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  • Journals
  • Articles  (213)
  • Escherichia coli  (136)
  • cell culture  (77)
  • Process Engineering, Biotechnology, Nutrition Technology  (213)
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 4 (1989), S. 1-5 
    ISSN: 1476-5535
    Keywords: Xylose isomerase ; Enzyme expression ; thermally inducible ; Hollow fiber bioreactor ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary TheEscherichia coli xylose isomerase (EC 5.3.1.5) has been expressed under the control of a thermal inverting promotor system (att-nutL-p-att-N block) and its performance in a hollow fiber bioreactor measured. The conversion of xylose to xylulose was inversely proportional to the flow rate and the system operated up to 60°C. The maximum conversion efficiency observed was 19.05% at 55°C.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 3 (1988), S. 21-28 
    ISSN: 1476-5535
    Keywords: Diaper ; Staphylococcus aureus ; Escherichia coli ; Candida albicans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Methods were developed to study the effects of absorbent materials from diapers on microbial survival, growth and toxic shock syndrome toxin-1 (TSST-1) production under specified in vitro conditions. Growth of representative skin and fecal flora organisms was equivalent in cultures in which materials from cotton cloth diapers, disposable diapers or disposable diapers containing absorbent gelling material were added as the sole carbon source. In urine used as an enrichment medium, growth of the test organisms in media containing material from the three diaper types was equivalent and no contribution to growth from the diaper material was detected. TSST-1 was not produced byStaphylococcus aureus under conditions in which urine was added to the diaper materials. Pathogenic strains of organisms purposefully introduced onto diapers failed to survive and the few microbial cells normally found in diaper material did not multiply when stored under conditions favorable to microbial growth. The data indicate that all three diaper types tested were the same with respect to growth and survival of representative skin and fecal organisms.
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  • 3
    ISSN: 0141-0229
    Keywords: 6-aminopenicillanic acid (6-APA) ; DH5 cells ; Escherichia coli ; constitutive; β-lactamase negative ; lactose broth (LB) ; optimization of enzyme production parameters ; pac gene ; penicillin acylase ; plasmid pUSAD2
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 4
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Enzyme and Microbial Technology 15 (1993), S. 730-735 
    ISSN: 0141-0229
    Keywords: Escherichia coli ; Molecular chaperones ; heat shock proteins ; protein folding ; recombinant DNA
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 5
    ISSN: 0141-0229
    Keywords: Escherichia coli ; computer control ; exponentially fed-batch culture ; penicillin acylase ; penicillin amidase ; plasmid ; recombinant
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 6
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Enzyme and Microbial Technology 16 (1994), S. 240-246 
    ISSN: 0141-0229
    Keywords: Bacillus subtilis ; Escherichia coli ; Fermentation ; Plasmid stability
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 7
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Enzyme and Microbial Technology 15 (1993), S. 652-656 
    ISSN: 0141-0229
    Keywords: Dihydrofolate reductase ; Escherichia coli ; continuous culture ; methotrexate
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 12 (1993), S. 256-262 
    ISSN: 1476-5535
    Keywords: Listeria ; Salmonella ; Shigella ; Aeromonas ; Staphylococcus ; Escherichia coli ; Bacillus cereus ; Clostridium botulinum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary MKES Tools is a microbial kinetics expert system for developing food production systems and assessing product safety. The specific information required as input are: (1) a flowchart of the production system, (2) the factors affecting the survival and growth of food-borne pathogens and (3) the ranges of variation for each factor's parameters. With this information, MKES Tools simulates the growth and survival of pathogenic microorganisms when subjected to many different factor/parameter situations. The responses obtained are then used to estimate the significance of each factor's parameters.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 1 (1986), S. 69-73 
    ISSN: 1476-5535
    Keywords: Escherichia coli ; Starvation ; Protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Escherichia coli bulk protein synthesis continued during the first 3–4 h of carbon starvation at 50–75% that of non-starved (growing) cells. Two-dimensional gel electrophoresis analysis of in vivo pulse-labelled proteins resolved at least 30 polypeptides with new or increased synthesis, relative to total protein synthesis, during this time. Among these polypeptides were several that were also synthesized by ethanol-treatedE. coli (heat-shock proteins). In addition, a number of unique polypeptides were synthesized by carbon-starved cells. These ‘starvation proteins’ may be involved in survival of the starving bacteria.
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  • 10
    ISSN: 1476-5535
    Keywords: Gene transfer ; Escherichia coli ; River water ; Indigenous bacteria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), fromEscherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1×107 CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated withE. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated withE. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms. The results of this particular assay indicate that pBGH1 or the portion of pBGH1 including the BST structural gene had not been transferred from W3110G [pBGH1] to indigenous microbial inhabitants of the Missouri River water flasks during this study.
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 16 (1996), S. 331-341 
    ISSN: 1476-5535
    Keywords: biofilms ; bacteriophages T4 and E79 ; Escherichia coli ; Pseudomonas aeruginosa ; SCLM ; fluorescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiccyanate (FITC), and by the addition of 4′6-diamidino-2-phenylindole (DAPI) to phage-infected host cells ofEscherichia coli andPseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rll-LacZ fusion), within alac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded β-galactosidase activity. Fluorescent antibodies were used to detect nonlabeled progeny phage. Phage T4 infected both surface-attached and surface-associatedE. coli while phage E79 adsorbed toP. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.
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  • 12
    ISSN: 1476-5535
    Keywords: fermentation ; fermentation monitoring ; fermentation control ; fermentation software ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The advent of inexpensive computers and associated control and data acquisition software makes possible the development of sophisticated, configurable, integrated monitoring and control systems for small-scale laboratory and pilot-scale fermentors at low cost. We describe here the implementation of such a system, the interfacing of off-line instruments to enhance real time data analysis, low level process control and several substrate feeding protocols.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 17 (1996), S. 151-158 
    ISSN: 1476-5535
    Keywords: bacteriocins ; colicins ; evolution ; ecology ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this review we focus on the ecological and evolutionary forces that determine the frequency and diversity of colicins inEscherichia coli. To begin, we describe that this killing phenotype is ubiquitous inE. coli, with as many as 50% of the isolates from a population producing colicin toxins, and that each population sampled has its own unique distribution of the more than 20 known colicin types. Next, we explore the dynamics of colicinogeny, which exhibits a typical form of frequency dependence, where the likelihood of successful colicin invasion into a population increases as the initial density of colicinogenic cells increases. We then incorporate thoughts on the evolution of chromosomal resistance to colicins and describe how resistance might influence the dynamics of colicinogen invasion and maintenance and the resulting colicin diversity. The final section deals with a genetic and phylogenetic characterization of colicins and a discussion of the evolutionary mechanisms responsible for generating colicin diversity. In this final section we provide details of the different molecular mechanisms known to play a role in generating colicin diversity, including the two most dominant forces in colincin evolution: recombination and positive, deversifying, selection.
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  • 14
    ISSN: 1476-5535
    Keywords: Recombinant human insulin-like growth factor ; Escherichia coli ; Fermentation ; Production ; Somatomedin C
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Two kinds of fed batch fermentation processes were compared at a 10-liter scale to examine their effect on recombinant human insulin-like growth factor (IGF-1) gene expression inEscherichia coli. The difference between the two processes was the feed medium composition and whether the process used a single or dual feed during the course of the fermentation. In the dual feed system, organic nitrogen was delivered at a higher rate (50 g/h) than in the single feed system (5 g/h). The dual feed process resulted in a significant increase in IGF-1 yield. 30 mg IGF-1/g dry cell weight was synthesized in the dual feed system compared to 3 mg IGF-1/g dry cell weight obtained in the single feed system. The presence of high levels of organic nitrogen during the induction period may enhance IGF-1 synthesis by protecting the IGF-1 from proteolytic degradation. The IGF-1 yield decreased to 17 mg/g dry cell weight when the glucose supply was decreased from 17 g/h to 8 g/h during the induction period; however, an increase in glucose supply from 17 g/h to 50 g/h during the induction period did not enhance the IGF-1 synthesis. Thus, the enhancement of IGF-1 gene expression in the dual feed process was mainly dependent on a high level of organic nitrogen and an appropriate level of glucose in the medium during the induction period.
