The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

The VCAM-1 gene that encodes the vascular cell adhesion molecule is a target of the Sry-related high mobility group box gene, Sox18

VCAM-1 (vascular cell adhesion molecule-1) and Sox18 are involved in vascular development. VCAM-1 is an important adhesion molecule that is expressed on endothelial cells and has a critical role in endothelial activation, inflammation, lymphatic pathophysiology, and atherogenesis. The Sry-related high mobility group box factor Sox18 has previously been implicated in endothelial pathologies. Mutations in human and mouse Sox18 leads to hypotrichosis and lymphedema. Furthermore, both Sox18 and VCAM-1 have very similar spatio-temporal patterns of expression, which is suggestive of crosstalk. We use biochemical techniques, cell culture systems, and the ragged opossum (RaOP) mouse model with a naturally occurring mutation in Sox18 to demonstrate that VCAM-1 is an important target of Sox18. Transfection, site-specific mutagenesis, and gel shift analyses demonstrated that Sox18 directly targeted and trans-activated VCAM-1 expression. Importantly, the naturally occurring Sox18 mutant attenuates the expression and activation of VCAM-1 in vitro. Furthermore, in vivo quantitation of VCAM-1 mRNA levels in wild type and RaOP mice demonstrates that RaOP animals show a dramatic and significant reduction in VCAM-1 mRNA expression in lung, skin, and skeletal muscle. Our observation that the VCAM-1 gene is an important target of SOX18 provides the first molecular insights into the vascular abnormalities in the mouse mutant ragged and the human hypotrichosis-lymphedematelangiectasia disorder.

A physiological product-release model for baculovirus infected insect cells

Existing models describe the product release from baculovirus infected insect cells as an unspecific protein leakage occurring in parallel with protein production. The model presented here shows that the observed product release of normally non-secreted proteins can be described through cell death alone. This model avoids the implicit non-physiological assumption of previous models that cells permeable to recombinant protein as well as trypan blue continue to produce protein. (c) 2005 Wiley Periodicals, Inc.

A phase-segregated model for plant cell culture: The effect of cell volume fraction

Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases, Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus, Large errors in the simulated results were observed if the phase-segregation was not considered.

Analysis and application of an equilibrium model for in vitro bioassay systems with three components: Receptor, hormone and hormone-binding-protein

A simple theoretical framework is presented for bioassay studies using three component in vitro systems. An equilibrium model is used to derive equations useful for predicting changes in biological response after addition of hormone-binding-protein or as a consequence of increased hormone affinity. Sets of possible solutions for receptor occupancy and binding protein occupancy are found for typical values of receptor and binding protein affinity constants. Unique equilibrium solutions are dictated by the initial condition of total hormone concentration. According to the occupancy theory of drug action, increasing the affinity of a hormone for its receptor will result in a proportional increase in biological potency. However, the three component model predicts that the magnitude of increase in biological potency will be a small fraction of the proportional increase in affinity. With typical initial conditions a two-fold increase in hormone affinity for its receptor is predicted to result in only a 33% increase in biological response. Under the same conditions an Ii-fold increase in hormone affinity for receptor would be needed to produce a two-fold increase in biological potency. Some currently used bioassay systems may be unrecognized three component systems and gross errors in biopotency estimates will result if the effect of binding protein is not calculated. An algorithm derived from the three component model is used to predict changes in biological response after addition of binding protein to in vitro systems. The algorithm is tested by application to a published data set from an experimental study in an in vitro system (Lim et al., 1990, Endocrinology 127, 1287-1291). Predicted changes show good agreement (within 8%) with experimental observations. (C) 1998 Academic Press Limited.

Multicultural career counseling: Theoretical applications of the systems theory framework

Increasing recognition of cultural influences on career development requires expanded theoretical and practical perspectives. Theories of career development need to explicate views of culture and provide direction for career counseling with clients who are culturally diverse. The Systems Theory Framework (STF) is a theoretical foundation that accounts for systems of influence on people's career development, including individual, social, and environmental/societal contexts. The discussion provides a rationale for systemic approaches in multicultural career counseling and introduces the central theoretical tenets of the STF. Through applications of the STF, career counselors are challenged to expand their roles and levels of intervention in multicultural career counseling.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

Investigation of candidate division TM7, a recently recognized major lineage of the domain bacteria with no known pure-culture representatives

