Determination of antibody content in live versus dead hybridoma cells: analysis of antibody production in osmotically stressed cultures

Hybridomas are known to exhibit increased specific antibody production rated when subjected to environmental stress. Under these conditions, viability is low so that population-average measurements do not properly reflect the state of viable cells. Even for flow cytometry, which gives a population distribution, special techniques must be used to discriminate between viable and nonviable cells. We describe the use of the vital stain ethidium monoazide (EMA) for independent measurement of intracellular antibody content in live and dead cells via flow cytometry. EMA is shown to be superior to light scattering techniques in identifying dead cells. We apply this technique to show that, in control batch culture, the specific antibody prodution rate and antibody content in live cells are constant during exponential growth, but decrease as cells enter the stationary phase. Antibody is retained in dead cells, but at a lower level than in live cells. We further show that, under hyperosmotic stress, the specific antibody production rate and antibody content in live both remain high during death phase. (c) 1992 John Wiley & Sons, Inc.     

Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488

Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed.     

Enumerative fluorescent vital staining of live and dead pathogenic yeast cells.

A quantitative technique is presented for differentiating live and dead yeast cells grown in culture through the use of the fluorescent dye acridine orange. The method gives results that correlate well with those of other commonly used vital staining techniques and is free of certain interpretative errors inherent in them. Vital staining of yeasts with acridine orange also allows for more precise assessment of the physiological state of individual cells and the culture as a whole. The progressive senescence of yeast cells in culture can be monitored by the changing staining characteristics of several subcellular organelles. The method is simple and reliable.     

Efficiency of potassium permanganate in differentiating between live and dead nematodes

An aqueous solution of potassium permanganate provided a simple and reliable means for differentiating live from dead nematodes. The cuticle and body content of dead nematodes were stained light amber to deep brown, whereas live nematodes were not affected. Taxonomic features of lightly or moderately stained specimens were not obscured.     

Comparison of Pb2 accumulation characteristics between live and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans

Pb2+ accumulation processes between live and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans are different. In the case of S. cerevisiae, the Pb2+ accumulation capacity of the live cells was higher than that of the dead cells but they showed reversed initial Pb2+ accumulation rates. On the contrary, A. pullulans used a different process due to the existence of extracellular polymeric substances, which allowed both the capacity and the initial rate of Pb2+ accumulation in the live cells to be higher than those in the dead cells. © Rapid Science Ltd. 1998     

Distribution of live and dead cells in pellets of an actinomycete Amycolatopsis balhimycina and its correlation with balhimycin productivity

Secondary metabolites such as antibiotics are typically produced by actinomycetes as a response to growth limiting stress conditions. Several studies have shown that secondary metabolite production is correlated with changes observed in actinomycete pellet morphology. Therefore, we investigated the correlation between the production of balhimycin and the spatio-temporal distribution of live and dead cells in pellets of Amycolatopsis balhimycina in submerged cultures. To this end, we used laser scanning confocal microscopy to analyze pellets from balhimycin producing and nonproducing media containing 0.2 and 1.0 g l^?1 of potassium di-hydrogen phosphate, respectively. We observed a substantially higher fraction of live cells in pellets from cultures yielding larger amounts of balhimycin. Moreover, in media that resulted in no balhimycin production, the pellets exhibit an initial death phase which commences from the centre of the pellet and extends in the radial direction. A second growth phase was observed in these pellets, where live mycelia are seen to appear in the dead core of the pellets. This secondary growth was absent in pellets from media producing higher amounts of balhimycin. These results suggest that distribution of live and dead cells and its correlation with antibiotic production in the non-sporulating A. balhimycina differs markedly than that observed in Streptomycetes.     

A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers

A method to simultaneously determine the relative numbers of live and dead cells in culture by introducing a combination of two fluorogenic substrates or a fluorogenic and a luminogenic protease substrate into the sample is described. The method is based on detection of differential ubiquitous proteolytic activities associated with intact viable cells and cells that have lost membrane integrity. A cell-permeable peptide aminofluorocoumarin substrate detects protease activity restricted to intact viable cells. Upon cell death, the viable cell protease marker becomes inactive. An impermeable peptide rhodamine 110 (or aminoluciferin) conjugated substrate detects protease activity from nonviable cells that have lost membrane integrity. The multiplex assay can detect 200 dead cells in a population of 10,000 viable cells. The protease substrate reagents do not damage viable cells over the course of the assay, thus the method can be multiplexed further with other assays in a homogeneous format. Ratiometric measurement of viable and dead cells in the same sample provides an internal control that can be used to normalize data from other cell-based assays.     

Sustainable coccidiosis control in poultry production: the role of live vaccines

The development of new methods of administering coccidiosis vaccines has facilitated their use in the hatchery and thereby improved prospects for the economic vaccination of broilers. The acquisition of protective immunity to Eimeria species is boosted by further exposure to infection after vaccination. Factors that affect the reproductive efficiency of non-attenuated and attenuated vaccines are considered and the key role that oocyst production plays in establishing and maintaining uniform immunity in a flock of chickens is discussed. In addition to immunisation, a possible advantage to the application of certain vaccines is that their use could repopulate poultry houses with drug-sensitive organisms. Theoretical rotation programmes in which the use of drugs is alternated with that of vaccines are described. Variability of the cross-protective immune response between strains of the same species should be considered during vaccine development and subsequent use. The significance of less common species of Eimeria, not included in all vaccines, also needs to be assessed. An important consideration is the occurrence of pathogens other than Eimeria (such as the bacterium Clostridium) in flocks given coccidiosis vaccines and the methods by which they might be controlled. More research is required into the relationship between bacterial and viral infections of poultry and coccidiosis vaccination. Vaccines need to be developed that are simple to apply and cost effective for use in areas of the world where small-scale poultry production is commonplace. In the near future it is likely that more live vaccines based upon oocysts derived from attenuated strains of Eimeria will be developed but in the longer term vaccines will be based on the selective presentation to the host of specific molecules that can induce protective immunity. This achievement will require significant investment from the private and public sectors, and, if successful, will facilitate the sustainable control of coccidiosis in poultry production.     

Succession of fungi on dead and live wood in brackish water in Brunei

We observed the sequence of fungi appearing on submerged wood of Hibiscus tiliaceus that initially was either dead or alive. Branches that were dead, but still attached to the tree, and live branches were cut from H. tiliaceus in the riparian vegetation in a brackish habitat on the Tutong River, Brunei. Branch segments were connected to the riverbank using monofilament line. Samples were examined for fungi before the branches were placed in the river and after the branches had been submerged 3 or 6 mo. Fifty taxa were found on the samples. Before being placed in the water different fungal assemblages were found on live as compared to deadwood. Branches that were alive when cut supported a distinctly different fungal assemblage after 3 mo in the water. Dead branches after 3 mo and both dead and initially live samples after 6 mo had been colonized by a fungal assemblage that is typical at this site. It is unknown whether the differences in colonization of dead and initially live wood can be attributed to differences in the substratum (i.e., the presence or absence of bark), inhibitory substances in more recently live wood or to assembly rules resulting from the different fungi that already were present in dead and live branches.     

Photocaged amplified FRET nanoflares: spatiotemporal controllable of mRNA-powered nanomachines for precise and sensitive microRNA imaging in live cells

There is considerable interest in creating a precise and sensitive strategy for in situ visualizing and profiling intracellular miRNA. Present here is a novel photocaged amplified FRET nanoflare (PAFN), which spatiotemporal controls of mRNA-powered nanomachine for precise and sensitive miRNA imaging in live cells. The PAFN could be activated remotely by light, be triggered by specific low-abundance miRNA and fueled by high-abundance mRNA. It offers high spatiotemporal control over the initial activity of nanomachine at desirable time and site, and a ‘one-to-more’ ratiometric signal amplification model. The PAFN, an unprecedented design, is quiescent during the delivery process. However, upon reaching the interest tumor site, it can be selectively activated by light, and then be triggered by specific miRNA, avoiding undesirable early activation and reducing nonspecific signals, allowing precise and sensitive detection of specific miRNA in live cells. This strategy may open new avenues for creating spatiotemporally controllable and endogenous molecule-powered nanomachine, facilitating application at biological and medical imaging.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.     

Folding, quality control, and secretion of pancreatic ribonuclease in live cells

Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t_½ < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t_½ = 16 min) to the extracellular space (t_½ = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn¹¹³, a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.     

