Influence of G2 arrest on the cytotoxicity of DNA topoisomerase inhibitors toward human carcinoma cells with different p53 status

We here report the influence of the cell cycle abrogator UCN-01 on RKO human colon carcinoma cells differing in p53 status following exposure to two DNA damaging agents, the topoisomerase inhibitors etoposide and camptothecin. Cells were treated with the two drugs at the IC90 concentration for 24 h followed by post-incubation in drug-free medium. RKO cells expressing wild-type, functional p53 arrested the cell cycle progression in both the G1 and G2 phases of the cell cycle whereas the RKO/E6 cells, which lack functional p53, only arrested in the G2 phase. Growth-arrested cells did not resume proliferation even after prolonged incubation in drug-free medium (up to 96 h). To evaluate the importance of the cell cycle arrest on cellular survival, a non-toxic dose of UCN-01 (100 nM) was added to the growth-arrested cells. The addition of UCN-01 was accompanied by mitotic entry as revealed by the appearance of condensed chromatin and the MPM-2 phosphoepitope, which is characteristic for mitotic cells. G2 exit and mitotic transit was accompanied by a rapid activation of caspase-3 and apoptotic cell death. The influence of UCN-01 on the long-term cytotoxic effects of the two drugs was also determined. Unexpectedly, abrogation of the G2 arrest had no influence on the overall cytotoxicity of either drug. In contrast, addition of UCN-01 to cisplatin-treated RKO and RKO/E6 cells greatly increased the cytotoxic effects of the alkylating agent. These results strongly suggest that even prolonged cell cycle arrest in the G2 phase of the cell cycle is not necessarily coupled to efficient DNA repair and enhanced cellular survival as generally believed.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Effect of increasing selenite concentrations, vitamin E supplementation and different fetal calf serum content on GPx1 activity in primary cultured rabbit hepatocytes

Primary rabbit hepatocytes from 6 week old female New Zealand White rabbits (3.0 x 10(6) viable hepatocytes per treatment) were incubated for 24 h or 48 h with two basic variants of the selenium and vitamin E free DMEM/F12-HAM nutrition medium containing 2.5% or 10% fetal calf serum (FCS). Selenium and vitamin E concentrations of the media were varied by the addition of 0, 10, 50 and 100 ng Se/mL medium as sodium selenite and 100 microg alpha-tocopheryl acetate/mL. Lactic dehydrogenase (LDH) leakage of the hepatocytes was not influenced by the various selenium concentrations of the media, whereas vitamin E addition significantly inhibited LDH release. The activity of cellular glutathione peroxidase (GPx1) was markedly induced by increasing the selenium supplementation of the culture media. Vitamin E supply further enhanced GPx1 induction. In hepatocytes cultivated at the lower serum concentration (2.5% FCS), increasing the selenite concentration of the media raised GPx1 and reduced the intracellular levels of the reduced tripeptide glutathione (GSH). No vectored relation between the selenium concentration of the media and the activity of superoxide dismutase (SOD) could be observed. After both incubation periods (24 h and 48 h) SOD activity was significantly higher in the cytosol of hepatocytes grown in media containing 10% FCS as compared to cells incubated at the 2.5% FCS level. Furthermore, SOD activity was reduced by the addition of vitamin E to the media. In conclusion the results indicate an effective metabolism of rabbit hepatocytes for selenite even in amounts as low as nanograms. A general cytoprotective role for vitamin E can be shown by its ability to decrease LDH leakage and by the reduction of SOD activity.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Flocculation in ale brewing strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: re-evaluation of the role of cell surface charge and hydrophobicity

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.     

Coevolution of bacteriophage PP01 and Escherichia coli O157:H7 in continuous culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h(-1) and by 3 orders of magnitude at a lower dilution rate (0.327 h(-1)). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h(-1) and persisted until the end of the experiment (approximately 200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

Free-flow electrophoresis. II. Analysis of the method with respect to preparative cell separation.

Electrophoretic cell separation by means of free-flow electrophoresis in an FF5 apparatus was investigated with respect to band resolution, separation capacity, reproducibility and influence on cell viability. Very sharp bands and a large separation capacity were achieved using triethanolamine/acetate buffered glycine media as liquid curtain. Acid buffer ions such as N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES) or phosphate produced broader bands. Osmotic expanders such as saccharides, though preserving cell viability excellently, decrease electrophoretic velocity and thus separation capacity. The decrease in cell viability observed in glycine media could be compensated for by addition of Ca2. Band broadening caused by methodologically specific velocity flow profiles could be reduced to a negligible level by coating the chamber walls with albumin and by appropriate adjustment of sample flow rate and liquid curtain velocity. Under the optimum conditions described, selective cell loss and artificial change in electrophoretic mobility of the cells during operation can be disregarded. The main reason for cell loss was cell aggregation at low ionic strength, which can be prevented or reversed by treatment of the cells with deoxyribonuclease.     

Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.     

Thecal cell 3-beta hydroxysteroid dehydrogenase activity: modulation by human chorionic gonadotropin, progesterone, estradiol-17 beta and dihydrotestosterone

Thecal cell steroidogenesis plays a major role in folliculogenesis within the porcine ovary. Accordingly, the effects of physiological concentrations of steroids on 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) were determined. Theca was excised from large porcine follicles and prepared in a monolayer culture in 1 ml of serum-free media. Cells were treated 24 h after culture as follows: (1) control, (2) hCG (5 IU); (3) progesterone (P, 3 micrograms); estradiol-17 beta (E, 4 micrograms); 5 beta-dihydrotestosterone (DHT, 1 microgram); (4) hCG + P or E or DHT. At 3, 6, 12, 24 and 48 h after treatment, media were assessed for P levels. For 3 beta-HSD activity, P formation by microsomal fractions incubated with 1 microM pregnenolone + 5 microM NAD+ for 1 h (37 degrees C) was monitored. Thecal cell P secretion increased from 27 to 72 h. hCG significantly (P less than 0.05) increased P levels after 36 h compared to controls. E or E + hCG decreased P levels at 36, 48, and 72 h and DHT prevented the hCG-induced increase in P secretion. 3 beta-HSD activity in thecal microsomes increased significantly from 27 to 72 h. hCG had little effect on 3 beta-HSD activity compared with controls from 27 to 36 h, but significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. However, P or P + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at all times. In addition, E or E + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. DHT prevented the hCG-induced decrease in 3 beta-HSD activity. In conclusion, porcine thecal secretion of P and microsomal 3 beta-HSD activity increased during 72 h of culture. Paradoxically, the addition of hCG to cultures enhanced media P concentrations but inhibited 3 beta-HSD activity. Further, the addition of E to cultures decreased media concentrations of P while P or E decreased 3 beta-HSD activity. Therefore, paracrine/autocrine effects of locally produced steroids may play a role in modulating thecal cell steroidogenesis.     

Selective degradation of insulin within rat liver endosomes

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.     

Modulation of porcine thecal cell aromatase activity by human chorionic gonadotropin, progesterone, estradiol-17 beta, and dihydrotestosterone

Porcine thecal cells synthesize estradiol, which may function as an intraovarian regulator of follicular growth. Production of estradiol by granulosa-cell aromatase is modulated by gonadotropins and local steroidal and nonsteroidal factors. Therefore, the effect of human chorionic gonadotropin (hCG) and physiological concentrations of steroids on aromatase activity of the thecal cells was determined. Theca was excised from large porcine follicles (greater than 10 mm diameter) and plated as monolayer cultures in 1 ml of serum-free medium. Twenty-four hours after culture, cells were treated as follows: 1) control; 2) hCG (5 IU); 3) progesterone (P, 3 micrograms), estradiol-17 beta (E, 4 micrograms), or dihydrotestosterone (DHT, 1 microgram); 4) hCG + P, E, or DHT. After 27, 30, 36, 48, and 72 h of culture, media were assessed for levels of P and E. Aromatase activity was determined by a radiometric assay. Levels of P in control media increased from 27 to 72 h. hCH significantly (p less than 0.01) increased P levels from 27 to 72 h of culture. Estrogen decreased (p less than 0.05) P levels at 36, 48, and 72 h compared to controls and also prevented the hCG-induced increase in P levels at these times. DHT significantly increased (p less than 0.05) P levels at 48 and 72 h. DHT + hCG reduced the hCG-associated increase in P concentration at 36 h and 72 h, but enhanced the hCG-induced increase in P levels at 48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21(WAF-1), and p27(KIP-1) levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G(1) arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.     

Induction of apoptosis in macrophage cell line,J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P. aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastase-positive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.     

Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality

Several small-scale Chinese hamster ovary (CHO) suspension cultures were grown in perfusion mode using a new acoustic filtration system. The separation performance was evaluated at different cell concentrations and perfusion rates for two different CHO cell lines. It was found that the separation performance depends inversely on the cell concentration and perfusion rate. High media flow rates as well as high cell concentrations resulted in a significant drop in the separation performance, which limited the maximal cell concentration achievable. However, packed cell volumes of 10% to 16% (corresponding to 3 to 6. 10(7) cells/mL) could be reached and were maintained without additional bleeding after shifting the temperature to 33 degrees C. Perfusion, up to 50 days, did not harm the cells and did not result in a loss of performance of the acoustic filter as often seen with other perfusion systems. Volumetric productivities in perfusion mode were 2- to 12-fold higher for two cell lines producing two different glycoproteins when compared to fed-batch or batch processes using the same cell lines. Product concentrations were in the range of 20% to 80% of batch or fed-batch culture, respectively. In addition, using the protease-sensitive product rhesus thrombopoietin, we could show that cultivation in perfusion mode drastically reduced proteolysis when compared to a batch culture without addition of protease inhibitors such as leupeptin.     

Effect of cytokines and growth factors on the expression of elastase activity by human synoviocytes, dermal fibroblasts and rabbit articular chondrocytes

Human synoviocytes, rabbit articular chondrocytes and human skin fibroblasts in culture were examined for their ability to express elastase activity. Latent enzyme activity degrading insoluble elastin was detected in the culture media of the three cell types and was completely abolished by metal chelating agents. Triton X-100 cell extracts were found to degrade a synthetic elastase substrate, N Succinyl-(Ala)3p-nitroanilide (SANA). The SANA-degrading activity of cell extracts could be attributed to a metalloprotease for fibroblasts and synoviocytes (100%) and to a metalloprotease associated with a cysteine protease for chondrocytes (70 and 30% respectively). This SANA-degrading activity was partly due to the combined action of an endo and an exopeptidase. Tumor Necrosis Factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) significantly enhanced the elastin degrading activity present in the culture media of both synoviocytes and chondrocytes. Interleukin-1 beta significantly increased the secretion of elastase by chondrocytes. By contrast, Transforming Growth Factor-beta (TGF-beta) reduced by 80 per cent the secretion of elastinolytic activity by chondrocytes but had not effect on other cell types.     

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. (c) 1993 John Wiley & Sons, Inc.     

Initiation and growth characterization of some hop cell suspension cultures

Cell suspension cultures of some hop (Humulus lupulus L.) cultivars were initiated from corresponding callus cultures induced on different media. Dissimilation curves were determined to characterize the growth of the suspension cultures maintained in Gamborg's B5 and in Murashige and Skoog's medium. Both media proved to be suitable; a comparison of the curves obtained for suspension cultures of the hop cultivar Wye Northdown grown in both media did not reveal striking differences. For four hop cell lines, the concentration of nitrate and sugar in the culture medium was analysed by HPLC during the growth of their suspension cultures. The cell suspension cultures of the various hop cultivars were also screened for the presence of bitter acids by HPLC and of volatile compounds by capillary GC. However, neither bitter acids nor volatile compounds could be detected; the addition of a non-toxic lipophylic phase (XAD-2, XAD-1180 or Miglyol 812) to the culture media did not help to induce the formation of volatile compounds.This paper will also appear as a chapter of the Ph.D. thesis of the first author; this thesis will be distributed among colleagues only.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels

The filamentous fungus Paecilomyces lilacinus is currently developed as a biocontrol agent against plant parasitic nematodes. Nematode eggs and cuticles are the infection sites for biocontrol agents that penetrate by the production of lytic enzymes. P. lilacinus was cultured in liquid media and proteases and chitinases were induced by the introduction of egg yolk and chitin, respectively. A serine protease was purified from a culture medium using Sepharose-bacitracin affinity column. The protease occurred in three forms, two of which were C-terminally truncated. Chitinase activity was also observed in the culture supernatant, and after separation by isoelectric focusing six proteins were detected that showed activity. Chitinase activity was further confirmed on non-denaturing one-dimensional (1D) and two-dimensional (2D) gels using a sandwich assay with glycol chitin as a substrate. Two of the proteins had similarities with endochitinases as shown by their N-terminal amino acid sequences.     

Enhancement of 2-phenylethanol productivity by Saccharomyces cerevisiae in two-phase fed-batch fermentations using solvent immobilization

The bioconversion of L-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae in fed-batch experiments has shown that concentrations of 2-phenylethanol of >2.9 g/L have a negative impact on the oxidative capacity of the yeast. Without tight control on ethanol production, and hence on the feed rate, ethanol rapidly accumulates in the culture media, resulting in complete inhibition of cell growth before the maximal 2-phenylethanol concentration of 3.8 g/L, obtained in the absence of ethanol production, could be achieved. This effect was attributed to a cumulative effect of ethanol and 2-phenylethanol, which reduced the tolerance of the cells for these two products. To enhance the productivity of the bioconversion, a novel in situ product recovery strategy, based on the entrapment of an organic solvent (dibutylsebacate) into a polymeric matrix of polyethylene to form a highly absorbent and chemically and mechanically stable composite resin, was developed. Immobilization of the organic solvent successfully prevented phase toxicity of the solvent and allowed for an efficient removal of 2-phenylethanol from the bioreactor without the need for prior cell separation. The use of the composite resin increased the volumetric productivity of 2-phenylethanol by a factor 2 and significantly facilitated downstream processing, because no stable emulsion was formed. The 2-phenylethanol could be backextracted from the composite resin, yielding a concentrated and almost cell-free solution. In comparison to two-phase extractive fermentations with cells immobilized in alginate-reinforced chitosan beads, the use of a composite resin was extremely inexpensive and simple. In addition, the composite resin was found to be insensitive to abrasion and chemically stable, such that sterilization with 2 M NaOH or heat was possible. Finally, the composite resin could be produced on a large scale using commercially available equipment.