Transgenic fish.

A range of transgenic animal species have been generated using DNA microinjection, and application of this technique to fish is now showing some degree of success. Studies to optimize microinjection techniques specifically for use with fish, and to investigate possible alternative methods for mass culture, should lead to the commercial production of transgenic fish able to transmit desirable characteristics, such as enhanced growth or disease resistance, to their progeny.


Enhanced transport and liquid membranes in bioseparations.

Membranes that use a reversible chemical reaction or sequestration to achieve high selectivities and productivities show great potential for use in bioseparations. The concept of liquid membranes, with and without a complexing agent (carrier), and the types of system configurations and carriers that may be used with these membranes, are discussed.


Deoxyribonuclease activity in rumen bacteria.

Deoxyribonuclease activity was surveyed in 22 strains belonging to 12 species of rumen bacteria, with lambda bacteriophage DNA as substrate. Activity was readily detected in broken cell preparations from 15 of these strains. Particularly high levels of activity were present in cells and culture supernatant of all 5 strains of Bacteroides succinogenes, and 2 out of 6 strains of Bacteroides ruminicola, examined.


The rapid detection of direct-acting DNA mutagens by electrical impedence with a DNA repair-deficient strain of Escherichia coli.

A differential killing assay using Escherichia coli WP2 (wild type) and WP67 (uvrA, polA) was combined with impedence microbiology to produce a rapid screening method for direct-acting mutagenic compounds. The assay showed that mitomycin C, N-nitroso guanidine, potassium dichromate, sodium azide and acridine orange were direct-acting mutagens. With this method results can be obtained within hours, as compared with two days for the standard Salmonella/microsome test.


Electrotransformation of intact cells of Brevibacterium flavum MJ-233.

Electroporation allowed transformation of intact cells of Brevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobase) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin in B. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was improved by the addition of 1.0 U/ml of penicillin G to the culture medium. The optimum yield of transformants per micrograms DNA was 5 x 10(4) when the cell suspension was pulsed at a cell density of 1 x 10(10)/ml and at a DNA amount of 1.0 micrograms.


Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor: 1. Effects of physical and chemical environments.

Chinese hamster ovary (CHO) cells were cultivated in a compact loop bioreactor using MEM-alpha medium supplemented with 10% fetal calf serum. Effects of physical and chemical environments, i.e., pH in the medium, stirring speed of impellers, temperature and partial pressure of oxygen (pO2) upon growth of suspended cells in the bioreactor were determined in batch cultures. Growth behavior was characterized by specific rates of growth (mu), glucose consumption (qG) and lactate production (qL), and the yield coefficients (cell yield from glucose, YX/G, and lactate yield from glucose, YL/G). An effect of medium osmolality was also evaluated with T-flask monolayer cultivation. The best growth was observed at pH 7.6, 37 degrees C, 400 rpm, 50-100% saturation with oxygen and 320 mOsmol kg-1. Corresponding to the previous work with a human melanoma cell line, the sophisticated cultivation and process control systems have been improved for CHO cells.


Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products.

Effects of biochemical factors, i.e., medium components and metabolic byproducts, on growth of Chinese hamster ovary (CHO) cells were investigated. Glucose and ammonia were found to inhibit the growth. Kinetic analysis gave the inhibition constants, 0.14 g l-1 for ammonia and 5.0 g l-1 for glucose. Since glutamine was unstable and was a main source of ammonia, precise studies on glutamine degradation and ammonia formation process were done. By evaluating the spontaneous reactions, net glutamine utilization and net ammonia production by the cells could be estimated. It became evident that asparagine could support the growth of CHO cells as a stable substitute for glutamine. Then, a glucose fed-batch culture was grown on a glutamine free and asparagine supplemented medium. Because of (1) low glucose concentration, but (2) no glucose limitation and (3) low ammonia accumulation, the maximum total cell concentration reached 3.4 x 10(6) ml-1, which was 1.8 times greater than that in the control experiment (initial 1.15 g l-1 glucose and 0.29 g l-1 glutamine, and no glucose feed).


Bacterial conjugation between Escherichia coli and Pseudomonas spp. donor and recipient cells in soil.

Experiments conducted in microcosms containing loam soil samples inoculated with either E. coli or Pseudomonas spp. donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period. E. coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples. In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation. Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil. The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10(-3) to 10(-4]. Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.


Properties of the biosurfactant produced by Bacillus licheniformis strain JF-2.

The activity of the biosurfactant produced by Bacillus licheniformis strain JF-2 was quantified using a unit defined as the amount of the acid-precipitated biosurfactant that lowered the surface tension by 10 mN/m. One unit was equivalent to 37 micrograms/ml of the acid-precipitated biosurfactant. Acid precipitation was very effective in the removal of the biosurfactant from the spent medium. Among the solvents tested methanol was the most efficient in extracting the surfactant activity from acid-precipitated material. Thin-layer chromatography of the acid-precipitated biosurfactant revealed four components, two of which contained a lipid moiety and one of which contained an amino group. The methanol-soluble fraction also contained these four components. Studies suggested that all four components were needed for full activity. The lowest interfacial tensions against octane were observed when NaC1 concentrations were 50 g/l or greater. Calcium concentrations greater than 25 g/l significantly increased the interfacial tension.


Serum-free media for lymphoid culture.

A serum-free medium is described that we have been using since almost ten years when growing a number of lymphoid cell lines. Serum-free media offer several advantages before media containing even well standardized animal sera. As an example of studies where serum-free media offer advantages we present data demonstrating the use of a defined medium when monitoring glycosidase enzyme levels in lymphoid cells.


Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilised in a fixed bed, porosphere culture system.

The aim of this study was to investigate the potential of fixed beds of macroporous glass spheres as a production process for animal cell products. The growth, metabolism and monoclonal antibody expression of a mouse-mouse hybridoma cell line was investigated in order to both test the potential of and to optimise the system. After the initial growth phase, the culture went into a steady-state phase brought on by glutamine limitation. An event occurred after 120-160 h of steady-state operation which destabilised the culture, causing a decline in productivity, after which the culture recovered. This event was analysed in detail to determine its cause, and whether a major switch in metabolic function had occurred. The parameter which correlated most closely to antibody production rate was oxygen, but as this was kept constant in the void medium of the bed it has to be concluded that oxygen diffusion into the spheres was the regulatory factor. A comparison of the fixed bed and a flask culture identified interesting differences in glucose metabolism between the two systems. The data gave strong indications as to how the productivity of the fixed bed system can be further improved. This includes optimisation of the glutamine concentration and modifying the porous structure of the spheres to improve diffusion characteristics.


The protective effect of serum against hydrodynamic damage of hybridoma cells in agitated and surface-aerated bioreactors.

The effect of serum on the growth rate and metabolism of CRL-8018 hybridoma cells in an agitated, surface-aerated bioreactor was examined. In the employed well-controlled bioreactors at high agitation rates, hybridoma cells in medium containing 1% fetal bovine serum rapidly die in the presence of a vortex with accompanying gas-bubble entrainment, whereas non-agitated control cultures grow normally in medium containing 1% serum. Serum levels greater than 5% counteract the detrimental hydrodynamic effects due to agitation and bubble entrainment. The protective effect is present after short-term (less than 1 h) exposure to 10% serum concentration, suggesting a protection mechanism which is, at least in part, of a physical nature. The apparent cell yields on glucose, lactate, and glutamine decreased with decreasing growth rate due to low serum concentrations. The results are incorporated into a simple model in which the apparent growth rate is the sum of an invariable growth rate and a changing death rate.


Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.


The effect of pH on the toxicity of ammonia to a murine hybridoma.

The growth inhibition of a murine hybridoma mediated by ammonium chloride was shown to vary with the pH of the culture medium. Values for the initial media concentration causing 50% growth inhibition (IC50) ranged from 4 mM to 7.6 mM as the pH was reduced from 7.8 to 6.8. A significant negative correlation was observed between the IC50 and the NH3 concentration of the medium, suggesting that ammonia and not ammonium may be the toxic species in the culture medium. The optimum initial pH for cell growth was 7.4. However, this optimum shifts to lower pH as ammonia accumulates in culture as a metabolic by-product. This suggests that in order to obtain high cell yields, it may be beneficial to adopt a culture strategy of lowering pH during cell growth to offset the inhibitory effects of accumulated ammonia.


