Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.     

Isolation and culture of pulmonary artery endothelial cells.

It has become increasingly evident that endothelial cells function as far more than a mechanical barrier between blood and parenchyma. Endothelial cells from one vesicular bed are known to differ structurally from those of another, and it has been suggested that they may differ functionally. Further to test the hypothesis that endothelial cells from one site may differ in terms of function from those of another site, it is necessary to test endothelium from various source after having obtained these cells in pure, well-characterized cultures. To facilitate such studies, we herein describe in detail means for the isolation, culture and characterization of endothelial cells from calf pulmonary artery. These cells may be of major interest in terms of specific metabolic activities as it has become evident that the lungs play a prominent role in determining the hormonal composition of systemic arterial blood.     

Progressive accumulation of epigenetic heterogeneity during human ES cell culture

Human embryonic stem cells (hESCs) can be maintained in culture over a large number of passages while maintaining apparently normal colony morphology. However, recent reports describe variability in epigenetic states in comparisons among different human ES cell lines. These epigenetic differences include changes in CpG methylation, expression of imprinted genes, and the status of X chromosome inactivation (XCI). We report here that the status of XCI in the female hESC line H9 (WA09) is hypervariable. We find that XIST expression can differ between individual culture isolates of H9. In addition, we find that XIST expression status can vary even between different colonies present within the same H9 culture, effectively rendering the culture mosaic. H9 cultures that lack XIST expression, but have cytological evidence of completed XCI, can also exhibit altered response to BMP4, a growth factor known to induce differentiation of hESCs to a trophectodermal lineage. In the same cultures we find biallelic expression of X-linked genes suggesting that these lines consist of mixtures of cells that retain inactivation of one of two X chromosomes following random choice. Prolonged culture of the XIST-negative isolates to high passage numbers did not result in changes in global epiproteomic signatures, demonstrating rather stable levels of post-translational nucleosome modifications within the culture-adapted hESC lines. The results show that epigenetic variants arise within human ES cell cultures after cell line derivation. In addition, the results indicate that apparently normal cultures of hESCs may contain mixtures of cells with differing epigenetic states. Assays of epigenetic integrity are warranted as quality control measures for the culture of hESCs.     

Modification of cell surface antigenicity as a function of culture conditions

Adaptation of monolayer cultures of a clonal line of rat glial cells to suspension culture resulted in the nearly complete loss of certain surface antigens. This change in surface antigenicity was paralleled by the loss of the ability of the cells to accumulate in vitro a protein specific to the nervous system (“S100-protein”). In contrast, when glial cells were co-cultivated in monolayer culture with another cell line apparently lacking these surface antigens, the number of these antigens was markedly increased. The possibility of a causal relationship between the changes in the surface antigenicity and the expression of differentiated function is considered.     

Correlation between culture of Mycobacterium tuberculosis and antimycobacterial antibody in lumbar, ventricular and cisternal cerebrospinal fluids of patients with tuberculous meningitis.

In this study positive culture for M. tuberculosis were obtained, 20% in lumbar cerebrospinal fluid (CSF), 75% in ventricular CSF and 87.5% in cisternal CSFs of patients with tuberculous meningitis. Low culture positivity in lumbar CSF is due to the low density of circulating tubercle bacilli in lumbar CSF than in cisternal or ventricular CSFs. However antimycobacterial antibody in lumbar, cisternal and ventricular CSFs circulate in significant titres and are not statistically different from one another. Since specimens of CSF can not be obtained from cisternal or ventricular routes for the routine bacteriological investigations in patients with tuberculous meningitis, detection of antimycobacterial antibody of M. tuberculosis antigen 5 in lumbar CSF by an indirect ELISA may be considered as an aid for the diagnosis of tuberculous meningitis, particularly when repeated CSF cultures are negative for M. tuberculosis.     

