Calcium transport by sarcoplasmic reticulum Ca(2+)-ATPase. Role of the A domain and its C-terminal link with the transmembrane region

After treatment of sarcoplasmic reticulum Ca(2+)-ATPase with proteinase K (PK) in the presence of Ca(2+) and a protecting non-phosphorylated ligand (e.g. adenosine 5'-(beta,gamma-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE(243) sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca(2+) and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADP-sensitive phosphoenzyme, Ca(2)E1P, with occluded Ca(2+). The ability of Ca(2)E1P to become converted to the Ca(2+)-free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca(2+). PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca(2+). It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca(2+) ions.     

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

FGF10 maintains stem cell compartment in developing mouse incisors

Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.     

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.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.