Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import

Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequence-containing proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.     

Ethanol-induced pseudohyphal transition in the cells of Candida tropicalis: participation of phosphoinositide signal transduction

Ethanol-induced pseudohyphal development in the cells of Candida tropicalis Pk233 was accompanied by the transient accumulation of inositol 1,4,5-trisphosphate (IP3) that occurred at an early growth stage. The concomitant addition of myo-inositol prevented the activation of IP3 accumulation and cancelled pseudohyphal development in the presence of ethanol. The addition of a specific phospholipase C inhibitor, U73 122, inhibited ethanol-induced pseudohyphal transition at the concentrations of subinhibitory levels of cell growth. Pseudohyphal development was also induced by the Ca2+ ionophore, A23 187 in the absence of ethanol. The effect of A23 187 on the development of pseudohyphae was little influenced by myo-inositol, but stimulated by concomitant addition of 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ethanol activated phospholipase C in competition with myo-inositol, and the resulting IP3-Ca2+ and protein kinase C pathways of PI signal transduction may work in pseudohyphal transition.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.     

Mathematical description of stress relaxation of bovine femoral cortical bone

In 1993 we proposed an empirical formula for describing the relaxation modulus of cortical bone based on the results of stress relaxation experiments performed for 1 x 10(5) sec: [E(t) = E0{A exp[ -(t/tau1)beta] + (1 - A) exp(-t/tau2)}, (0 < A, beta <1 and tau1 < tau2) where E0 is the initial value of the relaxation modulus, A is the portion of the first term, tau1 and tau2 are characteristic relaxation times, and beta is a shape factor [Sasaki et al., J. Biomechanics 26 (1993), 1369]. Although the relaxation properties of bone under various external conditions were described well by the above equation, recent experimental results have indicated some limitations in its application. In order to construct an empirical formula for the relaxation modulus of cortical bone that has a high degree of completeness, stress relaxation experiments were performed for 6 x 10(5) seconds. The second term in the equation was determined as an apparently linear portion in a log E(t) vs t plot at t>1 x 10(4) sec. The same plot for experiments performed for 6 x 10(5) seconds revealed that the linear portion corresponding to the second term was in fact a curve with a large radius of curvature. On the basis of this fact, we proposed a second improved empirical equation E(t) = E0{A exp [ -(t/tau1)beta] + (1 - A) exp[-(t/tau2)gamma]}, (0相似文献   

Essential role of phosphorylation of MCM2 by Cdc7/Dbf4 in the initiation of DNA replication in mammalian cells

We report the identification of Cdc7/Dbf4 phosphorylation sites in human MCM2 and the determination of the role of Cdc7/Dbf4 phosphorylation of MCM2 in the initiation of DNA replication. Using immunoblotting, immunofluorescence, and high-speed automated cell-imaging analyses with antibodies specific against MCM2 and Cdc7/Dbf4 phosphorylated MCM2, we show that the chromatin recruitment and phosphorylation of MCM2 are regulated during the cell cycle in HeLa cells. Chromatin-bound MCM2 is phosphorylated by Cdc7/Dbf4 during G1/S, which coincides with the initiation of DNA replication. Moreover, we show that baculovirus-expressed purified MCM2-7 complex and its phosphomimetic MCM2E-7 complex display higher ATPase activity when compared with the nonphosphorylatable MCM2A-7 complex in vitro. Furthermore, suppression of MCM2 expression in HeLa cells by siRNA results in the inhibition of DNA replication. The inhibition can be rescued by the coexpression of wild type MCM2 or MCM2E but not MCM2A. Taken together, these results indicate that Cdc7/Dbf4 phosphorylation of MCM2 is essential for the initiation of DNA replication in mammalian cells.     

High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder

The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.     

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.     

Photobiocatalyzed asymmetric reduction of ketones using Chlorella sp. MK201

Aromatic ketones were reduced using suspension culture of Chlorella sp. MK201 under fluorescent light illumination producing the corresponding chiral alcohols in high yields with excellent enantiomeric excess (ee). For example, 2′,3′,4′,5′,6′-pentafluoroacetophenone at 0.25?mg/ml was converted to the corresponding (S)-alcohol in 80?% yield with?>99?% ee by 1?mg dry wt of Chlorella/ml in 12?h illumination (2,000 lux).     

Nation-wide litter fall data from 21 forests of the Monitoring Sites 1000 Project in Japan

This data paper reports litter fall data collected in a network of 21 forest sites in Japan. This is the largest litter fall data set freely available in Japan to date. The network is a part of the Monitoring Sites 1000 Project launched by the Ministry of the Environment, Japan. It covers subarctic to subtropical climate zones and the four major forest types in Japan. Twenty-three permanent plots in which usually 25 litter traps were installed were established in old-growth or secondary natural forests. Litter falls were collected monthly from 2004, and sorted into leaves, branches, reproductive structures and miscellaneous. The data provide seasonal patterns and inter-annual dynamics of litter falls, and their geographical patterns, and offer good opportunities for meta-analyses and comparative studies among forests.