Effect of temperature, medium composition, and cell passage on production of herpes-based viral vectors

Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33 degrees C than at 37 degrees C and is further stabilized at 4 degrees C. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33 degrees C produced 2-fold higher amounts of vector than infected cells incubated at 37 degrees C, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33 degrees C than 37 degrees C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33 degrees C yielded similar levels regardless of passage number but was reduced at 37 degrees C as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.     

Effects of culture parameters on the production of retroviral vectors by a human packaging cell line

The use of retroviral vectors for human gene therapy requires the production of large quantities of high titer vector stocks. Maintaining high titers during the prolonged culture of packaging cells will require that critical parameters be controlled. The aim of this study was to determine which culture parameters critically affect the production/decay of retroviral vectors produced by the human packaging cell line FLYRD18/LNC-hB7. The stability of retroviral vectors released by this cell line was found to be temperature dependent (half-life of 6.9, 11.0, and 64.3 h when incubated at 37, 32, and 0 degrees C, respectively). Titers increased up to 10-fold when the packaging cells were cultured at 32 degrees C, compared to 37 degrees C, despite a decrease in cell yield (cell-specific titers were 20-fold higher). Virus titers were also over 10-fold higher when the packaging cells were cultured in a reduced serum concentration (1%) compared to 5%. Retrovirus production at a range of pH levels revealed a significant decrease in virus titer at pH levels below 6.8 and above 7.2, optimum titers being achieved in cultures at pH 7.2. Dissolved oxygen levels in the range 20-80% did not significantly affect titers under the conditions tested. Finally, a packed bed system containing the packaging cells immobilized on porous microcarriers was shown to sustain the production of active retroviral vectors for over 1 month, in relatively large volumes.     

Recombinant Fab expression and secretion in Escherichia coli continuous culture at medium cell densities: Influence of temperature

Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions.     

High-titer adenovirus vector production in 293S cell perfusion culture

Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 x 10(5) cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 x 10(6) cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 x 10(6) cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 x 10(6) cells/mL. One of the cultures was set at 37 degrees C and the two others at 35 degrees C. After a rapid initial cell loss, the average cell density stabilized at 5.75 x 10(6) cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 x 10(9) infectious viral particles/mL (IVP/mL) at 37 degrees C and 7.8 x 10(9) IVP/mL at 35 degrees C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 degrees C can improve viral titer by 2.4-fold compared to 37 degrees C, in accordance with a previous study on adenovirus batch production.     

Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions

Summary Genes encoding a light chain and an Fd region (a variable region and a CH1 domain of a heavy chain) of a mouse-human chimeric antibody with specificity for human carcinoembryonic antigen (CEA) were fused to a DNA segment coding for the signal peptide of Escherichia coli ompF. E. coli cells harbouring an expression vector containing these genes downstream of a tac promoter were able to secrete a Fab fragment of the antibody efficiently. When the cells were cultured at 37° C and the inducer (isopropyl-\-d-thiogalactopyranoside, IPTG) concentration was 1 mm (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; <90 ng/l culture). In order to optimize functional Fab production, we examined the influence of culture conditions (i.e. temperature and the inducer concentration) on secretion of the product. It was found that a 12.7-fold higher amount of Fab fragment could be produced at 30° C using 0.1 mm IPTG, as compared with standard conditions. Under these optimal conditions, functional Fab accumulated in the periplasm and culture medium for 10 h after induction and the total production level was found to reach approximately 4.5 mg/l culture. Correspondence to: T. Shibui     

Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium

Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.     

Antibody dependent cell-mediated cytotoxicity against Trypanosoma cruzi.

This paper describes in vitro antibody dependent cytotoxicity against Trypanosoma cruzi epimastigotes by normal mouse splenic lymphocytes. Cytotoxicity was expressed as the percentage reduction in the number of motile parasites upon incubation with lymphocytes at 37 degrees C in a defined medium. Failure of the non-motile parasites to regain motility and their ensuing degeneration of 28 degrees C in liver infusion tryptose (LIT) medium confirmed loss of motility as a criterion of cytotoxicity. Incubation of T. cruzi cruzi at 37 degrees C for 18 h in a defined medium per se did not interfere with motility but was followed by a lag phase of the growth curve in LIT medium at 28 degrees C. The lag phase was prolonged for T. cruzi which had previously been incubated at 37 degrees C in the absence of cells.     

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.     

The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium.

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.     

