Effect of exercise on tissue protein synthesis in rats. The effect of exercise on the protein metabolism in skeletal muscles (gastrocnemius and soleus), liver and small intestine was investigated in rats. Treadmill treatment for 7 d resulted in atrophy of the liver and small intestine, which was associated with a reduction in protein content. The rates of protein synthesis in the liver and small intestine were significantly suppressed in rats subjected to exercise. The change in protein synthesis in the visceral organs was mediated by the change in RNA activity (protein synthesis per unit RNA) but not by the change in RNA concentration. The tissue weight and the rate of protein synthesis in the gastrocnemius and soleus muscles were not affected by exercise. The results suggest that these changes in protein synthesis in the liver and small intestine may explain, at least partly, the atrophy of these organs which was observed after 7 d of exercise.


Metabolism of L-amino acids in a marine bacterium isolated from mackerel intestines in relation to eicosapentaenoic acid biosynthesis. Metabolism of glucose and L-amino acids in an obligately aerobic marine bacterium isolated from Pacific mackerel intestines was investigated for the mechanism and pathway of eicosapentaenoic acid (EPA) biosynthesis. This bacterium could not uptake glucose but the cell-free extract of this bacterium had the enzymatic activities of L-alanine oxidase (EC 1.4.3.2), L-alanine dehydrogenase (EC 1.4.1.1). L-serine dehydratase (EC 4.2.1.13), and malate dehydrogenase (EC 1.1.1.40), and of seven enzymes involved in the TCA cycle of the usual aerobes. On the other hand, the carbon-13 concentration in cellular fatty acids of the bacterium, especially that in their methyl carbon atoms in contrast to their carbonyl carbons, increased drastically when the bacterium was grown in the presence of 13CH3COONa. These results indicate that: (i) the TCA cycle works in this bacterium, (ii) glucose is not utilized and pyruvic acid is in vivo synthesized from L-alanine, L-serine, and malic acid, and (iii) EPA and other cellular fatty acids are in vivo synthesized from acetyl coenzyme A by the usual de novo synthesis route.


Inhibition of protein translocation in permeabilized cells of Schizosaccharomyces pombe by puromycin. To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-alpha-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.


Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist. In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.


Purification and some properties of chlorogenic acid oxidase from apple (Malus pumila). Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6-8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (-)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122 microM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity.


Isolation and synthesis of a new bio-antimutagen, petasiphenol, from scapes of Petasites japonicum. A new bio-antimutagen, petasiphenol [3-(3,4-dihydroxyphenyl)-2-oxopropyl caffeate] (1) was isolated from scapes of Petasites japonicum (AD50 = 95 micrograms/ml against UV-induced mutagenic E. coli WP2 B/r Trp-. Petasiphenol (1) and its isomer (2) were synthesized. The activity of 1 was observed in the presence of soybean oil (glyceride), although the isomer (2) did not show any activity in doses up to 300 micrograms/ml.


Uncoupling action of antibiotic sporaviridins with rat-liver mitochondria. The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.


Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli. We constructed the plasmid pTTB151 in which the E. coli bioB gene was expressed under the control of the tac promoter. Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E. coli carrying this plasmid. The requirements for this biotin-forming reaction included fructose-1,6-bisphosphate, Fe2+, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate. The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation. Our results suggest that an unidentified enzyme(s) besides the bioB gene product is obligatory for the conversion of dethiobiotin to biotin.


Hyperexpression and analysis of choB encoding cholesterol oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans. We examined the expression of choB, encoding cholesterol oxidase of Brevibacterium sterolicum ATCC 21387, in Escherichia coli JM105 and Streptomyces lividans TK23 using various deletion DNA fragments within the 5'-flanking region. The enzyme activity could be detected intracellularly in E. coli only when the 5'-flanking region was reduced to less than 256-bp and choB was transcribed by the lac promoter. A large amount of the enzyme were produced as inactive inclusion bodies when ChoB protein was fused with the NH2-terminal portion of LacZ protein. In contrast, choB with more than 256-bp of the 5'-flanking region was efficiently expressed in S. lividans TK23, and about 85 times as much of the active enzyme (170 U/ml) was secreted into the culture filtrate as with B. sterolicum in flask culture. These results suggest that the promoter of choB exist within 256-bp of the 5'-flanking region and can be efficiently recognized by the RNA polymerase of S. lividans. The characteristics of the enzyme purified from the culture filtrate of the S. lividans transformant and that of B. sterolicum were identical although the NH2-terminal amino acid sequence of the enzyme from the S. lividans transformant was 6 amino acids shorter than that from B. sterolicum.


Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp. No. 195. Newly isolated Bacillus sp. No. 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.


Purification and characterization of a DNA-dependent RNA polymerase from Pseudomonas putida. DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida. The enzyme had the typical composition of beta',beta,alpha, and sigma subunits of eubacterial RNA polymerases. The molecular masses of the subunits were 156,000 Da, 151,000 Da, 87,000 Da, and 42,000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The NH2-terminal amino acid residues of the alpha subunit had a marked homology with those of the alpha subunit of Escherichia coli RNA polymerase. The enzyme activity was dependent on ribonucleoside triphosphates, Mg2+, and a DNA template, and was inhibited in vitro by rifampicin. The enzyme activity was maximal in the presence of 10 mM MgCl2. In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P. putida initiated transcription at the same site as that of E. coli.


Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds. The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.


Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1. A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.


Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae. The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.


Calcium requirement for protoplast transfection mediated by polyethylene glycol of Lactobacillus casei by PL-1 Phage DNA. The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca2+. Sr2+ increased the transfection rate as well, but Ba2+, Mn2+, and Mg2+ did not. Co2+ and Zn2+ inhibited transfection. The simultaneous use of Ca2+ and Mg2+ increased the transfection efficiency. Impairment of transfection caused by lack of Ca2+ could not be reversed by the addition of Ca2+ later. A decrease in the Ca2+ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.


Biotechnology of the Archaea.