Why there is better evidence for culture in fish than chimpanzees

Fish have comparatively small brains and are not renowned for their intelligence. Yet a series of laboratory experiments on the guppy reveals that they can be surprisingly good at learning from each other, and that social learning processes can mediate behavioural traditions analogous to the tool using traditions of different populations of chimpanzees. Transmission chain experiments have established that arbitrary and even maladaptive information can be socially transmitted among shoals of fish. Studies of behavioural innovation in guppies are strikingly consistent with findings of equivalent studies in primates. There are strong sex differences in innovatory tendencies and social learning abilities in guppies, which also parallel observations of primates. These studies suggest that the adage ‘necessity is the mother of invention’ may be a characteristic feature of animal innovation. When considered in combination with the findings of transfer experiments carried out on natural populations of fish, it becomes apparent that fish are an excellent model system for studies of animal social learning and culture.     

The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions.     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Effects of cyclic AMP on limb bud chondrogenesis in low cell density culture

In a previous paper, it was shown that the limb bud mesodermal cells differentiated into cartilage even at low cell density by lowering the serum content in the culture medium (Hattori & Ide, Exp cell res 150 (1984) 338) [20]. The present paper describes the effects of cAMP on limb bud chondrogenesis at low cell density. cAMP promoted chondrogenesis at low cell density in cultures with various concentrations of serum. The limb bud cells differentiated into cartilage cells without forming aggregates. cAMP inhibited the loss of chondrogenic capability in serum-rich medium. The relationship between cAMP level and serum content is also discussed.     

Dynamics of recombinant protein production by mammalian cells in immobilized perfusion culture

In spite of the generally stable nature of immobilized perfusion culture, its profile of target protein production frequently shows variations. This might be explained by the drift in the metabolism of cultured cells. To address this issue, we performed a set of four Opticell bioreactor cultures producing recombinant anticogulant protein PCGFX. All the cultures lasted 40-50 days with the oxygen consumption rate (OCR) mostly around 10 μmol min⁻¹; nevertheless, glucose and lactate metabolism was fluctuated with a parallel fluctuation in the recombinant protein productivity (RPP). The mean productivity of recombinant PCGFX was determined to be about 1.0 mg day⁻¹ for all the cultures. The statistical analysis revealed a significant correlation between the lactate production rate (LPR) and RPP in two cultures. A significant correlation was further found between average OCR and RPP in another culture where OCR was exceptionally lowered under serum-free conditions. No parameter significantly correlated with RPP in the remaining one culture; thus, the overt drift of RPP resulted, at least partly, from that of the cell metabolic activity and the present data should be helpful to explore a strategy for maximizing productivity.     

Effect of steroids and antisteroids on human meningioma cells in primary culture

Human meningiomas are rich in progestin receptors but virtually devoid of oestrogen receptors. We have studied the hormonal sensitivity of meningioma cells in vitro during 8 days of primary culture in the presence of different steroids and antisteroids. On day 8 the thymidine labelling index (TLI) was determined as a measure of cell growth. To date 30 cultures have been established from 39 tissue specimens. 13 cultures had a TLI below 1.0 and their growth were not affected by hormones. The TLI of the other 17 cultures was 3.0 +/- 1.7 (mean +/- SD; range 1.2-7.7). Following culture in the presence of 1 and 10 nM progesterone TLI was 83 +/- 28% (n = 9) and 61 +/- 29% (n = 3) of that of the control cultures respectively. Although in individual cultures occasional differences were found, the overall values are not statistically different from 100. Similarly, 1 nM of oestradiol and testosterone had no effect on the TLI (n = 3). Tamoxifen at 1 nM increased the TLI to 138% in one culture and decreased it to 66% of the control in another. The antiprogestin mifepristone (RU 486) in concentrations of 0.1, 1.0, 10, 100 and 1000 nM decreased the TLI to 72 +/- 30; 54 +/- 20; 55 +/- 20; 59 +/- 18 and 65 +/- 10 respectively (n = 6-15; P less than 0.05 vs control). It is concluded that although a growth promoting effect of progestins on meningioma could not be shown, the therapeutic possibilities of antiprogestins warrant further investigation.     

