Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Biodegradation of 4-nitroanisole by two Rhodococcus spp.

Two Rhodococcus strains, R. opacus strain AS2 and R. erythropolis strain AS3, that were able to use 4-nitroanisole as the sole source of carbon and energy, were isolated from environmental samples. The first step of the degradation involved the O-demethylation of 4-nitroanisole to 4-nitrophenol which accumulated transiently in the medium during growth. Oxygen uptake experiments indicated the transformation of 4-nitrophenol to 4-nitrocatechol and 1,2,4-trihydroxybenzene prior to ring cleavage and then subsequent mineralization. The nitro group was removed as nitrite, which accumulated in the medium in stoichiometric amounts. In R. opacus strain AS2 small amounts of hydroquinone were produced by a side reaction, but were not further degraded.     

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).     

Frequency dependence of the action-perception cycle for postural control in a moving visual environment: relative phase dynamics

When standing human subjects are exposed to a moving visual environment, the induced postural sway displays varying degrees of coherence with the visual information. In our experiment we varied the frequency of an oscillatory visual display and analysed the temporal relationship between visual motion and sway. We found that subjects maintain sizeable sway amplitudes even as temporal coherence with the display is lost. Postural sway tended to phase lead (for frequencies below 0.2 Hz) or phase lag (above 0.3 Hz). However, we also observed at a fixed frequency, highly variable phase relationships in which a preferred range of phase lags is prevalent, but phase jumps occur that return the system into the preferred range after phase has begun drifting out of the preferred regime. By comparing the results quantitatively with a dynamical model (the sine-circle map), we show that this effect can be understood as a form of relative coordination and arises through an instability of the dynamics of the action-perception cycle. Because such instabilities cannot arise in passively driven systems, we conclude that postural sway in this situation is actively generated as rhythmic movement which is coupled dynamically to the visual motion. Received: 7 September 1993/Accepted in revised form: 2 May 1994     

Spotlight on phytochrome nomenclature

These recommendations for genes encoding phytochromes were developed independently by Quail et al., but are broadly consistent with the Commission's guidelines. Their original article, kindly provided in advance of publication, appeared as a Letter to the Editor inPlant Cell (6:468–471, 1994) and is published with permission of the American Society of Plant Physiologists.     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

Interpretation of gas residence time distributions in large airlift tower loop reactors

Gas-residence time distribution (RTD) response curves measured in a 23 m high pilot plant airlift tower loop reactor, which consisted of a riser, a special downcomer construction and at the top of the riser a large head. The measurements were evaluated by means of a deterministic dispersion model, which yielded the following particular parameters for the riser, downcomer and the head: Gas-Bo numbers, gas-mean residence times, gas holdups, liquid velocities, gas and liquid circulation times as well as a fraction of the large and small bubbles in a model medium (water) and during cultivation of baker's yeast.List of Symbols A cross section - Bo Bodenstein number - Bo _d (= l _d w _G,d /D _d ) - Bo _h (= l _h w _G,h /D _h ) - Bo _r (= l _r w _G,r /D _r ) - D longitudinal dispersion coefficient - E gas holdup - E(t) RTD-density function - L, l length parameter - q fraction of the gas throughput which is not recirculated (approximately equal to fraction of the large bubbles) - r fraction of the throughput which is recirculated (approximately equal to the fraction of the small bubbles) - t _c circulation time - t _cL liquid circulation time - t _c,L ^* liquid circulation time calculated from the measured w _Ld in the downcomer - V ^h hydrodynamical calculated gas-liquid volume - V _d ^h (=V _d+0.75/2 V _k ) - V _k ^h =(0.25V _k ) - V _r ^h = (V _r+0.75/2 V _k ) - V _L liquid volume - V _G dispersed gas volume - V _G ^* gas throughput at the gas distributor (given in m³/h) under standard conditions, 1 bar and 25°C) - V _G,d ^* gas throughput in downcomer (=V _G ^* ) - V _G,h ^* gas throughput in head (=V _G ^* ) - V _G,r ^* gas throughput in riser (V _G ^* (1+) - w _g gas velocity - w _G,rel relative gas velocity with respect to the liquid velocity w _L - w _G,d gas velocity in the downcomer (=w _G,rel –w _Ld ) - w _G,h gas velocity in the head (=w _G,rel ) (since wLh = o) - w _G,r gas velocity in the riser (=w _G,rel +w _Lr ) - w _L liquid velocity - w _L,d liquid velocity in the downcomer measured with mass flow meter - w _sg ·w _SL superficial gas and liquid velocities - first moment of the response curve - mean residence time Indices d downcomer - G gas phase - h head - L liquid phase - r riser - h hydrodynamic (upper position) Dedicated to the 65th birthday of Proffessor Fritz Wagner.The authors gratefully acknowledge the financial support by the Krupp Industrietechnik, Grevenbroich and the support of Pleser Co, Darmstadt. H. M. Rüffer thanks the Verband der Chemischen Industrie for a Fond der Chemie scholarship, and W. Liwei thanks the government of Lower Saxony for a graduate scholarship.     

Interpretation of the gas residence time distributions in large stirred tank reactors

The behaviour of dispersed gas in large aerated stirred tank reactors is modelled by means of a Markov-process, which distinguishes between small recirculation bubbles with stagnant gas content, large rising bubbles with active gas content and exchange of stagnant and active gas contents, the gas exchange region at the impeller. The measurements of the gas residence time distributions (RTDs) in an 1.5 m³ aerated stirred tank reactor with water and Penicillium chrysogenum cultivation medium are interpreted by this model.List of Symbols CPR CO₂ production rate - OTR oxygen transfer rate - PRS pseudo random signal - RTD residence time distribution - V gas volume - recirculation coefficient - mean gas residence time Indices act active gas - ex gas exchange - stagn stagnant gas - tot total gas Dedicated to the 65th birthday of Professor Fritz Wagner.The authors thank Hoechst AG for the strain and the medium components, the GBF for the support of the experiments and H.M. Rüffer thanks the Verband der Chemischen Industrie for a Fond-der-Chemie scholarship.     

Neutral adaptation of the genetic code to double-strand coding

Summary We lay new foundations to the hypothesis that the genetic code is adapted to evolutionary retention of information in the antisense strands of natural DNA/RNA sequences. In particular, we show that the genetic code exhibits, beyond the neutral replacement patterns of amino acid substitutions, optimal properties by favoring simultaneous evolution of proteins encoded in DNA/RNA sense-antisense strands. This is borne out in the sense-antisense transformations of the codons of every amino acid which target amino acids physicochemically similar to each other. Moreover, silent mutations in the sense strand generate conservative ones in its antisense counterpart and vice versa. Coevolution of proteins coded by complementary strands is shown to be a definite possibility, a result which does not depend on any physical interaction between the coevolving proteins. Likewise, the degree to which the present genetic code is dedicated to evolutionary sense-antisense tolerance is demonstrated by comparison with many randomized codes. Double-strand coding is quantified from an information-theoretical point of view.