Biotechnology of the Archaea. The Archaea, designated since 1979 as a separate Super-Kingdom (the highest taxonomic order), are a highly novel group of microorganisms which look much like bacteria but have many molecular and genetic characteristics that are more typical of eukaryotes. These unusual organisms can be conveniently divided according to their 'extreme' environmental niche, into three broad phenotypes: the thermophiles, methanogens and extreme halophiles. Each group has unique biochemical features which can be exploited for use in the biotechnological industries. The extreme molecular stability of thermophile enzymes, the novel C1 pathways of the methanogens and the synthesis of organic polymers by some halophiles are all currently or potentially valuable examples of the biotechnology of the Archaea.


Genetic engineering of beta-lactam antibiotic biosynthetic pathways in filamentous fungi. Recombinant DNA technology has facilitated a rapid increase in our knowledge of beta-lactam antibiotic biosynthesis. Using the tools of this technology, beta-lactam biosynthetic genes and proteins have been characterized at the molecular level, cephalosporin-C production has been improved, new beta-lactams produced, and novel beta-lactam biosynthetic pathways have been constructed.


Metabolic response to treatment with cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat. We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.


Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii. Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.


Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis. A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.


Highly probable active site of the sweet protein monellin. The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Synthetic monellin is 4000 times as sweet as sucrose on a weight basis, and the native conformation is essential for the sweet taste. Knowledge of the active site of monellin will provide important information on the mode of interaction between sweeteners and their receptors. If the replacement of a certain amino acid residue in monellin removes the sweet taste, while the native conformation is retained, it may be concluded that the position replaced is the active site. Our previous replacement studies on Asp residues in the A chain did not remove the sweet taste. The B chain contains two Asp residues at positions 7 and 21, which were replaced by Asn. [AsnB21]Monellin and [AsnB7]monellin were 7000 and 20 times sweeter than sucrose, respectively. The low potency of the [AsnB7]monellin indicates that AspB7 participates in binding with the receptor. AspB7 was then replaced by Abu. [AbuB7]Monellin was devoid of sweetness, and retained the native conformation. AspB7 is located at the surface of the molecule (Ogata et al.). These results suggest that Asp7 in the B chain is the highly probable active site of monellin.


Purification and primary structure of proteinous alpha-amylase inhibitor from Streptomyces chartreusis. A new polypeptide inhibitor, AI-409, that inhibits human salivary alpha-amylase, was purified from a fermentation broth of Streptomyces chartreusis strain No. 409. This protein consists of a single-chain polypeptide of 78 amino acid residues, and includes two disulfide bridges. The primary structure of AI-409 and the locations of the disulfide bridges were identified by enzymatic digestion and the automatic Edman technique. Enzymatic digestion was done with trypsin, carboxypeptidase Y, and chymotrypsin. One of the disulfide bridges was between Cys(10) and Cys(26), and the other between Cys(44) and Cys(71).


Overproduction of biologically-active human nerve growth factor in Escherichia coli. A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.


Primary structure of a base non-specific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinus edodes. The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase Le2) from the fruit bodies of Lentinus edodes was analyzed. The sequence was mostly determined by analysis of the peptides generated by V8 protease digestion and BrCN cleavage (including alpha-chymotryptic, and V8 protease digest of BrCN fragments). It consists of 239 amino acid residues. The molecular weight is 25831. The location of 10 half cystine residues were almost superimposable on those of known fungal RNases of the RNase T2 family. The sequence homologies between RNase Le2 and four known fungal RNases of the RNase T2 family, RNase T2, RNase M, RNase Trv, and RNase Rh, are 102, 103, 109, and 74, respectively. The homologous sequences are concentrated around the three histidines, which are supposed to form the active site of RNase T2 family RNases.


Effects of L-156,602, a C5a receptor antagonist, on mouse experimental models of inflammation. L-156,602, a C5a receptor antagonist, was found as an immunosuppressant with preferential effects on delayed-type hypersensitivity (DTH) in our screening program and it was shown that L-156,602 suppressed the efferent phase of DTH. Here, we tested its effects on experimental models of inflammation induced in mice. L-156,602 did not suppress serotonin- and carrageenan- induced inflammation while it completely suppressed concanavalin A-induced inflammation 4 h after elicitation. The inflammation appeared 24 h after the elicitation with concanavalin A and it was significantly suppressed by L-156,602. Muramyl dipeptide (MDP)-induced acute joint inflammation was also significantly suppressed by L-156,602. These results demonstrated the unique immunomodulating properties of L-156,602 in mouse experimental models of inflammation.


Effect of Pluronic F-68 on the mechanical properties of mammalian cells. The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility modulus of the cells were significantly greater (60% and 120%, respectively) in a medium with 0.05% (w/v) Pluronic F-68 compared to that without Pluronic. Pluronic F-68 therefore affected the strength of the membranes when the cells were exposed to it for a long period of time, i.e., in culture. The short-term effect of Pluronic F-68 on cell strength was also tested by its addition at various levels up to 0.2% (w/v) immediately before the mechanical property measurements. The resulting cell strength depended on the Pluronic concentration, but a significant short-term effect could only be detected above a threshold of 0.1% (w/v). Previous reports on the effect of Pluronic F-68 on animal cell culture are evaluated in the light of these observations.


Humans and viruses determine the nature of virus vaccine production processes. Vaccine production processes result from the interaction between humans with a particular cell and virus system. The factors that control progress lie not only in the nature of the virus and animal cell but also in the history of the environment in which the process is to be developed. This latter constraint strongly influences the nature of the technical process that is chosen for the production of the vaccine rather than the achievement of efficiency based on one or other of the many possible engineering parameters of the virus production process. In addition to this it is also clear that we have much to learn about the production of viruses from animal cells in culture and that we may be aided by changing our present paradigm of the "virus as a cellular enemy" to that of the "viruses are the cell's best friend".


Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity. Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U ml-1 (2 ng ml-1) of the interleukin.


