Oxygen uptake rate of immobilized growing hybridoma cells

Summary The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.     

Long-term cultivation of anchorage-independent animal cells immobilized within reticulated biomass support particles in a circulating bed fermentor

Summary Long-term cultivation of anchorage-independent animal cells immobilized within porous biomass support particles (BSPs) using a gas-stirred circulating bed fermentor (CBF) was investigated. Inoculation of mouse myeloma MPC-11 (ATCC CCL 167) cells into reticulated polyvinyl formal resin BSPs (3 × 3 × 3 mm; mean pore diameter, 60 m; porosity, 0.88) and the repeated batch culture of inoculated cells were performed under gentle circulation of BSPs, induced by sparging air from the base of the fermentor. The glucose uptake rate of cells decreased in the initial period just after the start of circulation, since a relatively large number of cells leaked from the BSPs. After that period, the uptake rate gradually increased and the leakage of cells diminished. In the meantime, when inoculated cells were incubated statically by introducing air into the upper part of the fermentor for the initial several days before circulating the BSPs, glucose consumption became very rapid and cell density in the BSPs reached at least 10⁷ cells/cm³ BSP. Thus, a long-term cultivation without significant leakage of cells and with high cell density in BSPs was successfully achieved in the CBF-BSP system.Offprint requests to: H. Yamaji     

Synergistic cytotoxic effect of tiazofurin and ribavirin in hepatoma cells

Tiazofurin, an anti-cancer drug, which induces remissions in human leukemia, and ribavirin, an anti-viral agent, bind at separate sites (NADH and IMP-XMP sites, respectively) on the target enzyme, IMP dehydrogenase. Now we show that the binding to IMP dehydrogenase of these drugs at two separate sites is translated into synergistic inhibition of de novo guanylate biosynthesis and synergistic toxicity in rat hepatoma 3924A cells. These results may be utilized in the chemotherapy of neoplastic diseases and in the treatment of hepatitis virus infection and hepatocellular carcinoma.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.     

A radioimmunoassay for'plasma progesterone

A radioimmunoassay for progesterone was developed which uses one micro-column chromatography for purification of hexane extracts of plasma.     

Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.     

Global bifurcation structure of a Bonhoeffer-van der Pol oscillator driven by periodic pulse trains

The Bonhoeffer-van der Pol (BVP) oscillator is a valuable dynamical system model of pacemaker neurons. Isochrons, phase transition curves (PTC), and two dimensional bifurcation diagrams served to analyze the neuron's response to periodic pulse stimuli. Responses are described and explained in terms of the nonlinear dynamical system theory. An important issue in the generation of spikes by pacemaker neurons is the existence of both slow and fast dynamics in the state point's trajectory in the phase plane. It is this feature in particular that makes the BVP oscillator a faithful model of living pacemaker neurons. Comparison of the model's responses with those of a living pacemaker was based also on return maps of interspike intervals. Analyzed in detail were the complex discharges called stammering which involve interspike intervals that arise unpredictably and exhibit histograms with several modes separated by the equal intervals.Supported by Trent H. Wells Jr. Inc.