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.     

Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.     

There are two major types of hepatitis C virus in Japan

The polymerase chain reaction (PCR) was used to detect hepatitis C virus (HCV) in plasma from chronic non-A, non-B hepatitis patients. By choice of adequate primers, 19 of 24 samples (79%) were found positive. Sequence analysis of amplified 400 bp cDNA fragments encoding a portion of NS5 gene suggested that HCV can be classified into two types (named K1 and K2) in Japan. Slot blot hybridization of the fragments indicated that 13 were HCV-K1 and 6 were HCV-K2, which show 80% and 67% nucleotide sequence homology, respectively, with that of the prototype.     

Cysteine proteinase inhibitor in the ascitic fluid of sarcoma 180 tumor-bearing mice is a low molecular weight kininogen. Partial NH2- and COOH-terminal sequences and susceptibility to various glandular kallikreins

It has been proposed that a cysteine proteinase inhibitor (CPI) found in the ascitic fluid of Sarcoma 180 tumor-bearing mice is a kind of kininogen (Itoh, N., Yokota, S., Takagishi, U., Hatta, A., and Okamaoto, H. (1987) Cancer Res. 47, 5560-5565). The first 40 NH2-terminal residues and 54 residues of the COOH-terminal sequence, including the bradykinin moiety of highly purified ascites CPI, were determined and compared with those of mammalian low molecular weight kininogens (LMWK). The significant identity between these amino acid sequences with those of other mammalian LMWKs suggests that ascites CPI corresponds precisely to mouse LMWK. This kininogen has a light chain composed of 43 amino acid residues, which contains a unique Met-Ala-Arg-bradykinin sequence. Hydroxyproline, which was recently identified in the bradykinin sequence of kininogen from the ascitic fluid of a cancer patient, was not found in the kinin moiety of this mouse kininogen. Among purified glandular kallikreins from human, hog, rat, and mouse, only mouse submaxillary gland kallikrein was able to release bradykinin from this kininogen. Kinetic studies using a newly synthesized fluorogenic substrate, N-t-butoxycarbonyl-Met-Ala-Arg-MCA, revealed that mouse kallikrein hydrolyzes this substrate approximately 80-fold faster than does hog kallikrein, suggesting that the unique Met-Ala-Arg-bradykinin sequence is responsible for the varied susceptibility of mouse kininogen to different kallikreins.     

Stimulating factor for calf thymus DNA polymerase β

A factor that stimulates purified DNA polymerase β about 2-fold was separated from DNA polymerase β activity on a DNA-cellulose column. During the early stage of purification, the factor may be associated with DNA polymerase β to form a complex that sediments at 3.9 S in sucrose gradients and behaved as a 52,000 dalton protein on a Sephadex G-100 column. The complex, which contains 40,000 and 12,500 dalton polypeptides, was insensible to the stimulator, and did not show any exonuclease activity.     

Three dimensional organization of mammalian adrenal cortex

Summary The adrenal cortex of different mammals was studied by SEM in order to demonstrate its actual three-dimensional organization. In the rat, as well as in the cat and pig, the adrenal cortex appeared as a tunnelled continuum of polyhedral cells arranged in plate-like structures (laminae). This laminar arrangement was more evident in the inner fasciculate and reticular zones where the cortex revealed a striking similarity to liver tissue. The polyhedral cells of all cortical zones possessed regular facets populated by small pits, larger invaginations and numerous microvilli with the exception of very short and smooth areas probably corresponding to attachment zones and/or gap junctions. This cellular architecture produced a labyrinthic system of intercellular channels or lacunae in which the capillaries were suspended.The pericapillary areas of this labyrinth contained microvilli, amorphous material, a delicate net of fibrils and occasional cells. The intercellular compartment of this lacunar system was mainly bordered by numerous microvilli arising from endocrine cells.The luminal surface of the capillary wall showed not only irregularly protruding margins (interpretable as endothelial junctions) but also clearly overlapping and flattened endothelial extensions.In all the animals and areas of the adrenal cortex examined, the endothelial wall was provided with abundant clusters of small fenestrations (about 50 nm in diameter) generally arranged in sieve plates. Larger fenestrations were noted mainly in the fasciculate and reticular zones of the cat and pig and occasionally in the rat.A final point related to the nature and significance of sinusoidal fenestrations was the occurrence of irregularly shaped and intracapillary located cells mainly noted in the deeper zones of the fasciculate and reticular zones of the gland. These elements — possessing the surface characteristics of macrophages — were observed, with their irregular and slender evaginations, in close proximity to the large fenestrations in a manner reminiscent of Kupffer cells within the lumen of liver sinusoids.     

Phototaxis in Cryptomonas sp. under condition suppressing photosynthesis

Effects of 3-(3, 4-dichlorophenyl)-l, 1-dimethylurea (DCMU)on photosynthetic oxygen evolution, respiratory oxygen uptake,phototactic response and swimming rate in Cryptomonas sp. weredetermined and compared. Photosynthetic oxygen evolution wascompletely inhibited in the presence of 10^–5 M DCMU. Thetreatment did not significantly affect the rates of respiratoryoxygen uptake, phototaxis, and swimming, indicating that directparticipation of photosynthesis in the phototaxis of this algacan be ruled out. Wavelength dependency of photosynthetic oxygen evolution wasalso determined in the range of 560 to 700 nm. The rate of photosyntheticoxygen evolution at 680 nm was as high as that at 560 nm, butno phototactic activity was seen at 680 nm although it was maximumat 560 nm. This is consistent with the above conclusion. (Received February 16, 1976; )     

Oxidative Degradation of Squalene by Arthrobacter Species

An organism isolated from soil and identified as Arthrobacter sp. was studied for its squalene degradation. The degradation product from squalene, which accumulated in the culture broth, was isolated and identified as trans-geranylacetone by mass spectrometry, gas chromatography, infrared spectrometry, and nuclear magnetic resonance spectrometry. Addition of a high concentration of K₂HPO₄ to the culture medium resulted in accumulation of fairly large amounts of carboxylic acids in addition to geranylacetone. These carboxylic acids were identified as isovaleric, β,β′-dimethylacrylic, geranic, and (+)-(R)-citronellic acids. Among these acids, α,β-saturated carboxylic acids were found to be predominant in quantity.