Continuous production of large amounts of monoclonal immunoglobulins in hollow fibers using protein-free medium. The performance of a protein-free medium was compared in culture flasks with a serum-supplemented medium and with a serum free medium in terms of cell growth and monoclonal antibody production by a murine hybridoma. We present results of continuous production in hollow fiber culture systems using serum-free medium and protein-free medium. In protein-free medium, it has been possible to produce large quantities of monoclonal antibody with a productivity similar to that obtained in serum-free medium. After a two steps purification process, monoclonal antibodies were characterized by SDS-PAGE, High Performance Size Exclusion Chromatography and Free Solution Capillary Electrophoresis. SDS-PAGE and high performance chromatography analysis have showed that purified monoclonal antibodies produced in serum-free medium or protein-free medium were similar. Furthermore, Capillary Electrophoresis characterization revealed that both MAbs were constituted by three isoforms with equivalent electrophoretic mobilities.


Characterization of macrophage cell line A640-BB-2: A640-BB-2 resembles peritoneal exudate macrophages in cell morphology, tumor cell recognition, responsiveness to immunomodulator OK-432 and lysosomal enzyme activity. Characteristics of mouse macrophage (MP) cell lines A640-BB-2, J774.1 and P388D1 and mouse peritoneal exudate MPs were studied and compared in cell morphology, ability to recognize tumor cells in the presence and absence of OK-432 known to activate MPs, and in lysosomal enzyme activity. In A640-BB-2 cells and exudate MPs, cell surfaces showed a few ridge-like processes and microvilli; spontaneous cytotoxicity was moderate against tumor target L929, and little or absent against targets SV3T3, B-16 and U937; and lysosomal enzyme activity of nonspecific esterase, acid phosphatase, and beta-glucuronidase was high. After culture in the presence of OK-432, A640-BB-2 cells and exudate MPs showed more extensive spreading with larger surface areas and with increased numbers of ridge-like processes and microvilli, and their cytotoxicity against target L929 became more extensive. The stable soluble factor did not participate in the mechanism of cytotoxicity against target L929 mediated by A640-BB-2 cells and exudate MPs. J774.1 and P388D1 cells were different from exudate MPs in cell morphology and ability to recognize tumor cells when cultured either with or without OK-432, and in lysosomal enzyme activity. A640-BB-2 cells seem to be useful in studying MP-tumor cell interaction and MP activation, and in detecting the trace biological activating factor of MPs.


Simulation of an iterative learning control system for fed-batch cell culture processes. This paper describes an iterative learning control scheme for fed-batch operation where repetitive trajectory tracking tasks are required. The proposed learning strategy is model-independent, and it takes advantage of the repetitive feature of system operations with a certain degree of intelligence and requires only small size of dynamic database for the learning process. The convergence of the learning process is proven. An example of simultaneously tracking two predefined trajectories by iterative learning control with two control inputs is given to illustrate the methodology. Satisfactory performance of the learning system can be observed from the simulation results.


Chemically defined medium for the production of biologically active substances of CHO cells. A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.


Genetic manipulation of milk proteins and its consequences for the dairy industry. Genetic selection of cattle by selective breeding patterns dates back to prehistoric times and has resulted in the diversity of breeds we see today. Selection in New Zealand has been for fat production earlier in the century, and more recently for protein production as well as fat. There is a lot of interest today in the naturally occurring variants of the milk proteins, as these can confer interesting differences in the molecular behaviour of the proteins as well as being correlated with compositional differences in the milk. Genetic modification holds great promise for the future in the dairy industry, but present constraints due to cost, lack of basic knowledge, and difficulty in producing genetically-modified calves, mean that only the biopharmaceutical area is likely to be affected in the near future. Coupled to this is an apparent lack of acceptance of food from genetically-modified animals by consumers. It will therefore need a change in public attitude as well as some development in science and technology before dairy products from genetically modified cattle become a commercial reality.


Mammalian sex-determining genes.

Mammalian sex-determining genes. The recent cloning of the Y-linked sex-determining gene SRY has ended one of the most notorious gene hunts in mammalian molecular genetics. Attention has now been turned to characterizing this gene further and studying how it acts as a switch in the choice of male or female developmental pathways.


The human genetic map.

The human genetic map. The introduction of new technology and increased effort from around the world is driving the completion of the human gene map. In parallel with the creation of the map, we are beginning to see the bio-medical benefits that are a direct consequence of learning more about our own genome.


DNA structure, mutations, and human genetic disease. The etiology of fragile X syndrome, myotonic dystrophy and Kennedy's disease has been attributed to the massive expansion of triplet repeat DNA sequences. This review details the relationships between the structural diversity of DNA, its secondary structure or DNA-directed mutagenesis, and the expansion of triplet repeats.


Molecular cytogenetics: diagnosis and prognostic assessment. This review describes molecular cytogenetic techniques for detection and characterization of genetic aberrations associated with human disease. The techniques of fluorescence in situ hybridization, primed in situ labeling and comparative genome hybridization are described, as are probes for repeated sequences, whole chromosomes and specific loci. Also reviewed are applications of these technologies to pre- and neonatal diagnosis and to the characterization of human malignancies.


Relevance of genomic imprinting to human diseases. The existence of functional differences between parts of the maternal and paternal genome has been demonstrated. The control of gene expression through genomic imprinting plays a significant role in normal developmental processes in mammals and is also instrumental in several human pathological conditions.


Transgenic animal models of cardiovascular disease. Transgenic experimentation has become a crucial part of hypertension and atherosclerosis research, and is growing more important in several other areas of cardiovascular disease. It has recently made a particular contribution to understanding the role of the renin-angiotensin system in controlling hypertension. The study of blood pressure regulation, cardiac hypertrophy, atherogenesis and thrombosis are also benefiting from the transgenic approach.


Immunotherapy of AIDS.

Immunotherapy of AIDS. The immunotherapy of AIDS encompasses a broad range of therapeutic possibilities. A number of stimulating and immunomodulating agents have been tried without any overt success. More recently, passive immunotherapy of AIDS using donated plasma and active therapeutic vaccine procedures have been reported. A number of clinical and laboratory observations together with preliminary results of clinical trials suggest that post-infection vaccines may be able to delay progression to disease in some individuals.


Oligonucleotide therapy.

