Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca²⁺, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca²⁺ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca²⁺ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X₃R > P2X_2b + X₄R > P2X_2bR > P2X_2a + X₄R > P2X₄R > P2X_2aR > P2X₇R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca²⁺ influx because of the coactivation of endogenously expressed Ca²⁺-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca²⁺ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca²⁺ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca²⁺ signals were not affected by voltage-gated Ca²⁺ influx and removal of extracellular sodium. Ca²⁺ signals reflected well the receptor-specific EC₅₀ values for ATP and the extracellular Zn²⁺ and pH sensitivities of P2XRs. These results indicate that intracellular Ca²⁺ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals     

Characterization of male meiotic germ cell-specific antigen (Meg 1) by monoclonal antibody TRA 369 in mice.

We have identified a male meiotic germ cell-specific antigen (Meg 1) with monoclonal antibody (mAb) TRA 369 in mice. The Meg 1 antigen was strongly expressed in specific steps of meiotic germ cells from pachytene spermatocyte to early spermatid, and not in other germ cells or somatic cells. Immunohistochemical examination revealed that the antigen was localized to the cytoplasm and was not distributed in the nucleus or on the cell surface. This antigen was demonstrated to have a molecular weight of 93 kDa and an isoelectric point of 5.2 by Western blotting. This molecule was first detected in the testis of 13-day-old mouse when pachytene spermatocytes first appeared. Thus this is a differentiation-specific antigen in male meiotic germ cells, and mAb TRA 369 is a useful tool to study the regulation of germ cell differentiation and to define germ cell development in a molecular level.     

Evaluation of a vector-control strategy of haemorrhagic thelohanellosis in carp,caused by Thelohanellus hovorkai (Myxozoa)

The life cycle of Thelohanellus hovorkai (Myxozoa), the causative agent of haemorrhagic thelohanellosis of carp Cyprinus carpio, involves the alternate oligochaete host Branchiura sowerbyi, which plays the role of vector in the parasite's transmission. Field investigations in carp farms suggested that oligochaete fauna were closely associated with the substrate type of the pond. The pond bottom in the enzootic farm consisted of clay soil and soft sediments comprised of organic mud, in which B. sowerbyi dominated in high densities, with a maximum of 5.6 ind. kg(-1) soil. In another case, in a carp farm with no previous history of the disease, the pond bottom was sandy soil, in which small-sized oligochaetes, composed mainly of Limnodrilus socialis, dominated. Laboratory studies on the substrate preference of oligochaetes proved that B. sowerbyi prefers mud to sand, whereas L. socialis has no tendency to substrate tropism. The delicate body surface of B. sowerbyi was subject to damage by rugged-edged sand particles, which inflicted severe injuries to the worms. Transmission experiments showed that L. socialis, which are non-susceptible to T. hovorkai, suppressed the production of T. hovorkai actinospores in B. sowerbyi in a mixed assemblage of oligochaetes. Field and experimental evidence in this study imply that substrate replacement in culture ponds might regulate the benthic oligochaete communities, resulting in minimization of the impact of haemorrhagic thelohanellosis. We propose that ecological control of oligochaete fauna by environmental management is a promising strategy against myxozoan diseases.     

Solid-liquid phase behavior of binary fatty acid mixtures. 1. Oleic acid/stearic acid and oleic acid/behenic acid mixtures

Solid-liquid phase behavior of binary fatty acid mixtures was investigated by means of differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) for the mixture composed of oleic acid (OA) and stearic acid (SA) and that composed of OA and behenic acid (BA). The DSC results provided a monotectic type T-X phase diagram for these mixtures, from which it was suggested that the two fatty acid species are completely immiscible in a solid phase regardless of the two polymorphs of OA, i.e., alpha-form or gamma-form. The solid phase immiscibility was confirmed by the FT-IR observation that the spectra obtained for the mixtures correspond to the superposition of the two spectra for respective components. Thermodynamic analysis of liquidus line demonstrated that OA and SA form an ideal mixture in a liquid phase, whereas the mixing of OA and BA in a liquid phase is slightly non-ideal.     

Characteristic rat tissue accumulation of nobiletin, a chemopreventive polymethoxyflavonoid, in comparison with luteolin

Nobiletin (NOB), a polymethoxyflavonoid, is an effective anti-inflammatory and chemopreventive phytochemical found in citrus fruits. We compared the absorption and metabolism characteristics of NOB with those of luteolin (LT) in male SD rats. Each flavonoid (67.1 micromol/kg of body weight) was given separately by gastric intubation, and then concentrations were measured at 1, 4, and 24 hours after administration. In the digestive organs, NOB showed a notable tendency for localizing into the mucous membrane and muscularis from 1 to 4 hours, in contrast to LT, though both NOB and LT were completely excreted within 24 hours. Further, significant amounts of NOB were detected in the whole liver and kidney specimens, whereas LT accumulation was slight. Although serum concentrations of NOB from 1 to 4 hours were comparable to those of LT, urinary concentrations of LT were significantly higher from 4 to 24 hours. Following glucuronidase/sulfatase treatments of urinary materials, we detected 3 types of mono-demethylated NOB, including 3'-demethyl-NOB, and two di-demethylated types, as well as 3'-demethyl-NOB alone in serum samples using liquid chromatography-mass spectral analysis. Our results suggest that the metabolic properties of polymethoxyflavonoids are distinct from those of other general flavonoids, because of their wide distribution and accumulation in tissue.     

Locoregional immunotherapy of head and neck cancers utilizing allogeneic spleen cells — a report of 2 cases

Frozen-stored human spleen cells (SC) cultured with streptococcus preparation OK-432 acquired direct cytotoxicity to autologous as well as allogeneic tumor cells. The activated cells started to produce cytocidal cytokine TGIF, which is distinct from previously known cytokines.We examined the possibility of allogeneic adoptive immunotherapy (AIT) using these OK-432-stimulated spleen cells (OK-SC) in two cancer patients. Rapid necrosis of cancer tissue and remarkable decreases of tumor markers in tumor effusion were observed. There were no severe side effects.     

Physiological activities and the active constituents of potentially medicinal plants used by wild chimpanzees of the Mahale mountains,Tanzania

Potential medicinal plants for wild chimpanzees have been studied in order to discover their physiologically active compounds. Tests of the physiological activity of 3 plant species—Vernonia amygdalina, Aspilia mossambicensis, andFicus exasperata—indicate that they contain a variety of active compounds. From one species,V. amygdalina, an antitumor agent and 2 possible antitumor promoters are identified. Furthermore, steroid glucosides were isolated as the bitter substances. These structurally new compounds are expected to exhibit a number of significant physiological activities. The chemical investigation of possible medicinal plants used by chimpanzees should be helpful in recovering naturally occurring compounds of medicinal significance for human use.     

Chalcone tetramers, lophirachalcone and alatachalcone, from Lophira alata as possible anti-tumor promoters.

Two chalcone tetramers were isolated as inhibitors of Epstein-Barr virus (EBV)-activation induced by a tumor promoter, teleocidin B-4, from a medicinal plant in tropical west Africa, Lophira alata (Ochnaceae). One of them was identified as lophirachalcone. The other, named alatachalcone, was new, and the structure was determined by spectral properties. Both compounds also showed potent inhibitory activities against teleocidin B-4-induced inflammation on mouse ear. In an initiation-promotion experiment on mouse skin, alatachalcone (16 nmol) significantly inhibited tumor promotion caused by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol).