Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Baculovirus production for gene therapy: the role of cell density,multiplicity of infection and medium exchange

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 × 10¹⁰ IP.mL⁻¹ were obtained in cultures infected at 3.5 × 10⁶ cells.mL⁻¹, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.     

Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus

The susceptibility of third instar larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Pyralidae) to ten distinct plaque purified genotypic variants of a selected isolate of the Anticarsia gemmatalis multiple-embedded nuclear polyhedrosis virus (AgMNPV), was compared. Despite the fact that this isolate, AgMNPV-Ds20, represents a wild strain of the AgMNPV selected for higher virulence to D. saccharalis, an alternate host, most of the variants are much more virulent to the original host Anticarsia than to Diatraea. Bioassays have shown an over one hundred-fold variation in LD₅₀ values ranging from 1700 polyhedron inclusion bodies (PIBs) to more than 200 000 PIBs/larva. The PIB production in infected larvae increased with the pathogenicity of the variant to the host, showing an average ten-fold reduction in Diatraea when compared to Anticarsia for the same variant. The virus particle yield ranged from 6×10⁷ to more than 10⁹ PIBs/g of infected larvae in Diatraea and from 8×10⁸ to more than 10¹⁰ PIBs/g of infected Anticarsia larvae. The data show a clear difference of the pathogenicity of the genotypic variants of AgMNPV in vivo both between the original and alternate host and between the individual variants for the same host. These differences found in vivo indicate that monitoring of shifts in variant frequency of wild and laboratory-propagated viral isolates in these highly heterogeneous populations would help ensure the efficacy of biological control programs.     

Adenovirus vector production using low-multiplicity infection of 293 cells

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001–0.1 transductional unit (TU) cell⁻¹. The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell⁻¹), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 × 10⁵ cells cm⁻³ were infected with Ad EGFP at a low MOI of 0.001 TU cell⁻¹, the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell⁻¹) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.     

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus–insect cell expression system

A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O₂ mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 10⁶ cells ml⁻¹) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 10⁶ cells ml⁻¹ were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell⁻¹, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L⁻¹ of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 10⁶ cells ml⁻¹. Received 09 December 1996/ Accepted in revised form 13 April 1997     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10^6.25 TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and −60 °C.     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products

The baculovirus expression vector system was employed to produce human apolipoprotein E and β-galactosidase in order to study the effect of multiplicity of infection on secreted and non-secreted recombinant protein production. Prior knowledge of the influence of other cell culture and infection parameters, such as the cell density at time of infection and the time of harvest, allowed determination of the direct and indirect influences of multiplicity of infection on recombinant protein synthesis and degradation in insect cells. Under non-limited, controlled conditions, the direct effect of multiplicity of infection (10⁻¹−10 pfu/cell) on specific recombinant product yields of non-secreted β-galactosidase was found to be insignificant. Instead, the observed increased in accumulated product was directly correlated to the total number of infected cells during the production period and therefore ultimately dependent on an adequate supply of nutrients. Only the timing of recombinant virus and protein production was influenced by, and dependent on the multiplicity of infection. Evidence is presented in this study that indicates the extremely limited predictability of post-infection cell growth at very low multiplicities of infection of less than 0.1 pfu/cell. Due to the inaccuracy of the current virus quantification techniques, combined with the sensitivity of post-infection cell growth at low MOI, the possibility of excessive post-infection cell growth and subsequent nutrient limitation was found to be significantly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination at low multiplicity of infection is investigated for a secreted recombinant form of human apolipoprotein E. Comparison of human apolipoprotein E production and secretion at multiplicities of infection of 10⁻⁴−10 pfu/cell revealed increased product degradation and contamination with intracellular proteins at low multiplicities of infection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.     