Monoclonal antibodies to bis-indole alkaloids of Catharanthus roseus and their use in enzyme-linked immuno-sorbent-assays.

High affinity monoclonal antibodies directed against bis-indole alkaloids from Catharanthus roseus were produced. Once characterized, they were used to develop sensitive enzyme-linked-immuno-sorbent-assays, enabling us to determine minute quantities of alkaloids related to vinblastine in plant material.


Biotransformation of 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra.

Two biotransformation products formed from 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra were isolated and their structures determined by chemical and spectral data as 3-O-[alhpa-L-arabinopyranosyl-(1----2)-beta-D-Glucuronopy ranosyl]-24- hydroxy-18 beta-glycyrrhetinic acid and 30-O-beta-D-glycopyrano-syl-18 beta-glycyrrhetinic acid. The formation of glycyrrhizin, the main triterpene glucuronide of the licorice root, was not detected among the biotransformation products. This is the first report of the glucuronylation of an exogenous triterpene in plant cell cultures.


Characterization of maize polyamine oxidase.

Some structural and biochemical characteristics of polyamine oxidase (PAO) purified from maize shoots have been examined. The enzyme has only alanine as N-terminal amino acid and its N-terminal sequence shows a significant degree of homology with tryptophan 2-monooxygenase from Pseudomonas syringae pv. savastanoi. The pH optimum for the stability of the native enzyme is 5, similar to that of the barley leaf enzyme. Calorimetric analysis shows a single two-state transition at pH 6 with Tm 49.8 degrees. At pH 5 the thermal stability is increased by more than 14 degrees. Amine oxidation products, delta 1-pyrroline and diazabicyclononane, are competitive inhibitors of PAO activity (apparent Ki = 400 and 100 microM respectively). Moreover these compounds improve the thermal stability of the enzyme. N1-Acetylspermine, which is a good substrate for mammalian PAO, acts as a non-competitive inhibitor for the plant enzyme.


Multiple forms of endopeptidase activity from jojoba seeds.

The cotyledons of 27 day post-germination jojoba seedlings (Simmondsia chinensis) contained five distinct endopeptidase activities separable by DEAE Bio-Gel and CM-cellulose ion exchange chromatography. The endopeptidases were purified 108- to 266-fold and their individuality was confirmed by activity-specific assays in native acrylamide gels along with differences in their Mr and catalytic properties. The five endopeptidases, which showed activity on model substrates and protein, were named EP Ia, EP Ib, EP II, EP III and EP IV. EP Ia was a serine proteinase with a pH optimum of ca 8 and Mr of 58,000. EP Ib, II and III were discrete cysteine proteinases showing pH optima of ca 6.8, 6.0 and 5.4 and Mr of 41,000, 47,000 and 35,000 respectively. EP IV was an aspartic acid proteinase with a ca pH optimum of 3.5 and Mr of 33,000.


ent-labdane glycosides from Baccharis pingraea.

The aerial parts of Baccharis pingraea, collected in Chile, afforded, in addition to two diterpene glycosides isolated previously, 14 new ones and two of the corresponding desacyl aglycones. The structures were elucidated by high field 1H NMR spectroscopy. The differences between the collections from Argentina and Chile are discussed.


Interfacial mass transfer in extraction of amino acid by ALIQUAT 336 in organic phase.

Reactive extraction of L-phenylalanine from alkaline aqueous solution into xylene in the presence of tri-octyl-methyl-ammonium chloride (ALIQUAT 336) as complexing agent was studied using a stirred transfer cell. The study investigated the effects of carrier concentration and temperature on mass transfer rates. Transfer rate across the interface in the presence of surfactant molecules was also studied. A two-film model was proposed by considering film mass transfer resistances at the aqueous and organic phases. The model predicted adequately the experimental time-concentration data at different carrier concentrations and temperatures. The model was modified to take into account the presence of surfactant in the organic phase.


Electrically enhanced extraction of penicillin G into dichloromethane.

The application of electrically enhanced liquid-liquid extraction techniques to the recovery and purification of penicillin G from aqueous solutions and from untreated mycelial culture broth is described. Experiments at laboratory scale have shown that extraction rates into dichloromethane at pH 4.0 may be increased by factors of up to five-fold by electrostatically spraying penicillin G solutions into a continuum of solvent held in electrical tension. The experimental studies revealed that in small spray column contactors, electrostatic spray conditions, once initiated at nozzle voltages in excess of 10 kV, could be sustained at nozzle voltages as little as 4 kV. The results demonstrate the potential of this technique as an alternative to mechanically augmented liquid-liquid contact for the intensification of whole broth extraction processes.


Transient response of a solid-liquid model biological fluidised bed to a step change in fluid superficial velocity.

The evolution of a solid-liquid model biological fluidised bed under a step change in fluid superficial velocity is described. During a transient step change, the fluidised bed divides into a top zone which remains at the initial porosity and a bottom zone which settles at the final porosity. The interface of discontinuity in porosity moves progressively upwards through the fluidised bed. The velocity at which the top of the fluidised bed expands or contracts and the upward velocity of the porosity transition interface depend only upon the initial and final states of the bed porosity and the fluid superficial velocity. This results in a linear evolution with time of the total bed height and the height of porosity transition interface. The proposed model is well suited to describe the transient response of low-density particles in a fluidised bed, such as encountered in biological systems, to a sudden change of liquid superficial velocity. The model was validated experimentally.


Thermodynamics and kinetics of lipase catalysed hydrolysis of oleyl oleate.

The kinetics of enzymatic hydrolysis of oleyl oleate in the boundary layer between stagnant organic and aqueous phases was studied using a commercial lipase preparation which was dissolved in the aqueous phase. Three aspects of the reaction were investigated. (1) The hydrolysis equilibrium in the organic phase cannot be expressed in terms of the concentrations of ester, alcohol and organic acid alone since the activity of the organic compounds changes dramatically with conversion, i.e. with the water content in the organic phase. An empirical correlation that accounts for the water activity and the unknown activity coefficients of the organic compounds is determined. (2) The influence of the interfacial area was examined, and it was found that the amount of ester converted per unit area of interface is independent of the available interfacial area and of the amount of ester. (3) The inhibition of the reaction by the hydrolysis products and by n-alkanes was measured. Both acid and alcohol inhibit the hydrolysis reaction while the influence of long-chained alkanes is very small. It is concluded that the reaction rate is determined by the interfacial concentrations, and that these concentrations differ from the bulk concentrations because of the different surface affinities of the components.


Folding of eukaryotic proteins produced in Escherichia coli.

Although intracellular expression in E. coli may result in accumulation of the eukaryotic protein in inclusion bodies, the protein may often be recovered by first solubilizing with denaturant followed by refolding. Some general guidelines for developing a refolding procedure are apparent but the specific protocol must be empirically determined for each protein. Convenient and rapid assays for detecting native protein are critical for developing a refolding procedure. Maintaining solubility during refolding is a common feature of recovery processes. Proper folding should be assessed by a number of methods including activity, spectroscopic and stability measurements. For some proteins, properly folded protein may be obtained by secretion from E. coli; however, secretion does not ensure correct folding and protection from proteolytic degradation.


Human retinoblastoma susceptibility gene.

It is clear that the RB-deficient tumor cells lost their tumorigenicity in nude mice after regaining the RB gene expression. However, the mechanism of tumor suppression by the RB gene is still unknown. More studies on the biological activities of RB protein, pp110RB, are necessary to answer this question. Recent studies have shown that several oncogenic viral proteins, such as SV40 large T antigen (47) and adenoviral E1A protein (48), bind to RB protein. The significance of these bindings remains unclear; nevertheless, they suggest that depletion of functional RB protein by viral proteins may provide another mechanism of RB inactivation. Continued study of naturally occurring as well as engineered RB mutants may give us some information on the biological activity of RB protein, and its roles in oncogenesis, differentiation, development and gene regulation. Additionally, direct detection of RB gene mutations would have great clinical utility. Probes for the RB gene and gene product will be useful for genetic diagnosis of cancer susceptibility in affected families. Therefore, antibodies to the RB protein will be excellent tools for diagnostic and/or prognostic application in clinical medicine.


The isolation and identification of cDNA genes by their heterologous expression and function.

Functional cDNA genes for cell surface proteins, receptors, growth factors and nuclear and cytoplasmic proteins can be isolated by a heterologous expression-function gene isolation strategy. The method of this strategy is dependent on highly efficient mammalian expression vectors, the properties of COS cells as hosts for SV40-origin-containing mammalian expression vectors, the well-established technology of constructing highly complex plasmid cDNA libraries and on the sensitivity of biological assays. These technologies are combined to identify a specific gene sequence by its biological phenotype. In theory this can be accomplished by individually testing each single gene of the cDNA library by expression in COS cells. In practice complex cDNA libraries are formed so that multiple cDNA genes can be tested and analyzed simultaneously. When the biological assay can discriminate phenotypic changes at the single cell level as typified by cellular morphology, cell membrane proteins or receptors or intracellular enzymes, then a reiterative expression screening method can be used. This was demonstrated by the isolation of cDNA genes encoding cell membrane proteins (7-10, 12). For the isolation of cDNA genes for growth factors and cytoplasmic enzymes by the expression-function cDNA gene isolation strategy, an alternative method is followed. The cDNA library is partitioned into sets of cDNA clones for transfection and analysis. The number of clones in a set is dependent on the efficiency of the expression vector used and on the sensitivity of the biological assay. For secreted cytokines and growth factors which can be simply detected by factor dependent cell lines, about 1,000 cDNA clones can be tested as a single set. By this strategy the genes for numerous hematopoietic growth factors, cytokines and interleukins have been isolated. With such numerous successes we can expect that the heterologous expression-function cDNA gene isolation strategy will become an important molecular biology method.


High performance isoelectric focusing using capillary electrophoresis instrumentation.

High performance isoelectric focusing in capillaries provides rapid, high resolution separation of proteins based on their isoelectric points. Results can be obtained in a matter of minutes with little or no sample preparation. The technique requires the use of coated capillaries to reduce electroendosmosis so that stable, focused zones can be attained. Once focused, protein zones may be mobilized by the addition of salt to the catholyte or anolyte buffer. On-tube UV monitoring enables direct detection of sample components during mobilization with mass sensitivity equal to that of silver staining. The linear relationship between mobilization time and isoelectric point allows the technique to be used for estimation of protein pI. Demonstrated applications include separation of proteins in biological fluids with possible clinical applications, and characterization of biopharmaceutical proteins and monoclonal antibodies.


The application of fungal protoplasts in biotechnology.

Fungi are organisms of enormous industrial importance, and are used for the production of compounds such as antibiotics, organic acids and immunosuppressants. Many studies have suggested that their industrial value may in some cases be increased by exploitation of technologies involving fungal protoplasts, where the cell wall has been removed, usually enzymically. This article critically examines what the advantages of using protoplasts are, the current methodology for the preparation, and how they behave subsequently.


Electron microscopy of hybridoma cells with special regard to monoclonal antibody production.

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.


Improvement in the antibody productivity of human-human hybridomas by transfection with Tac gene.

The presence of a highly purified recombinant interleukin-2 (rIL-2) increased the production of immunoglobulin (IgM or IgG) by human-human hybridomas to 1.5-2.0 times the production by untreated cells. However, these cells did not react with anti-Tac (IL-2 receptor alpha) antibody. To enhance the response of the hybridoma cells to rIL-2, Tac gene was introduced by co-transfection with Tac gene expression plasmid pTB459 and G418 resistant gene expression plasmid pRSVneo. Tac cDNA transfected hybridoma (HBW-4.16.459-6-126) was induced to produce 6 times as much IgG by rIL-2 as was the control. This antibody production promoting phenomenon mediated by rIL-2 was depressed by anti-Tac antibody.


Methods for determination of growth-rate-dependent changes in hybridoma volume, shape and surface structure during continuous recycle.

Hybridoma volume and surface membrane structure were found to vary as a function of specific growth rate using a method of cell recycle with continuous medium perfusion to vary growth rate. Mean hybridoma volume determined at constant osmolality by both electronic particle counting and scanning electron microscopic (SEM) methods indicated that rapidly growing cells are significantly larger than very slowly growing cells. We have previously determined that during both rapid and slow growth over a range of L-glutamine provision rates (Gln PR) that specific monoclonal antibody (MoAb) secretion rate was not changed. In this study a constant MoAb secretion rate per unit of membrane area was found which may indicate that changing membrane area is not a rate-determining factor in MoAb secretion. SEM methods were of limited use for accurate determination of cell volume due to cell shrinkage and large coefficients of variations. In spite of this limitation, SEM stereology methods were useful in confirming that cells remained spherical over a wide range of specific growth rates and that hybridoma cells were not circular. Sequential SEM observations also revealed that surface membrane structure of the 9.2.27 murine hybridoma investigated was correlated with growth rate. Under conditions of very slow growth, hybridoma surface microvilli density appeared to be significantly reduced.


A comparison of different culture methods for hybridoma propagation and monoclonal antibody production.

A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr-1.


Batch production and secretion kinetics of hybridomas: pulse-chase experiments.

Pulse chase experiments of two mouse hybridoma lines were conducted in order to elucidate the kinetics of monoclonal antibody (mAb) production and secretion during different stages of batch cultures. The results indicate that a stock of cytoplasmic IgG exists in hybridoma cells and that the concentration of this stored IgG depends on the cell line used and the stage of the culture. This stored IgG can be released by dying cells, and a certain quantity of the secreted IgG is derived from this source. However, only between 0.3 and 9.3% of the released IgG of U0208 (average: 2.08%) and between 2.08 and 25.8% of the IgG, released from I.13.17 (average: 6.95%), were of storage origin, calculated on culture viability and intracellular IgG-stock. Comparing the accumulation of radio-labelled IgG (IgG*) in the supernatant with the reduction of cytoplasmic IgG* during the chase experiments, the percentages range between 14 and 50%, somewhat higher values probably caused by changes in the culture conditions. These changes led to a release of IgG during the chase experiments, which accounts for about 20-25% of the totally secreted IgG. It could be established that during the logarithmic growth phase of batch cultures a certain percentage of synthesized IgG was not released but stored within the cells: for U0208: 0.3-4.5%, for I.13.17: 1-7.6%. During the stationary and death phase, this percentage ranged between 1.5 and 20% for U0208 and between 0.5 and 8.1% for I.13.17. Finally, the chase experiments also revealed that the time of synthesis, assembly, and secretion of mAbs does not vary much during the different phases of batch cultures, and is within the range of 1.5 and 3 hrs.


New tools for protein sequence analysis.

Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation which facilitate determination of protein primary structure have recently been developed.


Cryopreservation and the maintenance of cell lines.

Techniques for cell-line preservation which enable reliable and reproducible recovery of material with unchanged, defined characteristics are essential in many biotechnology industries. Cryopreservation is one such technique, and in this review we explore some of its problems, successes and potential future applications.


Antigen delivery systems for analysing host immune responses and for vaccine development.

Overall, the meeting was timely and worthwhile. It is evident that there is a need for comparative studies to establish a firm foundation for future work. This is particularly important since it appears that the responses observed are influenced by the particular strain used for attenuation, the means of attenuation, the antigen-presentation system employed and the animal most immunized. In this last regard, one must recognize that most work to date has been with inbred strains of mice which differ from the genetic diversity of most hosts that are the intended beneficiaries of the immunization strategies being developed.


Engineering enzymes for non-aqueous solvents.

Enzymes may be redesigned to permit catalysis in non-aqueous solvents by engineering their amino acid sequences, thereby altering their physical and chemical properties to suit the new solvent environment. The interactions that contribute to protein stability in non-aqueous solvents are discussed in the context of attempting to identify possible approaches to constructing enzymes which exhibit enhanced stability in non-aqueous media. These approaches are illustrated by several examples where protein engineering has resulted in enzymes that are better suited for catalysis in organic solvents.


Peptidomimetics and the template approach to nucleation of beta-sheets and alpha-helices in peptides.

Introducing structural constraints into engineered analogs of natural proteins is important for defining essential characteristics, such as specificity or stability, of the protein. This may be achieved by the use of peptidomimetics--elements which mimic the structure of natural peptide components, or conformational templates which induce specific structure formation in contiguous peptide sequence.


Antibody geometry and form: three-dimensional relationships between anti-idiotypic antibodies and external antigens.

Anti-idiotypes have been developed to mimic a wide variety of antigens in studies of antibody-antigen interactions. Molecular, biochemical and structural analysis of antibody-anti-idiotype pairs presents an excellent model for study of complex protein-protein interactions. An understanding of the ways in which anti-idiotypes mimic antigens and the features determining binding characteristics will facilitate rational design of compounds with biological, enzymatic, pharmaceutical and chemical applications.


Site directed mutagenesis: a tool for enzyme mechanism dissection.

Protein engineering has become the principle means of examining the active site of an enzyme to identify and quantify the roles of specific residues in ligand binding, specificity and catalysis. Site-specific mutagenesis has extended our knowledge gained from X-ray crystallography, and has provided striking proof that the intricate active-site geometry is supported by the remainder of the protein infrastructure for maximum catalytic efficiency.


An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus.

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.


Development of a highly specific diagnostic 23S rDNA oligonucleotide probe for Mycobacterium leprae.

A 21-mer DNA oligonucleotide probe targeting the 23S rRNA of Mycobacterium leprae was developed and its high specificity demonstrated by dot-blot hybridization. Even under relaxed hybridization and washing conditions (20 degrees C below Tm) the probe was highly selective in that positive signals were only detected with M. leprae, about half of the slow-growing and one of the fast-growing reference mycobacteria and Gordona bronchialis. At more stringent washing temperatures (6 degrees C below Tm) only the rRNA of Mycobacterium leprae was detectable.


Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis.

Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%-100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.


Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae.

The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a high-producing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paralleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at pH 8.0 and 30 degrees C. The pH had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity.


Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp. cremoris are related to secondary binding specificity.

Various chromophoric peptides have been tested as substates for two genetically related types (PI and PIII) of cell-envelope proteinases of Lactococcus lactis subsp. cremoris. The positively charged peptide methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide appeared to be cleaved with the highest catalytic efficiency by both enzymes, although in the case of PIII only at high ionic strength. A cation binding site in the PI-type proteinase that is not present in the related PIII-type appears to be mainly responsible for the difference between these enzymes with respect to the rate of conversion of this chromophoric substrate at relatively low ionic strength. This cation binding site most probably resides in the aspartic acid residue 166, which in PIII is substituted by asparagine. Substitution of the threonine residue 138 by lysine in PIII may also play a role. The binding step in the reaction pathway catalysed by PI at low ionic strength is governed mainly by an ionic interaction involving the cation binding site. In addition, hydrophobic interactions contribute to the binding process. Masking of the cation binding site only increases the Michaelis constant Km; the catalytic constant kcat is not affected. In the absence of the cation binding site (viz. in PIII) the free energy derived from the hydrophobic interactions only is too small to promote binding of the substrate effectively. High activities are measured only if a high ionic strength is introduced. Removal of electrostatic repulsion between the substrate and positively charged residues of the enzyme, among which is lysine 138, may contribute to this activation.(ABSTRACT TRUNCATED AT 250 WORDS)


Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor.

Glucoamylase production by Aureobasidium pullulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads.


A constitutive expression vector system driven by the deo P1P2 promoters of Escherichia coli.

The P1P2 promoters of Escherichia coli K12 deo operon, residing on an AvaII restriction fragment, were used to construct a new expression vector. To evaluate the potential of the P1P2-driven expression system we have inserted the sequence of human superoxide dismutase (hSOD) downstream of the deo ribosome binding site. Expression of hSOD was evaluated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzyme activity. In crude cell extracts hSOD expression levels were found to be high in hosts possessing no deoR or cytR repressors. Highest levels of hSOD expression were obtained with a high-copy-number plasmid regardless of the host used. Expressed hSOD can account for 35%-40% of total protein in E. coli.


Pigmented variants of Tolypocladium inflatum in relation to cyclosporin A production.

We have isolated four distinct colony types of the fungus Tolypocladium inflatum, the producer of the immunosuppressive agent cyclosporin A: morphologically normal white, red, and orange colonies and morphologically diverse tiny brown colonies. In liquid cultures, white normal and brown colonies developed into yellow broths. The broth of the brown colony had a low final pH and low cyclosporin production, whereas orange and red colonies had dark brown and even black broths with higher final pH and high cyclosporin production. The specific production of cyclosporin A by the red colony was three times that of the white normal colonies.


Storage and growth of neuroblastoma cells immobilized in calcium-alginate beads.

Mouse neuroblastoma cells (N18) were immobilized in calcium-alginate gel beads. Under standard culture conditions (37 degrees C; 5% CO2), cell growth was observed inside the beads. The number of cells increased threefold during 7 days of culture with cell division and differentiation visualized by electron microscopy. Cell properties maintained after short-term storage (2-3 days at 4 degrees C) included: (i) properties of voltage-dependent ionic channels tested by patch-clamp electrophysiological techniques; (ii) expression of cell-adhesion membrane proteins tested by immunohistochemistry (iii) morphological differentiation obtained by depletion of foetal calf serum in culture medium. The advantages of such an immobilization technique as applied to neurone cells are discussed.


Influence of the reaction medium on the product distribution of peroxidase-catalysed oxidation of p-cresol.

p-Cresol was oxidized by hydrogen peroxide in a reaction catalysed by horseradish peroxidase and the low molecular weight products were investigated. In aqueous media Pummerer's ketone (I) was the dominating product but in organic media the product distribution was quite different; 2,2'-dihydroxy-5,5'-dimethyldiphenyl (II) was the main low molecular weight product. Similar product distributions were obtained with peroxidase adsorbed on a solid support and suspended in toluene and with peroxidase solubilized in a microemulsion containing the same solvent. The best selectivity for the formation of (II) was obtained when the enzyme was adsorbed on Celite and suspended in water-saturated chloroform with 0.5% (v/v) extra water added. The yield of low molecular weight products in this case was 28%; of this fraction, 95% was (II).


Effects of lipids on thermophilic anaerobic digestion and reduction of lipid inhibition upon addition of bentonite.

The effect of bentonite-bound oil on thermophilic anaerobic digestion of cattle manure was investigated. In digestor experiments, addition of oil was found to be inhibitory during start-up and the inhibitory effect was less pronounced when the oil was added in the form of bentonite-bound oil compared to when the oil was added alone. After adaptation of the digestors, very rapid degradation of oil was observed and more than 80% of the oil was degraded within a few hours after daily feeding. In batch experiments, glyceride trioleate was found to be inhibitory to thermophilic anaerobic digestion when the concentrations were higher than 2.0 g/l. However, addition of bentonite (a clay mineral) at concentrations of 0.15% and 0.45% was found to partly overcome this inhibition. Addition of calcium chloride in concentration of 3 mM (0.033% w/v) showed a similar positive effect on the utilization of oil, but the effect was lower than with bentonite.


A control for fetal bovine serum.

The FBS Control is designed to assist cell culture scientists who have been frustrated with lack of consistency in FBS lots. Continued use will reduce the time and expense needed in classical lot selection methods.


Induction and purification of kievitone hydratase from Fusarium solani f. sp. phaseoli.

Fusarium solani f. sp. phaseoli is capable of detoxifying the major isoflavonoid phytoalexins produced by its host plant Phaseolus vulgaris. One of the enzymic activities involved is kievitone hydratase (KHase), a secreted glycoprotein which catalyses the conversion of kievitone to the less fungitoxic derivative, kievitone hydrate. Even under conditions of substrate induction, the enzyme is expressed at levels that are too low for satisfactory purification. Therefore, several other isoflavonoids were tested as inducers in culture. Among the phytoalexins produced by the host plant, phaseollinisoflavan was the best inducer, elevating the level of secreted enzyme eight-fold. Treatment with biochanin A, a product of chickpea, resulted in a 16-fold increase of secreted activity. The maximum rate of induction was observed 9-24 hr after addition of biochanin A, during which time several metabolites of the inducer were also present. KHase was purified from filtrates of biochanin A-induced cultures. Denaturing gel electrophoresis indicated that two species of Mr 47,000 and 49,000 copurified with the activity. N-Terminal sequence analysis indicated that the two species possessed related, or identical, polypeptide moieties. Comparison with the size of the non-denatured enzyme, previously determined to be ca 100,000, indicates that its native state is a dimer.


Carolinoside: a phytosteroidal glycoside from Solanum carolinense.

The glycoside of a new class of phytosteroids has been isolated from Solanum carolinense. The steroidal aglycone (carolinone) is alkylated at C-3 and is identified as C-[(2,4,5-trideoxy-3-keto-4,5-dehydro)-pentulopyranosyl]-(5----3)- (13,14- seco-14 beta,17 alpha-dihydroxy) estrogen. The hydrolytic labile glycosyl moiety is identified as O-(beta-D-glucopyranosyl) (1----1)-[L-(2,6-dideoxy-3-C-methyl)- arabinopyranose]. The linkage of this disaccharide in the steroidal glycoside (carolinoside) is shown to be O-(alpha-pentulopyranosyl)- (1----4)-O-(beta-L-arabinopyranosyl)-(1----1)-D-glucopyranose. Carolinoside occurs at concentrations of 10(-7)-10(-6) M in leaf tissue and was shown to be the host plant specific feeding induction factor for Manduca sexta.


CP-60,993, a new dianemycin-like ionophore produced by Streptomyces hygroscopicus ATCC 39305: fermentation, isolation and characterization.

CP-60,993, 19-epi-dianemycin, is a novel polycyclic ether antibiotic produced by Streptomyces hygroscopicus ATCC 39305. Fermentation recovery, purification and crystallization were achieved using standard procedures. CP-60,993 was characterized as a monocarboxylic acid. Elemental analysis suggested a molecular formula of C47H78O14 for the free acid and C47H77O14 Na for the sodium salt. Crystalline from CP-60,993 sodium salt shows the following properties: m.p. 193-205 degrees C, E1%(1 cm) = 157 at 232 nm, [alpha]25 degrees C(D) + 11.0 (c 1, methanol). The structure, determined by MS, PMR and CMR, differs from dianemycin only in the stereochemistry at position 19. This was confirmed by X-ray crystallography carried out on the rubidium salt of CP-60,993. It exhibited activity in vitro against Gram-positive and anaerobic bacteria, efficacy against Eimeria coccidia in vivo in poultry, and stimulation in vitro of rumen propionic acid production.


Introduction and maintenance of prokaryotic DNA in Ustilago violacea.

A strain of the basidiomycete, Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin. U. violacea transformants were selected at a frequency of 5 per microgram pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipient U. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction of U. violacea transformants failed to produce E. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction in U. violacea transformants produced E. coli transformants resistant to neomycin. The E. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harbor U. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryote U. violacea and acquire endogenous sequences from this organism.


Sizing biological samples by photosedimentation techniques.

The performance of the Joyce-Loebl disk centrifuge in the sizing of Escherichia coli cells, protein inclusion bodies, and cell debris is evaluated. The need for a density gradient that extends throughout the entire spin fluid is highlighted, and a set of standard conditions that fulfill this requirement is defined. E. coli cells experience a reduction in their Stokes diameter when exposed to ethanol, indicating that a spin-buffer fluid combination such as glycerol-water is to be preferred for the sizing of bacteria. The instrument baseline is influenced by the presence of particles, and a method of estimating the baseline is described. The sizing of small particles is further complicated by baseline drift due to temperature sensitivity of the optical yoke. An analysis of diffusion in the spin fluid is conducted, and an expression for the sedimentation:diffusive flux ratio is derived. For the current samples, it is shown that diffusion within the spin fluid does not lead to significant errors for 0.15-microns particles, whereas the phenomenon may be significant at the manufacturer's size limit of 0.01 micron.


Enhancing oxygen transfer in surface-aerated bioreactors by stable foams.

To enhance oxygen transfer in surface-aeration bioreactors, stabilized foams were generated to increase the gas-liquid interfacial area by slowly introducing coarse bubbles into media containing fetal bovine serum. The bubble sparging rates were so low (i.e., 20 and 50 mL/h) that the contribution to oxygen transfer from these bubbles was due to foaming instead of bubbling. Furthermore, no physical cell damage caused by bubble sparging was observed. Oxygen transfer coefficients, kLa, in the bioreactors were measured in cell-free media. Without the foam-stabilizing agent (i.e., serum), no appreciable change in kLa was observed due to the bubble sparging. On the other hand, with serum, kLa increased with increasing serum content and bubble sparging rate and corresponded well with the degree of foaming. With 10% fetal bovine serum and a bubble sparging rate of 50 mL/h, kLa increased approximately 90% compared with no foaming. The enhancing effect of foam on oxygen transfer in surface aeration bioreactors has been further demonstrated with hybridoma cultures simultaneously grown in three identical bioreactors with and without stabilized foams.


Representation of phase equilibrium behavior of antibiotics.

The phase equilibrium behavior of biomolecules is important both in understanding the partition mechanisms and in the design and optimization of downstream recovery processes. Chen et al. (1989) proposed a molecular thermodynamic framework that successfully represents the liquid-solid equilibrium behavior of amino acids and small peptides as functions of temperature, ionic strength, solvent compositions, and pH. Based on this theoretical framework, this paper presents recent results in representing the liquid-solid equilibrium behavior (solubilities) and the liquid-liquid equilibrium behavior (phase partitioning) of beta-lactam antibiotics, which are amino acid derivatives and important chemotherapeutic agents.


Detection of genetically engineered traits among bacteria in the environment.

The release of genetically engineered microorganisms (GEMs) into the environment has, as its main aims, the benefits of improved agricultural yield and control of environmental pollution. However, effective and safe release programmes necessitate the development of sensitive, selective detection methods to monitor the environmental impact of released organisms.


Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.


Toward a unified approach to genetic mapping of eukaryotes based on sequence tagged microsatellite sites.

The genomes of all eukaryotes appear to contain a special class of loci, termed microsatellites, which can serve, if sequenced and taken as the substrate for the polymerase chain reaction, as highly informative, locus-specific markers. By analogy to the "sequence tagged sites" recently proposed by Olsen et al. for standardizing the human physical gene map, these microsatellite markers are termed "sequence tagged microsatellite sites" (STMS). Genetic maps based on STMS will share with the Olsen physical maps the advantage that mapping vocabularies will be standardized to the DNA sequence base and that access to any particular locus will not require shipping or storing cloned probes. The species map will consist simply of a listing of nucleotide sequences. Reference populations for developing STMS maps can be chosen on the basis of biological or economic interest. It will not be necessary to maximize for genetic divergence.


Rattlesnake and scorpion antivenoms from the egg yolks of immunized hens.

Antivenoms used to treat poisonous bites and stings are usually derived from horse sera. Consequently, they contain horse immunoglobulins, which frequently cause complement mediated side effects, and other proteins that can cause serum sickness and, occasionally, anaphylactic shock. Here we describe a new, avian source of antivenoms that precludes these complications, and an efficient and gentle means for preparing antivenoms composed solely of venom-specific antibodies. We demonstrate that antivenoms purified from the egg yolks of laying hens immunized with Crotalus atrox rattlesnake venom and Leiurus quinquestriatus hebraeus scorpion venom neutralize the lethal effects of these venoms in vivo. Antivenoms purified from chicken eggs may be pharmaceutically safer and more economical to produce than current horse antivenoms.


Insect resistant cotton plants.

We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.


Effect of intercalating dyes on the production of antibiotics by Micromonospora rosaria and Micromonospora purpurea.

The effect of treatment with various intercalating dyes on the ability to produce antibiotics in Micromonospora rosaria and Micromonospora purpurea was studied. Treatment with acriflavine resulted in a high frequency loss of antibiotic productivity in both species. In M. rosaria, the loss of antibiotic-producing ability appeared to be strain-dependent. In M. purpurea, up to 90% of colonies were found to have lost gentamicin-producing ability when protoplasts were used in the test. These antibiotic-nonproducing strains were further studied. The following observations were made: (1) Unlike the producing ability, the resistance to the antibiotics is a very stable character in both species. (2) Protoplast fusion analysis indicates that rosamicin-nonproducing characteristics of MR 217-AF2 and MR 217-AF3 strains induced by the acriflavine treatment is due to chromosomal mutation or rearrangement but not to loss of a plasmid. (3) Gentamicin-nonproducing strains of M. purpurea responded differently to the supplementation of streptamine or DOS in the culture medium. When supplemented with streptamine or DOS, some of these strains regained the ability to produce antibiotic, showing that the biosynthesis of intermediate was affected in these strains.


Flow cytometric study of hybridoma cell culture: correlation between cell surface fluorescence and IgG production rate.

Flow cytometry was applied to measure the fluorescence of IgG molecules on hybridoma cell surface stained with FITC-conjugated anti-mouse IgG F(ab')2. Using light scattering to exclude the dead cell population from the analysis, the surface fluorescence intensity allows for the differentiation between producing and nonproducing cells. A linear correlation has been found between the intensity of mean surface fluorescence of the cell population and the specific antibody production rate.


Coenzymatic properties of low molecular-weight and macromolecular N6-derivatives of NAD+ and NADP+ with dehydrogenases of interest for organic synthesis.

The catalytic activity, expressed as Km and Vmax values, of 16 enzymes of practical interest with the macromolecular coenzymes poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ and their low molecular weight precursors N6-(2-aminoethyl)-NAD+ and N6-(2-aminoethyl)-NADP+, was investigated. The enzymes examined are of direct interest for organic synthesis (i.e. alcohol dehydrogenase from yeast, horse liver, or Thermoanaerobium brockii, lactic dehydrogenase, and several hydroxysteroid dehydrogenases) or are used for the regeneration of NAD+, NADP+, NADH, or NADPH (i.e. glutamate dehydrogenase from liver or Proteus, formate dehydrogenase, glucose dehydrogenase, and malic enzyme). The cycling efficiency of poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ was examined with coupled-enzymes or coupled-substrates systems. Poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and, even more so, poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ were excellent coenzymes with several dehydrogenases. In addition, the coenzymatic properties of N6-(3-sulfonatopropyl)-NAD+, an NAD+ derivative carrying a strong anionic group, were compared with those of the newly synthesized N6-(2-hydroxy-3-trimethylammonium propyl)-NAD+, an NAD+ derivative carrying a strong cationic group. It was expected that the presence of the sulfonic or quaternary ammonium group would enhance the residence time of the coenzyme inside continuous-flow reactors if membranes with anionic or cationic groups, respectively, were used.


Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.

We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.


Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila.

Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant kcat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the kcat and phenylmethylsulphonyl fluoride reactivity, but change the Km. The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins.


Entrapment vectors: a new tool for mammalian genetics.

The use of mouse embryonic stem (ES) cells has facilitated the creation of mouse strains carrying desired genetic alterations. Recently, "entrapment" vectors have been developed to identify and mutate genes active during embryogenesis. These vectors in combination with ES cells may provide an efficient screening strategy to find genes that control mouse development.


Inheritance and expression of chimeric genes in the progeny of transgenic maize plants.

We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.


A new vector for cloning large eukaryotic DNA segments in Escherichia coli.

We investigated the relationship between plasmid size and electroporation efficiency in E. coli and found that even very large plasmids can be transfected efficiently. The efficiencies are well above the minimum required to construct representative libraries of complex eukaryotic genomes. To exploit this observation we constructed a novel mammalian-E. coli shuttle vector whose replication in E. coli is driven by the F sex factor episome origin. This new vector system should accept inserts well in excess of 100 kb, thus putting the cloning of mammalian genes by direct phenotypic complementation within reach.


Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli.

We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form. Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid. Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged. This suggests that the net effect of VHb in E. coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism. We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis. These suggest other systems in which cloned VHb may enhance bioprocess productivity.


Cell culture on a thermo-responsive polymer surface.

We have used a thermo-responsive polymer, poly-N-isopropyl acrylamide (PNI-PAAm), as a substratum for the culture of human dermal fibroblasts by conjugating it with collagen. The cells attached well, spread, and grew on the substratum, indicating that the polymer has no toxicity towards the cells. PNIPAAm is insoluble in water over the lower critical solution temperature (LCST; about 32 degrees C) and reversibly solubilized below the LCST. Taking advantage of this conversion, monolayered fibroblasts cultured on the substratum containing the PNIPAAm over the LCST, were completely detachable from the substratum by simply lowering the temperature below the LCST, without the use of conventional detaching agents such as trypsin and EDTA. The detached cell sheet gradually aggregated and finally formed a multicellular spheroid. This polymer may provide a convenient and potentially useful technology for cell culture.


Development of a chromatographic process for large-scale purification of staphylococcal enterotoxin B.

A process for large-scale purification of staphylococcal enterotoxin B (SEB) is presented. The process consists of three chromatographic steps. The first two steps are based on ion-exchange chromatography and the final one on gel filtration. Diluted culture supernatant (2000 dm3) with a SEB content of 0.4 mg dm-3 were processed and purified. SEB was obtained in 1.5 dm3 of 0.1 mol dm-3 ammonium hydrogen carbonate buffer, pH 8.0. The final product produced a single band on SDS polyacrylamide gel electrophoresis and a single peak when subjected to analytical gel filtration. The overall recovery was 74%.


Ammonium effects on streptonigrin biosynthesis by Streptomyces flocculus.

A defined medium containing glucose and ammonium as the sole carbon and nitrogen sources was developed to support growth and streptonigrin production. In this defined medium, increased initial levels of ammonium resulted in increased growth suggesting that nitrogen is the growth limiting nutrient. In some cases, increased initial ammonium levels resulted in decreased specific streptonigrin productivity, suggesting that nitrogen regulatory mechanisms may adversely affect streptonigrin biosynthesis. This suggestion that nitrogen regulation adversely affects antibiotic biosynthesis is further supported by results from two studies in which the ammonium supply to the cells was controlled. In the first study, streptonigrin productivity and final titer were enhanced by the addition of an ammonium trapping agent. In the second experiment, when ammonium chloride was fed slowly throughout the course of cultivation, the production phase was lengthened and the maximum antibiotic concentration was enhanced compared to the batch controls containing either the same initial or the same total ammonium chloride levels. Although our results indicate streptonigrin production may be subject to nitrogen regulatory mechanisms, the effect of nitrogen on streptonigrin production cannot be strictly correlated to the extracellular ammonium concentration. In fact, we observed that when ammonium was depleted from the medium, streptonigrin production ceased.


Evaluation of infrared spectroscopy as a bacterial identification method.

A study motivated by the recent revival of interest in the use of IR spectroscopy to identify bacteria is reported. A library of FT-IR spectra of dried bacterial films was complied using 16 different strains. A test set was compiled from spectra of the same strains grown several months later. The test set was quantitatively compared with the library on the basis of spectral similarity in the region 980-1190 cm-1. Six of the strains in the test set were not matched with the correct strain in the library despite efforts to reproduce the conditions under which cells were grown and prepared. The results suggest that reproducibility of the bacterial spectra is a potential difficulty that must be addressed by any attempts to develop FT-IR spectroscopy as a bacterial identification method.


Optimization of the hardening treatment of S. cerevisiae bioparticles.

Immobilization of microorganisms for application in fermentation processes generates bioparticles whose mechanical properties can be greatly improved if they are subjected to a hardening process. In this work, the immobilization of Saccharomyces cerevisiae in K-carrageenan and their treatment with Al(NO3)3 as hardening agent have been optimized. The effect of several factors have been studied, including the cell concentration in the gel, the concentration of hardener, and time of contact of this latter with bioparticles. In repeated batch experiments, a factorial design technique has been employed, reaching high values of productivity and a good retention of particles in the matrix. Once optimal conditions were selected, continuous experiments were realized, in which the hardened beads showed significant advantages, such as increase of 20% in ethanol productivity, a better evacuation of gas from the reactor, and a higher cell retention.


