Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

Vitamin D and phosphate regulate fibroblast growth factor-23 in K-562 cells

Female rats were treated with FSH (40 IU/kg) on the first and second diestrus days (D1 and D2) and with LH (40 IU/kg) on the proestrus (P) day to synchronize and maximize ovarian changes. Follicle area increased by 50% from D1 to P, and the estrus (E) phase showed multiple corpora lutea and massive apoptosis. Increased oxygen uptakes (42-102%) were determined in ovary slices and in isolated mitochondria in active state 3 along the proliferation phase (D1-D2-P) that returned to initial values in the E phase. Mitochondrial content and the electron transfer activities of complexes I and IV were also maximal in the P phase (20-79% higher than in D1). Production of NO by mitochondrial nitric oxide synthase (mtNOS), biochemically determined, and the mtNOS functional activity in regulating state 3 oxygen uptake were also maximal at P and 79-88% higher than at D1. The moderately increased rate of NO in the proliferative phase is associated with mitochondrial biogenesis, whereas the high rate of NO generation by mtNOS at phase P appears to trigger mitochondria-dependent apoptosis. The calculated fraction of ovary mitochondria in state 3 was at a minimal value at the P phase. Mitochondrial oxidative damage, with increased thiobarbituric acid-reactive substances and protein carbonyls, indicates progressive mitochondrial dysfunction between phases P and E. The roles of mitochondria as ATP provider, as a source of NO to signal for mitochondrial proliferation and mitochondria-dependent apoptosis, and as a source of O(2)(-) and H(2)O(2) appear well adapted to serve the proliferation-apoptosis sequence of the ovarian cycle.     

Distinct nuclear factor-kappaB/Rel proteins have opposing modulatory effects in glutamate-induced cell death in HT22 cells

Members of the nuclear factor-κB (NF-κB)/Rel family (p50, p52, p65 (RelA), RelB and c-Rel) is sequestered in the cytoplasm through its tight association with the inhibitor of NF-κB (IκB). NF-κB has been shown to function as key regulators of either cell death or survival in neurons after activation of the cells by various extracellular signals. In the study presented here, we investigated whether the selective activation of diverse NF-κB/Rel family members in HT22 cells might lead to distinct effects on glutamate-induced cell death. Exposing HT22 cells to glutamate, which blocks cystine uptake into the cells via inhibition of the glutamate-cystine antiporter, resulted in a transient activation of IκB and NF-κB/Rel and caused delayed cell death. Aspirin, which has been shown to block phosphorylation of the IκB component of the cytoplasmic NF-κB complex, significantly suppressed glutamate-induced cell death, whereas the NF-κB decoy oligonucleotide potentiated it. The inhibition of NF-κB/Rel protein expression by antisense oligonucleotides showed that p65 is involved in glutamate-mediated cell death, whereas p50 is involved in inhibitory pathways of the cell death. These findings suggest that in HT22 cells, the balance between promoting and presenting cell death to glutamate-induced oxidative stress relies on the activation of distinct NF-κB proteins.     

Development of a monoclonal antibody against Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) that can detect EBNA-LP expressed in P3HR1 cells

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) fused to glutathione-S-transferase. The antibody detected endogenous and exogenous EBNA-LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA-LP. While another monoclonal antibody to EBNA-LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA-LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA-LP, especially investigations into the phenotypes of mutant EBNA-LP expressed in P3HR1 cells.     

Effects of ICI 182780 on estrogen receptor expression,fluid absorption and sperm motility in the epididymis of the bonnet monkey

Background  

The importance of estrogen in regulation of fluid absorption and sperm maturation in the rodent epididymis has been established from studies on estrogen receptor-alpha knockout mice. However, functional studies on the role of estrogen in primate epididymis have been few. The main objective of this study was therefore to extend these observations and systematically analyze the presence and function of estrogen receptors in modulating the function of the primate epididymis, using the bonnet monkey (Macaca radiata) as a model system.     

A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes—usually used for molecular analysis—were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.