Characterisation of intact microorganisms using electrospray ionisation mass spectrometry

We report the first application of electrospray ionisation mass spectrometry (ESI-MS) for the reproducible characterisation of strains of intact Gram-negative and Gram-positive bacteria. Electrospray ionisation was performed in both the positive and negative ion modes and the spectra obtained from Escherichia coli and Bacillus cereus were very information rich. Several of the observed negative mass ion fragments from E. coli could be assigned to specific fragmentation from bacterial phospholipids.Cluster analyses of these spectra showed that ESI-MS could be used to discriminate between these microorganisms to below species level. Therefore we conclude that ESI-MS constitutes a powerful approach to the characterisation and speciation of intact microorganisms.     

An EPR investigation of surfactant action on bacterial membranes

The effects of the surfactants, alcohol ethoxylate, amine ethoxylate, amine oxide and SDS on cell membranes were investigated using the lipid soluble spin label 5-doxyl stearic acid (5-DS). Electron paramagnetic resonance (EPR) spectroscopy revealed that the action of the surfactants was to significantly increase membrane fluidity of Proteus mirabilis, Staphylococcus aureus and Saccharomyces cerevisiae. The action of these surfactants as biocides was investigated and found to be dependent on the type of organism tested. There was, however, no direct correlation between enhanced membrane fluidity observed due to the action of the surfactants and biocidal activity. Data presented suggest that perturbing the fluidity of the cytoplasmic membrane is not immediately responsible for cell death.     

Characterization of exopolymers of aquatic bacteria by pyrolysis-mass spectrometry

Exopolymers from a diverse collection of marine and freshwater bacteria were characterized by pyrolysis-mass spectrometry (Py-MS). Py-MS provides spectra of pyrolysis fragments that are characteristic of the original material. Analysis of the spectra by multivariate statistical techniques (principal component and canonical variate analysis) separated these exopolymers into distinct groups. Py-MS clearly distinguished characteristic fragments, which may be derived from components responsible for functional differences between polymers. The importance of these distinctions and the relevance of pyrolysis information to exopolysaccharide function in aquatic bacteria is discussed.     

ESR characteristics of Photosystem I in deuterium oxide: Further evidence that electron acceptor A1 is a quinone

The appearance of ESR signals from Photosystem I (PS I) electron acceptors A₁ and A₀ in water or deuterium oxide suspension was followed using a low-temperature photoaccumulation technique. In deuterated samples the A₁ signal was narrowed by a factor of 0.66 compared with the control. This effect was fully reversible upon resuspension of treated samples in H₂O. The narrow ESR signal from deuterated A₁ had similar power saturation characteristics to the normal signal; however, a signal from a second component resolved by deuteration was saturated at higher microwave powers than the control. The power saturation behaviour of A⁻₁ in un-modified reaction centres indicated that it is an anionic semiquinone in a ‘protic’ environment. Deuteration reversibly modified the relative extents of reduction of iron sulphur electron acceptors A and B such that centre B became the more stable electron acceptor. The g-value and line-width of iron sulphur centre X was not modified by deuteration although it appeared to become more efficiently reduced. These results are discussed in the light of current evidence from optical, electron spin polarisation and extraction experiments that suggest that A₁ is a quinone, probably vitamin K-1.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Production of antigen-specific human monoclonal antibodies from in vitro-primed human splenocytes

We have developed culture conditions for human lymphocytes that support primary in vitro immune responses to protein Ag of either human or nonhuman origin. We now show that these primed B cells can be efficiently immortalized by fusion with a heterohybrid fusion partner to generate human, Ag-specific IgM or IgG antibody-producing heterohybridomas at a rate of 17 to 50 hybrids/10(6) lymphocytes fused. Approximately 50% of the Ig-secreting clones were stable with respect to Ig secretion. Levels of secretion attained with terminal cultures ranged from less than 1 to 100 micrograms/ml. Fusions of cells between 2 and 5 days after initiation of in vitro exposure to Ag produced more Ag-reactive and Ag-specific antibodies than fusions at 1 day or fusions performed after 5 days. Ag-reactive hybrids could be isolated at frequencies of 3 to 10%, depending on antigenicity of the immunogen. Foreign proteins, horse spleen ferritin, and a murine monoclonal Ig, induced higher percentages of Ag-reactive mAb than immunization with the human-derived ferritin. Ag-reactive IgG mAb were produced at relatively high frequency, depending on immunization conditions and the nature of the Ag. The strategy for identification of the best hybrids included early elimination of unstable hybridomas and of hybridomas producing broadly cross-reactive antibody, followed by evaluation of units of Ag reactivity/micrograms Ig. Ferritin-specific mAb selected according to these criteria showed immunocytochemical reactivity with ferritin-containing tissues and apparent affinities in the range of 10(7) to 10(8)/mol.     