Protease-catalysed peptide synthesis in aqueous-organic two-phase systems: reactant precipitation and interfacial inactivation.

Peptides can be efficiently synthesized by the proteases thermolysin and pepsin acting in aqueous-organic two-phase systems similar to those reported previously. However, a number of problems can prevent successful reactions in these systems, with these and other proteases: (1) Complexes between amino acid reactants that are sparingly soluble in both phases may be precipitated, though their solubility behavior is complex; (2) the enzyme may be subject to rapid inactivation by contact with the phase interface, and the rate of this is unusually dependent on the nature of the solvent; and (3) the reaction rate is usually slow.


Immobilization of lipase from Candida cylindraceae and its use in the synthesis of menthol esters by transesterification.

Lipase (EC 3.1.1.3) from Candida cylindraceae has been immobilized by the cellulose-titanium chloride method, and on EP-400 polyethylene, with and without glutaraldehyde crosslinking, to give active preparations when assessed by their ability to catalyse the hydrolysis of tributyrin. In both cases, the use of glutaraldehyde crosslinking was shown to improve the stability of the preparations for repeated use. The lipase immobilized on EP-400 polyethylene was found to be effective in transesterification using tributyrin or triacetin as acyl donors with l-menthol as acceptor. The production of methyl butanoate and of methyl acetate using this immobilized preparation was in each case enhanced in the presence of Amberlite IR 47 Anion exchange resin (OH form).


Microbial sensor for penicillins using a recombinant strain of Escherichia coli.

E. coli harboring the multicopy plasmid pBR327, which codes for beta-lactamase synthesis, was immobilized on acetylcellulose membranes. These were placed in the vicinity of a flat pH electrode, thus constituting a penicillin biosensor based on the detection of changes in pH using a reference pH electrode. A response time of 8 min was obtained with at least 2 mg of cells cm-2 of membrane. A linear detection range between 5 and 30 mM of penicillin was achieved. Buffering capacity decreased the sensitivity, but to a lower extent as when compared with probes using purified enzyme. The sensor was completely stable for at least 13 days and proved to be useful for penicillin estimation in complex media such as fermentation broths and milk.


Harvesting recombinant microbial cells using crossflow filtration.

A contained, crossflow filtration (CFF) membrane system is described for harvesting Saccharomyces cerevisiae and Escherichia coli cells. This system is portable and can be cleaned and sanitized in place. Low- and high-cell density (LCD, HCD) fermentations of recombinant cells in 10- to 200-l volumes were used as the starting material. LCD fermentations, up to 8.3 g l-1 dry weight (dcw) of S. cerevisiae, with volumes of 10 to 200 l were harvested and diafiltered in 0.5 and 1.5 h, respectively. HCD 200-l fermentations of S. cerevisiae (47-63 g l-1 dcw) were harvested and diafiltered in approximately 2 h. E. coli fermentations, LCD and HCD (up to 16.2 g l-1 dcw), of 200-l volumes were harvested and diafiltered in 2.3 h while employing 14 and 75 ft2 of membrane area, respectively. Using hollow fiber or flat sheet membranes from different sources, cell harvesting times were less than 2.5 h. These studies demonstrate that CFF is an efficient method for harvesting and diafiltering recombinant S. cerevisiae and E. coli cells from fermentation broth.


Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii.

Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501. The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation. For example, Log, 4.44 transformants microgram-1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 microF did not produce any transformants. Cell survival was also poor at high voltages. A. vinelandii transformants were not recovered on N-free agar medium. In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 microF; 2500 V, 3 microF; and 1500 V, 25 microF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.


Endoglucanase E, produced at high level in Escherichia coli as a lacZ' fusion protein, is part of the Clostridium thermocellum cellulosome.

The Clostridium thermocellum celE gene was expressed at high level in Escherichia coli TG1 when placed under the transcriptional and translational control of lacZ' in pUC18; in the presence of a multicopy plasmid (pNM52) containing the lacIq gene, expression of full-length and truncated forms of celE was regulated by isopropyl-beta-D-thiogalactopyranoside. Catalytically active endoglucanase E (EGE) produced by E. coli was subject to proteolytic processing. The main protein species produced from full-length and truncated forms of celE was around 40 kDa in size and had an N-terminal amino acid sequence corresponding to that derived for mature EGE from the nucleotide sequence; in addition, larger species of about 75 kDa, presumably corresponding to full-size EGE, were produced by E. coli containing the full-length celE gene. Even after removal of the signal peptide sequence, EGE produced by E. coli was secreted into the periplasm. Up to 157 bp could be deleted from the 5' end of the celE gene without affecting the catalytic activity of EGE produced by E. coli. A polypeptide of Mr 86 kDa, immunoreactive with anti-EGE antiserum, was demonstrated in the high-molecular-weight, cellulose-binding multiprotein aggregate recoverable from C. thermocellum culture supernatant.


Growth and product inhibition kinetics of T-cell hybridomas producing lymphokines in batch and continuous culture.

The kinetics of growth and lymphokine formation by T-cell hybridomas were investigated in batch and continuous culture. Product inhibitions by ammonia and lactic acid were considered in the proposed model and experimental data were used to determine the kinetic and inhibition constants. The effect of dilution rate on specific substrate consumption and product formation rates was investigated.


Transient behavior of T-cell hybridomas in response to changes in metabolite concentrations in continuous culture.

Effects of pulse and step changes in major metabolite concentrations such as glucose, glutamine, lactic acid, and ammonium on behavior of T-cell hybridomas in continuous culture were investigated in order to develop a strategy to maximize specific rate of lymphokine formation. Step increases in glucose, lactic acid, and ammonium concentrations decreased, and a step increase in glutamine concentration increased, the specific rate of lymphokine formation. Effects of step and pulse changes on specific rates of glucose and glutamine consumption and viability of cells were also investigated.


Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel beads: I. Effect of immobilization on the primary and ultimate biodegradation of SDS, and redistribution of bacteria within beads during use.

The surfactant-degrading bacterium Pseudomonas C12B was immobilized in polyacrylamide gel beads. Conditions were established for minimizing the apparent loss of sodium dodecyl sulfate (SDS)-degrading activity accompanying polymerization, while still retaining durable gel beads. Apparent losses in SDS-degrading activity compared with free untreated bacteria were attributed largely to substrate diffusion limitations imposed by the gel matrix. Changes in the rate and extent of conversion of radiolabel from [1-14C]SDS to 14CO2 were attributed to diffusional restrictions on O2 availability within the gel beads. Scanning electron microscopy was used to show that beads (3 mm3) repeatedly exposed to SDS for 35 days contained a high cell density in a sub-surface layer 0.4-0.7 mm deep, with relatively few bacteria either at greater depth or at the bead surface.


Immobilization of microbial cells by adsorption.

Immobilized cells cover a wide area of applications and are essential components of many biotechnological processes. In general it can be distinguished between two immobilization methods: (1) entrapment into polymers and (2) natural adsorption onto porous and inert support materials. The immobilization by adsorption is discussed by the following criteria: biomass loading, strength of adhesion, enzymatic stability/specific activity of the biocatalyst, effectivity/reaction engineering and operational stability.


Construction of vector of Brevibacterium lactofermentum and study of its stability in continuous culture.

A 5.7-kb vector plasmid pBK2 was constructed by ligating the kanamycin resistance gene from Escherichia coli plasmid pACYC177 to an endogenous cryptic 4.4-kb plasmid of Brevibacterium lactofermentum ATCC 21086. The vector replicates efficiently and is stably maintained in the host and other coryneforms. However, the copy number varied from 50 to 10 per chromosome-equivalent under different culture conditions. Continuous culture studies showed instability when low dilution rates were used. Co-culture experiments were performed at various dilution rates to measure the growth rate ratio (alpha) of the plasmid-free cells to the plasmid-containing cells. It was observed that at low dilution rates the value of alpha was higher than that at high dilution rates. Thus, the instability of the plasmid can be attributed to the increase in alpha at low dilution rates. Modelling of instability using a random partitioning model of plasmid segregation and experimentally obtained values of alpha showed agreement with experimental data. This demonstrated that active partitioning is not the operative mechanism for plasmid segregation in this case.