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  • 15
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 19 (1995), S. 95-105 
    ISSN: 1573-0778
    Keywords: antibiotics ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This article describes the historical development of the prophylactic use of antibiotics in cell culture as well as their effects on cells. The influence of antibiotics on cell morphology, cellular degeneration and cell death and cellular function is summarized. Cellular DNA as well as protein synthesis are affected which can lead to interference with, or even changes in, metabolic processes. Such effects must be considered in cell culture research. As antibiotics are used in multifold ways, the otherwise standardized conditions in cell culture are no longer comparable. The prophylactic use of antibiotics is rejected for scientific reasons.
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  • 16
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 2 (1989), S. 181-185 
    ISSN: 1573-0778
    Keywords: culture ; media ; skin ; hypertrophic scar ; cell culture ; electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Twenty-five human or mouse tissue samples, some up to 8×4×2 cm, were immersed in a special transport medium (TM), L15M15, up to 7 days before being processed or placed in tissue culture. To test the efficacy of this medium, we concurrently placed pieces of the same tissues in a sterile phosphate buffered solution (PBS). We also tested the preservative capabilities of TM and PBS at room temperature and with refrigeration. Differences between TM and PBS are demonstrated, which are more pronounced using room temperature up to 4 days time. The tissues stored in TM show fewer degenerative or autolytic changes than the same tissue stored in PBS under identical conditions. Using regrigeration further enhanced the preservative qualities of TM up to 4 days, but not PBS. There were no obvious differences between tissues stored in TM and PBS with refrigeration after 7 days. We conclude that transport medium L15M15 is a useful medium for preserving tissue viability, especially large tissue samples, up to 4 days, especially if refrigerated.
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  • 17
    ISSN: 1573-0778
    Keywords: anticancer drugs ; at-MDR ; cell culture ; DNA topoisomerase II ; drug resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo IIα gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 11 (1993), S. 169-174 
    ISSN: 1573-0778
    Keywords: cell culture ; hybridoma ; monoclonal antibody ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.
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  • 19
    Electronic Resource
    Electronic Resource
    Springer
    Cytotechnology 11 (1993), S. 219-231 
    ISSN: 1573-0778
    Keywords: cell culture ; chelators ; metal ion buffer ; serum-free medium ; serum replacement (serum substitute) ; trace elements
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum. Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.
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  • 20
    ISSN: 1573-0778
    Keywords: cell culture ; porous microcarrier ; prourokinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A recombinant DNA CHO cell line secretingurokinase-type plasminogen activator (u-PA) wascultivated with Cytopore cellulose porousmicrocarriers in a 30l Biostat UC stirred tankreactor. After 26 days of culture, using a spinfilter toretain cells in bioreactor, the cell density couldreach 1.33 × 107 ml-1. The maximal u-PAactivity in supernatant was 7335 IU·ml-1, and204l supernatant containing 7.1 g u-PA was harvested.After 100 days of culture with 0.1% fetal bovineserum medium, a modified cell retention system whichcan be washed-out backward, substituted thespinfilter to prevent filter clogging. The maximalcell density was over 107 ml-1, the maximalu-PA activity in supernatant reached 6250IU·ml-1, and 1604l supernatant containing about51 g u-PA was harvested. Compared to perfusionculture, batch medium-replaced culture could raiseutilizing efficiency of the medium, increase cell specificproductivity and improve the quality of the product which wasnot steady in a 37 °C environment. Cells can movefrom seed porous microcarriers occupied by cells tovacant microcarriers spontaneously, withouttrypsinization, and continue to grow until all microcarriers contained cells. It shows that Cytoporeporous microcarriers are very useful and convenient toscale up cultivation step by step.
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  • 21
    ISSN: 1573-0778
    Keywords: cell culture ; flow injection analysis ; glycosylation ; macro-heterogeneity ; prolactin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A flow injection analysis (FIA) system is presented for a twostep immunoassay-based determination of the total humanprolactin (hPRL) concentration along with its degree ofglycosylation. Separate measurement of total hPRL and nonglysosylated human prolactin (nG-hPRL) were made using twoflow-through cartridges each containing immobilized antibodiesof different specificity. The antibodies are immobilized on thesurface of a carrier. Glycosylated hPRL (G-hPRL) and, thus, thedegree of glycosylation were calculated by the differencebetween the two specific determinations. Enhanced specificityfor the determination of nG-hPRL was obtained using unfavorablebinding conditions through incorporation of alkaline pH andchaotropic agents into the carrier/dispersion buffer. The assayfor total hPRL and nG-hPRL were each found to be linear withinthe relevant concentration range. The results of the two-stepFIA method were found to agree with those obtained by thestandard methods of ELISA and western blotting while offeringthe advantage of minimal analysis time (10 min) and eliminationof manual manipulations.
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  • 22
    ISSN: 1573-0778
    Keywords: cell culture ; half-life ; packaging cells ; retrovirus ; titer ; ΨCRIP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/ΨCRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/ΨCRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.
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  • 23
    ISSN: 1573-0778
    Keywords: cell culture ; lymphocyte ; lymphokine-activated killer cell ; recombinant interleukin 2 ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (3H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.
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  • 24
    ISSN: 1573-0778
    Keywords: adoptive immunotherapy ; cell culture ; cell culture apparatus ; Interleukin-2 ; lymphokine-activated killer cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7×107 cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity. The JCC-device was able to reduce the consumption of basal medium, rIL-2 and serum by 20%, 84% and 96%, respectively compared to the conventional tissue culture systems. The JCC-device improved the routine performance of adoptive immunotherapy with LAK cells and rIL-2.
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  • 25
    ISSN: 1573-0778
    Keywords: cell culture ; enhanced production ; fibroblast cells ; interferon-β ; Langmuir-Blodgett film
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Normal human skin (NB1-RGB) cells were cultured in the presenceof polyinosinic and polycytidylic acids, diethylaminoethyldextran, cycloheximide and actinomycin D, which induced humaninterferon-β. The simplest induction method, that requiredonly polyinosinic and polycytidylic acids and diethylaminoethyldextran was found to give the highest production ofinterferon-β by the cells. The cell growth and productionof interferon-β were investigated for NB1-RGB cellscultured on silk fibroin, poly(γ-methyl-L-glutamate),poly(γ-benzyl-L-glutamate) and collagen films prepared bythe Langmuir-Blodgett (LB) and casting methods. The cell densityof NB1-RGB cells cultured on the LB films was found to be higherthan that on the cast films made of the same polymer. Thisindicates that not only the chemical structure of the polymersused for the preparation of the films but the preparationmethods of the films, i.e., casting and LB methods, are also astrong factor affecting the cell growth. The production ofinterferon-β per unit number of cells was found to behigher on the cast films than that on the LB films made of thesame polymer. This is explained by the fact that the optimalsuppressed growth of NB1-RGB cells on the cast films leads tothe enhanced production of interferon-β on the cast filmscompared to those on the LB films prepared by the same polymer.
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  • 26
    ISSN: 1573-0778
    Keywords: cell culture ; FGF ; growth factors ; protein sequencing ; zwitterionic detergent
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.
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  • 27
    ISSN: 1573-0778
    Keywords: hybridoma ; cell volume ; cell culture ; flow cytometry
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    Notes: Abstract Light scattering properties of hybridoma cells were examined with flow cytometry. Viable and dead cells form two distinct populations. The distribution of the two populations changes during a batch culture. the concentration of dead cells measured by flow cytometry correlates well to that measured by hemacytometer. The distribution based on small-angle light scattering is similar to the distribution based on volume as measured by Elzone particle counter. It thus appears that viable cells form the population with a larger mean cell volume. The results also indicate that the volume of viable cells decreases during the cultivation while that of dead cells remains relatively constant.
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  • 28
    ISSN: 1573-0778
    Keywords: cell culture ; mixing time ; oxygen demand ; oxygen transfer ; pH and dO2 sensitivity ; scale-up
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    Notes: Abstract Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K L a and of mixing (via pH measurements) in bioreactors up to 8 m3 at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO2 has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.
