Activation of membrane-associated procaspase-3 is regulated by Bcl-2.

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.

Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells.

CHO SSF3 cells grow as a suspension culture in unmodified commercial medium with only low-molecular weight ingredients. Continuous serum-free culture unexpectedly induced expression of a low dihydrofolate reductase activity in the originally dhfr- CHO cells. Nevertheless, it was possible with methotrexate to induce amplification of a gene coding for the hybrid plasminogen activator K2tu-PA cotransfected with a dhfr gene. Expression of K2tu-PA expression was proportionally increased to that of dhfr, which was measured with fluorescent methotrexate. Because no serum proteases were present, secreted K2tu-PA was not converted to the enzymatically active form, but was exclusively recovered in proenzyme form.

Scaled-up production of recombinant human renin in CHO cells for enzymatic and X-ray structure analysis.

A process was developed to produce recombinant human renin for X-ray analysis and enzyme inhibition studies. An expression vector containing a human prorenin cDNA and expressing a mouse dihydrofolate reductase selection marker was transfected into dhfr-minus Chinese hamster ovary cells. After selection of cell strains with an increased gene copy number with methotrexate, cultures of the recombinant cells were scaled-up in serum-free media. Major improvements in cellular productivity were achieved by using continuous suspension cultures with cell recycling instead of an adherent culture system or batch-mode suspension cultures. The recombinant zymogen prorenin was purified and preparatively activated with trypsin. Enzymatic properties of the recombinant active renin are described.

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

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

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

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

Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement.

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

Amyloid deposition in Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, accounts for approximately 60 percent of all victims of dementia and affects greater than 10 percent of the population over 65 years old. Although the cause is unknown, there is evidence that beta-amyloid plays an important role in its pathogenesis. The deposition of this type of amyloid in the brain and its implications in AD are discussed.

Production of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium.

The growth-factor prototrophic Chinese hamster ovary (CHO) SSF3 cell line was previously adapted for growth in serum-free media. Here we present a newly designed medium which allows these cells to grow in the absence of any exogenously added growth factors. To investigate the capacity of CHO SSF3 cells for the efficient production of recombinant proteins in protein-free media, expression plasmids containing either a human single chain urokinase-type plasminogen activator (uPA)-encoding cDNA or a humanized immunoglobulin G (IgG) kappa light chain cDNA were introduced by transfection. The tryptophan synthase (trpB) gene of Escherichia coli was used as a dominantly acting selection marker allowing the cells to survive in a medium containing indole in place of tryptophan. Some of the clones obtained exhibited a stable uPA expression over a period of several months under selective conditions and the yields were up to 74 mg of uPA/l in a bioreactor and the productivity was around 40 mg/day per 10(9) cells. The yields of IgG light chains were up to 118 mg/l and the productivity was in the order of 56 mg/day per 10(9) cells in a bioreactor. These results demonstrate the potential of CHO SSF3 cells for the efficient production of recombinant proteins under protein-free conditions.

Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture.

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were alpha-modified Eagle's minimum essential medium (MEM-alpha) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.

Cell-cell adhesion and aggregation: Influence on the growth behavior of CHO cells.

The influence of cell-cell adhesion on the growth behavior of Chinese hamster ovary (CHO) cells in suspension culture was investigated. CHO cells form aggregates under suboptimal growth conditions. Clusters are formed around decaying and dead cells. The deoxyribonucleic acid (DNA) released from these cells was found to mediate the cells was found to mediate the cell-cell adhesion. Cluster formation dramatically influenced the growth behavior of the cells. First, cells within aggregates showed a strongly reduced specific proliferation rate, and second, shear forces exerted on large aggregates caused a considerable higher specific death rate than those exerted on single cells. These factors led to a reduction of the specific growth rate up to 50%. This decrease could be avoided by addition of DNase 1 to the medium. It is shown that the separate determination of the specific proliferation and death rates is not feasible with state-of-the-art methods. To achieve a more profound and precise description of the growth pattern of animal cells, we propose an extended Monod model and describe the relevant methods.

Investigation of protein patterns in mammalian cells and culture supernatants by matrix-assisted laser desorption/ionization mass spectrometry.

The direct protein profiling of mammalian cells and bacteria has a growing influence in biotechnology as a high information bearing method for characterization of cells and cell states. Monitoring of proteins excreted in culture media not only serves to produce data on product yield and quality but provides important information on cell viability and nutrient supply that forms the basis for future process and expression optimization. Fast and simple MALDI mass spectrometry approaches were developed to efficiently characterize such complex biological systems. Several mammalian cell lines including CHO DXB11, CHOSSF3, and hybridomas were investigated; the lysis process, the sample pretreatment, and the matrix preparation were optimized for MALDI conditions. Initial experiments to observe the success of protein translation in gene expression experiments were performed. Using MALDI-compatible detergents, it was possible to extend the mass range detectable by MALDI mass spectrometry from the current range of 16,000 to 75,000 Da. In this mass range, the data are complementary (offering a better mass accuracy) to those obtained by SDS-PAGE electrophoresis experiments. These new methods were used to monitor a large-scale cultivation of hybridoma cells expressing an antibody of the IgG type. The increase in whole antibody and antibody light-chain protein, 8650 Da, and the decrease of insulin were followed during the monitoring period. Quantitative measurements of the IgG level during the cultivation compared favorably with those obtained by affinity HPLC.

The transfusion needs of an autologous bone marrow transplant patient with IgA deficiency.

BACKGROUND: Transfusion management of the patient who is undergoing a marrow or peripheral blood stem and progenitor cell transplantation is often challenging. The situation is further complicated when the patient is IgA deficient with circulating anti-IgA. CASE REPORT: This report describes an approach to transfusion therapy primarily using red cells washed by automated techniques and cryopreserved autologous plateletpheresis components. Additional platelet support was provided with manually washed allogeneic plateletpheresis components. Autologous fresh-frozen plasma was collected concurrently, and IgA-deficient allogeneic units were ordered and kept in storage, but they were not needed during transplantation. The patient experienced no transfusion sequelae as a result of the IgA deficiency. CONCLUSION: With this approach, the transfusion needs of an IgA-deficient patient were adequately met during bone marrow transplantation.