Ca2+ elevation. The number of factors involved (both AMPK and Ca2+, or Ca2+ only) in exercise-induced PGC-1alpha expression may differ among muscles.