ARF tumor suppressor induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family proteins

A tumor suppressor gene product, ARF, sensitizes cells to apoptosis in the presence of appropriate collateral signals. In this study, we analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytochrome c release from mitochondria, decreases mitochondrial membrane potential, and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the proapoptotic Bcl-2 family proteins Bax and Bim and down-regulation of antiapoptotic Bcl-2 in mitochondrial fractions. Additionally, ARF seems to down-regulate Bcl-2 in a p53-dependent manner while up-regulating Bax/Bim via a p53-independent pathway.     

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg-EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf-dependent, antibody-independent and complement-mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.     

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.     

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.     

Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopy

The effects of exposure to a 50 Hz magnetic field (maximum of 41.7 to 43.6 mT) on the membrane protein structures of living HeLa cells were studied using attenuated total reflection infrared spectroscopy. One min of such exposure shifted peak absorbance of the amide I band to a smaller wave number, reduced peak absorbance of the amide II band, and increased absorbance at around 1600 cm(-1). These results suggest that exposure to the ELF magnetic field has reversible effects on the N-H inplane bending and C-N stretching vibrations of peptide linkages, and changes the secondary structures of alpha-helix and beta-sheet in cell membrane proteins.