Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells

Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of beta-l,3-glucan (callose) and fibrils of beta-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.     

An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions

We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer. A genomewide search showed that E. histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E. histolytica. The EhNifS protein expressed in E. coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms. Eh-NifU protein existed as a tetramer and contained one stable [2Fe-2S]2+ cluster per monomer, revealed by spectroscopic and iron analyses. Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase. Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes.     

Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype

To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (betaR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the betaR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the betaR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo.     

Mechanism of the generation of autonomous activity of Ca2+/calmodulin-dependent protein kinase IV

Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr196 by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr196 phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr177 phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296-469 and 296-350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304-325) containing minimum autoinhibitory sequences (residues 314-321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of approximately 50 microm, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr196-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr196 phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity.     

Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives

Embryonic stem (ES) cells deficient in the enzyme poly(ADP-ribose) polymerase (Parp1) develop into teratocarcinoma-like tumors when injected subcutaneously into nude mice that contain cells with giant cell-like morphology. We show here that these cells express genes characteristic of trophoblast giant cells and thus belong to the trophectoderm lineage. In addition, Parp1(-/-) tumors contained other trophoblast subtypes as revealed by expression of spongiotrophoblast-specific marker genes. The extent of giant cell differentiation was enhanced, however, as compared with spongiotrophoblast. A similar shift toward trophoblast giant cell differentiation was observed in cultures of Parp1-deficient ES cells and in placentae of Parp1(-/-) embryos. Analysis of other cell lineage markers demonstrated that Parp1 acts exclusively in trophoblast to suppress differentiation. Surprisingly, trophoblast derivatives were also detected in wildtype tumors and cultured ES cells, albeit at significantly lower frequency. These data show that wildtype ES cells contain a small population of cells with trophectoderm potential and that absence of Parp1 renders ES cells more susceptible to adopting a trophoblast phenotype.     

Identification of the 58-kDa phosphoprotein associated with motility initiation of hamster spermatozoa

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that the 58-kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues in association with the start of motility. In the present experiments, we identified and localized the 58-kDa protein. The 58-kDa protein was assumed to exist in the acrosomal region domain of the sperm head and the whole sperm flagellum. In particular, a large amount of 58-kDa protein was localized in the equatorial segment of the acrosomal region domain of the sperm head and the middle piece of the sperm flagellum. In the next step, the 58-kDa protein was identified by peptide mass finger printing and LC-MS/MS analysis. The results suggested that the 58-kDa protein was ATP synthase H(+) transporting F1 beta, which is one of the mitochondrial components. Therefore, it is likely that the 58-kDa protein is associated with ATP production in the mitochondrial sheath in the middle piece of the sperm flagellum, and H(+) transport in the sperm head and the sperm flagellum except for the middle piece, since ATP synthase also acts as an H(+) pump.     

Oxygen affinity of hemoglobin regulates O2 consumption,metabolism, and physical activity

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O(2) delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the alpha-globin gene or Presbyterian mutation in the beta-globin gene. The mutant mice showed increased O(2) consumption and CO(2) production in tissue metabolism, suggesting enhanced O(2) delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O(2) consumption, physical activity, and behavior in mice.