Oligonucleotide therapy. Rapid progress in oligonucleotide therapeutics has continued over the past year as major programs established in the past four years have grown and begun to be productive. Important advances were reported in the medicinal chemistry of oligonucleotides and in understanding their pharmacodynamic properties. Significant progress was made in understanding the pharmacokinetic and toxicologic properties of first generation analogs, particularly phosphorothioates and one oligonucleotide, ISIS 2105, entered clinical trials. Additionally, combinatorial approaches designed to identify oligonucleotides that may bind to a variety of targets were reported.


Therapeutic immunosuppression of T cells. Current immunosuppressive therapy carries a range of unwanted side effects, and tends to penalize the whole immune system. It is desirable to develop therapies that are more selective for antigen-reactive cells. As T lymphocytes use a wide range of surface receptors to interact with antigen-bearing cells and with each other, much interest is devoted to trying to develop agents that selectively block the interaction of these receptors with their ligands, and others that could be used to reprogram the immune system so that it might become tolerant to the antigens rather than attack them.


Families of twelve transmembrane domain transporters. Several functionally distinct families of transport proteins share the general structural motif of twelve transmembrane domains. The number of membrane proteins known to possess this common feature continues to expand with the cloning of transporters for various neurotransmitters, nucleosides, osmolytes and basic amino acids, in addition to the previously defined families of facilitative and sodium-driven sugar transporters.


Alzheimer's disease: transgenic models to test new chemicals and pharmaceuticals. Alzheimer's disease is the most common form of senile dementia and is predicted to become even more prevalent as the proportion of elderly in the population increases over the next few decades. As yet, there are no effective treatments for the disorder. A major limitation to identifying new drugs and therapeutic targets for Alzheimer's disease has been the absence of an animal model displaying typical Alzheimer's pathology. Transgenic technology is now providing a powerful new approach for the development of animal models of Alzheimer's disease.


Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK 226. A nitrilase was purified, apparently to homogeneity, from a cell extract of Acinetobacter sp. AK 226, which converts racemic 2-(4'-isobutylphenyl)propionitrile (Ibu-CN) to S-(+)-2-(4'-isobutyl-phenyl)propionic acid (S-(+)-ibuprofen). The molecular weight of the native enzyme was estimated as 580,000 upon gel filtration. The nitrilase hydrolyzed many kinds of nitrile compounds such as aliphatic, aromatic, and heterocyclic mononitriles or dinitriles. The amino-terminal amino acids were sequenced and found to be partly homologous to a nitrilase from Klebsiella pneumoniae subsp. ozanae. The purified enzyme had a pH optimum of 8.0 and a temperature optimum of 50 degrees C. The enzyme was not affected by chelating reagents, carbonyl reagents, reductants, most metal ions, or thiol reagents except silver ion, p-chloromercuribenzoate, and phenylmercuribenzoate. The reaction with racemic Ibu-CN resulted in the preferential production of S-(+)-ibuprofen, demonstrating that the nitrilase is highly enantioselective to S-(-)-Ibu-CN. In fact, the enzyme showed a 180-fold higher activity for racemic Ibu-CN than that for R-(+)-Ibu-CN.


Mass measurement at high molecular weight--new tools for biotechnologists. It has long been possible to analyse small molecules accurately and reproducibly by mass-spectrometric techniques. Two new techniques extend the application of mass spectrometry to proteins and other biopolymers of high molecular mass. The accuracy, sensitivity and resolving power of these new methods permits the detection of minor, but biologically significant protein modifications.


Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography. Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.


Microbes and microbial enzymes for cyanide degradation. Cyanide is an important industrial chemical produced on a grand scale each year. Although extremely toxic to mammalian life, cyanide is a natural product generated by fungi and bacteria, and as a result microbial systems have evolved for the degradation of cyanide to less toxic compounds. The enzymes which utilize cyanide as a substrate can be categorized into the following reaction types: substitution/addition, hydrolysis, oxidation, and reduction. Each of these categories is reviewed with respect to the known biochemistry and feasibility for use in treatment of cyanide containing wastes.


Safe biotechnology (4). Recommendations for safety levels for biotechnological operations with microorganisms that cause diseases in plants. The Working Party on Safety in Biotechnology of the European Federation of Biotechnology has proposed a classification of microorganisms that cause diseases in plants. In this paper appropriate safety levels are proposed for these classes of microorganisms in order to ensure that research, development and industrial fermentation work with plant pathogens will limit the risk of outbreaks of diseases in crops that could result from work with such microorganisms when they are cultivated in laboratories, glasshouses and biotechnology installations.


Continuous high-cell-density fermentation of the ciliated protozoon Tetrahymena in a perfused bioreactor. An efficient method of growing the protozoon Tetrahymena to high cell densities in a 2-1 bioreactor is described. The first phase of the fermentation is a batch phase with minimum generation times (1.4 h). During the next phase medium is exchanged continuously using a perfusion module based on microporous hollow fibres while cell are retained. Compared to standard batch fermentations of this organism 30- to 40-fold higher cell concentrations and dry weights were achieved routinely. A maximum cell concentration of 2.2 x 10(7) cells/ml and a dry weight of 54 g/l have been obtained. As estimated from isocitrate dehydrogenase activity in the culture medium, no cell damage occurred even at high agitation rates. In addition, the cells remained viable for several weeks. Temporal limitation of the process was due to a decrease in the perfusion rate caused by blocking of the membranes. By X-ray microprobe analysis calcium phosphate depositions were detected in the pores of the clogged hollow-fibre membranes. However, even a T. pyriformis strain possessing mucocysts, dense core secretory organelles that may lead to early membrane clogging, was cultivated successfully for 3 weeks. Additionally, the consumption of nutrient protein and carbohydrates during fermentation was investigated and the effect of different perfusion rates and of glucose was studied in order to increase the efficacy of the system.


Introduction of sulphhydryl groups into the crystalline bacterial cell surface layer protein from Bacillus stearothermophilus PV72 and its application as an immobilization matrix. The crystalline cell surface layer (S-layer) from Bacillus stearothermophilus PV72 was used as a matrix for reversible immobilization of beta-D-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2'-dipyridyldisulphide, 550 micrograms beta-D-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one beta-D-galactosidase molecule [relative molecular mass (M(r)), 116,000] per two S-layer subunits (M(r), 130,000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies beta-D-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.


Influence of pH, nitrogen and phosphorus sources on the production of hepatitis B virus pre-S2 antigen by Hansenula polymorpha. Experimental design techniques were used to study the influence of the composition of the culture medium on the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha. pH, phosphoric acid, ammonium chloride and yeast extract concentrations were selected as experimental factors and their influence was investigated using Central Composite design techniques. The results indicated that antigen yield was maximized at high pH and in a culture medium containing both ammonium chloride and yeast extract. Phosphoric acid was found to have a detrimental effect on antigen production. This study allowed a 50% increase in antigen production in a medium in which the yeast extract concentration was decreased to 32 g/l. These optimal conditions have been confirmed with an octagonal design experiment. Moreover, it was shown that the antigen produced was very stable up to at least 8 days after induction and that the yeast extract concentration could be lowered to 22 g/l without appreciable effect on antigen yield. The increase in antigen production was not due to an increase in cell biomass, since no correlation could be found between these two parameters. The newly defined culture medium should allow a greatly increased antigen production at the fermentor level, at a lower cost and with minimal operational problems.


Low-serum medium development for human diploid fibroblast microcarrier cultures. A new medium supplement mixture, PPRF92, has been developed to enable the serial subculture of human diploid fibroblasts (MRC-5 cells) on microcarriers. Furthermore, the PPRF92 supplements enable cell growth at serum levels as low as 1%. Through an optimization programme, the PPRF92 supplements have evolved into a simple mixture with the concentrations of key components at a level that makes the overall cost very competitive with medium containing 10% foetal bovine serum (FBS). Furthermore, the PPRF92 supplement mixture is most efficacious when FBS is replaced with the cheaper, and more widely available, adult bovine serum (ABS). Although medium exchange with serum is necessary in order to achieve confluence on microcarriers, the PPRF92 mixture is only necessary at the initiation of each passage. Using the medium replenishment protocol that has been developed in our laboratory, MRC-5 cells were successfully serially passaged through 13 bead-to-bead transfers on microcarriers in DMEM/F12 medium enriched with the PPRF92 supplement mixture reported here, and 1% ABS.


Kinetic resolution of organosilicon compounds by stereoselective dehydrogenation with horse liver alcohol dehydrogenase. Stereoselective dehydrogenation of three isomers of trimethylsilylpropanol was carried out with horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1.) and optically active organosilicon compounds were obtained in a water-organic solvent two-layer system with coenzyme regeneration. Furthermore, we examined the effects of the silicon atom on stereoselectivity of HLADH compared to the corresponding carbon compounds. Substitution of the silicon atom for the carbon atom was found to improve the stereoselectivity of HLADH. For example, the optical purity of the remaining 1-trimethylsilyl-2-propanol was higher than 99% enantiomeric excess (ee) at 50% conversion, whereas that of the carbon analogue was 84% ee. This phenomenon was probably ascribable to the bulkiness of the organosilicon compounds derived from their longer Si-C bond. Kinetic analysis in an aqueous monolayer system demonstrated that the specific properties of the silicon atom greatly affected the reactivity of these substrate compounds.


Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography. The optimization of the production of recombinant DNA-derived proteins in Escherichia coli was investigated. We chose restriction endonucleases EcoRI and EcoRV from E. coli as model proteins, despite the observation that overproduction can result in a toxic effect to the cells. The enzymes were expressed as fusion proteins consisting of protein A from Staphylococcus aureus and the desired enzyme in order to facilitate purification. The expression of the fusion protein was induced by a temperature shift using the pR promoter of phage lambda regulated by the repressor plasmid pRK248cI. Data from batch fermentations provided the basis for planning a continuous two-stage fermentation. The EcoRI enzyme activity was investigated as a function of the induction time after cell disintegration and allowed an estimation of yield of the continuous culture. Plasmid instability, which was only observed under continuous conditions, could be prevented by adding tetracycline (resistance of the repressor plasmid) to the medium. We established a continuous cell disintegration system and purified the fusion protein semicontinuously by affinity chromatography. The biological activity of the fusion protein was the same as the native endonuclease so there was no need for cleavage of the fusion protein and the product could be used without further processing.


Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis. In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5'-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the alpha-amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33-120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.


Cloning and heterologous expression of a novel arylmalonate decarboxylase gene from Alcaligenes bronchisepticus KU 1201. We have cloned and sequenced a DNA fragment that encodes the arylmalonate decarboxylase (AM-Dase) gene from Alcaligenes bronchisepticus KU 1201. The AMDase gene consists of an open reading frame of 720 nucleotides, which specifies a 240-amino-acid protein of relative molecular mass (M(r)) 24734. The M(r) deduced from the AMDase gene is in good agreement with that of the AMDase isolated from A. bronchisepticus. No TATA or TTGA sequence was observed within the cloned DNA fragment, but the fragment was expressed in Escherichia coli by the lac promoter of pUC19. The enzyme produced in E. coli has the same M(r) and the same enzyme activity as that purified from A. bronchisepticus. Comparison of the DNA sequence and the deduced amino acid sequence of AMDase with available DNA and amino acid sequence data bases revealed that there are no significant sequence homologies.


Selection and characterization of alpha-amylase-overproducing recombinant Escherichia coli containing the bacterial hemoglobin gene. We previously reported that the presence of the bacterial (Vitreoscilla) hemoglobin gene enhances alpha-amylase production in recombinant Escherichia coli strain MK79. Using the growth of MK79 on starch as a selective method we have produced a mutant strain (BSC9) that produces up to four times as much alpha-amylase as MK79. Both MK79 and BSC9 produce the most alpha-amylase (per cell and per milliliter) in the stationary phase; almost all of the enzyme is intracellular in both strains. Modification of the standard alpha-amylase assay increases the amount of amylase detected about sixfold. BSC9 has about five to nine times as many copies per cell as MK79 of the recombinant plasmid, which carries both the amylase and hemoglobin genes, but both strains produce about the same amount of hemoglobin. While MK79 respiration decreases upon going from log to stationary phase, BSC9 respiration increases during the same period. The two latter results may be of particular importance in determining the way in which hemoglobin enhances the production of cloned protein products in recombinant bacteria.