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  • 29
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    Cytotechnology 22 (1996), S. 3-16 
    ISSN: 1573-0778
    Keywords: cell culture ; process monitoring ; oxygenation ; CO2 transfer ; aggregation ; segregation ; diffusion, on-line monitoring
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO2, a significant accumulation of dissolved CO2 is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.
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  • 30
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    Cytotechnology 31 (1999), S. 3-8 
    ISSN: 1573-0778
    Keywords: cell culture ; livestock ; milk ; nuclear transfer ; transgenic
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Transgenesis may allow the generation of farm animals with altered phenotype, animal models for research and animal bioreactors. Although such animals have been produced, the time and expense involved in generating transgenic livestock and then evaluating the transgene expression pattern is very restrictive. If questions about the ability and efficiency of expression could be asked solely in vitro rapid progress could be achieved. Unfortunately, experiments addressing transcriptional control in vitro have proved unreliable in their ability to indicate whether a transgene will be transcribed or not. However, initial studies suggest that cell culture may be able to predict in vivo post-transcriptional events. We review these issues and propose that strategies which engineer the transgene integration site could enhance the probability for efficient expression. This approach has now become feasible with the development of techniques allowing animals to be generated from somatic cells by nuclear transfer. The important step in this procedure is the use of cells grown in culture as the source of genetic information, allowing the selection of specific transgene integration events. This technology which has dramatically increased the potential use of transgenic livestock for both agricultural and biotechnological applications, is based on standard cell culture methodology. We are now at the start of a new era in large animal transgenics.
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  • 31
    ISSN: 1573-0778
    Keywords: heartwater ; cell culture ; Cowdria ruminantium ; bovine vascular endothelial cells
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    Notes: Abstract The causal agent of heartwater disease of domestic ruminants,Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0–6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.
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  • 32
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    Cytotechnology 15 (1994), S. 87-94 
    ISSN: 1573-0778
    Keywords: Adaptation ; ammonia ; cell culture ; glutamine ; glutamate ; dipeptides
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Although glutamine is used as a major substrate for the growth of mammalian cells in culture, it suffers from some disadvantages. Glutamine is deaminated through storage or by cellular metabolism, leading to the formation of ammonia which can result in growth inhibition. Non-ammoniagenic alternatives to glutamine have been investigated in an attempt to develop strategies for obtaining improved cell yields for ammonia sensitive cell lines. Glutamate is a suitable substitute for glutamine in some culture systems. A period of adaptation to glutamate is required during which the activity of glutamine synthetase and the rate of transport of glutamate both increase. The cell yield increases when the ammonia accumulation is decreased following culture supplementation with glutamate rather than glutamine. However some cell lines fail to adapt to growth in glutamate and this may be due to a low efficiency transport system. The glutamine-based dipeptides, ala-gln and gly-gln can substitute for glutamine in cultures of antibody-secreting hybridomas. The accumulation of ammonia in these cultures is less and cell yields in dipeptide-based media may be improved compared to glutamine-based controls. In murine hybridomas, a higher concentration of gly-gln is required to obtain comparable cell growth to ala-gln or gln-based cultures. This is attributed to a requirement for dipeptide hydrolysis catalyzed by an enzyme with higher affinity for ala-gln than gly-gln.
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  • 33
    ISSN: 1573-0778
    Keywords: Baculovirus ; cell culture ; Drosophila ; gene expression ; insect cell ; metallothionein promoter ; recombinant protein
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.
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  • 34
    ISSN: 1573-0778
    Keywords: Mycoplasma ; cell culture ; clinical testing ; microbial screening ; PCR
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    Notes: Abstract The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.
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  • 35
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    Cytotechnology 16 (1994), S. 147-150 
    ISSN: 1573-0778
    Keywords: Hybridoma ; peptone ; monoclonal antibody ; cell culture
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0–10 g l−1 range. It was found that 1–5 g l−1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3–5 g l−1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3–5 g l−1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l−1.
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  • 36
    ISSN: 1573-0778
    Keywords: Escherichia coli ; functional expression ; protein folding ; phage display system
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    Notes: Abstract Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods (“panning”). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l−1 of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.
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  • 37
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    Cytotechnology 7 (1991), S. 121-130 
    ISSN: 1573-0778
    Keywords: cell culture ; cellulose sulphate ; encapsulation ; monoclonal antibodies ; poly-dimethyl-diallyl-ammoniumchloride
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A new encapsulation method was developed for the cultivation of mammalian cells. The capsules were produced using a solution of sodium cellulose sulphate (CS)(1.5%) and poly-dimethyl-diallyl-ammoniumchloride (PDMDAAC). When CS droplets fell into the precipitation bath consisting of a 2% solution of PDMDAAC, immediately a membrane at the interphase was built up. The influences of varying encapsulation process parameters on capsule characteristics, cell growth, and monoclonal antibody production were tested. This new method showed advantages when compared to other methods mainly due to time simplicity of the whole process.
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  • 38
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    Cytotechnology 4 (1990), S. 181-189 
    ISSN: 1573-0778
    Keywords: cell culture ; microperfusion ; luminal-antiluminal media gradients ; specific support ; extracellular matrix proteins
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    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The lack of a suitable system to culture epithelial cells for a long period under a luminal-antiluminal medium gradient, was the reason to develop a new system. It consists of an interchangeable sheet of permeable support material, which is set in place by two tight fitting holding rings. For special demands the supports can be coated with extracellular matrix proteins improving cellular attachment and terminal differentiation. The handling of the sheets by forceps proceeds easily and quickly, thus fastening the transfer of cultured cells without additional manipulations. The sheets can be transferred to a newly developed microperfusion chamber on which an apical and a basal perfusion over a long culture period parallel to a transepithelial electrophysiological registration becomes possible. The chamber has an extremely low amount of fluid dead space. The separate perfusion of cultured cells under isotonic, hypotonic or hypertonic conditions opens new possibilities. Thus, culture can be performed under most natural conditions e.g., that found within the kidney.
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  • 39
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    Cytotechnology 5 (1991), S. 31-46 
    ISSN: 1573-0778
    Keywords: serum-free ; cell culture ; carcinoma
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  • 40
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    Journal of industrial microbiology and biotechnology 1 (1987), S. 335-340 
    ISSN: 1476-5535
    Keywords: Escherichia coli ; Outer membrane permeability ; Porin
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary We describe the construction and analysis of an isogenic series ofEscherichia coli K12 strains that vary in their outer membrane permeability. They carry mutations that alter the amount and the type of porin present in the outer membrane. The permeability profiles of these strains suggest that both the amount and the type of porin present in the outer membrane affects permeability. Several of the strains carry mutations that extend the permeability of the outer membrane to include a variety of compounds that are normally excluded from entering the cell.
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  • 41
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    Journal of industrial microbiology and biotechnology 14 (1995), S. 218-225 
    ISSN: 1476-5535
    Keywords: Caesium ; Potassium ; Toxicity ; Accumulation ; Escherichia coli ; Bacillus subtilis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs+ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs+ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K+. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K+ and Na+ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs+ toxicity towards the bacterial strains it was therefore necessary to define the K+:Cs+ ratio in the external medium. The minimum inhibitory K+:Cs+ concentration ratio for theBacillus strains tested was in the range 1∶2–1∶3 whileE. coli had a minimum inhibitory K+:Cs+ concentration ratio of 1∶6.
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  • 42
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 57-61 
    ISSN: 1476-5535
    Keywords: Escherichia coli ; hypochlorites ; chlorine ; inactivation kinetics
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The kinetic inactivation parameters of four wild strains and two enterotoxigenic strains ofEscherichia coli exposed to commercial calcium hypochlorite were determined. The four wild strains (1A, 3C, 4D and 8H) were isolated from lettuce bought in São Paulo (Brazil), and the two enterotoxigenic strains (TR69 and TR101) were originally isolated from human patients. Decimal reduction time ‘D’, for 10 mg L−1 available chlorine at pH 6.8, varied between 71.4 s for the wild strain 4D and 31.3 s for the toxigenic strain. The ‘D’ values obtained for wild strain 1A exposed to 5.0 mg L−1 available chlorine at pH 6.8 varied between 111.1 s and 41.7 s. The ‘D’ values obtained forE. coli strain TR69 exposed to 10 mg L−1 available chlorine varied from 15.2 s at pH 5.4 up to 83.3 s at pH 8.2. The use of the most resistant wild strain ofE. coli as a biological standard assures maximal effectiveness in controlling water contamination by chlorination.
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  • 43
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 364-369 
    ISSN: 1476-5535
    Keywords: biornass ; growth ; phosphate uptake ; Pseudomans fluorescens ; Escherichia coli ; Acinetobacter radioresistens
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (102–105 cells ml−1) were used. At a high initial biomass concentration (108 cells ml−1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (107 cells ml−1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (106, 107 cells ml−1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L−1 compared to average ranges of 4.92–17.14 mg L−1 forA. radioresistens and to average ranges of 0.50–8.50 mg L−1 forE. coli.
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  • 44
    ISSN: 1476-5535
    Keywords: Fermentation ; High cell density ; Escherichia coli ; RecombinantE. coli ; Atrial natriuretic factor (ANF) ; Fusion protein expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.
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  • 45
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    Journal of industrial microbiology and biotechnology 2 (1987), S. 87-95 
    ISSN: 1476-5535
    Keywords: Fermentation ; Process control ; Expression of recombinant proteins ; Escherichia coli ; Malaria vaccine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary A variety of feeding strategies have been described for attaining high cell densities in fed-batch fermentors. Although cell density is an important component in the produtivity of recombinant fermentations, it must be achievable with high product expression levels. Experiments were conducted to study the influence of fermentation feeding strategies on the production of a recombinant malaria antigen inEscherichia coli. C-source feeding profiles were calculated to maintain specific growth rates at 0.1, 0.2, 0.35, and 0.5 l/h prior to induction in defined and complex media using an exponential growth model. Fed-batch fermentations employing these feeding profiles effectively controlled the specific growth rates prior to induction. Antigen yields per dry cell weight did not vary with specific growth rate. Antigen yields from fed-batch fermentations achieving high cell densities were similar to batch fermentations achieving low cell densities. These results show that C-feeding policies can limit growth without reducing expression levels in some systems, and suggest applications in managing oxygen demand and catabolic by-product formation during process scale-up.
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  • 46
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    Journal of industrial microbiology and biotechnology 1 (1987), S. 377-381 
    ISSN: 1476-5535
    Keywords: Antimicrobial ; Metal salt ; Lanthanum ; Escherichia coli ; Staphylococcus aureus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Rare earth metal cations are antibacterially synergistic with anionic surfactants, yielding mixtures that have bactericidal activity against a variety of gram-positive and gram-negative bacteria at minimum concentrations ranging from 16 to 125 μg/ml. Uptake of surfactant byEscherichia coli increases in the presence of lanthanum, suggesting that the role of rare earth metal cations is to reduce the net negative surface charge on the bacteria, thereby increasing the affinity between the negatively charged surfactant and the bacterial surface.
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  • 47
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    Journal of industrial microbiology and biotechnology 11 (1993), S. 253-257 
    ISSN: 1476-5535
    Keywords: Colonization ; Escherichia coli ; Gastrointestinal ; Environmental ; Survival
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The colonizing potential ofEscherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 μg/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any rat from any group. Thus, theE. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.
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  • 48
    ISSN: 1573-0972
    Keywords: Acetic acid production ; carbon metabolism ; continuous culture ; Escherichia coli ; metabolic engineering
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    Notes: Abstract The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO2 production, and in oxygen consumption.
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  • 49
    ISSN: 1573-0972
    Keywords: Bacterial adherence ; bromodeoxyuridine (BrdU) ; Escherichia coli ; fimbria ; immunomax technique
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    Notes: Abstract Fimbriated and fimbria-less strains of Escherichia coli were isolated from urine of pyelonephritis patients, labelled with bromodeoxyuridine and their adhesion to human umbillical vein endothelial cells was studied employing ELISA and immunocytochemistry. No significant differences were noted in adhesion of the two types of strains.
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  • 50
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    World journal of microbiology and biotechnology 13 (1997), S. 159-161 
    ISSN: 1573-0972
    Keywords: Escherichia coli ; plasmid-containing bacteria ; pond water ; survival
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The survival of Escherichia coli K-12 J62-1 containing the antibiotic-resistance plasmid R1 and an isogenic plasmid-free strain were studied in pond water microcosms. The number of plasmid-containing cells recovered from the microcosms remained constant over a sampling period of 31 days whereas plasmid-free cell numbers declined.
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  • 51
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    World journal of microbiology and biotechnology 9 (1993), S. 34-36 
    ISSN: 1573-0972
    Keywords: Bacteriophages ; Escherichia coli ; Salmonella ; sewage ; wastewater
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    Notes: Abstract A new method for quantifying F-specific bacteriophages in wastewater is described. Somatic coliphages were also determined. Host-strainSalmonella typhimurium WG 49 was sensitive to only a few bacteriophages and this could have arisen from infection by F-RNA phages. Host-strainEscherichia coli ATCC 9723 C, however, supported multiplication of a wide range of bacteriophages present in sewage, giving plaque counts one to three orders of magnitude greater than those on F+ and F- salmonellas.
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  • 52
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    World journal of microbiology and biotechnology 9 (1993), S. 609-610 
    ISSN: 1573-0972
    Keywords: Antibiotics ; Escherichia coli ; resistance ; river water
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Of 107 Escherichia coli strains isolated from the water, sediment and fish of the Bhavani River, all of which are considered potential causes of human enteric disease, 62% were resistant to more than four antibiotics. Levels of resistance to bacitracin, penicillin, and novobiocin were generally high whereas those to polymyxin-B and chloramphenicol were much lower. A high incidence of multiple antibiotic resistant E. coli was noted in all samples and the multiple antibiotic resistance index of the strains showed that 95% of the strains originated either from man or cattle.
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  • 53
    ISSN: 0006-3592
    Keywords: Escherichia coli ; Chloramphenicol Acetyltransferase (CAT) ; Culture Redox Potential (CRP) ; Dithiothreitol (DTT) ; reducing agents ; molecular chaperones ; proteases ; heat shock ; stress response ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/μg total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/μg CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (σ32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/μg CAT min.) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 248-259, 1998.
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  • 54
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    Biotechnology and Bioengineering 51 (1996), S. 399-409 
    ISSN: 0006-3592
    Keywords: cell damage ; cell culture ; bubble aeration ; agitation ; bubble coalescence and breakup ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. © 1996 John Wiley & Sons, Inc.
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  • 55
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    Biotechnology and Bioengineering 51 (1996), S. 434-438 
    ISSN: 0006-3592
    Keywords: polyphosphate ; Escherichia coli ; phosphate starvation ; gene expression ; heterologous ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of intracellular polyphosphate on the phosphate-starvation response in Escherichia coli was studied by genetically manipulating the intracellular polyphosphate levels and by performing phosphate shifts on the genetically engineered strains. Strains that produced large quantities of polyphosphate and were able to degrade it induced the phosphate-starvation response to a lesser extent than wild-type strains, whereas strains that were unable to degrade a large intracellular polyphosphate pool induced the phosphate-starvation response to a greater extent than wild-type strains. These results have important implications for expression of heterologous genes under control of the phoA promoter. © 1996 John Wiley & Sons, Inc.
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  • 56
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    Cytotechnology 2 (1989), S. 135-140 
    ISSN: 1573-0778
    Keywords: hepatoma ; cell culture ; microcarrier ; agitation rate ; cell inoculation density ; growth rate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Hepatoma cells, HepG2, grew normally on microcarriers even at a relatively high agitation rate if sufficient time was allowed for cell attachment and adhesion. However, if a high agitation rate was applied shortly after initial cell attachment, the growth rate was retarded. This sensitivity to mechanical agitation appears to be dependent on the inoculation cell density.
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  • 57
    ISSN: 1573-0778
    Keywords: apoptosis ; bcl-2 ; cell culture ; chloramphenicol acetyltransferase ; recombinant protein ; Sindbis virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2 cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h-1). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neo cells, which were all dead by three days post-infection. Though infected AT3-neo cells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2 cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2 cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.
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  • 58
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    Cytotechnology 22 (1996), S. 263-267 
    ISSN: 1573-0778
    Keywords: biodegradable ; bone regeneration ; cell culture ; human cell osteoblasts ; polymers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The care of patients with a skeletal deficiency currently involves the use of bone graft or a non-biologic material such as a metal or polymer. There are alternate possibilities in development which involve the growth of bone cells (osteoblasts) on degradable polymer scaffolds. These tissue engineering strategies require production of the polymeric scaffold, cellular harvest followed by either ex vivo or in vivo growth of the cells on the scaffold, and exploration of the interaction between the cell and scaffold. Research into these strategies utilizes cells from a variety of species, but clinical applications will likely require human osteoblasts. This study explores the process whereby human osteoblasts are harvested under sterile conditions during joint replacement surgery from normally discarded cancellous bone, transported from the operating room to the lab, and grown in culture. This process is feasible, and the cells express their phenotype via the production of alkaline phosphatase and collagen in culture.
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  • 59
    ISSN: 1573-0778
    Keywords: CHO ; IGF-I ; serum-free ; autocrine growth ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
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  • 60
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    Cytotechnology 3 (1990), S. 157-169 
    ISSN: 1573-0778
    Keywords: acid proteinase ; cell culture ; hybridoma ; immunoglobulin cleavage ; lysosomal proteinases ; recycling reactor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An acid proteinase has been detected in culture supernate of the 9.2.27 murine hybridoma. This enzyme extensively degrades albumin and transferrin during short incubations at pH 3 and below. Limited proteolysis of the 9.2.27 IgG2a appears to occur in the culture supernate. Proteolysis is enhanced at low pH in the presence of urea or 1 M acetic acid. The proteinase activity accumulates in continuous perfusion, total cell recycle cultures, beginning during exponential growth of the hybridoma. It is destroyed by boiling and blocked by pepstatin, but not by inhibitors of cysteine or serine proteinases or by EDTA. The low pH optimum may distinguish this enzyme from the known rat and mouse aspartic acid proteinases including cathepsin D and cathepsin E.
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  • 61
    ISSN: 1573-0778
    Keywords: cell culture ; polymerase chain reaction ; retrovirus ; reverse transcriptase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Reverse transcriptase (RT) is a good diagnostic tool for the detection of retroviruses. We have developed a simple and rapid assay for RT activity in culture supernatants. A 370-base RNA sequence from the tetracycline-resistance gene in pBR322 plasmid DNA was used as a template for RT-mediated cDNA synthesis. To detect the resultant cDNA, we used the nested polymerase chain reaction. A sensitivity test using purified recombinant RT of human immunodeficiency virus type 1 demonstrated that the detection limit of this method was 10-7–10-8 units of RT activity in 20 μl of a test sample (2 × 10-9–2 × 10-10 units ml-1). This method detected RT activity in unconcentrated supernatants of cell cultures infected with human T-cell leukemia virus, Moloney murine leukemia virus, Moloney murine sarcoma virus, or Rous sarcoma virus. This nonisotopic method provides results within 10 h and is useful for quality control to detect retroviruses in cell cultures.
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  • 62
    ISSN: 1573-0778
    Keywords: cell culture ; cell culture apparatus ; dialysis membrane ; perfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We recently developed a new dialysis culture system (termed LIFROC-device) for the cultivation of lymphokine-activated killer cells (Murataet al., 1990, 1991). In the present study, we applied the LIFROC-device (400 ml culture vessel) to the cultivation of mammalian cells for the production of biologically active substances. We cultured mouse-mouse hybridoma TP-709, secreting anti-tissue plasminogen activator (tPA) monoclonal antibody (mAb), recombinant CHO GT19, secreting hGH, and human melanoma Bowes cells, secreting tPA. With the LIFROC-device, TP-709 grew to a maximal cell density of 3.8×106 cells/ml and and produced 480 μg/ml (192 mg in total) of mAb. GT19 reached a cell density of 2.2×106 cells/ml and produced 302 μg/ml (120 mg in total) of hGH. Bowes cells expanded to 4.4×106 cells/ml and secreted 8.5 μg/ml (3.3 mg in total) of tPA. The protein concentration in the culture broths of the LIFROC-device became 7–200 times higher than that of batch culture. Thus, the LIFROC-device can be applied to protein production as well as cell growth with high efficiency.
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  • 63
    ISSN: 1573-0778
    Keywords: cell culture ; colorectal cancer ; microbial contamination ; stroma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Development of primary colorectal cancer cell lines ishampered by contamination from regional microbes, overgrowthof stromal cells, and purported genetic drift from selectionpressures in vitro. We initiated 32 primaryadenocarcinomas, 3 recurrences and 6 distant metastases incell culture. Twelve cell lines from eleven tumors weregenerated (26.8%) overall. Nine of 32 primary tumorsyielded 10 cell lines, 5 were lost to contamination, 13 wereoverwhelmed by stromal cells, and 5 demonstrated no growth.Addition of isobutyl methyl xanthine (IBMX) to culturelimited fibroblastoid growth. There was no associationbetween tumor location (p = 0.535, mid-P), degree ofdifferentiation (p = 0.850, mid-P) or clinicopathologic stage(p = 0.400, mid-P), and the ability of cells to becomeestablished in culture. The majority of cell lines hadsimilar nuclear DNA content and expression of cell-surfaceantigens compared with their parent tumors. Microbialcontamination and stromal cell overgrowth present thegreatest obstacle to capturing a representative bank ofcolon tumors in vitro.
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  • 64
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    Cytotechnology 5 (1991), S. 15-30 
    ISSN: 1573-0778
    Keywords: high density ; cell culture ; serum-free medium ; hybridoma ; CHO cells ; virus production ; insect cells ; adoptive immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conclusion At the 1989 annual meeting of the U.S. Tissue Culture Associations, Ricahrd am, a leading investigator in the serum-free nutrient requirements of cultured cells, commented on the process of medium development. He noted that a survey of major media manufacturers revealed that, among the top selling mammalian cell culture media formulations, most were nearly thirty years old. This commentary is noteworthy considering the tremendous changes in cell culture understanding and derived applications which have emerged over these three decades. Fastidious cell types relatively unknown to investigators of the 1950s and 1960s are now being cultivated in defined, serum-free environments. Culture environments range from limiting dilution clonal recoveries to maintenance cultures approaching tissue densities. While research applications continue to predominate, applications of cell culture have expanded to the engineered production of biopharmaceuticals, to replacement of animal models for toxicology testing, and to the preservation, activation and expansion of human cells, tissues and organs. It is likely that future nutrient medium development will be predicated upon the design of a minimal number of defined formulations of relatively generic utility to a broad class of cell types. Analytical techniques derived from those described herein will be exploited in the user laboratory and in collaboration with the supplier to optimize the nutrient composition for the desired biological response.
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  • 65
    ISSN: 1573-0778
    Keywords: bronchus ; cell culture ; cytology ; morphometry ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Malignant A-549 lung carcinoma and adenovirus-12 SV40 hybrid virus transformed non-tumorigenic human bronchial epithelial cells (BEAS-2B) were objectively discriminated from normal bronchial epithelial (BE) cells on the basis of Papanicolaou stained nuclear features (e.g. shape, chromatin texture, hyperchromasia) and nucleolar morphology (e.g. number per cell, irregular contours). Morphometric analysis indicated that significant differences in cellular morphology existed between BE, BEAS-2B, and A-549 cells. Similar analyses of transformed, tumorigenic cell lines demonstrated that nuclear features (i.e., chromatin texture, clearing of parachromatin, hyperchromasia, variation in thickness of the nuclear envelope, sharp indentations in the nuclear envelope), and nucleolar features (i.e., degree of roundness, presence of angular projections, number per cell) discriminated chemically and virally transformed cells from spontaneously transformed cells. Nuclear and nucleolar features were correlated with the growth rate of tumorigenic cell lines. These analytical approaches will be helpful in studies of the effects of various factors (e.g. vitamin A, phorbol ester, oncogene transfection) on cellular proliferation and/or differentiation.
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  • 66
    ISSN: 1573-0778
    Keywords: proinsulin processing ; CHO ; mutant human proinsulin ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.
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  • 67
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    Cytotechnology 30 (1999), S. 149-158 
    ISSN: 1573-0778
    Keywords: bioreactor ; cell culture ; disposable ; wave agitation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications.
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  • 68
    ISSN: 1573-0778
    Keywords: cell culture ; carcinoembryonic antigen ; aspirin ; enhanced production ; Langmuir-Blodgett film
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human colorectal adenocarcinoma tumor (CW2) cells were cultivated in RPMI 1640 media containing 0–7.5 mM aspirin and 10% fetal bovine serum for the production of carcinoembryonic antigen (CEA). By adding aspirin to the media, the production of CEA per cell increased by up to one hundred fold compared to cultivation in normal media containing no aspirin, even though the total cell concentration decreased with the increase in aspirin in the media. The production of CEA was also investigated for CW2 cells cultured on silk fibroin, poly(γ-benzyl-L-glutamate) and poly(γ-benzyl-L-glutamate)/poly(ethylene oxide) diblock copolymer films prepared by the Langmuir-Blodgett and casting methods. The highest production of CEA per cell was observed for the CW2 cells on poly(γ-benzyl-L-glutamate) and its diblock copolymer films prepared by the Langmuir-Blodgett method in the medium containing 5 mM aspirin after 168 hr of inoculation. This originates from the fact that the cell density on the films in the medium containing 5 mM aspirin was the lowest under these conditions. It is suggested that CW2 cells produce CEA more effectively when the cell growth is suppressed by addition of toxic chemicals such as aspirin or by culture on unfavorable films for cell growth.
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  • 69
    ISSN: 1573-0778
    Keywords: cell culture ; hollow fiber bioreactor ; hybridoma ; micro bioreactor ; optimization ; T-flask
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.
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  • 70
    ISSN: 1573-0778
    Keywords: anchorage-dependent cell ; cell culture ; packed-bedreactor ; retroviral vector ; viral production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract To indentify a plausible large-scale production system forretroviral vector, three culture systems, i.e., batch culturewith medium exchange, microcarrier culture, and packed-bedreactor culture were compared. In batch cultures with mediumexchange, high cell concentrations were maintained for about amonth, and the harvested retroviral titer remained constant. Inmicrocarrier cultures, although cell growth was rapid, theretroviral titer was unexpectedly low, suggesting that the lowtiter was due either to serious damage to the retroviral vectoror to a reduction in the production rate of retroviral vector,caused by mechanical shear forces. Although the retroviral titer(maximum titer, 1.56 × 106) in the packed-bedreactor was a little bit lower than that obtained in the batchculture with medium exchange (maximum titer, 1.91 ×106), continuous production made it possible to increasethe cumulative titer up to 16-fold of that from the batchculture with medium exchange. Moreover, as the packed-bedreactor system requires less labor and shows excellentvolumetric productivity in comparison to batch cultures withmedium exchanges, it will be an appropriate production systemfor retroviral vector in large quantities.
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  • 71
    ISSN: 1573-0778
    Keywords: acridine derivative ; cell culture ; fluorescence microscopy ; mycoplasma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.
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  • 72
    ISSN: 1573-0778
    Keywords: cell culture ; kinetics ; Ig promoter/enhancer ; plasmacytoma ; recombinant protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.
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  • 73
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    Cytotechnology 6 (1991), S. 49-54 
    ISSN: 1573-0778
    Keywords: Bivalvia ; cell culture ; embryo ; mitosis ; scallop
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months. Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end. A possibility of growing marine invertebrates cells in long term primary culture is discussed.
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  • 74
    ISSN: 1573-0778
    Keywords: cell culture ; endosteal human osteoblasts ; maxilla ; mandible ; titanium ; biocompatibility ; alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Fragments of cancellous and cortical bone from human maxilla and mandible were cultured by the explant technique. Cells isolated by trypsinization of primary cultures were characterized as osteoblasts on the basis of intracellular alkaline phosphatase activity, the constituents of the extracellular matrix, and response to human parathormone (PTH). In culture, the osteoblasts often gave rise to superposed clumps of large cells whose cytoplasm contained endoplasmic reticulum, numerous mitochondria, vacuoles, and a dense network of intermediate filaments, often at the level of the plasma membrane. In the presence of vitamin C and 1,25-dihydroxyvitamin D3, the osteoblasts produced an extracellular matrix composed of collagen type I and various non-collagenous proteins, including osteocalcin. Biochemical test results were comparable to those reported for osteoblasts of other origins (rat calvaria, human iliac crest), and namely elevated intracellular alkaline phosphatase activity and cAMP accumulation in response to stimulation by human PTH (1–34). Osteoblasts isolated in this manner were cultured in the presence of pure titanium disks to determine the effects of exposure to this metal. Electron microscopy revealed few significant differences in cell growth and specific enzyme activity compared to control osteoblasts grown on plastic dishes, reflecting the excellent biologic and biochemical relationship between the osteoblasts and pure titanium. This experimental system thus appears suitable for biocompatibility studies, and in particular, evaluation of dental implants.
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  • 75
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    World journal of microbiology and biotechnology 14 (1997), S. 263-269 
    ISSN: 1573-0972
    Keywords: Escherichia coli ; fluorescence ; NAD(P)H ; nitrate reduction ; nitrite reduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract When nitrate was added to anaerobic resting cultures of Escherichia coli, two different profiles of NAD(P)H fluorescence were observed. E. coli is known to reduce nitrate to ammonia via nitrite as an anaerobic respiration mechanism. The profile showing single-stage response corresponded to situations where the nitrite formed from nitrate reduction was immediately converted to ammonia. The other profile showing two-stage response resulted from a much slower reduction of nitrite than nitrate. Nitrite thus accumulated during the first stage and was gradually reduced to ammonia when nitrate was depleted, i.e. in the second stage. An undamped oscillation of NAD(P)H fluorescence was also observed in the cultures showing the two-stage response. The oscillation was always detected during the second stage and seldom during either the first stage or the recovered anaerobic stage (after complete nitrite reduction). It never occurred in the cultures showing the single-stage response. The period of oscillation ranged from 1 to 5min. The possibility of the common glycolytic oscillation being responsible is low, as judged from the current knowledge of the nitrate/nitrite reductases of E. coli and the observations in this study. This is the first report on the occurrence of oscillatory NAD(P)H fluorescence in E. coli.
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  • 76
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    World journal of microbiology and biotechnology 13 (1997), S. 619-625 
    ISSN: 1573-0972
    Keywords: Alcohol ; biofuel ; corn fibre ; corn germ ; Escherichia coli ; fermentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Corn hulls and corn germ meal were both evaluated as feedstocks for production of ethanol for biofuel. Currently, these fibrous co-products are combined with corn steep liquor and the fermentation bottoms (if available) and marketed as cattle feed. Samples were obtained from wet and dry corn mills. The corn hulls and germ meal were evaluated for starch and hemicellulose compositions. Starch contents were 12 to 32% w/w and hemicellulose (arabinoxylans) contents were 23 to 64% w/w. Corn fibrous samples were hydrolysed, using dilute sulphuric acid, into mixed sugar streams containing arabinose, glucose and xylose. Total sugar concentrations in the hydrolysate varied from 8.4 to 10.8% w/v. The hydrolysates were fermented to ethanol using recombinant E. coli strains K011 and SL40. Ethanol yields were 0.38 to 0.41g ethanol produced/g total sugars consumed and fermentations were completed in 60h or less. However, residual xylose was detected for each hydrolysate fermentation and was especially significant for fermentations using strain SL40. Strain K011 was a superior ethanologenic strain compared with strain SL40 in terms of both ethanol yield and maximum productivity.
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  • 77
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    World journal of microbiology and biotechnology 15 (1999), S. 635-637 
    ISSN: 1573-0972
    Keywords: Anthocyanin ; cell culture ; conditioned medium ; strawberry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A conditioned medium (CM) prepared from strawberry suspension cultures greatly stimulated anthocyanin accumulation. CM separated by dialysis membrane showed a significant increase (p 0.05) in anthocyanin synthesis at a fraction smaller than 10,000 Da. The stimulation by CM was eliminated when the CM was treated with alkali.
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  • 78
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    World journal of microbiology and biotechnology 10 (1994), S. 346-347 
    ISSN: 1573-0972
    Keywords: Chromatography ; Escherichia coli ; plasmid ; transfection
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract A simple procedure to obtain plasmid preparations, suitable for transfecting mammalian cell lines using a calcium phosphate co-precipitation technique, is described. The protocol is based on the purification of plasmid DNA by double gel-filtration chromatography on Sephacryl S-1000 and additional slight modifications to the original transfection procedure. The purity of plasmid preparation was verified by analytical methods. The resulting preparation efficiently transfected NIH-3T3 cells.
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  • 79
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    World journal of microbiology and biotechnology 12 (1996), S. 585-588 
    ISSN: 1573-0972
    Keywords: Aerobactin ; colicin ; Escherichia coli ; growth ; plasmid profile
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified cases of urinary tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed an association of aerobactin and colicin production with the distribution of plasmid bands having a median value of 2.33 and 1.72 (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For colicin producers, the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains.
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  • 80
    ISSN: 1573-0972
    Keywords: Escherichia coli ; diamines ; homospermidine synthase ; polyamines ; spermidine deficiency ; spermidine synthase
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract The Escherichia coli mutant speE deficient in the gene encoding for spermidine synthase has no absolute requirement for spermidine but shows a retarded growth rate. This growth retardation could be unspecifically restored to the respective wild type level by exogenously supplied polyamines such as spermidine, spermine and homospermidine as well as the diamines putrescine and cadaverine. In comparison to the respective wild type, the mutant shows a two-fold increased level of endogenous putrescine but displays a reduced ability to accumulate the diamines putrescine and cadaverine. The ability to accumulate polyamines is not affected. The deleted spermidine synthase gene of the mutant was substituted by heterologous expression of the hss gene from Rhodopseudomonas viridis encoding homospermidine synthase.
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  • 81
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    World journal of microbiology and biotechnology 15 (1999), S. 65-71 
    ISSN: 1573-0972
    Keywords: Escherichia coli ; fedbatch cultivation ; growth rate ; high cell density ; plasmid stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effect of higher cell densities on the expression and segregational stability of a recombinant E. coli- B. subtilisshuttle plasmid coding for carboxymethylcellulase (CMCase) activity, was studied in E. coli DH5α. Of the various feeding policies adopted for maximal expression and stability, exponential feeding resulted in the highest biomass of 15g dry cell weight (DCW) l−1 and plasmid stability of 45%. A CMCase activity of 11400 Uml−1 was achieved as compared to 230 Uml−1 during batch cultivation. In the case of other feeding strategies viz., constant feeding, linear feeding or intermittent feeding, the plasmid stability varied between 20% to 60%. Biomass achieved ranged from 5.0 g DCW l−1 to 9.0 g DCW l−1 and enzyme activities were between 2550 Uml−1 and 6000 Uml−1.
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  • 82
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    World journal of microbiology and biotechnology 15 (1999), S. 475-480 
    ISSN: 1573-0972
    Keywords: α-Amylase ; Bacillus laterosporus ; cloning ; Escherichia coli ; secretion
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract α-Amylase gene from Bacillus laterosporus P3 was cloned and expressed in Escherichia coli HB101 and DH5α. Up to 92% of the cloned gene product was secreted into the medium by the recombinant E. coli. The recombinant crude enzyme showed improved functionality in terms of activity at a wider pH range and at higher temperature, as compared to the crude enzyme from the donor strain. The improved functionality of the cloned enzyme was due to the absence of a contaminating protease which was co-produced in the donor strain. Sub-cloning of the α-amylase gene using the promoter-probe vector, pKT240 in E. coli DH5α indicated the presence of a promoter of B. laterosporus P3 in the cloned fragment.
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  • 83
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    World journal of microbiology and biotechnology 15 (1999), S. 497-499 
    ISSN: 1573-0972
    Keywords: Amylose ; amylopectin ; Escherichia coli ; β-glucuronidase ; pullulan ; starch
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Synthesis of β-glucuronidase in starch-degrading Escherichia coli (S1) was induced by amylose, amylopectin and pullulan supplied in mineral medium as the sole carbon source (1%, w/v). The maximum activity occurred after 4 days when cultures reached the stationary phase of growth, but induction was also evident during log-phase. The effects obtained with amylose, amylopectin and pullulan were higher than that obtained with maize starch.
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  • 84
    ISSN: 1573-0972
    Keywords: Escherichia coli ; plasmids ; verotoxin
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract Three strains of verotoxin-producing Escherichia coli isolated from patients with haemorrhagic colitis harboured plasmids ranging in size from 2.7 kb to 91.2 kb. Those plasmids ranging from 2.7 kb to 6.8 kb hybridized to Shiga-like toxin I and Shiga-like toxin II gene probes.
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  • 85
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    World journal of microbiology and biotechnology 16 (2000), S. 719-724 
    ISSN: 1573-0972
    Keywords: Escherichia coli ; haemolytic uraemic syndrome ; haemorrhagic colitis ; pathogenicity ; Verocytotoxin ; VTEC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In 1977, Konowalchuk and colleagues (Konowalchuk, J., Speirs, J.I. & Stavric, S. 1977 Infection and Immunity 18, 775–779) were the first to describe Verocytotoxin-producing strains of Escherichia coli or VTEC. The surveillance of infection caused by VTEC demonstrated strains of E. coli belonging to serogroup O157 as the main cause of human infection capable of causing haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Infection with O157 VTEC results in a range of disease manifestations including abdominal cramps, vomiting and fever. This frequently leads to cases with bloody diarrhoea and HC, and approximately 10% of patients develop HUS. The symptoms of disease caused by VTEC O157 have been well documented and the pathogenic mechanisms expressed by VTEC have been the focus of considerable attention. However, the role of putative pathogenic mechanisms in the pathogenesis of disease is not fully understood. The aim of this review is to consider the clinical aspects of infection with strains of VT-producing E. coli O157 in terms of the putative pathogenic mechanisms expressed by these bacteria.
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  • 86
    ISSN: 0006-3592
    Keywords: homogenization, high-pressure ; cell disruption ; inclusion bodies ; size distribution ; centrifuge, analytical ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20°C to 5°C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4°C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.
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  • 87
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    Biotechnology and Bioengineering 39 (1992), S. 663-671 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; acetic acid ; methionine ; yeast extract ; continuous fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acetic acid formation in Escherichia coli fermentation has been studied in continuous cultures. Experimental results suggest that the limited capacity of the oxidative metabolism (perhaps the limited capacity of TCA cycle) may be responsible for acetic acid formation. At low growth rates, both anabolic and catabolic requirements may be satisfied by the oxidative metabolism. However, at high growth rates these two demands may exceed the capacity of the oxidative metabolism alone. It is proposed that under these circumstances, E. coli reorganizes the oxidative metabolism to first meet the anabolic requisition and then supply the necessary amount of energy using both the remaining capacity of the oxidative metabolism and acetic acid formation metabolism. Escherichia coli selects acetic acid synthesis as the aerobic energy source because it generates the second largest amount of ATP and NADH2. According to our proposition, acetic acid formation could be reduced by decreasing the anabolic requirement, i.e., reducing glucose uptake, or by increasing the capacity of the oxidative metabolism. These two approaches were experimentally confirmed by observing reduced acetic acid formation by reducing the glucose uptake with a yeast extract addition and enhancing the capacity of oxidative metabolism with a methionine addition.
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  • 88
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    Biotechnology and Bioengineering 40 (1992), S. 1115-1118 
    ISSN: 0006-3592
    Keywords: microencapsulation ; poly(vinylamine) ; cell culture ; mechanical strength ; erythropoietin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Poly(vinylamine) was synthesized and used to replace poly-L-lysine in forming microcapsule with alginate. Test results indicated that capsules with good mechanical strength and permeability could be obtained under the controlled treatment conditions of poly(vinylamine) and alginate. Application of the current microcapsular system to cell culture was demonstrated by the usage of erythropoietin- (EPO-) producing IW32 mouse erythroleukemia cells. The encapsulated IW32 cells grew to a density of 8 × 107 cells/mL, two times that found in the corresponding poly-L-lysine/alginate capsules. The EPO accumulation inside the microcapsule with the current encapsulation system was also higher. A concentration of 7.3 U/mL was attained as compared to 4.3 U/mL in the poly-L-lysine/alginate microcapsule. © 1992 John Wiley & Sons, Inc.
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  • 89
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    Biotechnology and Bioengineering 39 (1992), S. 732-740 
    ISSN: 0006-3592
    Keywords: cell disruption ; chemical permeabilization ; Escherichia coli ; fermentation ; protein recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Notes: Overall protein release greater than 75% in less than 1 h can be attained by exposing exponentially growing Escherichia coli cells to 0.4 M guanidine plus 0.5% Triton X-100 at 37°C in medium. Cell growth stops immediately upon addition of the chemicals, but the cells are not lysed. Guanidine concentrations lower than 0.2 M, in conjunction with 0.5% Triton X-100, do not release significant intracellular protein, nor do they inhibit cell growth. Under these conditions, the cells undergo an adaptation that confers resistance to protein release by further treatment with guanidine and Triton X-100. Cells treated with 0.2 M guanidine plus 0.5% Triton X-100 display intermediate behavior. Protein release is approximately 35%, and growth is temporarily interrupted by an extended lag phase. Subsequent resumption of cell growth results in resistant cells and no additional protein release. This resistance is shown to be reversible and is most likely due to physiological adaptation rather than genetic mutation.
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  • 90
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    Biotechnology and Bioengineering 39 (1992), S. 775-780 
    ISSN: 0006-3592
    Keywords: formate ; Escherichia coli ; formate hydrogenlyase ; cell immobilization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH2 (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, Km, and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.
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  • 91
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    Biotechnology and Bioengineering 41 (1993), S. 316-324 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; acetic acid ; inhibition ; glycine ; methionine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more β-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h-1, the methionine addition enhanced β-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h -1, the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. © 1993 John Wiley & Sons, Inc.
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  • 92
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    Biotechnology and Bioengineering 42 (1993), S. 30-36 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; fiber optic ; firefly luciferase ; on-line ; viability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 106 cells/mL, or an OD600 of 0.004, are easily detectable and concentrations greater than 1010 cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. © 1993 John Wiley & Sons, Inc.
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  • 93
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    Biotechnology and Bioengineering 42 (1993), S. 59-73 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; amino acids ; nucleotides ; biosynthesis ; linear optimization ; metabolic fluxes ; metabolic engineering ; stoichiometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. © 1993 John Wiley & Sons, Inc.
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  • 94
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    Biotechnology and Bioengineering 40 (1992), S. 271-279 
    ISSN: 0006-3592
    Keywords: carbon starvation ; Escherichia coli ; growth control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of glucose starvation to uncouple the production of recombinant β-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control β-galactosidase synthesis. β-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of β-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombiant β-galactosidase occurred continuously throughout the experiment. The cst-1 controlled β-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.
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  • 95
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    Biotechnology and Bioengineering 41 (1993), S. 221-230 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; medium optimization ; chemostat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h-1 for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. © 1993 John Wiley & Sons, Inc.
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  • 96
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    Biotechnology and Bioengineering 43 (1994), S. 399-410 
    ISSN: 0006-3592
    Keywords: lac-based promoters ; Escherichia coli ; genetic control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model that describes induction of protein synthesis from lac-based promoters has been developed and incorporated into the single-cell model of Escherichia coli with transcriptional and translational modifications. Unlike previous models of lac-based promoters, this model allows a priori prediction of the intracellular parameters controlling transcription from lac-based promoters with only the extracellular levels of substrate and inducer as inputs. Because of the structural detail of the model, it is possible to simulate different genetic constructions for comparison, such as Laclq strains versus wild-type cells, or including lacl on a multicopy plasmid. Expression from lac to tac promoters is predicted to yield 5% and 30% of the total cellular protein, respectively, with a pBR322-type plasmid. The model predicts the experimental observation that the Laclq strain is not as fully induced as the wild-type strains, even at higher inducer concentrations. Additionally, the model predicts the right order of magnitude of protein production from lac and tac promoters when mechanisms for attenuation of transcription at lower translational efficiency are considered. Finally, the model predicts that for high copy number systems ribosomes become limiting in the synthesis of plasmid-encoded proteins. © 1994 John Wiley & Sons, Inc.
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  • 97
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    Biotechnology and Bioengineering 44 (1994), S. 132-139 
    ISSN: 0006-3592
    Keywords: glycogen ; Escherichia coli ; cell growth ; acetate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations. © 1994 John Wiley & Sons, Inc.
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  • 98
    ISSN: 0006-3592
    Keywords: taxol production ; Taxus cuspidata ; cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cell culture of Taxus cuspidata represents an alternative to whole plant extraction as a source of taxol and related taxanes. Feeding phenylalanine to callus cultures was previously shown to result in increased taxol yields, probably due to the involvement of this amino acid as a precursor for the N-benzoylphenylisoserine side chain of taxol. Inthis study, we have examined the effect of various concentrations of phenylalanine, benzoic acid, N-benzoylglycine, serine, glycine, alanine, and 3-amino-3-phenyl-propionic acid on taxol accumulation in 2-year-old cell suspensions of Taxus cuspidata, cell line FCL1F, and in developing callus cultures of T. cuspidata. All compounds tested were included in media at stationary phase (suspensions) or after the period of fastest growth (calli). Alanine and 3-amino-3-phenyl-propionicacid were tested only in callus cultures and did not affect taxol accumulation. Significant increases or trends toward increases in taxol accumulationin callus and suspensions were observed in the presence of phenylalanine, benzoic acid, N-benzoylglycine, serine, and glycine. The greatest increases in taxol accumulation were observed in the presence of various concentrations of phenylalanine (1 mM for callus; 0.05, 0.1, and 0.2 mM for suspensions) and benzoic acid (0.2 and 1 mM for callus and 0.05, 0.1, and 0.2 mM for suspensions). Increases in taxol yields of cell suspensions in the presence of the most effective precursors brought taxol amounts at stationary phase from 2 μg · g-1 to approximately 10 μg . g-1 of the extracted dry weight. The results are discussed in termsof possible implications to taxol biosynthesis and in terms of practical applications to large-scale cell culture systems for the production ofthis drug. © 1994 John Wiley & Sons, Inc.
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  • 99
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    Biotechnology and Bioengineering 43 (1994), S. 275-285 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; amino acids ; linear optimization ; metabolic fluxes ; metabolic engineering ; culture stability ; oxygen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. © 1994 John Wiley & Sons, Inc.
    Additional Material: 11 Ill.
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    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 43 (1994), S. 388-398 
    ISSN: 0006-3592
    Keywords: ribosome synthesis ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Details of the mechanism for ribosome synthesis have been incorporated in the single-cell Escherichia coli model, which enable us to predict the amount of protein synthesizing machinery under different environmental conditions. The predictions agree quite well with available experimental data. The model predicts that ribosomal protein limitations are important when the translational apparatus is in high demand. Ribosomal RNA synthesis is induced by an increase in translational activity, which, in turn, stimulates ribosomal protein synthesis. However, as the demand increases still more, the ribosomal protein mRNA must compete with the plasmid mRNA for ribosomes, and the efficiency of translation of ribosomal proteins is reduced. © 1994 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
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