Two-stage cultivation of Digitalis lanata cells: semicontinuous production of deacetyllanatoside C in 20-litre airlift bioreactors.

A two-stage cultivation method was employed to develop a semicontinuous biotransformation process for the production of deacetyllanatoside C, a cardenolide of the important digoxin series. Digitoxin was used as the substrate for biotransformation. The process was optimized in 1-l shake flasks and then established on the 20-l scale using two airlift bioreactors, one for cell growth (working volume 12 litres) and another for deacetyllanatoside C production (working volume 18 litres). Growth and production phases were synchronized and the process finally ran semicontinuously in 7-d cycles. Six consecutive production runs were performed yielding a total of 43.8 g deacetyllanatoside C.


Environmental carcinogenesis and biotechnology.

Numerous environmental and host factors, some of which are known and some unknown, contribute to cancer development. While data and studies abound, our current understanding of the relation between cancer and the environment is still very limited. Understanding environmental carcinogenesis is critical to its effective management. Biotechnology has revolutionalized the study of biological and biomedical sciences. This minireview provides an overview of environmental carcinogenesis with emphasis on the contributions and prospects of biotechnology in advancing an understanding of environmental carcinogenesis for its prevention and intervention.


Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures.

The kinetics of monoclonal antibody synthesis and secretion have been studied in synchronous and asynchronous mouse hybridoma cell cultures. Pulse-labelling of IgG followed by immunoprecipitation and quantitation of synthesized and secreted IgG in synchronous cultures show maximum production during G1/S phases. Secretion takes place through exocytotic release of vesicle contents. Pulse-chase experiments show that 71% of the synthesized IgG is secreted within 8 h of the pulsing period and only a further 4% is secreted by 22 h. Higher specific antibody production (QA) is obtained if (a) cells are arrested and then maintained in G1/S phases, (b) viability is decreased during the death phase of batch culture, (c) the dilution rate is decreased in continuous culture or (d) cells are subjected to hydrodynamically induced stress. The increase in QA in all these cases is mainly due to the passive release of the accumulated intracellular antibody. DNA and protein synthetic activity peak during the early exponential phase and decline rapidly during mid and late exponential and death phases. Metabolic activity however peaks up to 20 h after the peak in DNA synthesis, and declines similarly during the death phase. The data are consistent with the idea that slow growth and higher death rates increase QA and that Ig secretion is probably subject to complex intracellular control.


Hyperproduction of a bifunctional hybrid protein, metapyrocatechase-protein A, by gene fusion.

A hybrid protein between metapyrocatechase and Staphylococcal protein A was produced by recombinant DNA techniques. A plasmid carrying the fusion gene that encodes the hybrid protein was constructed and expressed in E. coli. Over 70% of soluble proteins of the cell extracts was estimated to be the hybrid protein. This fusion protein is about 65,000. Both the IgG-binding activity of protein A and the metapyrocatechase activity were found in the hybrid protein. The optimum pH of metapyrocatechase in the fusion protein was at around 6.5 and Km was 1.3 X 10(-5) M. A simple immuno-enzymometric assay was developed for anti-BSA antibody using the fusion protein.


Proteins as biological effectors.

This chapter presented an overview of the role of proteins as biological effectors. From a simplistic point of view, based solely on comparison of the structural diversity of immunoglobulins, blood coagulation proteins, gonadotropins, growth factors, and antiproteins, it could be concluded that the functional mechanisms of these protein families bear little or no relationship. Despite this enormous divergency in structure-function relationship, there are in fact elements of commonality in their effector roles arising as a direct consequence of the ability of these classes of protein effectors to act as exquisite examples of the processes of biorecognition. All these case histories, and the numerous other familial case studies of protein effectors which could have been employed to illustrate the different functional roles of proteins, owe their biological properties to their primordial protein antecedents which have traversed the harsh wilderness of evolution in biorecognition phenomena and survived to elicit specific effector roles. Dictated by underlying physicochemical constraints, deceived at times by the lulling tones of the siren entropy, and constantly vulnerable to the vagaries of other more pervasive forms of biological networking and information transfer encoded in the genes of virus and invading microorganisms, protein biorecognition in higher life forms, and particularly in mammals, represents the finely tuned molecular avenues for the genome to transfer its information to the next generation. The examples summarized in this chapter illustrate the complex, and in disease states imperfect, functional potential of proteins to be manifested in the jigsaw of biorecognition and be realized in the network of nature's biological effectors. Proteins thus represent a diverse range of effector molecules whose properties are totally dependent on their conformational or topographic status. The three-dimensional structure defines active sites on the molecule through which intermolecular interaction and biorecognition phenomena can occur. The cipher for this surface topography is, of course, coded in the primary amino acid sequence. Much experimental work is being directed in these and other laboratories at elucidating the principles governing the folding of unique peptide sequences into three-dimensional structures. Further advances in the theoretical understanding of the thermodynamics of protein folding as observed by x-ray crystallography, nuclear magnetic resonance and other spectroscopic techniques will greatly aid this quest. In addition, more comprehensive computer-aided algorithms for structure simulation, improved models of protein conformational behavior, and greater insight into the molecular forces which control sequence nucleation will also be required.(ABSTRACT TRUNCATED AT 400 WORDS)


Development of new thrombolytic agents using recombinant DNA technology.

The increasing incidence of thromboembolic diseases has sustained the search for new agents able to stimulate the natural fibrinolytic system. The first generation of antithrombotic agents include bacterial streptokinase and human urine urokinase. Because these molecules lack specificity for the fibrin clot, important efforts have been made to produce, using recombinant DNA technology, agents presenting higher fibrin clot selectivity such as t-PA (tissue-type plasminogen activator) and scu-PA (single chain urokinase-type plasminogen activator). In parallel, several laboratories are presently attempting to create mutants and hybrids plasminogen activators displaying improved thrombolytic properties with respect to the natural molecules. In this paper, we describe briefly the mechanisms of fibrinolysis and the role of the different natural thrombolytic agents. In addition, we review the possibilities of genetic engineering for the production of natural and novel plasminogen activators.


Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).


Optimal temperature control policy for a two-stage recombinant fermentation process.

The optimal temperature control policy to be followed in the operation of a two-stage fermentation system in which gene expression is induced by a temperature-sensitive gene switching system was studied. A genetically structured model was used to describe product formation, and kinetic equations based on experimental data were used to quantify the specific gene expression rate and parameters that affect plasmid instability. A constant temperature control policy and temperature profiling control policy including temperature cycling were studied and compared. Maximum average production rate was obtained from a temperature control policy in which the second stage was operated initially at about 40.5 degrees C and the temperature decreased slightly to a constant value at 40.0 degrees C. The maximum average production rate, which corresponds to the optimal temperature control policy, for an operation of 180 h was 29.7 units of protein (mg of cells)-1 h-1.


Optimal chemostat cascades for periplasmic protein production.

This theoretical work predicts the optimal system design for the steady-state production of secreted protein in a chemostat cascade, using bakers' yeast (Saccharomyces cerevisiae) as the host organism. The protein of interest, mutant invertase, is secreted to the periplasmic space instead of the culture medium on account of its large size. This work uses the secretion model developed and tested by Park and Ramirez (1988). It is shown that the highest productivity is achieved when the chemostat cascade contains two stages, although the improvement over the single-stage productivity is small. When no recycle is used, the advantage of two stages results from the tradeoff between maximizing the cell concentration and maximizing the rate of protein production per cell. When recycle is used, the cell concentration and protein productivity are increased, and the advantage of two stages results from the tradeoff between maximizing the specific protein production rate and maximizing the specific protein secretion rate. Cascades with three stages were also investigated, but these were found to have no improvement over the corresponding two-stage cascades.


Plant cell culture using a novel bioreactor: the magnetically stabilized fluidized bed.

A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture.


Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.