Cloning and expression of a thermostable exo-alpha-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli. The gene coding for a thermostable exo-alpha-1,4-glucosidase (alpha-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo-alpha-1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62,000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.


Purification of lipases.

Purification of lipases. Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.


Characterization of two differently glycosylated molecular species of yeast-derived hepatitis B vaccine carrying the pre-S2 region. Modified hepatitis B virus surface antigen M protein particles (HBsAg M-P31c) produced in yeast is mainly composed of two differently glycosylated proteins, GP37 and GP34. GP37 has an N-linked sugar chain and O-linked sugar chains; and GP34 has an N-linked sugar chain bound to the peptide backbone P31. Although M-P31c vaccine elicits both anti-S and anti-pre-S2 antibodies, whether there are any differences between GP37 and GP34 in the ability to elicit these antibodies is still unknown. To clarify this issue, we prepared particles which were composed solely of GP37 or GP34 by affinity chromatography, using polymerized human serum albumin as a ligand and digestion with alpha-mannosidase. We also prepared particles composed solely of P31 by successive digestion with alpha-manosidase and endo-beta-N-acetylglycosaminidase H. The vaccines derived from these three kinds of particles elicited both anti-S and anti-pre-S2 antibodies in mice to the same extent as the original M-P31c vaccine. These results suggest that the N- and O-linked sugar chains of M-P31c component proteins produced in the host yeast cells have no effect on the ability to elicit anti-S and anti-pre-S2 antibodies and that there are no differences with respect to antibody response in mice between the two major components of M-P31c, GP37 and GP34.


Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola. Using whole cells of H. anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue. The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess (DE). In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with << 1% yield only. In contrast, H. silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H. anomala. The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield. A single enzyme of 54 kDa reduces the substrate with 97.4% DE. Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone. It was demonstrated that the enzymes often discussed for the reduction of beta-ketoesters with yeast e.g. L-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the beta-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The physiological role of the new enzyme is still unknown.


Selective protein separations using Formed-In-Place anion exchange membranes. Anion exchange membranes prepared by adsorption of polymers on Formed-In-Place microfiltration substrates were formed and ion-exchange separations of solutions containing two proteins were determined by ion exchange membrane sequential separation procedures, similar to affinity membrane separation procedures. Representative ion exchange separation processes utilizing adsorbed poly(ethylene imine) (PEI) as the ion exchange membrane for the separation of the components of solutions containing two proteins, bovine serum albumin (BSA) and lysozyme and ovalbumin and lysozyme, are described. The stability of the PEI adsorbed layer, binding characteristics of the BSA to the membrane and purification properties of the procedure were determined.


Process development for the recovery and purification of recombinant protein G. The domains of protein G from streptococcus which bind immunoglobulin G have been cloned and expressed in Escherichia coli (Fahnestock et al., 1986). Because protein G binds to several animal immunoglobulin G's, it has many immunochemical applications. This report describes process development for large-scale production of this recombinant protein G (also known as GammaBind G). In 200 l cultures of E. coli, this protein G variant was released from the cell into the culture medium by heating at 80 degrees C for 10 min. The concentration was monitored by either a competitive enzyme-linked immunoassay or a liquid chromatographic assay. Cross-flow microfiltration with 0.22 micron membrane was used to remove the cells. The protein G-rich permeate from the cross-flow microfilter was purified by affinity chromatography using a 5 l column of IgG-Sepharose 6 Fast Flow, which yielded 16-18 g of protein G per column cycle. The pools of purified protein G were concentrated and desalted using ultrafiltration. The salt-free protein G was then lyophilized as bulk product. The overall recovery through the entire process was 50-64%. The analysis of the final product included sodium dodecyl sulfate polyacrylamide gel electrophoresis, UV-visible spectrum, high performance gel filtration, endotoxin level and binding efficiency to human IgG Sepharose.


Influence of constant and oscillating dissolved oxygen concentrations on keto acid production by Gluconobacter oxydans subsps. melanogenum. Gluconobacter species are known to oxidise glucose via a direct oxidation pathway which is distinct from the pentose phosphate pathway. In the present communication results of an investigation on the influence of different dissolved oxygen concentrations (DO) on the production of 2,5-diketogluconic acid in batch and chemostat cultures are given. DO of 30% relative to air at 1 bar was found as a threshold level for optimum productivity. The positive influence of continuous availability of dissolved oxygen on the process of rapid glucose oxidation was unambiguously shown as the result of induction of membrane bound dehydrogenases involved in direct glucose oxidation. Furthermore data of scale-down experiments in which the organism was cultivated under oscillations of dissolved oxygen, are given. The influences of such oscillations of DO in the region of the established threshold (30% saturation) were found to result in a prolonged lag phase for growth and product formation. The data obtained in this study revealed critical residence times at low DO that could be employed as a criterion for scale up of this aerobic process.


Improving the production of E. coli beta-lactamase in Bacillus subtilis: the effect of glucose, pH and temperature on the production level. Bacillus subtilis has been considered a promising host for the production of foreign proteins. However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins. Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E. coli TEM beta-lactamase secreted from B. subtilis, when applying an expression system based on B. amyloliquefaciens alpha-amylase. The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium. The promoter of B. amyloliquefaciens alpha-amylase gene is repressed by glucose. However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression. We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors. Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.


12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors. A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.


