A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

A new DNA profiling system for cell line identification for use in cell banks in Japan

Summary Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and λTM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique.     

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites

The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca²⁺ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca²⁺. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.     

An anchorage-dependent locus in the cell cycle for the growth of 3T3 cells

Mouse fibroblasts, 3T3 cells, require a solid surface for continuous growth, but when 3T3 cells, during their exponential phase in Petri dishes, were transferred to a suspension culture, the number of cells roughly doubled by 30 h. During the suspension culture the number of pairing cells (c₂) increased, but that of the single cells decreased. When cells synchronized at mitosis or at the G1-S boundary were transferred to the suspension culture, the number of pairing cells peaked at 30 min and at 10 h, respectively. DNA synthesis began immediately after the cells, which were cultured for 16 h in the suspension, had settled onto the surface of the Petri dishes. When cells in a confluent culture were arrested at an early G1 period and were suspended, the number of pairing cells did not increase. These results indicate that the most important locus for anchorage growth seems to be at a late G1 period of the cell cycle.     

On the nature of the three intermediate species formed after reaction of reduced cytochrome oxidase with oxygen.

Spectral examinations of the reaction of reduced cytochrome oxidase with molecular oxygen has revealed the formation of at least three intermediates, which are designated as Compounds I, II, and III according to the order of their appearance. From the difference spectrum against the oxidized oxidase, Compound I is characterized by a maximum at 605 nm, Compound II at 578 nm, and Compound III by double peaks at around 600 and 580 nm. In the Soret region, Compound I shows a peak at 435 nm and a trough at 412 nm, Compound III exhibits a peak at 442 to 443 nm and a trough at 418 nm. In the absence of cytochrome c, the spontaneous decay of Compound I precedes that of Compound II; the first order rate constants have been found to be 4 X 10(-3) s(-1) and 8 X 10(-4) s(-1) for Compounds I and II, respectively. Compound III, however, does not revert back to the oxidized form even after several hours. The decay of Compound I is accelerated in the presence of ferrocytochrome c by a factor of 10(3) to 10(4) depending on the concentration of the latter. The time for sequential differentiation between Compound I and Compound II becomes less clear in the presence than in the absence of ferrocytochrome c. On the contrary ferricytochrome c does not show such an accelerating effect. These and other observations lead us to postulate Compound I as an active intermediate, the true oxygenated compound in the cytocchrome oxidase reaction.     

Synthesis of 6-Sulfur Analogues of Oxanosine and Closely Related Derivatives Thereof

Abstract

Novel β-D-ribofuranosides having a 5-substituted imidazo [4,5-d] [1,3]thiazine ring, including the S⁶-congener 3 of oxanosine 2, were synthesized for screening their anticancer and antiviral activities.     

Determination of absolute configuration of photo-degraded catechinopyranocyanidin A by modified Mosher's method

Catechinopyranocyanidins A and B (cpcA and cpcB) are two purple pigments present in the seed-coat of red adzuki bean, Vigna angularis, of which cpcA is the major pigment, containing two chiral carbons in the catechin part. Their absolute configurations were determined by comparison of their experimental and quantum chemical calculated electronic circular dichroisms (ECDs). These purple pigments are labile on light irradiation and easily decompose to photo-degraded catechinopyranocyanidins A and B (pdcpcA and pdcpcB), while retaining the stereostructure of the catechin residue. We applied modified Mosher's method for determining the chirality of the secondary alcohol in pdcpcA. Hexamethylation of pdcpcA by diazomethane followed by esterification using (S)- and (R)-MTPACl gave (R)- and (S)-MTPA esters, respectively. By analysis of the NMR spectra of (R)- and (S)-MTPA esters of tetramethylated (+)-catechin, the chirality of pdcpcA was determined to be 2R, 3S, same as the absolute configuration of cpcA.     

Burr marigold (Bidens tripartita L.) roots directly and immediately scavenge rhizosphere methane with highly exuded hydrogen peroxide via a rhizosphere Fenton reaction

Plant and Soil - The major factors controlling the soil methane (CH4) concentration and CH4 emissions of various plant (mainly wetland) species were identified. Five plant species (Oryza sativa,...     

Unweighted state as a sidestep preparation improve the initiation and reaching performance for basketball players

The preparatory motion of a defensive motion in contact sport such as basketball should be small and involve landing on both feet for strict time and motion constraints. We thus proposed the movement creating a unweighted state. Ten basketball players performed a choice reaction sidestepping task with and without the voluntary, continuous vertical fluctuation movement. The results indicated that the preparatory movement shortened the time of their sidestep initiation (301 vs. 314 ms, p = 0.011) and reaching performance (883 vs. 910 ms, p = 0.018) but did not increase their peak ground reaction force or movement velocity. The mechanism of the improvement was estimated to be the following: in the preparation phase, the vertical body fluctuation created the force fluctuation; after the direction signal, the unweighted state can shorten the time required to initiate the sidestepping (unweighted: 279 ms; weighted: 322 ms, p = 0.002); around the initiation phase, the dropping down of the body and weighted state can contribute to the reaching performance. We conducted additional experiment investigating muscle–tendon-complex dynamics and muscle activity using ultrasound device and electromyography. The result suggests that the building up of active state of muscle might explain the improvement of sidestepping performance.