Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

Penetration of Eimeria tenella sporozoites under different oxygen concentrations in vitro.

Sporozoites of Eimeria tenella (Wisconsin strain) were inoculated onto monolayers of normal chicken kidney fibroblasts and cultured in RPMI-1640 supplemented with 5% fetal bovine serum, sodium bicarbonate, and gentamicin under either aerobic, 5% CO2/95% air, or anaerobic conditions. Penetration of fibroblasts by sporozoites under CO2 or anaerobic conditions at 2 and 24 hr postinoculation was 3-4 times greater than that in the aerobic atmosphere. Effect of reduced oxygen concentrations, i.e., 20.0, 12.5, and 5.0% oxygen, was also investigated in an N2-O2-CO2 incubator. Under 5.0 and 12.5% oxygen at 2 and 24 hr postinoculation, the number of sporozoites that penetrated was about 4 and 2 times greater, respectively, than under 20.0% O2. These results indicate that lower oxygen concentrations provide for greater penetration by E. tenella sporozoites in cultured cells.     

Adenosine deaminase deficiency due to heterozygous abnormality consisting of a deletion of exon 7 and the absence of enzyme mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BADO5 derived from a Japanese patient with severe combined immunodeficiency disease and two B lymphoblastoid cell lines, BAMO5 from his mother and BAFO5 from his father, were characterized. To identify mutations affecting ADA activity, we prepared cDNAs to ADA mRNAs of the BADO5 cell line for nucleotide sequencing. Sequence analysis of one of the BADO5 ADA cDNA clones revealed deletion of exon 7, and one point mutation of base 629 from G to A that did not affect the amino acid sequence. All clones of the BADO5 cell line so far examined showed the absence of exon 7 by Southern blotting analysis. Ribonuclease protection assay with an RNA probe spanning from exon 5 to exon 11 showed that the BADO5 ADA mRNA had a deletion of exon 7, the BAMO5 mRNA had normal length, and the BAFO5 mRNA had two species with a deletion of exon 7 and with normal length. Consequently, the patient's ADA genes resulted from one allele of the BAMO5 ADA gene that did not produce a detectable mRNA, and the other allele of the BAFO5 ADA gene producing an aberrant mRNA without exon 7.     

Thymidine triphosphate dephosphorylating activity in regenerating rat liver

The enzyme activity of dephosphorylation of thymidine triphosphate was found in microsomal fraction of rat liver. The enzyme activity decreased at the time when [³H]thymidine incorporation into DNA of regenerating liver increased. When the [³H]thymidine incorporation was suppressed by 1,3-diaminopropane, the enzyme activity remained elevated. These results suggest that the enzyme activity appears to be closely linked to DNA synthesis.     

Changes in spatial distribution pattern during the larval stage of the fall webworm,Hyphantria cunea drury (Lepidoptera: Arctiidae)

Summary The changes in spatial distribution pattern during larval stage of the fall webworm,Hyphantria cunea were quantitatively investigated in the field experimental populations. The female adult deposits eggs as a cluster and the hatchlings make a compact colonial-web. In this period, the all-or-none type mortality which is characteristic in gregarious insect species was occasionary recognized before spinning a compact colonial-web. Once making a compact colonial-web, the larvae feed the leaves in the colonial-web up to about 5th instar. In this period, the movement of larvae occurred due to the local food shortage in a colonial-web and the expansion of colonial-web. As the larvae developed, the colonial-web was separated into several small groups. These larvae began to disperse about 5th instar. In this period, the local food shortage seems to be an important trigger for the larval dispersal. The mean concentration of larvae on leaves abruptly decreased, and finally the larvae became solitary at the 6th or 7th instars. The dispersal process in later larval stage is not necessarily due to the complete food shortage. The dispersal prior to the occurrence of food shortage may be a safety mechanism to protect the larvae from the food shortage.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Characterization of O-trimethylsilyl derivatives of d-glucose,d-galactose,and d-mannose by gas-liquid chromatography-chemical-ionization mass spectrometry with ammonia as reagent gas

The per(trimethylsilyl) ethers of d-glucose, d-galactose, and d-mannose were analyzed by g.l.c.-c.i.m.s. with ammonia as the reagent gas. C.i.m.s. gave simple fragmentation and fragment ions of high intensity in the high-mass range where the QM⁺ ion is also detected. The β-d anomers gave ions at m/e 558 showing intensities 3–12 times those of the α-d anomers. The epimers could be distinguished by differences in the intensities of the ions and by the observation that d-glucose gave a base peak at m/e 198, d-galactose at m/e 468, and d-mannose at m/e 204. The pyranose and furanose structures could be distinguished by comparing the ion intensities at m/e 198, m/e 271, m/e 361, m/e 396, and m/e 451. A similar analysis was also performed with 2-methylpropane as the reagent gas.     

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid