Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis. By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.


A rapid, quantitative microplate assay for NAD-linked D-mannitol dehydrogenase. A 96-well microtitre plate assay for NAD-linked D-mannitol dehydrogenase based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by reduced NAD is described. The assay allows rapid measurement of D-mannitol dehydrogenase in crude bacterial extracts derived by sonic disruption, in acetone permeabilized cells and in column eluates during enzyme purification. The absorbance of reaction mixtures in a microtitre plate is measured at 620 nm over a 3-4 min period using a programmable microplate reader. The rate of increase in absorbance is directly proportional to the amount of enzyme present and there is excellent correlation between activities derived using the microplate assay with those determined using conventional spectrophotometric methods.


Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum. Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.


Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1. The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.


Coiled tube membrane bioreactor for cultivation of hybridoma cells producing monoclonal antibodies. A coiled tube membrane reactor was developed for the cultivation of mouse-mouse hybridoma cells producing monoclonal antibodies. The cell and antibody concentrations obtained in the membrane reactor were higher than that obtained in a batch spinner flask without a membrane. A mathematical model has been developed to describe the membrane transport coupled growth and product formation, and the physical and kinetic constants of the system were determined.


Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system. The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses. Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion). Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system. This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate. Influence of heat loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods.


Application of carboxypeptidase C for peptide synthesis. Carboxypeptidase C partially purified from the flavedo of citrus fruit by a new, simple procedure was studied as a catalyst for peptide-bond formation. Dipeptides were obtained in high yields (80-95%) with Bz--Tyr--OEt as carboxyl-compound, and amino acid amides and amino acid alkylesters as nucleophiles. To characterize the synthesis reaction, a number of parameters such as pH, excess of the nucleophile, and the molarity of the buffer were evaluated. The yield of dipeptides depends on the side chain of the amino acid alkylester used as the carboxyl component as well as on the N-terminal protecting group. Esterase activity was minimal in the absence of a nucleophile, suggesting a modified mechanism for the synthesis reaction compared to other serine proteases. No secondary hydrolysis of the peptides formed was observed.


Optimal feed temperature for an immobilized enzyme packed-bed reactor. Optimal feed temperature was determined for a nonisothermal immobilized enzymatic reaction with enzyme deactivation in a packed-bed reactor. The optimal feed temperature was obtained by maximizing the average substrate conversion over a given reaction period. Simulation showed the optimal feed temperature to be strongly dependent on the flow dispersion, the reaction activation energy, the corresponding enzyme inactivation energy and the heat of reaction. It was also observed that in a plug flow reactor the enzyme reaction generally exhibited a lower optimal feed temperature and higher substrate conversion than in a continuously stirred tank reactor.


Recycling of salts in partition protein extraction processes. Aqueous two-phase extraction systems were developed to separate intracellular proteins from cell debris. To economize on the use of chemicals as well as to minimize environmental pollution, a closed mode of operation was sought. Various approaches to achieve recycling of salt from the primary lower phase containing 30-60% (w/w) of the added salt together with cell debris, proteins and nucleic acids were studied. Techniques assessed included microfiltration, heat treatment and the extraction with phase systems formed by aliphatic alcohols. With 20% (w/w) primary lower phase it was found to be possible to separate 95% of the salt in 3-4 theoretical steps using counter-current extraction.


Continuous separation of particles from macromolecules in split-flow thin (SPLITT) cells. A split-flow thin (SPLITT) cell with a perpendicular driving force of one gravity has been utilized for the rapid separation of micron-sized particles from macromolecules. The procedure involves the simultaneous use of two transport mechanisms and thus two operating modes: a sedimentation process controls the displacement of the particles across the thickness of the thin channel, while diffusion controls the displacement of macromolecules. The theoretical equations for these two operating modes are summarized and it is shown how the two modes can be combined to yield specified recovery factors. The theory was tested on a mixture of 10 microns polystyrene latex beads and three different proteins. The observed separation was in excellent agreement with theory. Attempts to fractionate red blood cells and plasma proteins from whole blood were only partially successful as a consequence of the weak sedimentation of red blood cells. Various remedies to this problem are suggested, the most promising of which is the use of a SPLITT cell subject to mild centrifugal forces.


Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins in Escherichia coli. The major leftward early promoter of phage lambda pL, has frequently been used to drive expression of heterologous genes in Escherichia coli. pL is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressing pL involves exposing cells to nalidixic acid, which results in the "activation" of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of the E. coli LexA protein, resulting in induction of the SOS regulon (at least 15 E. coli genes, including recA). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind-) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying a cl+ defective lysogen. Synthesis of two heterologous proteins, human alpha 1-antitrypsin and a fusion protein partially derived from the Plasmodium falciparum circumsporozoite surface antigen, was examined in the wild-type and mutant strains. The maximum alpha-1 antitrypsin concentration achieved was improved by 50% when the recA o strain was used rather than the wild-type; however, only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein were observed. Use of the lexAind- strain resulted in a decrease in the maximum concentration attained for both heterologous products.


Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors. Production of heterologous proteins by yeast secretion imposes additional factors that need to be considered, which do not appear with production by direct expression. These include additional intracellular polypeptide processing dynamics through the secretory organelles and the protein concentration in the culture medium, which is the usual final destination of the product. Optimal control theory is applied to optimize fed-batch production of secreted protein. We maximize an objective function that includes both total production rate and product concentration. A mutant invertase is chosen as the model heterologous secretory protein. Optimal control control strategies have been obtained for the use of two different promoters for the gene transcription, a dere-pressible SUC2 promoter and a strong glycolytic GPD promoter. With the use of the strong GPD promoter, achieving maximum production occurs on the singular arc of maximum specific growth rate. As the object switches to maximum product concentration, operation occurs for longer periods of time at a slow glucose singular arc condition. The optimal control for maximizing protein production with the weak SUC2 promoter requires transitions between high and low glucose concentrations associated with multiple distinct singular arc conditions. For maximum product concentration, the high concentration branches of the singular arc supporting maximum growth rate and maximum secretion rate disappear. Operation stays essentially on the low glucose concentration branch of the singular arc, which maximizes the protein production rate and minimizes the dilution of the broth product concentration.


Enzymatic cell lysis for product release. The development of expression systems for recombinant proteins and recombinant protein particles which cannot be secreted and are located in specific cell locations necessitates the development of novel, more selective techniques for cell lysis and or product release. Mechanical cell disruption methods do not discriminate the release of the desired product from among a host of other contaminating molecules and cell debris, and they may also damage the protein product. In contrast, the use of lytic enzyme systems, which can provide biological specificity to the process of cell lysis and product release, shows an interesting potential for controlled lysis. In this chapter we have reviewed the enzymes presently available for cell lysis of bacteria and yeast and have described mathematical models developed to describe the process of lysis, particularly of yeast cells, including simple, structured, and population balance models. Factors affecting the kinetics of lytic enzyme reactions, enzyme recovery, and the effect on downstream operations as well as conditions for use have been discussed. Sources of lytic enzymes and latest developments on enzyme production have been reviewed. Finally, the potential for large-scale use of lytic enzyme technology has been discussed. The design and use of lytic enzyme systems for differential product release from microbial cells have been reviewed. Lytic enzyme systems are usually specific either for yeast or for different types of bacteria. The activity profile of a lytic system will have an effect on the product distribution. This profile can be manipulated at the genetic, physiological, production reactor, enzyme purification, and lysis reactor levels. Alternative process designs that will allow the sequential release of products from different cell locations are discussed. Alternatives are explored by process modeling, process simulation, and optimization techniques. These studies show that the use of lytic enzyme systems has tremendous promise as a method of controlled lysis and differential product release.


Immobilization of microbial cells in a mixed matrix of silicone polymer and calcium alginate gel: epoxidation of 1-octene by Nocardia corallina B-276 in organic media. Immobilization of Nocardia corallina B-276 was examined for production of 1,2-epoxyoctane from 1-octene in the presence of n-hexadecane as the organic solvent. Hydrophobic silicone polymer was the most suitable material for entrapment of the cells. Coentrapment of aqueous reaction medium into the silicone polymer matrix improved the epoxide productivity. It was also effective to immobilize the cells in a mixed matrix composed of silicone polymer and calcium alginate gel involving the reaction medium. In the organic monophase, the amount of epoxide accumulated with the cells immobilized in an almost equivolumetric composite of both materials was 2 and 7 times the amounts in the silicone and alginate single matrices, respectively, and it became larger than with the free cells in the aqueous-organic two-liquid phase after a longer period of batch operation. The use of such an optimized composite matrix enabled us to perform a relatively simple operation of the continuous three-phase bioreactor.


Defining an optimal carbon source/methionine feed strategy for growth and cephalosporin C formation by Cephalosporium acremonium. The effect of the method of methionine addition, growth-limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose-limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to mumax (mu approximately 0.036 h-1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (mu approximately 0.01 h-1) with sucrose for the duration of the fermentation.


Chemical, physical, and biochemical concepts in isolation and purification of proteins. In this chapter the physicochemical basis of protein retention in adsorption chromatography has been examined. It is evident from this treatment that significant developments are in process with regard to both the theory and practice of high-resolution chromatographic methods, particularly at the preparative level with adsorptive stationary phases. The recognition that most purification strategies must be based on multidimensional multistage procedures presents numerous challenges for the protein chemist, the chromatographic scientist, and the biochemical engineer. The urgency for these developments has been stressed by the potential of modern biotechnology to produce large quantities of new proteins that must be obtained in highly purified form. The capabilities that these advances present will prove catalytic in the materials sciences as new solid phases are examined with particular properties for large-scale utilization. Similarly, new initiatives in protein engineering will lead to greater utilization of the fusion handle approach for selective protein isolation and recovery. The realization of these capabilities will represent an enthusiastic and innovative method that will revolutionize the role of bioprocess development over the next decade.


Fluidized bed adsorption for whole broth extraction. Fluidized bed adsorption using a high-density synthetic resin has proven to be an invaluable technique for separating novel compounds from unfiltered fermentation broths during the very early stages of fermentation development, where product concentrations are typically in the parts per million range. Previous initial downstream processing strategies consisted of cell separation from whole broth or direct extraction with water-immiscible solvents, both of which resulted in lengthy time cycles, conflicts with existing operations requiring the use of high-cost centrifugal separators, and environmental/solvent recovery concerns. Laboratory and subsequent pilot plant process development work along with concomitant improvements in yield, quality, and time cycles are presented for one of several fluidized bed processes piloted in Merck's Natural Product Isolation facility.


Separation processes in biotechnology. Ion-exchange processes. Through the use of several differentiating mechanisms, ion exchangers can separate ionic and nonionic materials, solutions containing only ionic species, and even completely nonionic mixtures. Although the mechanisms are distinct in their mode of operation, the resin characteristics that influence the results are largely the same. A practical understanding of the resin properties involved is all that is necessary to begin to use ion-exchange resins successfully. Ion exchange owes most of its history to water treatment, which has provided the economic and technological driving force in the past for the development of improved resins. However, specialty applications such as those in biotechnology are steadily becoming major factors in industry, perhaps not in shear volumes of resin used, but certainly in the value added by the process. The field of biotechnology no doubt holds many of the exciting new applications for ion exchange.


Practical considerations in the measurement of culture fluorescence. We have examined practical considerations associated with the use of a commercially available fluorescence probe for in situ measurements in bioreactors. The optical path length of the measurement was first determined and a flow cell subsequently designed. The environment (agitation/aeration rates) of the probe was found to have a significant influence on the measurement. These effects were eliminated by placing the probe in a recycle loop using the flow cell. Fluorescence measurement in the recycle loop was verified to be representative of the cell sample and to not affect cell metabolism.


Separation processes in biotechnology. Aqueous two-phase separations. Aqueous two-phase systems are useful for separation of a wide range of water-compatible substances (from peptides to cells). The selectivity of the separation normally increases with the size of the partitioned molecules or particles. The partition and separation capacity can be influenced in a number of ways, including electric charge, hydrophobicity, or specific ligand binding. Because of the simpleness in operation and high capacity, aqueous two-phase systems are well suited for large-scale purification of biomaterials such as enzymes and other specific proteins.


Separation processes in biotechnology. Precipitation. Precipitation has a clear role in downstream processing for product concentration, and ongoing developments promise a more important role in fractionation. Its advantages include adaptability to continuous processing and larger scales, the wide variety of possible precipitants, and the ability to retain biological activity. The focus here has been on precipitation of proteins, but many of the principles apply to nucleic acid removal and precipitation of lower-molecular-weight biological products, such as pharmaceuticals.


Ion exchange in purification.

Ion exchange in purification. Biochemicals have large complex three-dimensional structures with multiple weak acid and weak base groups on their surfaces. The ionization of these groups results in heterogeneous charge structures and complex ion-exchange equilibria. In this chapter the ion-exchange equilibria of amino acids are reviewed first. The ionization of the alpha-COOH group has a strong effect on the apparent equilibrium affinities of amino acids. Salt concentration and pH can also significantly affect the apparent affinities and valences. The implications on the ion-exchange equilibria of peptides and proteins are discussed. The second focus of this chapter is on the local equilibrium models of multicomponent chromatographic processes. The differences between unidirectional and two-way flow conditions, linear and nonlinear systems, elution and displacement processes, and isocratic and gradient operations are discussed. Fundamental analysis of these phenomena is applied to formulate strategies to shorten cycle time, improve resolution, raise product concentration, and increase throughput.


Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection. Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.


Protein secretion systems in microbial and mammalian cells. Secretion is an attractive production mode for proteins that require posttranslational modifications carried out in the secretory pathway. For example, amino acid chains fold properly, disulfide bonds form correctly, and glycosylation occurs accurately as the protein is secreted. In addition, recovery of secreted proteins is simplified by the fact that cells need not be broken and the product may be a major species present in a minimal synthetic culture medium. The current state of the art permits the production and secretion of proteins by heterologous cells. While future efforts will be required to improve the efficiency of secretion, current methods yield commercially interesting levels of secretion from E. coli, B. subtilis, S. cerevisiae, Aspergilli, and many mammalian cell types. Each of these systems offers certain advantages, and the choice of system depends on the specific protein to be secreted. High-value human therapeutic products such as plasminogen activators are reasonable candidates for secretion from mammalian cell lines, while industrial proteins such as calf prochymosin require a more economical host, such as baker's yeast or Aspergillus. The wide variety of posttranslational modifications observed in nature may prevent any single secretion system from dominating the field for many years to come.


Altered gene expression in plants due to trans interactions between homologous genes. We have attempted to explain co-suppression in terms of a hypothesis whereby homologous sequences are able to interact somatically in trans, in a manner influenced by sequence context or location. We have speculated that there might be mechanistic similarities between co-suppression and some other trans interaction and epigenetic phenomena in plants, fungi and animals; and that it might be the sequence context in which a gene lies, or its location in the genome that influences the likelihood that it will participate in these phenomena. We suspect that elucidation of the mechanisms behind these phenomena will play a role in developing a better understanding of the relationship between nuclear architecture and gene expression. This, in turn, will be helpful in understanding the developmental regulatory mechanisms that exert control over somatic trans interactions in plants, and perhaps in understanding some aspects of the basis of cellular differentiation in development.


New approaches to the synthesis of antiviral nucleosides. There is a pressing, worldwide need for new antiviral agents. The chemical synthesis of novel nucleosides for chemotherapeutic screening usually involves multistage processes which can be time consuming. The application of enzymatic methods for the synthesis and modification of antiviral nucleosides shows great promise because of the simplicity and high specificity of enzymatic reactions.


Hen egg white lysozyme expressed in, and secreted from, Aspergillus niger is correctly processed and folded. We transformed Aspergillus niger with the full length cDNA gene encoding hen egg-white lysozyme (HEWL) and its secretion signal sequence. Lysozyme levels up to 12 mg/l were secreted when expression was controlled by the A. awamori glucoamylase (GAM) promoter and 1 mg/l when controlled by the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. N-terminal sequence analysis of the recombinant protein indicated that the signal peptide was correctly processed by the A. niger secretory apparatus. The specific catalytic activity of the recombinant protein was identical to that of authentic hen lysozyme. The recombinant HEWL was examined by 2D 1H-NMR spectroscopy and shown to have a spectrum identical to that of authentic HEWL indicating that the protein was correctly folded.


Applications of imaging spectroscopy in molecular biology. II. Colony screening based on absorption spectra. Digital imaging spectroscopy has been used to obtain the grayscale spectrum of colored bacterial colonies directly from petri dishes. Up to 500 individual colony spectra can be simultaneously recorded and processed from a single plate. Spectra can be obtained in the visible to near infrared region (400nm-900nm) with 10nm resolution. Instrument response is normalized through run-time radiometric calibration such that each grayscale spectrum can be converted to the ground-state absorption spectrum of the colony. In this study, mutants of the photosynthetic bacterium Rhodobacter capsulatus have been differentiated by the absorption spectra of their pigment-protein complexes. This imaging technique is applicable to chromogenic systems in which colony and/or media color (e.g. indicator plates) provides a quantitative indicator of gene expression.


Controlled release of interleukin-2 from biodegradable microspheres. We have evaluated the use of biodegradable poly(DL-lactide-co-glycolide) microspheres for the controlled release of interleukin-2 (IL-2) and its modified forms: succinyl IL-2 (SIL-2) and polyethylene glycol-modified IL-2 (PEG IL-2). We show that a microsphere formulation can be prepared from PEG IL-2 using HSA as an excipient which, after an initial burst, releases 2-3% PEG IL-2 per day in a bioactive form continuously over a 20- to 30-day period.


Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents. The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.


Immobilization of genetically engineered cells: a new strategy for higher stability. The r-DNA clones improve the bioprocess and provide better economics, if and when properly developed. In recent times, many approaches were made to improve the stability of recombinants in a reactor which includes both genetic and environmental methods, but many of them were proved to be unsuccessful in the scale-up process. The immobilization technique, exploited recently for the cultivation of recombinants, in many cases gave high cell concentrations, better expression of cloned gene products and also maintained plasmid stability for longer periods in a host under continuous operation in comparison to a free cell system. Many plasmids and hosts were tested for improved stabilities. So far, no explanation was provided for higher stability in the immobilized system. However, it was observed to reduce the competition between the plasmid harboring and plasmid free cells in a matrix. The stability of recombinant strains under immobilization technique is affected by various factors, and these are important parameters for the commercial process. Thus, the immobilization system is promising for the successful cultivation and scale-up of genetically engineered cells.


Recombinant brewer's yeast strains suitable for accelerated brewing. Four brewer's yeast strains carrying the alpha-ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The alpha-ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae. In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.


Enzymatic synthesis of polyaniline film using a copper-containing oxidoreductase: bilirubin oxidase. Electroactive polyaniline films have been synthesized by using a copper-containing oxidoreductase, bilirubin oxidase (BOD). Enzymatic polymerization took place on the surface of BOD-adsorbed solid matrix which was in contact with a buffer solution containing aniline. Optimum conditions for enzymatic polymerization of aniline were investigated. Elemental analysis and IR spectroscopy indicated that the enzymatically synthesized film was polyaniline. The cyclic voltammetric studies demonstrated that the polyaniline film was electrochemically reversible in the redox properties in acidic aqueous solutions. Since the film retained enzymatic activity of BOD which was employed as a catalyst for polymerization, enzymatic polymerization seems promising in preparation of immobilized enzyme membranes.


Electrical effects on the proliferation of living HeLa cells cultured on optically transparent electrode surface. Low d.c. potential application induced changes of cellular morphology and growth of living cells on a potential-controlled electrode. At a potential range higher than +0.7 V (vs. Ag/AgCl), serious electric effects on cell viability, membrane permeability, and cytoskeletal morphology of HeLa cells were observed. On the other hand, at lower than +0.5 V no effect was observed. At the boundary potential range between +0.5 V to +0.7 V, where HeLa cells were cultured on the potential-controlled optically transparent In2O3 electrode (OTE) surface, intriguing effects on HeLa cells appeared. At this potential range, where HeLa cells cultured on a potential-applied OTE, all the cells were alive accompanying morphological change. The morphology of HeLa cells returned to their normal spindle shape, when potential application to the electrode was cut off. At a potential of +0.65 V, cell proliferation ratio of cultured cell on an electrode was about one-fifth of that on a non-controlled electrode. These results suggest that low d.c. electrical effects induce significant change in cellular morphology and function.


Cell growth and enzyme synthesis of a mutant of Arthrobacter sp. (DSM 3747) used for the production of L-amino acids from D,L-5-monosubstituted hydantoins. A microorganism with the ability to form L-tryptophan from D,L-5-(3-indolyl-methyl)hydantoin (D,L-5-IMH) was isolated and identified as Arthrobacter sp. (DSM 3747). After isolation of a mutant with high tryptophan production activity but low tryptophan degradation, cultural conditions were optimized to achieve high amounts of biomass with good specific activities concerning the enzymatic hydantoin-cleaving reactions. The ability of the microorganism to perform these bioconversions was found to be inducible by D,L-5-IMH as well as to be dependent on the presence of Mn2+. The highest specific D,L-5-IMH-cleaving activity of the cells was observed in the exponential phase of growth. The addition of yeast extract to the mineral salts medium was found to be essential for obtaining biomass concentrations of about 25 g l-1 cell dry mass by bioreactor cultivations. In order to obtain a constantly high growth rate, feeding of the C-source was pO2-controlled. The inducer D,L-5-IMH had to be continuously fed to prevent a decline of the L-tryptophan-forming enzyme activities, because it was subjected to degradation with the enzymes induced and higher concentrations of D,L-5-IMH aggravated the growth significantly. The synthesis of the enzymes was also inducible, when inducer and Mn2+ were not added until the late growth phase. Using this process, the consumption of D,L-5-IMH was reduced remarkably. So, under these conditions biomass concentrations of 25 g l-1 cell dry weight with a specific enzymatic activity of 0.20 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 13 h. Using 1 g l-1 of the chemically modified inducer D,L-5-(3-indolylmethyl)-3-N-methylhydantoin, which was not degradable by the microorganisms, a biomass concentration of 28 g l-1 cell dry weight with a specific activity of 0.34 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 28 h.


Production of L-tryptophan from D,L-5-indolylmethylhydantoin by resting cells of a mutant of Arthrobacter species (DSM 3747). The reaction parameters and the stereospecificity of the enzymatic cleavage of D,L-5-indolylmethylhydantoin in producing L-tryptophan with resting cells of Arthrobacter sp. DSM 3747 were studied. When intact cells were tested, the optimal pH was between 8.5 and 9.0 and the optimal temperature was 50 degrees C. Both, L-N-carbamoylase and hydantoinase could be stabilized over 24 h at 30 and 40 degrees C by the addition of D,L-5-indolylmethylhydantoin. Furthermore, the hydantoinase was stable over 24 h at 50 degrees C by the addition of 0.5 mM Mn2+ ions. The treatment with sodium desoxycholate turned out to be successful in overcoming the poor availability of D,L-5-indolylmethylhydantoin for the cells. The optimal temperature with permeabilized cells decreased to 30 degrees C and therefore ensured a good enzyme stability. While the L-N-carbamoylase proved to be absolutely L-specific, the hydantoinase led to a mixture of enantiomers of N-carbamoyltryptophan. The produced D-N-carbamoyl-tryptophan caused an inhibition of the L-N-carbamoylase. The transformation yield from D,L-5-indolylmethylhydantoin always reached 100%.


Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium. For the production of recombinant human interleukin-2 (IL-2) two different culture processes, a 1-2 liter homogeneous stirred bubble-free aerated system and a dense cell hollow fibre bioreactor were compared. Cultivations were carried out with serum- or protein-free medium formulations. In the stirred culture 0.75 mg IL-2 were produced with 1 l of perfused medium at a maximum cell number of 3 X 10(10). The product yield in the hollow fibre module was only 0.23 mg l-1 at a maximum cell number of 6 X 10(10). In contrast to results with hybridoma or EBV-transformed cell lines, in which hollow fibre bioreactors showed comparable efficiency to perfused stirred tank reactors, the tissue-like cell density is disadvantageous as adherent cells tend to stick together leaving insufficient intercellular space for removal of product.


A yeast system for stable expression of hepatitis B surface antigen. We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.


Differential stability of recombinant human insulin-like growth factor II in Escherichia coli and Staphylococcus aureus. Recombinant human insulin-like growth factor II (IGF-II), produced as a soluble extracellular fusion protein, was shown to be proteolytically degraded in Escherichia coli. In contrast, the fusion protein secreted from Staphylococcus aureus was stable and the full length product could be recovered by affinity chromatography. After site specific cleavage of the fusion protein, soluble IGF-II with biological activity was obtained without refolding procedures. These results demonstrate that a eukaryotic protein unstable in E. coli can be stabilized by expression in a Gram positive host. The full-length fusion protein from S. aureus was used to characterize the protease responsible for the degradation in E. coli. Biochemical and genetic analysis suggests a specific degradation by the outer membrane protease (OmpT).


Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride. A physical method for immobilization of liver alcohol dehydrogenase (ADH) by hydrophobic adsorption onto a supporting membrane of polyvinylidene difluoride (PVDF) was performed. Simultaneously, a physicochemical characterization of the immobilized enzyme regarding its kinetic behaviour was performed. The activity/pH profile observed points to an effect of pH on activity that is completely different from the case of ADH in solution. The disturbance in the typical bell-shaped profile owing to the fact that the enzyme was immobilized is explained on the basis of a potent limitation to the diffusion of the protons in the support. The findings of the present work also reveal the existence of an effect that limits free external diffusion of the substrate towards and/or the product from the support; this effect seems to be the determinant of the overall rate of the enzymatic reaction and is thus of great importance in the effective kinetic behaviour (v([S])) of immobilized ADH, whose kinetic behaviour is complex (non-Michaelian), as may be seen from the lack of linearity observed in the corresponding double reciprocal and Eadie-Hofstee plots. By non-linear regression numerical analysis of the v([S]) data and application of the F-test for model discrimination, the minimum rate equation necessary to describe the intrinsic kinetic behaviour of PVDF-immobilized ADH proved to be one of the polynomial quotient type of degree 2:2 (in substrate concentration).


Microwave hydrolysis of peptides and proteins for amino acid analysis. The speed, accuracy, and convenience of microwave hydrolysis make it a promising new technique in the protein chemistry field. Acid hydrolysis of protein samples has traditionally been the rate limiting step in amino acid analysis. Complete hydrolysis and analysis of proteins and synthetic peptides is now possible in less than an hour using microwave hydrolysis. This represents a significant improvement in quality control monitoring capability during the synthesis of high purity peptides. Microwave hydrolysis will also facilitate basic research in molecular biology and streamline sample preparation procedures for amino acid assays in the food science area.


CP-82,996, a novel diglycoside polyether antibiotic related to monensin and produced by Actinomadura sp. A new polyether antibiotic CP-82,996 (C50H86O16) was isolated by solvent extraction from the fermentation broth of Actinomadura sp. (ATCC 53764). Following purification by silica gel column chromatography and crystallization, the structure of CP-82,996 was determined by a single crystal X-ray analysis. The structure is closely related to monensin, but is unique in that it contains two sugar groups, whereas monensin has none. The 1H and 13C NMR chemical shifts and assignments for CP-82,996 were elucidated, and they were compared with those determined previously for monensin. CP-82,996 is active against certain Gram-positive bacteria, and is a very potent anticoccidial agent. It effectively controlled chicken coccidiosis caused by several Eimeria species at 5-10 ppm in feed, and is 10-20 times more potent than monensin.


Extending the chemistry of enzymes and abzymes. Selective chemical modification can be used to create novel proteins, particularly enzymes and antibodies, with altered specificities and catalytic activities in vitro. Modification strategies now being developed should soon yield a wide spectrum of novel biomolecules whose activities are optimized for specific industrial processes or therapeutic applications.


Towards an understanding of the genetics of bacterial metal resistance. Many bacteria show great promise for use in metal recovery. However, the genetics of metal-leaching, accumulation-resistance, and oxidation/reduction mechanisms of these bacteria is still an area of research in its infancy. The introduction of such genes into bacteria of economic importance would have application in biomining and environmental bioremediation.


Progress towards rabies control.

Progress towards rabies control. Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the world still depends on vaccines derived from neural tissue which were introduced half a century ago. Considerable advances have been made in our understanding of the molecular biology of rabies virus, and genetically engineered recombinant viruses (vaccinia-rabies virus glycoprotein) have been developed. These may facilitate the control of rabies in some species by oral vaccination campaigns.


Gene replacement in Bordetella pertussis by transformation with linear DNA. We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.


Protective surface antigen P69 of Bordetella pertussis: its characterization and very high level expression in Escherichia coli. The surface antigen, P69 of Bordetella pertussis, an N-terminal fragment of the precursor protein, P93, is likely to be an important component of future subunit vaccines against whooping cough. We have expressed several defined N-terminal fragments of P93 in E. coli and compared their electrophoretic mobilities with that of purified P69 from B. pertussis. These experiments show that P69 is considerably smaller than the 69 kD originally estimated from its gel mobility and is probably 60.4 kD in size. Our initial plasmids expressed only very low levels of this antigen. We diagnosed the limiting factor to be a poor ribosome binding site (RBS) by demonstrating a large stimulation of expression on a two-cistron plasmid. The limitation of expression could be completely overcome by only two base changes close to the initiation codon, resulting in a further increase in expression of P69 at levels to 30-40% total cell protein. Although the protein accumulated as insoluble inclusion bodies, it could be solubilized by guanidinium chloride.


High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product. We have developed a direct expression system for high-level production of recombinant human interleukin-6 (rhIL-6) in Escherichia coli. In this system, (i) the natural N-terminal coding region of the hIL-6 gene was replaced by a synthetic sequence containing A-T rich codons, (ii) dual Shine-Dalgarno (SD) sequences were employed, (iii) an A-T rich segment was inserted in front of the initiation codon to avoid putative mRNA secondary structure in the region and (iv) the natural amber termination codon of the hIL-6 gene was changed to an ocher stop codon. The hIL-6 polypeptide, synthesized at a high level, formed cytoplasmic inclusion bodies. After refolding, the N-terminal methionine was removed by aminopeptidase-P in vitro. The purified recombinant hIL-6 had B-cell differentiation activity equivalent to natural IL-6 from a human T-cell culture.


Immobilized and free cell continuous cultures of a recombinant E. coli producing catechol 2,3-dioxygenase in a two-stage chemostat: improvement of plasmid stability. The immobilization of recombinant strains of E. coli W3110/pTG205 in K-carrageenan gel beads improves the plasmid stability during continuous cultures in the absence of selection pressure. Since, xyl E gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida) transcription is controlled by the trp promoter, the effects of tryptophan (repressor) and 3 beta-indolyl acrylic acid (derepressor) on pTG 205 stability and enzyme production have been studied in both free and immobilized cell cultures. A two-stage continuous culture system running for 150 h is described. In the first stage an immobilized culture is performed in the presence of tryptophan with a significant plasmid stability. The cells released from the gel beads are continuously transferred in the second stage reactor where expression is induced by 3 beta-indolyl acrylic acid. In these conditions an efficient production of catechol 2,3-dioxygenase is observed.


Selective adherence of neurons and glial cells from dissociated cerebral and spinal cord microcarrier cultures. In stationary cultures of dissociated brain and spinal cord grown on microcarriers (MCs), the neuronal and ependymal cells attached to the MCs forming floating aggregates in which they grow in a three-dimensional pattern. The glial and meningeal elements on the contrary, tend to dissociate from the aggregates and adhere to the plastic dish where they divide to form a monolayer. This different behavior of CNS components is not observed in rotating cultures in which all CNS cells remain attached to the MCs and develop into mature floating structures. This cell separation in stationary MC-cultures which is documented here by SEM and immunocytochemistry, may be useful for analysis and evaluation of the metabolic biochemical events of each of the cellular components derived from the same culture.


Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement. Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium.


Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production. A murine hybridoma cell line (167.4G5.3) was cultivated in batch mode with varying inoculum cell densities using IMDM media of varying fetal bovine serum concentrations. It was observed that maximum cell concentrations as well as the amount of monoclonal antibody attainable in batch mode were dependent on the inoculum size. Specifically, cultures with lower inoculum size resulted in lower cell yield and lower antibody concentrations. However, in the range of 10(2) to 10(5) cells per ml, the initial cell density affected the initial growth rate by a factor of only 20%. Furthermore, specific monoclonal antibody production rates were independent of initial cell density and the serum concentration. Glutamine was the limiting nutrient for all the cultures, determining the extent of growth and the amount of antibody produced. Serum was essential for cell growth and cultures with initial cell concentrations up to 10(6) cells per ml could not grow without serum. However, when adapted, the cells could grow in a custom-made serum-free medium containing insulin, transferrin, ethanolamine, and selenium (ITES) supplements. The cells adapted to the ITES medium could grow with an initial growth rate slightly higher than in 1.25% serum and the growth rate showed an initial density dependency-inocula at 10(3) cells per ml grew 30% slower than those at 10(4) or 10(5). This difference in growth rate was decreased to 10% with the addition of conditioned ITES medium. The addition of conditioned media, however, did not improve the cell growth for serum-containing batches.


Two stage cultures for the production of berberine in cell suspension cultures of Thalictrum rugosum. Two stage culture systems were examined in cell suspensions of Thalictrum rugosum which produce berberine in a growth associated manner. The utilization of indole 3-acetic acid (IAA) as a growth regulator, in place of 2,4-dichlorophenoxyacetic acid (2,4-D), at various growth phases was investigated. Although the enhancing effect was clear when it was used separately, in combination with 2,4-D effect of IAA was suppressed and medium change from 2,4-D to IAA was detrimental to product formation. The decrease of berberine production in this two stage culture may be due to the loss of conditioning factors associated with medium replacement.


Purification of the glycoprotein allergen Ag7 from mugwort pollen by concanavalin A affinity chromatography. Two affinity columns comprising immobilized concanavalin A (Con A), Con A-Sepharose and Con A-XP3507, were evaluated for their purifying ability for the glycoprotein allergen Ag7 from a partially purified extract of mugwort pollen. The most pronounced difference between the two columns was the nature of their nonspecific interactions; hydrophobic interactions were dominant with Con A-XP3507, whereas ionic interactions were dominant with Con A-Sepharose. Both Con A-columns were effective for purifying Ag7 with a recovery of 50% after specific elution with displacing sugars. The inclusion of 1.0 M NaCl and 20% ethylene glycol in the elution medium was useful for desorbing nonspecifically bound material, prior to specific elution of adsorbed Ag7 in the presence of the displacing sugars, alpha-methyl glucoside and alpha-methyl mannoside. The most efficient purification of Ag7 was achieved with Con A-Sepharose at room temperature rather than at 4 degrees C. Affinity chromatography with Con A-XP3507 resulted in a slightly more contaminated product (purity 54%) than with Con A-Sepharose (purity 64%).


Measurement of oxygen concentration gradients in gel-immobilized recombinant Escherichia coli. In this study, an oxygen microsensor was used to measure oxygen concentration profiles in carrageenan gel particles containing growing, immobilized Escherichia coli B (pTG201). Profiles, which were measured at intervals during continuous culture of gel slabs and beads, became increasingly steep with time. The oxygen penetration depth in the gel decreased with time, eventually reaching a steady state value of approximately 100 microns for both gel beads and slabs. A reaction-diffusion model employing zero-order cell growth kinetics was found to provide an excellent fit to the experimental concentration data. Growth rates estimated from profiles obtained during the first few hours of culture were 0.24h-1 (gel slabs) and 0.18h-1 (beads), compared to a value of 0.30 h-1 measured in free-cell suspensions at 25 degrees C.


Effect of free-cell growth parameters on oxygen concentration profiles in gel-immobilized recombinant Escherichia coli. Oxygen concentration profiles in immobilized recombinant cells were measured using a microsensor. Experimental results showed that the final depth of oxygen penetration in the carrageenan gel was always in the range 80-100 microns for the strains and media used, although profiles of oxygen concentration during the early stages of cell growth depended on the strain and nutrient medium used. Variations in the oxygen profiles corresponded to differences in kinetic parameters measured for the same strains and media in free-cell experiments.


Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme. A yeast lytic enzyme was covalently immobilized on an enteric coating polymer, Eudragit S, that is reversibly soluble and insoluble (S-IS) depending on the pH of the reaction medium. The yeast lytic enzyme immobilized on Eudragit S (Y-E) showed a sharp response of solubility to slight changes in pH without decrease in enzymatic activity. The specific activity per amount of enzyme protein of Y-E for dry yeast cells was about two-thirds that of the native enzyme. In both lysis reactions of dry and pressed baker's yeast cells, changing the pH of the reaction medium from 7.0 to 4.8 at an appropriate interval allows the insoluble Y-E and the reaction products (soluble protein for dry yeast cells and invertase and soluble protein for pressed baker's yeast cells) to be repeatedly separated. The reaction method using a reversible S-IS enzyme is a promising procedure for repeated use of the enzyme in a heterogeneous reaction system containing yeast cells as a substrate.


Cloning of a gene coding for phosphotransacetylase from Escherichia coli. A phosphotransacetylase gene (pta) has been cloned from a genomic DNA library of Escherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities of pta+ plasmid-containing strains were amplified about 150-fold under control of the lac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pta gene was found to be downstream of ackA by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of the purF-folC-hisT region of the chromosome.


Reconstituted viral envelopes--'Trojan horses' for drug delivery and gene therapy? Reconstituted viral envelopes (RVEs) are formed by solubilizing intact virus in detergent and reassembling the envelope on removal of detergent. RVEs can be formed in the presence of agents that become encapsulated and can then be utilized in vitro and in vivo for drug delivery, cell destruction, transfer of membrane components, and as vectors for genetic engineering. The problems with biotechnological applications of RVEs and possible strategies for overcoming them are discussed in this article.


Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases. Recent developments in DNA and RNA amplification technology are enabling the design of ultra-sensitive diagnostic assays for infectious diseases. The leading amplification technology is the polymerase chain reaction (PCR). An alternative approach, Q-beta amplification, also promises remarkable speed and precise quantification of assay results.


Repacking protein interiors.

Repacking protein interiors. Several goals of protein engineering may be achieved through redesign and repacking of protein interiors. The effects of interior apolar substitutions on protein stability depend strongly on the site of the substitution. One reason for this is that protein interiors have properties both of apolar liquids and of crystalline solids. Substitutions at interior sites affect the stability of a protein by changing the hydrophobicity, but each site in a protein has a characteristic energy associated with introducing packing changes, and the net stability depends on both of these factors.


Vision assisted robotics and tape technology in the life-science laboratory: applications to genome analysis. Recent proposals to analyze a number of major genomes have created a need for methods of rapidly manipulating and assaying very large numbers of specimens containing DNA fragments. Systems for sampling semi-solid biological material from mixed disordered arrays are required. These samples need to be sorted in an ordered format and stored in minimum space at known locations. It should be possible to recall them quickly from stock, individually or collectively, for duplication or re-ordering into new arrays for appropriate analysis. This article reviews a range of problems associated with high speed multiple specimen handling and assay in the molecular biology laboratory and outlines the solutions currently available to address these difficulties. Designs for automatic systems to manipulate biosamples and analyze DNA are presented.


A novel, quantitative bioassay for type I interferon using a recombinant indicator cell line. We describe a specific and quantitative novel assay for biologically active human type I interferon (IFN), the MxR assay. It is based on a Vero cell line containing multiple copies of a hybrid gene consisting of the murine Mx promoter, which is responsive to type I IFN, linked to the human growth hormone (hGH) transcription unit. Exposure of this cell line to IFN-alpha or -beta for 12-48 hours results in the production of hGH that is measured by a commercially available radio-immune assay. The response to IFN-alpha is dose-dependent between 3 and 1000 units/ml. There is no response to TNF, IL-1 and a number of other cytokines and growth factors, and only a negligible response to IFN-gamma.


Development of expression vectors for transgenic fish. Genetic alteration of fish is important for aquatic biotechnology as well as for investigating molecular interactions that occur during vertebrate development. The numerous, large, transparent, and externally fertilized eggs of many fish species make them ideally suitable for genetic manipulation, especially for production of transgenic animals. Genetic engineering of fish requires suitable expression vectors. Accordingly, we developed two fish expression vectors, FV-1 and FV-2, which contain the proximal promoter and enhancer regulatory elements of the carp beta-actin gene and the polyadenylation signal from the salmon growth hormone gene. The two fish expression vectors were tested in microinjected fish eggs and in tissue cultured fish and mammalian cells. These two "all-fish" expression vectors should be useful for genetic engineering of fish and have been used with growth-enhancing genes in transgenic fish.


A nuclear magnetic resonance technique for determining hybridoma cell concentration in hollow fiber bioreactors. We have developed a technique for determining cell concentration in a hollow fiber bioreactor based on 23Na nuclear magnetic resonance (NMR) spectroscopy. Cell concentrations determined with this method agreed closely with concentrations calculated from 31P NMR nucleoside triphosphate (NTP) measurements and oxygen consumption rate measurements. Oxygen transfer limitations, which can complicate cell mass determinations based on oxygen consumption rates, were shown to be negligible for the bioreactor used. Specific antibody production rates in hollow fiber culture, calculated from these cell number estimates, were similar to those found in suspension culture for this cell line.


Biosorption of radionuclides by fungal biomass. Four kinds of bioreactor were evaluated for thorium removal by fungal biomass. Static-bed or stirred-bed bioreactors did not give satisfactory thorium removal probably because of poor mixing. An air-lift bioreactor removed approximately 90-95% of the thorium supplied over extended time periods and exhibited a well-defined breakthrough point after biosorbent saturation. The air-lift bioreactor promoted efficient circulation and effective contact between the thorium solution and the mycelial pellets. Of several fungal species tested, Rhizopus arrhizus and Aspergillus niger were the most effective biosorbents with loading capacities of 0.5 and 0.6 mmol g-1 respectively (116 and 138 mg g-1) at an inflow thorium concentration of 3 mmol dm-3. The efficiency of thorium biosorption by A. niger was markedly reduced in the presence of other inorganic solutes while thorium biosorption by R. arrhizus was relatively unaffected. Air-lift bioreactors containing R. arrhizus biomass could effectively remove thorium from acidic solution (1 mol dm-3 HNO3) over a wide range of initial thorium concentrations (0.1-3 mmol dm-3). The biotechnological application and significance of these results are discussed in the wider context of fungal biosorption of radionuclides.


Recovery of metal ions by microfungal filters. Many microfungi contain chitin/chitosan as an integral part of the cell wall structure. The binding of toxic and heavy metal ions by chitosan or partly deacetylated chitin is a direct consequence of the base strength of the primary amine group and is most effective for those metals that form complexes with ammonia. Of the microfungi studied, hyphae from Mucor mucedo and Rhizomucor miehei, after treatment with hydroxide to expose the chitin/chitosan, were found to be most effective in the capture of metal ions. Chemically treated mycelia have so far been shown to bind silver, zinc, lead, copper, nickel, cobalt, cadmium, iron and chromium, with the efficiency of metal-ion binding apparently being inversely proportional to the valency state of the metal ions to be bound. Wet-laid papers produced from mixed slurries of treated mycelia and various conventional paper-making and textile fibres have exceptionally good tensile- and bursting-strength properties, particularly in the wet state. Papers containing 1 g treated mycelia removed up to 90% of various metal ions in solution (50 cm3, 1.5 mmol dm-3) with flow rates of 0.5 cm3 cm-2 min-1. However, the total metal-ion binding capacities of single-thickness microfungal papers are limited under constant flow conditions. The total volume flowing through the system before metal-ion breakthrough occurs increases in direct proportion to the number of sheets of microfungal paper but the concomitant reduction in flow rates becomes a limiting factor. Mycelia-containing structures that allow efficient metal-ion binding at commercially acceptable flow rates are currently being investigated.


An immobilized cell bioprocess for the removal of heavy metals from aqueous flows. Microorganisms can be used to remove toxic heavy metals from liquid industrial wastes. In addition to the chemical toxicity of many of the latter, the production of long-lived nuclides from nuclear power programmes has introduced additional radiotoxicological hazards. Associated problems of the presence of contaminating, non-metal co-pollutants and the presentation of dilute, high-volume wastes have received little attention. Traditional biotechnological waste treatments have relied either on the use of non-living biomass ('biosorption') or on the accumulation of metals by living cells with the associated problems of metal toxicity effects and the requirements for cell viability or growth. Identification of an enzymically-mediated metal accumulation step can permit decoupling of cell growth from metal accumulation. Using pre-grown biomass immobilized in a flow-through filter ('bioreactor') the metal-accumulative bioprocess can be described accurately applying traditional Michaelis-Menten kinetics. The effect of co-pollutants can be then quantified in order to run the bioreactor in the most efficient way.


Review of biotechnology applications to nuclear waste treatment. This paper gives an overview of the feasibility of the application of biotechnology to nuclear waste treatment. The contents are based on a report which PA Technology carried out for the Department of the Environment (DoE Reference: DoE/RW/88.008 Sector No 2.3). Many living and dead organisms accumulate heavy metals and radionuclides. The controlled use of this phenomenon forms the basis for the application of biotechnology to the removal of radionuclides from nuclear waste streams. Indeed, biotechnology offers a series of new opportunities for removal of radionuclides from dilute aqueous process effluents. Such technology is already used for heavy metal removal on a commercial basis and could be optimised for radionuclide removal. An overview of biotechnology areas, namely the use of biopolymers and biosorption using biomass applicable to the removal of radionuclides from industrial nuclear effluents is given. The potential of biomagnetic separation technology, genetic engineering and monoclonal antibody technology is also to be examined. The most appropriate technologies to develop for radionuclide removal in the short term appear to be those based on biosorption of radionuclides by biomass and the use of modified and unmodified biopolymers in the medium term.


Metal recovery using chitosan.

Metal recovery using chitosan. Chitosan is a natural polycationic polymer which possesses valuable properties as a metal recovering and water purifying agent. Applications are waste water treatment for heavy metal and radio isotope removal and valuable metal recovery, potable water purification for reduction of unwanted metals, agriculture--controlled release of trace metals essential to plant growth, food--complex binding of iron in precooked food to reduce 'warmed-over flavour'. The interactions of metals with chitosan are complex, probably simultaneously dominated by adsorption, ion-exchange and chelation. To study this it is of utmost importance to work with well characterized chitosans. This has been a problem as available characterizing methodology is limited. Degree of polymerization and deacetylation and the distribution of acetyl groups along the polymer chain is of crucial importance for chitosan metal interacting characteristics. Making chemical derivatives is a way to alter the metal interacting characteristics of chitosan. Chitosan possesses general coagulant/flocculant characteristics towards bio-molecules and surfaces.


Effects of oxygen supply on the production of nikkomycin with immobilized cells of Streptomyces tendae. For continuous production of the antibiotic nikkomycin immobilized cells have been used in a fluidized bed bioreactor. Cells of Streptomyces tendae were immobilized on sintered glass particles. Different biomass concentrations on the particles correspond to different thicknesses of mycelial layers because growth occurs only on the outer surface of the particles. The antibiotic productivity decreased with increasing layer thickness. In fermentations with higher concentrations of both biomass on the particles and dissolved oxygen levels of about 70% the productivity was also limited because of limited oxygen diffusion in the layers.


Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system. The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP). The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation. CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp. TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer. Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate. The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system.


Quantification and identification of the main components of the Trichoderma cellulase complex with monoclonal antibodies using an enzyme-linked immunosorbent assay (ELISA). An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies has been developed to measure the concentration of three main cellulase components from Trichoderma reesei, cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and endoglucanase I (EG I), in both commercial enzyme preparations as well as in samples from laboratory fermentations. The sensitivity of the assay is 1-10 ng protein, depending on the type of cellulase. The coefficient of variability is between 10% and 20%. By a combination of two different domain-specific monoclonals against CBH I or II it is also possible to quantify the concentration of intact and truncated forms of these two enzymes, respectively. The use of the ELISA to quantify the formation of the three cellulase components under different cultivation conditions is described.


Aryl acylamidase from Rhodococcus erythropolis NCIB 12273. A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a Km for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen.


High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1. A defined medium was developed which, by means of a specific fed-batch mode, allows growth of the recombinant Escherichia coli strain TG1 (pBB210) up to a cell density of 60 g dry weight/l. Apart from glucose and aqueous ammonia fed as carbon and nitrogen sources, it was necessary to supply other nutrients or O2-enriched air. Aqueous ammonia also served for pH control. The pO2 level was kept at 20% saturation via closed-loop controls operating the two output variables of stirrer speed and glucose feeding rate. This fed-batch method prevented significant accumulation of acetate and other metabolic by-products. The recombinant E. coli expressed interferon alpha 1 more efficiently at a lower specific growth rate (muPr approximately 0.15 h-1) than at the maximum specific growth rate (mu max = 0.45 h-1). Therefore, fermentation in the batch phase at mu max was only allowed to continue up to a medium cell density. In the succeeding fed-batch phase, the specific growth rate was reduced to muPr by increasing the stirrer speed according to an empirically developed time scale.


Improved transformation frequency and heterologous promoter recognition in Aspergillus niger. Wild-type strains of Aspergillus niger were transformed with integrative vectors. The number of stable transformants varied from approximately 20-30/micrograms up to 17,000/micrograms using acetamide and hygromycin B selection, respectively. The introduction of deletions of 5' and 3' non-coding regions of the acetamidase gene (amdS+) revealed that these sequences influenced the number of transformants. The molecular characterization of A. niger transformants revealed that several copies of the vectors were tandemly integrated into the nuclear DNA. These oligomers were stably inherited, even after 100 days of growth on non-selective medium. The expression of the vector-encoded genes was confirmed by evidence from the mRNAs and corresponding proteins encoded by the selectable marker genes.


Peptide and amino acid transport in Streptococcus bovis. In amino acid transport studies with Streptococcus bovis using 14C-labelled amino acids, it has been shown that between 87% and 95% of cell-associated radioactivity was located in the cytosol. In similar studies with unlabelled peptides, most test peptide associated with S. bovis was truly intracellular. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the proteolytic activity in S. bovis was found to be largely cell-associated and of the serine-protease type, but stimulated by dithiothreitol. A wide range of extracellular peptide hydrolysing activities was demonstrated against the pentapeptide Leu-Trp-Met-Arg-Phe, which was completely hydrolysed to eight products after 10 min incubation. Some of this pentapeptide was transported intact, indicating the existence of mechanisms for the transport of peptides up to 751 Da. In studies with Arg-Phe-Ala, only Phe (F) and Ala (A), and to a much lesser extent Phe-Ala (FA) were transported after extracellular hydrolysis to FA, Arg (R), F and A. In this case, amino acid transport was much more predominant than peptide transport. The extent and nature of peptide transport was affected by the addition of protease inhibitors.


High-level secretion of human apolipoprotein E produced in Escherichia coli: use of a secretion plasmid containing tandemly polymerized ompF-hybrid gene. A gene encoding the mature form of human apolipoprotein E (h-apoE) was fused to the secretion signal coding sequence of the Escherichia coli major outer membrane protein F (ompF) which was preceded by a consensus Shine-Dalgarno sequence. Two copies of this hybrid gene were inserted tandemly into an expression vector and expressed in E. coli under the transcriptional control of two tac promoters regulated by lac repressors. By the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to the growth media, cells synthesized h-apoE at the level of 27.2 micrograms per A600 and up to 22% of the total cellular protein. The h-apoE produced by E. coli was processed precisely, secreted into the periplasmic space and formed protein aggregates there. However, despite aggregation, they were easily dissolved in water and actively formed protein-lipid complexes with dimyristoyl phosphatidyl choline (DMPC). These results demonstrated that E. coli cells are able to synthesize and secrete a large amount of active h-apoE using a prokaryotic signal sequence.


Influence of medium exchange schedules on metabolic, growth, and GM-CSF secretion rates of genetically engineered NIH-3T3 cells. The metabolic and secretory characteristics of NIH-3T3 fibroblasts transfected with a cDNA encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were examined as a function of the culture medium exchange schedule. The rates of glucose and glutamine consumption and of lactate and ammonia production were measured over exchange schedules ranging from complete medium replacement weekly (1/week) to complete medium replacement daily (7/week). All measured metabolic rates increased with increased medium exchange rates and accelerated sharply between exchange rates of 3.5/week and 7/week. The lactate/glucose and ammonia/glutamine yield coefficients, however, remained invariant at about 1.9 and 1.0 mol/mol, respectively, under all medium perfusion conditions. A shift-up in medium perfusion rates from 3.5/week to 7/week resulted in increased metabolic rates that resembled those observed in the cultures that were exchanged at the 7/week rate throughout, showing that the metabolic rates could be directly controlled by the perfusion rate. Differential regulation of medium versus serum perfusion demonstrated that increased NIH-3T3 cell metabolism was directly proportional to the serum flux to which the cells were exposed. Thus a limiting serum component is responsible for the altered metabolic and growth rates. The GM-CSF production by the transfected 3T3 cells was stable but exhibited substantial transient increases during periods of cell proliferation, demonstrating that the secretion of transfected gene products can be highly modulated even when the cDNA is driven from a constitutive promoter. These studies show that the metabolic and secretory behavior of genetically engineered cells is influenced by the medium exchange schedule.


Monitoring cell concentration and activity by multiple excitation fluorometry. Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.


Pilot scale production of a Trichoderma reesei endo-beta-glucanase by brewer's yeast. Endo-beta-glucanase I (EGI) of Trichoderma reesei was produced in laboratory and pilot scale using recombinant strains of "bottom-fermenting" Saccharomyces cerevisiae. The gene eg/1 was integrated in the chromosome or an expression cassette was inserted on a multicopy plasmid. Expression levels were compared in a laboratory scale bioreactor. The best EGI-producing strain was cultivated in pilot scale. Adsorbent treatment was used to remove endogenous yeast proteins and other impurities from the culture filtrate during concentration. Effective pilot scale one-step purification of the EGI protein was obtained using DEAE-Sepharose, on which EGI was weakly bound. The purified enzyme reacted with antibodies prepared against T. reesei EGI and catalyzed the hydrolysis of both insoluble and soluble substrates.


Effect of free fatty acids and phospholipids on growth of and product formation by recombinant baby hamster kidney (rBHK) and Chinese hamster ovary (rCHO) cells in culture. Recombinant BHK and CHO cells producing human antithrombin III (rh ATIII) were used to investigate the utilization of phospholipids and free fatty acids from low-serum (0.1% FBS) culture medium. Both cell lines show distinctly different patterns of fatty acid utilization. For rBHK ATIII cells it is shown that under low serum conditions several different combinations of free fatty acids (bound to bovine albumin) elicit an identical growth stimulatory effect although individual consumption and production rates of fatty acids are different. Increased fatty acid concentrations lead to increased uptake rates without any further effect on growth rate being observed. Recombinant antithrombin III formation is found to be a function of combinations and concentrations of fatty acids present in the culture medium.


Macroreticulate buffers: a novel approach to pH control in living systems. It is possible to control the pH of growing living systems in vitro by adding, to the growth media, macroreticulate buffers, i.e. amphoteric resins made with buffering and titrant groups simultaneously affixed to the matrix. Such beads possess a very precise isoelectric point (pI) and are able to maintain the solutions' pH close to their pI values for extended growth periods. These pearls are made of a neutral polyacrylamide backbone containing up to 200 mM grafted weak acrylamido acids and bases. It is possible to produce such buffers with any desired pH value in the pH 2.5-11 scale. An example is given of conditioning the pH of endive plants grown hydroponically.


Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli. A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isolated. Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes. DNA fragments contained in all of the endoglucanase cosmids hybridized to T. curvata chromosomal DNA. The cellulase genes were expressed in E. coli, but at rather low levels.


The effect of pH on the production of pertussis toxin by Bordetella pertussis. The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields.


Rapid immunoassay technique for process monitoring of animal cell fermentations. A calibration and quality control technique suited to process monitoring with immunoassay is demonstrated. The particle concentration fluorescence immunoassay (PC-FIA) is shown to provide a sensitive and rapid method for the quantification of specific biomolecules in cell cultures. Smoothing of linear calibration parameters is performed by forming weighted averages of standard points as the run progresses. These estimates are then used to determine slope and intercept values for improved calibration. The nonuniformity of the fluorescent signal variance is also considered, and a weight model is developed to describe the relationship between signal fluorescence and signal variance for weighted linear curve fitting. Pooling calibration results over the process run improves overall assay performance as determined by using standard control chart analysis. This method is suitable for semicontinuous monitoring of animal cell fermentations and has been used here to measure cell-associated and culture supernatant concentrations of monoclonal antibody (Ab) from hybridoma cells. The cell-associated Ab concentration correlates with cell-specific production rate. Assay times on the order of 10 min for supernatant and 25-30 min for cell-associated Ab concentrations can be achieved, making this procedure suitable for process monitoring and control. Under these conditions the assay has a detection limit of approximately 10 ng/mL, providing a sensitive and specific method for the quantification of cell culture constituents.


High cell density suspension culture of mammalian anchorage independent cells: oxygen transfer by gas sparging and defoaming with a hydrophobic net. Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs. During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 l reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l.h equivalent to a consumption rate of 1 X 10(8) cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net. Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 5 l scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7 X 10(7) cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4 X 10(7) cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.


Strategies for the recovery of chemicals from fermentation: a review of the use of polymeric adsorbents. Many different compounds can be produced by using microorganisms or enzymes. An important element in the design of a viable biotechnological process is the selection of an economical and efficient separations train. Production of chemicals via biotechnology generally requires isolation and purification from dilute, aqueous solution. A general framework for separation process design relies on exploiting a unique molecular physicochemical property (or properties) for separating the molecule of interest from water and the other species in solution. Important properties that can be utilized for the recovery of low molecular weight polar compounds are molecular charge, hydrophobicity, Lewis acidity or basicity, volatility, and limited solubility. In turn, it can be useful to characterize molecular properties by using separation processes, such as, for example, hydrophobicity by measuring octanol/water partition coefficients. This paper reviews the use of adsorption onto hydrophobic, nonpolar macroreticular polymers and Lewis acid-base complexation by using functionalized polymers for the recovery of amino acids, carboxylic acids, alcohols, and ketones from dilute aqueous solution. The focus will be on utilizing physical and chemical properties to predict uptake capacity. This information will be relevant to separation process development and will help to characterize molecular properties in aqueous solution.


Application of population balance model to the loss of hybridoma antibody productivity. A simple dynamic model has been applied to explain the population dynamics of monoclonal antibody (MAb) producing (producer) and nonproducing hybridoma cells (nonproducer) coexisting in culture. The events of mutation or loss of genes associated with antibody synthesis have been incorporated into the model to account for the conversion of a producer to a nonproducer. The model shows that the cell population is not necessarily dominated by the nonproducer, and a steady balance of producer and nonproducer populations can be achieved. A nonproducer population is undesirable, and cultivation strategies to maximize MAb production are suggested, taking into account the dynamics of a nonproducer population.


Regulation and expression of transforming growth factor type-beta during early mammalian development. We have examined the effect of differentiation on the expression of different members of the transforming growth factor type-beta (TGF-beta) family using embryonal carcinoma (EC) cells and early mammalian embryos. We determined that TGF-beta activity increases approximately 25-100% when the mouse EC cell line, F9, is induced to differentiate with retinoic acid (RA). Interestingly, the increased TGF-beta activity reflects the induction of TGF-beta 2 secretion following differentiation of both F9 EC cells and the human EC cell line, NT2/D1. Using the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have verified that differentiation induces the expression of TGF-beta 2 as well as a distant member of the TGF-beta family, Vgr-1. Transcripts for TGF-beta 2 and Vgr-1 were readily detected in the differentiated cells of F9 and PC-13 but not in their undifferentiated counterparts. Moreover, TGF-beta 2 mRNA was readily detected in NT2/D1 cells following differentiation. In addition, transcripts for TGF-beta 2 were detected by RT-PCR in mouse morulae, preimplantation blastocysts and cultured blastocysts. Based on the data presented, it appears that the expression of both TGF-beta 2 and Vgr-1 is closely associated with the induction of differentiation during early development.


Membrane modifications in the course of hepatocyte isolation. A transmission E/M, scanning E/M and freeze fracture ultrastructural study has been performed on the rat hepatocyte in the course of isolation from the liver parenchyma. The cell submicroscopic aspect indicates a good morpho-functional preservation from the liver perfusion to the final stages of cell isolation. The freeze fracture membrane analysis evidentiates the constant presence of gap junctions and tight junctions, characterized by particular structural alterations, probably due to progressive functional uncoupling. The persistence of these cell differentiations until complete cell isolation may be considered a further morphological expression of the maintenance of the differentiated stage of the hepatocyte. Fragments of membranes from adjacent cells, still adherent to isolated hepatocyte surfaces, can also be occasionally detected by freeze-fracture techniques.


Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures. The aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.


Antibody production in packed bed reactors using serum-free and protein-free medium. The present work demonstrates the utility of packed bed reactors for the production of monoclonal antibody. We present data from a continuous process run for the production of over 100 grams of antibody, using serum-free medium. An additional pilot run also demonstrates the potential for continued antibody production under protein-free conditions, using a standard basal medium.


Improved culture conditions for Cowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants. 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.


Bioconversion of avermectin into 27-OH avermectin. The bioconversion of avermectin to its 27-hydroxy derivative is achieved with Nocardia autotrophica subsp. canberrica. The approach of increasing bioconversion productivity rather than efficiency was adopted in these studies. Process improvement studies focused on the physico-chemical conditions of the fermentation, examined initially at the shake-flask scale. Bioconversion yields were affected by pH, substrate concentration, time of substrate addition, substrate solubilization, carbon to nitrogen ratio, and medium strength. Optimization of these parameters resulted in a 8-fold process improvement. During pre scale-up studies, the sensitivity of this bioconversion to the antifoam employed was demonstrated and lard oil was selected as giving the best results. Additional process changes were required during scale-up efforts in larger vessels, including replacement of the original substrate solvent with dimethylsulfoxide.


DNA sequence analysis of endoglucanase genes from Pseudomonas fluorescens subsp. cellulosa and Pseudomonas sp. NCIB 8634. The DNA of two previously isolated recombinant clones, one from Pseudomonas sp. NCIB 8634 (= Cellvibrio mixtus) (pPC71) and another from Pseudomonas fluorescens subsp. cellulosa (pPFC4) that express endoglucanase activity in E. coli was sequenced. Plasmid pPC71 had three open reading frames, two of which include portions of plasmid pBR322. The third open reading frame occurs entirely within the Pseudomonas DNA insert and encodes a protein with a molecular mass of 5845 Da. The DNA insert in pPFC4 was found to contain an open reading frame (PFC-ORF) that encodes a protein of 32189 Da. The major endoglucanase produced in E. coli cells carrying pPFC4 is about 30,000 Da. It is concluded that PFC-ORF encodes this endoglucanase. Both ribosome and catabolite gene activator protein binding sites lie upstream from the initiating codon of PFC-ORF. An interesting feature of the PFC-ORF protein is the presence of amino acid motifs Val-Ser-Ser-Ser-Ser and Val-Val-Ser-Ser-Ser-Ser-Ser that occur within a 25 amino acid span.


Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis. Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-14C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin by Saccharothrix aerocolonigenes.


Biochemical fingerprints of Legionella spp. by the BIOLOG system: presumptive identification of clinical and environmental isolates. Six Legionella strains were characterized with the BIOLOG identification system. A specific metabolic pattern for the six Legionella strains was observed after 24 h. Several positive reactions were detected intermittently in all six strains tested. The possible application of the BIOLOG system for identification of Legionella spp. from environmental or clinical samples is discussed.