Differentiation of live, dead and treated cells of Escherichia coli O157:H7 using FT-IR spectroscopy

Aims: To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol. Methods and Results: Fourier transform‐infrared (FT‐IR) spectroscopy was used to analyse E. coli O157:H7 cells, after filtration or immunomagnetic collection. Partial least squares analysis of the spectra quantified live E. coli O157:H7 in the presence of dead cells with an R² > 0·996. Canonical variate analysis (CVA) not only differentiated between spectra of 100% dead and 100% live cells but also between 1% live : 99% dead and 100% dead. CVA using principal components also differentiated between the spectra of the differentially treated cells at a 95% confidence level, and Cooman plots showed clear separation between clusters of spectra of bacteria exposed to the different inactivation treatments. Mahalanobis distances (MD) corroborated the results of CVA. Conclusions: These results demonstrated the effectiveness of rapid cell collection and FT‐IR spectroscopy techniques to differentiate between live and dead E. coli O157:H7 cells. Significance and Impact of the Study: This technique has potential applications for use with foods subjected to various inactivation treatments.     

Diversity and ubiquity of xylariaceous endophytes in live and dead leaves of temperate forest trees

To test the hypothesis that xylariaceous endophytes were ubiquitous on live and dead leaves of various tree species in the field, xylariaceous fungi were isolated from live leaves and bleached and nonbleached portions of dead leaves of a total of 94 tree species in a cool temperate forest in Japan. The biodiversity of xylariaceous endophytes was evaluated as the richness of operational taxonomic units (OTUs) determined by phylogenetic analysis of the nucleotide sequence of the D1/D2 region of the LSU rDNA of fungal isolates. A total of 326 isolates of xylariaceous fungi were isolated from live and dead leaves and classified into 15 OTUs. The three major OTUs, Xylaria sp.1, Nemania sp., and Biscogniauxia sp., accounted for 94% (308 isolates) of the total number of isolates, and were isolated from various live and dead leaves. Xylaria sp.1 was frequently encountered on bleached portions (which were produced due to the selective decomposition of lignin) of dead leaves of broad-leaved deciduous tree species. The results suggest that xylariaceous endophytes did not show host specificity and had a saprobic phase on dead leaves in their life cycles and that Xylaria sp.1 was capable of decomposing lignin in the field conditions.     

A first attempt at determining larval growth in three Antarctic fish from otoliths and RNA/DNA ratios

Otolith growth and RNA/DNA ratios of larval stages of Notolepis coatsi, Gobionotothen gibberifrons and Trematomus scotti, three Antarctic fish, were studied during early 1998. RNA/DNA ratios were significantly different between the notothenioids and paralepidids (P<0.01), but similar among notothenioids (P>0.05). The ratios were independent of larval total length and water temperature. Recent otolith growth (based on the last five increments) and biochemical indices correlated slightly, but not significantly. N. coatsi inhabited deeper and colder waters of the Weddell Sea and showed less growth than the other species, which are associated with the Antarctic Peninsula. In all three species, some larvae had very good growth rates though most were rather poor. Recent growth indices might allow the detection and back-dating of growth changes in Antarctic larvae, and provide insight into a crucial yet poorly understood life-phase of these fish.     

An attempt at comparison of the morphology of normal and RSV--transformed cells in a scanning microscope

The electronic scanning microscope (SEM) was first used to analyze biological material in 1965 (Hollenberg M. et al. The Journal of Histochem. and Cytochem. 21, 109-130, 1973). It has since been possible to collect specific data on cell morphology and the changes in cell surface and structure caused by external factors, such as oncogenic viruses.     

Effect of precise control of flux ratio between the glycolytic pathways on mevalonate production in Escherichia coli

Mevalonate is a useful metabolite synthesized from three molecules of acetyl-CoA, consuming two molecules of NADPH. Escherichia coli ( E. coli) catabolizes glucose to acetyl-CoA via several routes, such as the Embden–Meyerhof–Parnas (EMP) and the oxidative pentose phosphate (oxPP) pathways. Although the oxPP pathway supplies NADPH, it is disadvantageous in terms of acetyl-CoA supply, compared with the EMP pathway. In this study, the optimal flux ratio between the EMP and oxPP pathways on the mevalonate yield was investigated. Expression level of pgi was controlled by isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter in an engineered mevalonate-producing E. coli strain. The relationship between the flux ratio and mevalonate yield was evaluated by changing the flux ratio by varying IPTG concentration. At the stationary phase, the mevalonate yield was maximum at an EMP flux of 39.7%, and was increased by 25% compared with that with no flux control (EMP flux of 70.4%). The optimal flux ratio was consistent with the theoretical value based on the mass balance of NADPH. The flux ratio between EMP and oxPP pathways affects the synthesis fluxes of mevalonate and acetate from acetyl-CoA. Fine tuning of the flux ratio would be necessary to achieve an optimized production of metabolites that require NADPH.