Recovery of photosynthetic systems during rewetting is quite rapid in a terrestrial cyanobacterium, Nostoc commune

Recovery processes of photosynthetic systems during rewetting were studied in detail in a terrestrial, highly drought-tolerant cyanobacterium, Nostoc commune. With absorption of water, the weight of N. commune colony increased in three phases with half-increase times of about 1 min, 2 h and 9 h. Fluorescence intensities of phycobiliproteins and photosystem (PS) I complexes were recovered almost completely within 1 min, suggesting that their functional forms were restored very quickly. Energy transfer from allophycocyanin to the core-membrane linker peptide (L(CM)) was recovered within 1 min, but not that from L(CM) to PSII. PSI activity and cyclic electron flow around PSI recovered within 2 min, while the PSII activity recovered in two phases after a time lag of about 5 min, with half times of about 20 min and 2 h. Photosynthetic CO(2) fixation was restored almost in parallel with the first recovery phase of the PSII reaction center activity. Although the amount of absorbed water became more than 20 times the initial dry weight of the N. commune colony in the presence of sufficient water, about twice the initial dry weight was enough for recovery and maintenance of the PSII activity.     

Low-molecular-mass polypeptide components of a photosystem II preparation from the thermophilic cyanobacterium Thermosynechococcus vulcanus

Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.     

Efficient biallelic mutagenesis with Cre/loxP-mediated inter-chromosomal recombination

The isolation of mutant cells with phenotypes caused by random mutagenesis has been hampered in mammalian cells because there are two alleles per gene and the disruption of both alleles is extremely rare. We describe a method for the efficient biallelic mutagenesis in embryonic stem cells. loxP sites were introduced near the centromeric regions of a pair of chromosome 1s. A mutant neo gene was inserted at the distal part of one of the loxP sites so that biallelic mutants would be selected by high-dose G418. Expression of Cre induced the recombination between homologous chromosomes and led to an elevation in the number of biallelic mutants. This system will facilitate phenotype-driven gene function study in the mammalian system.     

Highly sensitive time-resolved fluorometric determination of estrogens by high-performance liquid chromatography using a beta-diketonate europium chelate

A new fluorescent europium chelate labeling reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2-pentafluoro-3,5-pentanedione (CDPP), was synthesized for the time-resolved fluorometric detection of HPLC. The label can be directly bound to amino or phenolic hydroxyl groups of analytes with its chlorosulfonyl group, and the labeled analytes are separated on a HPLC column. After separation, EuCl(3), TOPO (tri-n-octylphosphine oxide), and Triton X-100 were added by post-column introduction to the eluent, and the fluorescence of the europium chelate was measured with the time-resolved fluorometric detector. Estrone (E1), 17beta-estradiol (E2), ethynylestradiol (EE2) and estriol (E3) were measured with the detection limits of 0.65, 0.65, 0.65 and 0.60 ng/ml, respectively. The recovery for river water samples was in the range of 86.0-105.1% with the RSD of 1.9-5.8%. The method was applied to the analysis of a river water sample and estrone (E1) was determined to be 2.1 ng/l. The results and processing have been compared with those of a GC-MS method and a high degree of correlation (r> or =0.98) was observed.     

IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells

We examined whether interleukin (IL)-6 exerts the stimulatory effects on the secretion of histamine from human mast cells triggered by crosslinking of the high affinity IgE receptor (FcepsilonRI) with IgE and anti-IgE. As target cells, we used peripheral blood-derived cultured mast cells grown with SCF, because they were superior in FcepsilonRIalpha expression to cord blood-derived mast cells. Incubation with SCF+IL-6 for 1 week increased the IgE-dependent release as well as intracellular content of histamine in the cultured mast cells, as compared with the values obtained by incubation with SCF alone. The magnitude of these increases was higher than that for priming with SCF+IL-4. A striking difference was also found in the expression of FcepsilonRIalpha between the two-factor combinations. The addition of IL-6 during FcepsilonRI crosslinking with IgE/anti-IgE in the presence of SCF did not influence histamine secretion. When SCF, IL-6 and IL-4 were used together, a further increase was observed in the anti-IgE-dependent liberation of histamine from the cultured mast cells, compared with the two-factor combinations. These results suggest that IL-6 functions as a secretagogue for the inflammatory mediator of human mast cells in the presence of SCF.     

A method for estimating torque-vector directions of shoulder muscles using surface EMGs

In this study, a new method is proposed to estimate the torque-vector directions of each shoulder muscle. The method is based on a multiple regression model that reconstructs shoulder torque, which is calculated from the hand force and posture, from the surface EMG of many muscles recorded simultaneously. The torque-vector directions of eleven shoulder muscles of four subjects were obtained at up to 30 different arm postures with this method. The mean confidence interval (p < 0.05) of the estimated torque-vector direction of each subject was 7.7-10.6 degrees. The correlation coefficient between the measured shoulder torque and reconstructed shoulder torque was between 0.76-0.84. The results for majority of the muscles were in accordance with previous studies, and reasonable from the viewpoint of anatomy. The torque-vector directions of a muscle, which are estimated with this method, have more of a functional meaning than a pure anatomical or mechanical one. These indicate the direction of the shoulder torque accompanying the muscle activation for a normal shoulder action that involves the cooperative contraction of many muscles.     

Horticultural characterization of<Emphasis Type="Italic"> Angelonia salicariifolia</Emphasis> plants transformed with wild-type strains of<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis>

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.     

Metabolism of amyloid precursor protein in COS cells transfected with a β-secretase candidate

Thimet oligopeptidase (TOP) is a thiol- andmetallo-dependent peptidase and has been shown to beone of the -secretase candidates. TOPexpressed in COS cells cleaved amyloid precursorprotein (APP) at the -secretase site, and wefound a proteolytic product of APP called secretedform of APP by -secretase (sAPP) in theconditioned media. Here we demonstrate thatsAPP was increased in conditioned media whenTOP was coexpressed in COS cells with APP and treatedwith an ADAM inhibitor SI-27. In addition, althoughTOP expressed in COS cell was localized at nuclei orGolgi apparatus, it exclusively colocalized at Golgiapparatus when APP was coexpressed with TOP.