Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture     

Immobilized mitochondrial electron transport particle for NADH determination

Nonphosphorylating electron transport particles (ETP) prepared from beef heart mitochondrion were immobilized in agar gel. The immobilized ETP showed an oxidase activity to both NADH and succinate. The immobilized ETP was reusable. An electrochemical device for the determination of either NADH or succinate was assembled consisting of the membrane-bound ETP and an oxygen probe. The response to succinate was specifically inhibited by the addition of malonate.     

Isolation and culture of protoplasts from moss protonemata

Protoplats were enzymatically prepared from protonemata of the mossPlagiomnium vesicatum, which was cultured on a Murashige-Skoog's medium supplemented with 0.2% yeast extract and solidified with agar. The protoplasts incubated in soft agar medium containing sucrose began to divide after 15 days of incubation, and after 50 days 10- to 15-cell aggregates were present. When transferred to a sugar-free medium, the cells in the aggregates grew into filaments.     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

Inhibitory effects of steroidal allenes on growth,development, and steroid metabolism of the silkworm,Bombyx mori

Steroidal allenes, stigmasta-5,24(28),28-trien-3β-ol (allene-I) and cholesta-5,23,24-trien-3β-ol (allene-II), were tested for their inhibitory effects on growth, development, and steroid metabolism in the silkworm, Bombyx mori. The allenic analogue (I) of stigmasta-5,24(28)-dien-3β-ol (2) was found to be a specific inhibitor for the conversion of stigmast-5-en-3β-ol (1) to stigmasta-5, 24(28)-dien-3β-ol (2) and/or stigmasta-5,24(28)-dien-3β-ol (2) to 24,28-epoxy-stigmast-5-en-3β-ol (3) This inhibitor held the larvae in the second instar for more than 20 days without developing to the third instar, when administered alone or with the dietary sterols of stigmast-5-en-3β-ol (1) or stigmasta-5,24(28)-dien-3β-ol (2). The second allene (II) with a similar structure to cholesta-5,24-dien-3β-ol (4) was also found to be an inhibitor for insect growth and development, but it appeared not to be acting via inhibition of sterol dealkylation.     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.M. Matsuo and K. Koizumi contributed equally to this work. This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats

We examined, by using a specific PGE receptor subtype EP4 agonist and antagonist, the involvement of EP4 receptors in duodenal HCO(3)(-) secretion induced by PGE(2) and mucosal acidification in rats. Mucosal acidification was achieved by exposing a duodenal loop to 10 mM HCl for 10 min, and various EP agonists were given intravenously 10 min before the acidification. Secretion of HCO(3)(-) was dose-dependently stimulated by AE1-329 (EP4 agonist), the maximal response being equivalent to that induced by sulprostone (EP1/EP3 agonist) or PGE(2). The stimulatory action of AE1-329 and PGE(2) but not sulprostone was attenuated by AE3-208, a specific EP4 antagonist. This antagonist also significantly mitigated the acid-induced HCO(3)(-) secretion. Coadministration of sulprostone and AE1-329 caused a greater secretory response than either agent alone. IBMX potentiated the stimulatory action of both sulprostone and AE1-329, whereas verapamil mitigated the effect of sulprostone but not AE1-329. Chemical ablation of capsaicin-sensitive afferent neurons did not affect the response to any of the EP agonists used. We conclude that EP4 receptors are involved in the duodenal HCO(3)(-) response induced by PGE(2) or acidification in addition to EP3 receptors. The process by which HCO(3)(-) is secreted through these receptors differs regarding second-messenger coupling. Stimulation through EP4 receptors is mediated by cAMP, whereas that through EP3 receptors is regulated by both cAMP and Ca(2+); yet there is cooperation between the actions mediated by these two receptors. The neuronal reflex pathway is not involved in stimulatory actions of these prostanoids.     

The secretory response through electric stimulation of differentiated PC12 rat pheochromocytoma cells transfected with neuropeptide Y fused with enhanced green fluorescent protein

Exocytosis in pheochromocytoma cells was induced by electric stimulation. To chase the movement of vesicles by electric stimulation, dense-core secretory vesicles were visualized by expression of the fusion protein between neuropeptide Y and enhanced green fluorescent protein (EGFP) in these differentiated PC12 rat pheochromocytoma cells. When the cells were stimulated with constant voltage potential at –300 mV, the movement of dense-core secretory vesicles could be regulated.