Growth and phosphate uptake of a high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6 in continuous culture

A high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6, was grown in a chemostat under glucose-limitation. Two different growth patterns at steady state with various dilution rates were obtained. In one case, cells having a coccus shape tended to washout at a low dilution rate, 0.2 (h(-1)). In another, cells with a rod shape grew faster and gave a good steady-state growth at a dilution rate of 0.4. Such a close relationship between growth rate and cell morphology was found both in continuous and batch cultures. The amount of phosphate uptake per cell mass was almost constant irrespective of the dilution rate, but the rate of the uptake was maximum at about the dilution rate of 0.4. A clone of PAB-6 was isolated from the continuous culture with high dilution rate and had maximum specific growth rate of 0.7 in a simple glucosesalt medium.     

An evolutionary view of plant tissue culture: somaclonal variation and selection

Plants regenerated from in vitro cultures possess an array of genetic and epigenetic changes. This phenomenon is known as 'somaclonal variation' and the frequency of somaclonal variation (SV) is usually elevated far beyond that expected in nature. Initially, the relationship between time in culture and detected SV was found to support the widespread belief that SV accumulates with culture age. However, a few studies indicated that older cultures yielded regenerants with less SV. What leads to this seemed contradiction? In this article, we have proposed a novel in vitro callus selection hypothesis, differentiation bottleneck (D-bottleneck) and dedifferentiation bottleneck (Dd-bottleneck), which consider natural selection theory to be fit for cell population in vitro. The results of multiplication races between the cells with the true-to-type phenotype and the deleterious cells determine the increase/decrease of SV frequencies in calli or regenerants as in vitro culture time goes on. The possibility of interpreting the complex situation of time-related SV by the evolutionary theory is discussed in this paper. In addition, the SV threshold, space-determined hypothesis and D-bottleneck are proposed to interpret the loss of the regenerability through a long period of plant tissue culture (PTC).     

Sucrose stimulates branching morphogenesis of embryonic mouse lung in vitro: a problem of osmotic balance between lumen fluid and culture medium

In organ cultures of lung rudiments from 11-day mouse embryos, it was found that addition of sucrose to the culture medium stimulated branching morphogenesis and reduced lumen distension. Two possible roles of sucrose were postulated: one as a nutrient and another as a generator of osmotic pressure inducing osmosis of water from the lumen fluid to the culture medium across a simple columnar epithelial cell layer. To assess which was the case, branching morphogenesis was investigated in lung rudiments cultured in medium in which osmotic pressure was increased by the addition of lactose or NaCl rather than sucrose: similar acceleration of branching was observed in both. In another experiment, lumen fluid of cultured lung rudiments was mechanically drained each day, and significantly stimulated branching morphogenesis was observed even when sucrose was not added to the culture medium. Heparin is known to induce abnormal lumen distension and inhibits branching morphogenesis. Heparin-induced abnormal morphogenesis was prevented either by the addition of sucrose to the culture medium or by the mechanical drainage of lumen fluid. These results suggest that lumen distension caused by the accumulation of lumen fluid disrupts lung branching morphogenesis in vitro, even when the mechanism of branching morphogenesis is intact.     

Microcalorimetric study on the growth and metabolism of microencapsulated microbial cell culture

The rate of heat output is one of the suitable measurements of metabolic activity of the organism or its parts, down to the cellular or even the sub-cellular levels. In this paper, microcalorimetry was first applied to study the metabolic activity of microbial in both alginate-polylysine-alginate and alginate-chitosan-alginate microencapsulated cultures as well as in free non-encapsulated culture. The organisms used for the measurements were Escherichia coli and Saccharomyces cerevisiae. As a result of this work, it was found that, despite E. coli cell in free non-encapsulated culture has the highest metabolic rate due to the highest value of heat output, the proliferation of the cells terminates quickly with a lowest biomass formed. And we found also an obviously longer stationary phase in microencapsulated culture. As far as S. cerevisiae was concerned, it was found that there was also the highest value of heat output in free non-encapsulated culture, but the cell density was lower than that in microencapsulated culture. On account of the microcalorimetric and metabolic measurements, it can be concluded that more substrate can be used to convert to biomass in microencapsulated culture which means a higher biomass yield existed.     

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.     

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h^-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article no. 95-07-064.