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C. Born M. Biselli C. Wandrey J. Thömmes M. -R. Kula 《Bioprocess and biosystems engineering》1996,15(1):21-29
Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.The help of H. Schmitz, A. Bader, J. Gätgens and M. Halfar during the experiments is gratefully acknowledged. This work was partially funded by the ministry of science and research of the Federal Republic of Germany within the project Stoffumwandlung mit Biokatalysatoren. 相似文献
4.
van der Pol JJ Machnik M Biselli M Portela-Klein T de Gooijer CD Tramper J Wandrey C 《Cytotechnology》1997,24(1):19-30
The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored
on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method
(ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns.
After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the
column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure
was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and
antibody concentrations measured by HPLC was linear.
A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the
circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre,
situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system
was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter,
which had a surface of 4.8 cm2, resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical
device functioned without failure during this long-term culture.
The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous
increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium
stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth
rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates
also increased in the temperature range between 38 to 40 °C.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
5.
Variation in heat shock proteins within tropical and desert species of poeciliid fishes 总被引:8,自引:0,他引:8
Norris CE; diIorio PJ; Schultz RJ; Hightower LE 《Molecular biology and evolution》1995,12(6):1048-1062
The 70-kilodalton heat shock protein (hsp70) family of molecular
chaperones, which contains both stress-inducible and normally abundant
constitutive members, is highly conserved across distantly related taxa.
Analysis of this protein family in individuals from an outbred population
of tropical topminnows, Poeciliopsis gracilis, showed that while
constitutive hsp70 family members showed no variation in protein isoforms,
inducibly synthesized hsp70 was polymorphic. Several species of
Poeciliopsis adapted to desert environments exhibited lower levels of
inducible hsp70 polymorphism than the tropical species, but constitutive
forms were identical to those in P. gracilis, as they were in the
confamilial species Gambusia affinis. These differences suggest that
inducible and constitutive members of this family are under different
evolutionary constraints and may indicate differences in their function
within the cell. Also, northern desert species of Poeciliopsis synthesize a
subset of the inducible hsp70 isoforms seen in tropical species. This
distribution supports the theory that ancestral tropical fish migrated
northward and colonized desert streams; the subsequent decrease in
variation of inducible hsp70 may have been due to genetic drift or a
consequence of adaptation to the desert environment. Higher levels of
variability were found when the 30- kilodalton heat shock protein (hsp30)
family was analyzed within different strains of two desert species of
Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In
both cases the distribution of hsp30 isoform diversity was similar to that
seen previously with allozyme polymorphisms.
相似文献
6.
Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP 总被引:8,自引:5,他引:3
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We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase. 相似文献
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8.
A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant 总被引:3,自引:0,他引:3
Takken FL Thomas CM Joosten MH Golstein C Westerink N Hille J Nijkamp HJ De Wit PJ Jones JD 《The Plant journal : for cell and molecular biology》1999,20(3):279-288
The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E. 相似文献
9.
PJ?MumbyEmail author JD?Hedley JRM?Chisholm CD?Clark H?Ripley J?Jaubert 《Coral reefs (Online)》2004,23(2):171-183
Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m2 pixels and 6 spectral bands with 0.25-m2 pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead
Porites (total colony mortality within 6 months), old dead (>6 months) Porites,
Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites
colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m2 plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m2 grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m2 quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of
Porites spp.). At the scale of the reef (all ten 25-m2 quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living
Porites, recently dead Porites
and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m2 of reef, which represents 3,700 plots of 25 m2 each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m2 and 0.25-m2 image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m2 pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey). 相似文献
10.
In vitro effect of indomethacin and interferon-alpha on Th1 and Th2 cytokine synthesis in patients with chronic hepatitis C 总被引:1,自引:0,他引:1
Andreone P Gramenzi A Loggi E Favarelli L Cursaro C Margotti M Biselli M Lorenzini S Bernardi M 《Cytokine》2004,26(3):95-101
Current evidences suggest that non-steroidal anti-inflammatory drugs could enhance the antiviral activity of interferon-alpha in chronic HCV infection. In this study, we investigated the effect of indomethacin, a non-steroidal anti-inflammatory drug, and interferon-alpha on cytokine production by peripheral blood mononuclear cells from 12 untreated patients with chronic hepatitis C. We evaluated the effect of incubation with indomethacin, interferon-alpha or both on synthesis of Th1- (interleukin-2, interferon-gamma) and Th2-associated cytokines (interleukin-4, interleukin-10), and of the antiviral protein 2',5'-oligoadenylate synthetase. Interferon-alpha induced a significant increase in production of interleukin-2. Smaller increases were also seen in the presence of indomethacin, while incubation with both indomethacin and interferon-alpha leads to a synergistic effect. Incubation with indomethacin decreased both interleukin-4 and interleukin-10, whereas interferon-alpha increased these cytokines. The addition of indomethacin to interferon-alpha significantly reversed this interferon-induced increase. Finally, both indomethacin and the association interferon-alpha plus indomethacin determined a significant increase in 2',5'-oligoadenylate synthetase production compared to both baseline and interferon-alpha alone. In conclusion, indomethacin was able to enhance the antiviral activity of interferon-alpha and to modulate the interferon-induced Th1 and Th2 cytokine response by increasing the Th1-response, fundamental for sustained clearance of HCV, and by decreasing the Th-2 type response, associated with HCV persistence. 相似文献