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°C followed by virus replication at 27°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°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°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×10⁷ cells.ml⁻¹, and a β-gal concentration of up to 104,000 unit.ml⁻¹ were achieved.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Toxicity assessment of paraverine hydrochloride and papaverine-derived metabolites in primary cultures of rat hepatocytes

Summary The present study was undertaken to assess and compare the toxic effects of papaverine hydrochloride and its metabolites. Primary cell cultures of rat hepatocytes were treated with papavarine (papaver), 3′-O-desmethyl (3′-OH), 4′-O-desmethyl (4′-OH), and 6-O-desmethyl (6-OH) papaverine at 1×10⁻⁵, 1×10⁻⁴, and 1×10⁻³ M for 4,8, 12, and 24-h periods. Cell injury was determined by: a) cell viability using the trypan blue exclusion test; b) cytosolic enzyme leakage of lactate dehydrogenase and aspartate aminotransferase; c) morphologic alterations; and d) lactate: pyruvate (L:P) ratios. Cell cultures showed concentration-and time-dependent responses. For example, a decrease in cell viability and an increase in enzyme leakage were observed after cell treatment with 1×10⁻⁴ and 1×10⁻³ M papaver for 8 h; 1×10⁻³ M 6-OH papaverine for 8 h and 1×10⁻⁴ M for 24 h; and 1×10⁻³ M 4′-OH papaverine for 24 h (P<0.05). Furthermore, changes in morphology correlated to cell viability and enzyme release in those cultures treated with papaver, 4′-OH and 6-OH papaverine. Some of these changes included size deformation, cell detachment from the dishes, and cell necrosis. On the other hand, an increase in L:P ratios (P<0.05) was detected with papaver as early as 8 h with 1×10⁻⁴ and 1×10⁻³ M and 12 h with 1×10⁻⁵ M; 6-OH showed an increase, in L:P ratios at 8 h with 1×10⁻³ M and 12 h with 1×10⁻⁴ M; these changes were evident with 4′-OH at 12 h with 1×10⁻³ M. In contrast, cells treated with 3′-OH papaverine did not show significant damage with any time period and concentration used in this study. The results of this study indicate that papaverine-derived metabolites are less cytotoxic than its parent compound, papaver. The toxicity was ranked as follows: papaver>6-OH>4′-OH>−3′-OH. This work was supported in part by grant ES04200-02 from the National Institute of Environmental Health Sciences, Bethesda, MD. Presented in part at the fall ASPET meeting in Salt Lake City, August, 1989. Daniel Acosta is a Burroughs Wellcome Scholar in Toxicology.     

Regeneration through organogenesis in rattan

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l⁻¹ glutamine, 100 mg l⁻¹ arginine, 100 mg l⁻¹ asparagine, 60 g l⁻¹ sucrose, 8 g l⁻¹ Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Cultivation ofLactobacillus crispatus KLB46 isolated from human vagina

Bacterial vaginosis can be treated by restoring the normal vaginal flora using lactobacilli.Lactobacillus crispatus KLB46 that was isolated from the human vagina has a strong antimicrobial activity and was grown in a batch and in a continuous fermentor. During batch cultivation, the maximum specific growth rate ofL. crispatus KLB 46 was 0.63 h⁻¹ and the highest viable cell count (1.9×10⁹ CFU/mL) was obtained at pH 5.5.L. crispatus KLB 46 did not grow well at either pH 3.5 or 7.5. During continuous cultivation, the highest viable cell count (1.53×10⁹ CFU/mL) was obtained at a dilution rate of 0.32 h⁻¹. However, the maximum productivity of viable cells was obtained at a dilution rate of 0.52 h⁻¹, and was 7.33×10¹¹ CFU L⁻¹ h⁻¹, that is approximately 5 times higher than that obtained from batch culture.     

Metabolic flux analysis of a poly-β-hydroxybutyrate producing cyanobacterium,Synechococcus sp. MA19, grown under photoautotrophic conditions

To understand the utilization property of light energy,Synechococcus sp. MA19, a poly-β-hydroxybutyrate (PHB) producer, was cultivated at the different incident light intensities of 15.3, 50.0 and 78.2 W/m² using media with and without phosphate. From the results of metabolic flux analysis, it was found that the cell yield based on ATP synthesis was estimated as 3.5×10⁻³ kg-biomass/mol-ATP in these cultures. Under the examined conditions, there were no significant differences in the efficiency of light energy conversion to chemical energies estimated as ATP synthesis and reducing potential (NADH+NADPH) formation whether the PHB synthesis took place or not. The energy converted from light to ATP was kept relatively high around the energy absorbed by the cells of 2.5–3.0×10⁶ J h⁻¹ kg⁻¹, whereas the energy of reducing potential was hardly changed in the examined range of the energy absorbed by the cells.