Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Day-night changes in production of carbohydrate and protein by natural phytoplankton population from Lake Biwa, Japan

The ¹³C and gas chromatography-mass spectrometry (¹³C-GC-MS)method was applied to determine the day-night changes in thecomposition of photosynthetic products of the natural phytoplanktonpopulation from Lake Biwa, Japan. Glucose is the most abundantmonosaccharide in acid-hydrolyzable carbohydrate. The contributionof glucose was high in incubatesd samples in daytime and decreasedduring the night. Other monosaccharides (rhamnose, fucose, ribose,arabinose, xylose, mannose and galactose) and amino acids tendedto be produced throughout both day- and night-time. These resultssuggested that the carbon flows from glucose, which might constitutereserve glucan, to other monosaccharides and amino acids duringnight-time. The disproportionate production of glucose (reservedglucan) during daytime was thus partly cancelled out at night.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Development of a gene cloning system for the hydrogen-producing marine photosynthetic bacterium Rhodopseudomonas sp.

Seventy-six strains of marine photosynthetic bacteria were analyzed by agarose gel electrophoresis for plasmid DNA content. Among these strains, 12 carried two to four different plasmids with sizes ranging from 3.1 to 11.0 megadaltons. The marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 had two plasmids, pRD06S and pRD06L. The smaller plasmid, pRD06S, had a molecular weight of 3.8 megadaltons and was cut at a single site by restriction endonucleases SalI, SmaI, PstI, XhoI, and BglII. Moreover, the marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 containing plasmid pRD06 had a satisfactory growth rate (doubling time, 7.5 h), a hydrogen-producing rate of 0.96 mumol/mg (dry weight) of cells per h, and nitrogen fixation capability. Plasmid pRD06S, however, had neither drug resistance nor heavy-metal resistance, and its copy number was less than 10. Therefore, a recombinant plasmid consisting of pRD06S and Escherichia coli cloning vector pUC13 was constructed and cloned in E. coli. The recombinant plasmid was transformed into Rhodopseudomonas sp. NKPB002106. As a result, Rhodopseudomonas sp. NKPB002106 developed ampicillin resistance. Thus, a shuttle vector for gene transfer was constructed for marine photosynthetic bacteria.     

Nonradioactive detection of nucleic acid by the universal probe system

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.     

Regulation of angiotensin converting enzyme activity in cultured human vascular endothelial cells

Angiotensin converting enzyme (ACE) of vascular endothelial cells is suggested to control vascular wall tonus through the conversion of angiotensin I (AI) to angiotensin II (AII) and the degradation of bradykinin. To obtain more insight into the pathophysiological significance of ACE of vascular endothelial cells, we studied the regulation of ACE produced by cultured human umbilical vein endothelial cells (EC). Phorbol 12-myristate 13-acetate (PMA) increased the cellular and medium ACE activity, accompanied by a marked morphological change in EC. N'-O'-dibutylyladenosine 3';5'-cyclic monophosphate (db-cAMP) increased only the cellular ACE activity and not the medium ACE activity. The effect of isoproterenol with 0.1mM theophylline mimicked that of db-cAMP. These findings suggest that PMA and cAMP-related agents participate in the control of vascular wall tonus through the positive regulation of ACE produced by vascular endothelial cells.     

Ceramide-induced intracellular oxidant formation, iron signaling, and apoptosis in endothelial cells: protective role of endogenous nitric oxide

Sphingolipid ceramide (N-acetylsphingosine), a bioactive second messenger lipid, was shown to activate reactive oxygen species (ROS), mitochondrial oxidative damage, and apoptosis in neuronal and vascular cells. The proapoptotic effects of tumor necrosis factor-alpha, hypoxia, and chemotherapeutic drugs were attributed to increased ceramide formation. Here we investigated the protective role of nitric oxide (.NO) during hydrogen peroxide (H(2)O(2))-mediated transferrin receptor (TfR)-dependent iron signaling and apoptosis in C(2)-ceramide (C(2)-cer)-treated bovine aortic endothelial cells (BAECs). Addition of C(2)-cer (5-20 microm) to BAECs enhanced .NO generation. However, at higher concentrations of C(2)-cer (> or =20 microm), .NO generation did not increase proportionately. C(2)-cer (20-50 microm) also resulted in H(2)O(2)-mediated dichlorodihydrofluorescein oxidation, reduced glutathione depletion, aconitase inactivation, TfR overexpression, TfR-dependent uptake of (55)Fe, release of cytochrome c from mitochondria into cytosol, caspase-3 activation, and DNA fragmentation. N(w)-Nitro-l-arginine methyl ester (l-NAME), a nonspecific inhibitor of nitricoxide synthases, augmented these effects in BAECs at much lower (i.e. nonapoptotic) concentrations of C(2)-cer. The 26 S proteasomal activity in BAECs was slightly elevated at lower concentrations of C(2)-cer (< or =10 microm) but was greatly suppressed at higher concentrations (>10 microm). Intracellular scavengers of H(2)O(2), cell-permeable iron chelators, anti-TfR receptor antibody, or mitochondria-targeted antioxidant greatly abrogated C(2)-cer- and/or l-NAME-induced oxidative damage, iron signaling, and apoptosis. We conclude that C(2)-cer-induced H(2)O(2) and TfR-dependent iron signaling are responsible for its prooxidant and proapoptotic effects and that .NO exerts an antioxidative and cytoprotective role.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.