A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca(2+)](i) response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH(2)O (hypotonic) evoked transient increase in [Ca(2+)](i) of principal cells of rat CCDs. The [Ca(2+)](i) increase was markedly attenuated by the removal of extracellular Ca(2+)(.) The application of a P(2) purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca(2+)](i) increase. The [Ca(2+)](i) increase in response to extracellular hypotonicity was not influenced by application of Gd(3+) and ruthenium red. On the other hand, a voltage-gated Ca(2+) channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca(2+)](i) increase in the principal cells. In order to assess Ca(2+) entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn(2+). The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn(2+), indicating that a Ca(2+)-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn(2+) quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca(2+) entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca(2+)](i) elevation of principal cells in rat CCD.     

Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression

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Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.     

Cell Structure of Yeasts Grown Anaerobically in Aspergillus orpzae-Proteolipid-supplemented Media

Effects of Aspergillus oryzae-proteolipid (PL) on the formation of high concentrations of alcohol, more than 20% as in sake brewing, have been studied. Electron microscopy revealed that there were no vacuoles but many lipid deposits in cytoplasm of the alcoholtolerant cells of Saccharomyces sake Kyokai No. 7, which were obtained by the anaerobic culture supplemented with PL. Sphaeroplasts from the alcohol-tolerant cells were stable in 20% alcohol, whereas those from the untolerant cells grown anaerobically in the PL-unsupplemented media were ruptured. The cell membrane became alcohol-endurable in the anaerobic cultures with PL. Synthetic phospholipid promoted yeast growth and the fermentative activity, whereas a small amount of sterol ester enhanced the alcohoi-endurability. The supplementation of both lipids anaerobically induced physiological properties characteristic of the alcohol-tolerant cells.     

Cloning and sequencing of the human homeobox gene HOX4A.

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60c-src, Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60c-src. A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.     

Recovery of phosphorous from swine wastewater through crystallization

All the phosphate rock Japan needs must be presently imported from abroad because the country has no subterranean phosphorous resources. Therefore, there is a need to accelerate the development of and establish the technologies for phosphorous recovery from waste and wastewater. Swine wastewater has a high potential for phosphorous recovery in Japan. A reactor for removing and recovering phosphorous from swine wastewater was designed with dual functions, crystallization through aeration and separation of formed struvite by settling. However, a dehydration, composting and characterization process was first needed before using sediment sludge, including struvite, on farmland, since the struvite will settle along with huge amounts of other suspended solids (organic matter). For the recovery of pure struvite, an accumulation device was designed and its efficiency examined. The device has a struvite-accumulation face made of stainless steel wire mesh (1 mm in diameter, 1 cm(2) square) to reduce its total weight. During submergence in the aeration column of the demonstration reactor, struvite cross-bridged and accumulated on the face of the device. The struvite could be scraped off easily with only a light brushing, and was found to be approximately 95% pure. Because this device is a very simple structure, it is thought to be acceptable to swine farmers.     

Inter- and intramolecular stacking interaction between indole and adeninium rings

Crystals of 1,9-dimethyladeninium-indole-3-acetate (1:1) complex (I) and 9-(3-indol-3-ylpropyl)-1-methyladeninium iodide (II), an inter- or intramolecular model for the stacking interaction between the tryptophanyl residue and the methylated (or protonated) adenine base, were subjected to X-ray analyses. Nearly parallel stacking and interplanar spacing near to 3.4 A were observed between the indole and adeninium rings of both crystals. In particular, one of the two stacking pairs formed in I showed the existence of a partial charge-transfer interaction in their ground states. On the basis of the molecular orbital consideration, the mutual orientation between these stacked aromatic rings is considerably governed by the orbital interaction between the highest occupied molecular orbital of the indole ring and the lowest unoccupied one of the adeninium ring. The ring stacking observed in II was stabilized by the strong coupled dipole-dipole interaction. Absorption, fluorescence, and proton nuclear magnetic resonance spectra indicated the existence of a stacking interaction in the aqueous solutions of I and II, as well as in their crystalline states. The biological implication for the observed stacking interactions has been discussed.     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Antitumor effect of photodynamic therapy with zincphyrin, zinc-coproporphyrin III, in mice

We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptomyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78-100 microg/ml with visible light of 27.2 mW x min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5-50 mg/kg with light irradiation of 75.48 mW x min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 microg/ml within 20 min, and the concentration remained at 4.2 microg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW x min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.