Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

The secondary coordination sphere and axial ligand effects on oxygen reduction reaction by iron porphyrins: a DFT computational study

Oxygen reduction reaction (ORR) catalyzed by a bio-inspired iron porphyrin bearing a hanging carboxylic acid group over the porphyrin ring, and a tethered axial imidazole ligand was studied by DFT calculations. BP86 free energy calculations of the redox potentials and pK _a’s of reaction components involved in the proton coupled electron transfer (PCET) reactions of the ferric-hydroxo and -superoxo complexes were performed based on Born–Haber thermodynamic cycle in conjunction with a continuum solvation model. The comparison was made with iron porphyrins that lack either in the hanging acid group or axial ligand, suggesting that H-bond interaction between the carboxylic acid and iron-bound hydroxo, aquo, superoxo, and peroxo ligands (de)stabilizes the Fe–O bonding, resulting in the increase in the reduction potential of the ferric complexes. The axial ligand interaction with the imidazole raises the affinity of the iron-bound superoxo and peroxo ligands for proton. In addition, a low-spin end-on ferric-hydroperoxo intermediate, a key precursor for O–O cleavage, can be stabilized in the presence of axial ligation. Thus, selective and efficient ORR of iron porphyrin can be achieved with the aid of the secondary coordination sphere and axial ligand interactions.     

Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.     

A gene trap approach to identify genes that control development

One methodology called gene trap represents a versatile strategy by which murine genes that control developmental events can be captured and identified with corresponding mutants produced at the same time. Gene trap methodology has been developed and several genes and their mutants have been analyzed, but almost all of the genes reported are those already known or murine homologs of other species. In this study, the efficiency of the gene trap methodology was improved and a novel mutant mouse strain named jumonji established which displayed an intriguing defect. Homozygous fetal mice died in utero and a significant proportion of the homozygotes showed abnormal groove formation on the neural plate and a defect in neural tube closure with a mixed genetic background of 129/Ola and BALB/c. The trapped gene believed to be responsible for these phenotypes encodes a novel nuclear protein. The results reveal that the gene trap approach can identify unknown interesting genes in murine development. The gene trap strategy, however, has several problems, the greatest of which is the difficulty in prescreening embryonic stem (ES) cells for interesting trapped genes. Recent studies are solving this problem and show that the prescreening of ES cells for genes with several characteristics is possible.     

A possible role of Atg8 homologs as a scaffold for signal transduction

Atg8 and its homologs are essential for autophagosome formation in various species. In animal cells, Atg8 homologs have an additional function in clearance of damaged organelles and bacteria, acting as a landmark for selective autophagy. We have recently shown that OATL1, a Rab-GTPase-activating protein (Rab-GAP), is a novel binding partner of Atg8 homologs in mammalian cells, but to our surprise, it is not a substrate of autophagy. Further analysis indicates that OATL1 is involved in the fusion between autophagosomes and lysosomes through its GAP activity and its Atg8 homolog binding activity. Our findings suggest a novel function of Atg8 homologs as a scaffold for signal transduction that regulates autophagosomal maturation.