米曲霉果胶酸酯裂解酶（pectin lyase 1）在原核系统中重组表达

来自米曲霉(Aspergillus oryzae)和黑曲霉(Aspergillus niger)的果胶酸酯裂解酶(pectinlyase)一直被用于传统发酵食品的生产,但自然条件下A.oryzae和A.niger的果胶酸酯裂解酶产量较低。通过RT-PCR的方法,获得不含信号肽的A.oryzaePel1cDNA,将Pel1cDNA连入pET-28a( )载体,构建pET-28a( )-pel1质粒。pET-28a( )-pel1转化Turner(DE3)placⅠ细胞,得到转化子pET-28a( )-pel1-Turner(DE3)placⅠ,表达与6个组氨酸融合的Pel1。进一步对Pel1在E.coli系统中表达的条件进行了研究,在37℃,220r/min条件下,培养pET-28a( )-pel1-Turner(DE3)placⅠ细胞,当OD600至0.8左右时,用500μmol/Lisopropylβ-D-thiogalactogalactop-yranoside(IPTG)进行诱导表达,在15℃和170r/min条件下,继续培养60h后,表达效果最好,产酶可达到400u/mL,是A.oryzae自然条件下产酶量的4000倍,也高于已报道的真菌果胶酸酯裂解酶在真菌体系中重组表达的效果。     

Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.     

Development of a fed-batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat

To effectively achieve tight regulation and high-level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30 degrees -->41 degrees C stepwise upshift, the shake-flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30 degrees C. By exploiting the fed-batch fermentation approach, the recombinant cell batch initially kept at 30 degrees C in a lab-scale fermentor was exposed to 41 degrees C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37 degrees C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880-fold over that obtained at the shake-flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale-up of recombinant proteins.     

Involvement of protease in L-glutamine control of nucleic acid and polyphosphate metabolism in cells transformed by 9,10-dimethyl-1,2-benzanthracene, SV40 and H-ras oncogene

Mammalian cells transformed with either 9,10-dimethyl-1,2-benzanthracene, SV40 or H-ras oncogene dramatically changed their ability to synthesize DNA and RNA and metabolize polyphosphate when L-glutamine was withdrawn from the growth medium or when heat shocked (growth at 42 degrees C). Untransformed, DNA and RNA synthesis decreased by 50-80% when glutamine was withdrawn, but polyphosphate accumulated whether or not glutamine was supplied. Heat shock did not alter this response. Transformed isogenic cells responded differently; at 37 degrees C, they decreased their synthesis of DNA and RNA if starved for glutamine, whereas at 42 degrees C, synthesis was optimal without glutamine. Transformed cells accumulated polyphosphate at 37 degrees C when starved for glutamine, but at 42 degrees C, no polyphosphate accumulated. This apparent non-dependence on glutamine by transformed cells when heat shocked was found to be due to the production of glutamine from serum proteins through induction of a protease(s).     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

A fluorescence study on the mobility of surface antigens of untreated tumor cells and of tumor cells undergoing cell-mediated lysis

Heterologous (rabbit) antibodies were raised against murine P-815 mastocytoma cells of DBA/2 origin. Antisera and IgG preparations were highly cytotoxic, whereas Fab fragments thereof lost all activity. Fab fragments also showed a much lower avidity than IgG, both for tumor and normal DBA/2 and C57 spleen cells as measured by the release of iodinated Fab and IgG. Both preparations bound specifically to P-815 cells since they were capable of inhibiting T cell-mediated target cell lysis. The binding of IgG and monovalent Fab fragments was studied by fluorescence. Rhodamine-coupled IgG bound homogeneously in the cold and quickly formed patches upon warming but did not form caps even after prolonged incubation at 37 degrees C. Rhodamine-coupled Fab fragments also bound homogeneously. Their distribution was unaltered after incubation at 37 degrees C even when tumor cells formed uropod-like tails. Fab fragments, however, could be induced to cap with a second and third antibody layer. P-815 cells labeled with rhodamine-coupled Fab fragments were incubated with cytolytic T cells (CTL). The conjugates formed between CTL and fluorescent target cells were observed. No gross redistribution of surface antigens on target cells was observed even at late stages of the lytic process. CTL, therefore, do not seem to operate via a redistribution of surface antigens.     

The effect of elevated temperature on the aggregation of African green monkey kidney cells

The aggregation kinetics of African green monkey kidney cells CV1 and of the SV40 transformed derivative COS1 cells that had been incubated at 37 degrees C or 43.5 degrees C was studied using the shaking flask system. COS1 cells show a three fold decrease in aggregation rate compared to CV1 cells when both cell types were incubated and aggregated at 37 degrees C. When these cell types were incubated at 43.5 degrees C for 5 hours, then aggregated at 37 degrees C showed a faster aggregation kinetics than before. Their aggregation at 43.5 degrees C with prior incubation at 37 degrees C or 43.5 degrees C reached the aggregation kinetics of 43.5 degrees C incubated cells aggregated at 37 degrees C. The addition of serum in the aggregation medium did not influence extensively the aggregation rates of both cell types.     

Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C

To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.     

Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes

The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which [14C]acetylcholine had been accumulated previously. The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C. Inhibition of [14C]acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C. Both Fabs reversed the acetylcholine release inhibition by the toxin. However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the "lag time."     

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.