Expression in E. coli and purification of intracellular proteins by fusion to cyclomaltodextrin glucanotransferase. A plasmid expression vector was constructed to direct the synthesis of foreign proteins in Escherichia coli as fusions with cyclomaltodextrin glucanotransferase (CGT) with cytoplasmic location (delta ssCGT). The ability of CGT to bind to covalently immobilized cyclodextrins was utilized in purifying fused target proteins. A large proportion of the cytoplasmically synthesized delta ssCGT formed inclusion bodies which adopted the active conformation at considerably high refolding concentration (67 microM delta ssCGT solution). By lowering the cultivation temperature the proportion of the soluble delta ssCGT was slightly increased. Intracellularly expressed delta ssCGT provides a potential affinity handle which forms easily refoldable inclusion bodies increasing the yield and stability, and possibly allows the expression of lethal target proteins. Interestingly, the interaction between one model fusion protein delta ssCGT-CAT (CAT, chloramphenicol acetyltransferase) and the E. coli heat shock protein GroEL was observed.


Gene expression following transfection of fish cells. Various genes containing different transcriptional regulatory elements (TRE) and the bacterial marker gene coding for chloramphenicol acetyl transferase were transfected into several fish cell lines to evaluate the efficiency of expression in comparison with mammalian cells. The CMV and RSV TRE were the most efficient non-inducible promoters in directing reporter gene expression. RSV and CMV appeared of similar potency in a stable fish cell line. The human HSP-70 promoter showed high potency in a carp and in a trout cell line after thermal induction. This promoter also induced the synthesis of human growth hormone directed by the corresponding cDNA, but not by the gene. RSV TRE was also able to drive the synthesis of bovine growth hormone when attached directly to the cDNA but not to the gene. These data suggest that non-fish gene TRE can be used to express foreign genes in fish cells or transgenic fish; however, in most cases they are relatively inefficient. The data also suggest that the translation and secretion machinery of fish cells can express efficiently foreign genes but that mammalian introns might be not processed properly in some cases.


Intergeneric protoplast fusion between Gluconobacter oxydans and Corynebacterium species. Intergeneric protoplast fusion between 2,5-diketo-gluconic acid producing Gluconobacter oxydans (ATCC 9937) and a mutant strain of Corynebacterium species (ATCC 31090), capable of reducing 2,5-diketo-gluconic acid to 2-keto-L-gulonic acid, a penultimate step in vitamin C production) resulted in viable recombinants. Some of the fusion products exhibited the capacity to convert D-glucose to 2-keto-L-gulonic acid, but the conversion rate is low.


Production of antimicrobials by Bacillus subtilis MIR 15. We report the isolation and characterization of a strain of Bacillus, designed MIR 15, which appears to produce and excrete antimicrobials active against Gram-negative bacteria, but not against fungi. B. subtilis MIR 15 varied its antimicrobial profiles and production with the cultivation temperature.


Influence of the temperature on the shear stress sensitivity of adherent BHK 21 cells. The influence of temperature on the shear sensitivity of anchorage-dependent baby hamster kidney (BHK) cells was investigated. The temperature effect in general was compared for stressed and unstressed cells. Both the growth rate as well as the shear sensitivity are temperature-dependent. Decreasing the temperature lowered the growth rate and increased the ability of the BHK cells to withstand shear stress.


Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans. The alpha-amylase gene (amy) from Streptomyces griseus IMRU 3570 and the beta-galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P2 or P3. The alpha-amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum.


Cloning, sequence analysis and yeast expression of the egl1 gene from Trichoderma longibrachiatum. A gene (egl1) encoding an endoglucanase (EGL1) from Trichoderma longibrachiatum has been cloned and sequenced. This gene, homologous to the T. reesei egl1 gene, differs from it in the length of the introns (particularly the first one) and encoded protein. A cDNA fragment obtained by the rapid amplification of cDNA ends method, which takes advantage of the polymerase chain reaction, has been expressed in yeast under control of the cyc-gal inducible promoter and yeast clones able to secrete active enzyme have been obtained.


Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers. Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulate in vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.


An engineering analysis of rotating sieves for hybridoma cell retention in stirred tank bioreactors. The use of internal rotating sieves for perfused hybridoma culture offers unique advantages but has been up to now largely empirical. Calculations have been performed on a 15 l spinfilter stirred tank in order to have an idea of hydrodynamic conditions inside and outside the rotating sieve. The large peripheral velocity value, resulting from sieve rotation (compared to axial and radial velocities) is expected to affect strongly sieve surface colonization by cells; this is confirmed by lab scale experiments, showing that cell colonization is prevented providing sieve rotation exceeds a defined value (around 0.6 m.s.1 tip speed); the fluid removal force calculated under these conditions appears to be in the range of 10 pN, similar to the adhesion force already reported for mammalian cells attached to inorganic substrata.


Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical "ladder" of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.


Physiology of myeloma cells grown in glucose-limited chemostat cultures. A recombinant myeloma NS1-derived clone was grown in chemostat cultures in Dulbecco's MEM/Ham's F12 (1:1) medium containing various concentrations of glucose, at a dilution rate of 0.028 h-1. Serum-supplemented cultures were virtually glucose-limited at a large range of glucose feed concentrations (0.7-5 mM). True glucose-limited cultures, however, were only established at low glucose supply levels to 1.3 mM at a maximum. In cultures obtained at higher glucose concentrations methionine was shown to be the growth-limiting compound. The pattern derived for serum-free chemostat cultures was similar, except that growth yields on glucose were much lower. Glucose was shown to be the growth-limiting substrate in cultures fed with media containing less than 4.5 mM glucose. Upon supplying glucose at higher concentrations such cultures presumably run into methionine and/or tryptophan limitation.


Enhancing monoclonal antibodies and hybridoma cell lines. We are interested in determining the range of variants present in a cell population that can actually be isolated. We have used subcloning and sublining to search for variants with increased antibody stability, increased cell line stability to freezing and defrosting, increased cell population viability, increased antibody production and the ability to grow in simpler media. This paper presents the case histories of several different hybridoma cell lines which required some property changed before they became production ready clones. We found that switching the class of an antibody from IgG3 to IgG1 did increase its stability, decrease its tendency to aggregate and allowed it to be used in a commercial diagnostic kit. We could isolate subclones that produced twice the level of antibody with a frequency of 1-3%. It was straight forward to isolate clones that were stable to freezing and defrosting or grew in a simpler media. We were not successful in increasing the maximum viability of a cell line. In conclusion, we have found that any population of hybridoma cells has natural variants with significantly enhanced properties that can be isolated.


Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus. Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33 degrees C followed by virus replication at 27 degrees C 3-4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27 degrees C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33 degrees C (6 degrees C above the optimal 27 degrees C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (14 1 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27 degrees C) to produce recombinant protein (beta-galactosidase). A cell density as high as 1 x 10(7) cells.ml-1, and a beta-gal concentration of up to 104,000 unit.ml-1 were achieved.


Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture. A mathematical model has been developed to describe the growth and infection of insect cells by recombinant baculoviruses. The model parameters were determined from a series of independent experiments involving batch suspension culture. The profiles generated by the model for cell growth, virus production and protein production agree with those observed in experiments. Presently, the model simulates only systems where cells are not growth-limited. The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.


Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates. mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575-582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.


The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function. We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a more in vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%-h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used for in vitro research and testing and is also being tested in clinical applications.


Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies. Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells, i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal antibodies to remove and inactivate murine retrovirus. Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an unknown virus be excluded. Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological manufacturing processes yielding a purified antibody for use in man.


Regulatory considerations when developing biological products. Report of the Center for Biologics Evaluation and Research. The Center for Biologics Evaluation and Research, whose regulatory authority includes monoclonal antibodies, cytokines, vaccines, toxins and somatic cellular therapies, communicates to sponsors issues for consideration in the development of biological products through the publication of "Points to Consider" and "Guideline" documents. This paper summarizes the available "Points to Consider" and "Guideline" documents and outlines recommendations from these documents for characterizing the cells used to produce biological products.


Avoiding rapid growth at high cell densities: a potentially important optimisation criterion for hybridoma cultures. Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002-0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4 x 10(9) cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.


DNA fingerprinting--a valuable new technique for the characterisation of cell lines. DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic "snap-shot" of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.


Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level. I. Steady-state responses to different glutamine feed concentrations. A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.


Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells. Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which codes for beta-galactosidase (beta-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10(-7), and 10(-6) M methotrexate (MTX), and the beta-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplified dhfr gene content and foreign beta-gal gene expression in the cell populations. A biochemical assay for beta-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10(-7) M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10(-6) M MTX was 20% lower than that of recombinant CHO cells at 10(-7) M MTX. There was no effect of 10(-5) M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect of dhfr and foreign gene amplification and increased beta-galactosidase expression.


Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads. Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.


Design and performance of a trickle-bed bioreactor with immobilized hybridoma cells. A trickle-bed system employing inert matrices of vermiculite or polyurethane foam packed in the downcomer section of a split-flow air-lift reactor has been developed for hybridoma culture to enhance antibody productivity. This quiescent condition favoured occlusion and allowed the cells to achieve densities twelve fold greater (12.8 x 10(6) cells/ml reactor for polyurethane foam) than in free cell suspension. The reactor was operated in a cyclic batch mode whereby defined volumes of medium were periodically withdrawn and replaced with equal volumes of fresh medium. The pH of the medium was used as the indicator of the feeding schedule. Glucose, lactate and ammonia concentrations reached a stationary value after 5 days. With vermiculite packing, a monoclonal antibody (MAb) concentration of 2.4 mg/l was achieved after 12 days. The MAb concentration declined then increased to a value of 1.8 mg/l. In the polyurethane foam average monoclonal antibody (MAb) concentrations reached a stationary value of 1.1 mg/l in the first 20 days and increased to a new stationary state value of 2.1 mg/l for the remainder of the production. MAb productivity in the trickle-bed reactor was 0.3 mg/l.d (polyurethane foam) and 0.18 mg/l.d (vermiculite) in comparison to 0.12 mg/l.d for free cell suspension. This trickle-bed system seems to be an attractive way of increasing MAb productivity in culture.


Maximisation of perfusion systems and process comparison with batch-type cultures. Maximisation of perfusion cultures. A comparison of cell yields and monoclonal antibody productivity from the same hybridoma has been made in a wide range of cell bioreactors including both batch and continuous perfusion cultures. The most productive systems were based on porous microcarriers in fixed and fluidised beds which can be operated with a high degree of efficiency and reliability from the physico-chemical engineering point of view. Further improvements should be possible by improving the physiological environment in dense cell cultures, as indicated by the preliminary studies that are described. These include experimental data showing the relationship between monoclonal antibody production rates with glucose, glutamine, ammonia, and oxygen levels in microporous beads. The results strongly indicate that perfusion processes that are scaleable in both volume and cell density can significantly reduce production costs. Manufacturers of biologicals from animal cells now have a choice between the proven batch-type processes and reliable perfusion systems based on microporous beads.


Modified CelliGen-packed bed bioreactors for hybridoma cell cultures. This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG1 monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 10(8) cells per cm3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1%) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.


The membrane dialysis bioreactor with integrated radial-flow fixed bed--a new approach for continuous cultivation of animal cells. A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l-1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran carriers with 3-5 mm diameter showed an advantage compared to those with 1-2 mm diameter. For the 3-5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s-1 and 0.75 mm s-1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.


Optimal medium use for continuous high density perfusion processes. For maintenance of high cell density in continuous perfusion processes not only feeding with substrates but also removal of inhibitors and toxic waste products are of special interest. High perfusion rates cause large volumes of product containing medium which have to be processed in product isolation. In order to minimize these volumes concentrated feed solutions of optimized medium are used. On the other hand, such media may cause high concentrations of toxic or inhibitory metabolites which can negatively influence cell growth and product formation. Especially, if the spent medium (or special parts of it) is used again after product isolation, the removal or even better the control of inhibitor production is of highest importance. We have developed a continuous fermentation concept and system (continuous medium cycle bioreactor, cMCB) in which both limitation and inhibition effects can be generated to identify special substances as limiting or inhibitory components. With the results from those experiments it was possible to lower the total perfusion rate during serum-free perfusion cultures of hybridoma cells and to obtain an optimal substrate utilization. The advantages for decreasing the production costs (for media, special supplements and product isolation) are obvious. The other aim of this study was to identify secreted metabolic waste products as inhibitor or toxic metabolite.


Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines. We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.