A molecular approach was used to investigate a recently described candidate division of the domain Bacteria, TM7, currently known only from environmental 16S ribosomal DNA sequence data, A number of TM7-specific primers and probes were designed and evaluated. Fluorescence in situ hybridization (FISH) of a laboratory scale bioreactor using two independent TM7-specific probes revealed a conspicuous sheathed-filament morphotype, fortuitously enriched in the reactor. Morphologically, the filament matched the description of the Eikelboom morphotype 0041-0675 widely associated with bulking problems in activated-sludge wastewater treatment systems. Transmission electron microscopy of the bioreactor sludge demonstrated that the sheathed-filament morphotype had a typical gram-positive cell envelope ultrastructure. Therefore, TM7 is only the third bacterial lineage recognized to have gram-positive representatives. TM7-specific FISH analysis of two full-scale wastewater treatment plant sludges, including the one used to seed the laboratory scale reactor, indicated the presence of a number of morphotypes, including sheathed filaments. TM7-specific PCR clone libraries prepared from the two full-scale sludges yielded 23 novel TM7 sequences. Three subdivisions could be defined based on these data and publicly available sequences. Environmental sequence data and TM7-specific FISH analysis indicate that members of the TM7 division are present in a variety of terrestrial, aquatic, and clinical habitats. A highly atypical base substitution (Escherichia coli position 912; C to U) for bacterial 16S rRNAs was present in almost all TM7 sequences, suggesting that TM7 bacteria, like Archaea, may be streptomycin resistant at the ribosome level.

Model-based analysis of anaerobic acetate uptake by a mixed culture of polyphosphate-accumulating and glycogen-accumulating organisms

An increasing number of studies shows that the glycogen-accumulating organisms (GAOs) can survive and may indeed proliferate under the alternating anaerobic/aerobic conditions found in EBPR systems, thus forming a strong competitor of the polyphosphate-accumulating organisms (PAOs). Understanding their behaviors in a mixed PAO and GAO culture under various operational conditions is essential for developing operating strategies that disadvantage the growth of this group of unwanted organisms. A model-based data analysis method is developed in this paper for the study of the anaerobic PAO and GAO activities in a mixed PAO and GAO culture. The method primarily makes use of the hydrogen ion production rate and the carbon dioxide transfer rate resulting from the acetate uptake processes by PAOs and GAOs, measured with a recently developed titration and off-gas analysis (TOGA) sensor. The method is demonstrated using the data from a laboratory-scale sequencing batch reactor (SBR) operated under alternating anaerobic and aerobic conditions. The data analysis using the proposed method strongly indicates a coexistence of PAOs and GAOs in the system, which was independently confirmed by fluorescent in situ hybridization (FISH) measurement. The model-based analysis also allowed the identification of the respective acetate uptake rates by PAOs and GAOs, along with a number of kinetic and stoichiometric parameters involved in the PAO and GAO models. The excellent fit between the model predictions and the experimental data not involved in parameter identification shows that the parameter values found are reliable and accurate. It also demonstrates that the current anaerobic PAO and GAO models are able to accurately characterize the PAO/GAO mixed culture obtained in this study. This is of major importance as no pure culture of either PAOs or GAOs has been reported to date, and hence the current PAO and GAO models were developed for the interpretation of experimental results of mixed cultures. The proposed method is readily applicable for detailed investigations of the competition between PAOs and GAOs in enriched cultures. However, the fermentation of organic substrates carried out by ordinary heterotrophs needs to be accounted for when the method is applied to the study of PAO and GAO competition in full-scale sludges. (C) 2003 Wiley Periodicals, Inc.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhIRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown OS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Delta/as mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-1) could not be detected when any of the lasRI or rhIRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of OS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a CS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

The prevalence and concentration of Escherichia coli O157 in faeces of cattle from different production systems at slaughter

Aims: To determine the prevalence and concentration of Escherichia coli O157 shed in faeces at slaughter, by beef cattle from different production systems. Methods and Results: Faecal samples were collected from grass-fed (pasture) and lot-fed (feedlot) cattle at slaughter and tested for the presence of E. coli O157 using automated immunomagnetic separation (AIMS). Escherichia coli O157 was enumerated in positive samples using the most probable number (MPN) technique and AIMS and total E. coli were enumerated using Petrifilm. A total of 310 faecal samples were tested (155 from each group). The geometric mean count of total E. coli was 5 x 10(5) and 2.5 x 10(5) CFU g(-1) for lot- and grass-fed cattle, respectively. Escherichia coli O157 was isolated from 13% of faeces with no significant difference between grass-fed (10%) and lot-fed cattle (15%). The numbers of E. coli O157 in cattle faeces varied from undetectable (