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442. © 2010 Wiley Periodicals, Inc.     

Monitoring the Effects of Chiral Pharmaceuticals on Aquatic Microorganisms by Metabolic Fingerprinting

The effects of the chiral pharmaceuticals atenolol and propranolol on Pseudomonas putida, Pseudomonas aeruginosa, Micrococcus luteus, and Blastomonas natatoria were investigated. The growth dynamics of exposed cultures were monitored using a Bioscreen instrument. In addition, Fourier-transform infrared (FT-IR) spectroscopy with appropriate chemometrics and high-performance liquid chromatography (HPLC) were employed in order to investigate the phenotypic changes and possible degradation of the drugs in exposed cultures. For the majority of the bacteria studied there was not a statistically significant difference in the organism''s phenotype when it was exposed to the different enantiomers or mixtures of enantiomers. In contrast, the pseudomonads appeared to respond differently to propranolol, and the two enantiomers had different effects on the cellular phenotype. This implies that there were different metabolic responses in the organisms when they were exposed to the different enantiomers. We suggest that our findings may indicate that there are widespread effects on aquatic communities in which active pharmaceutical ingredients are present.Active pharmaceutical ingredients (APIs) and their metabolites are ubiquitous in the environment (12), and the occurrence of APIs in the aquatic environment is a growing concern (13). There are a number of routes by which APIs and their metabolites and degradation products may enter these ecosystems, and a common avenue is through excretion of the APIs and their metabolites in urine and feces. It is known that APIs have different rates of metabolism in humans. For example, the β-blocker propranolol is almost completely metabolized in the liver, and only 1 to 4% of an oral dose is excreted as the unchanged API and its metabolites. In contrast, 40 to 50% of an oral dose of atenolol (also a β-blocker) is excreted as the API or its metabolites (2, 6, 7). Subsequent degradation of the APIs and their metabolites may also occur at sewage treatment plants (STPs); this degradation is usually substrate specific and varies greatly for different APIs. The rates of adsorption to activated sewage sludge during treatment differ for different APIs and are dependent on the hydrophobic and electrostatic interactions of the APIs with the particulates and microorganisms in the activated sewage sludge (13). Any remaining APIs and relevant metabolites are diluted in the surface water when the effluent is released from the STP. Hence, many APIs are present at low concentrations (ng liter⁻¹ to μg liter⁻¹) in aquatic environments, such as rivers, streams, and estuaries (2, 6, 12). The majority of APIs are neither persistent nor highly bioaccumulative; however, the continuous release of APIs into the aquatic environment poses a potential risk to aquatic organisms even though the concentrations of APIs in the receiving waters are quite low (12).Despite the fact that little is known about the effects of APIs in the environment, the fact that they are designed to have a specific mode of action in humans must be taken into account (12). Adverse side effects may occur in humans at higher doses of the APIs, and it can be expected that any beneficial or adverse effect may also be observed in aquatic organisms with similar biological functions or receptors. It must also be noted that similar targets may control different metabolic processes in different species (43), and therefore APIs and their metabolites may have additional modes of action in aquatic organisms. The effects of the APIs may be subtle due to the very low concentrations observed in the aquatic environment, and as a result these effects may go unnoticed (12). It is also likely that the effect of an API has an impact on the local population dynamics in the whole ecosystem, from bacteria to higher organisms. To explore the effects of APIs on biological systems, a wide range of concentrations should be employed along with appropriate analytical platforms to profile the complement of biochemical components in cells. Indeed, it is known that APIs can become concentrated in the benthic environment of river beds, and as bacteria inhabit this niche, the bacterial community may be exposed to higher-than-expected levels of these compounds (16, 37, 49).While the effect of APIs in the environment is currently a growing area of research, there is very little understanding of the environmental effects of chiral pharmaceuticals (5, 14). A chiral molecule is a molecule that lacks an internal plane of symmetry. The nonsuperimposable mirror images are termed enantiomers and are labeled (R) or (S) according to a priority system (Cahn Ingold Prelog priority rules) based on the atomic number of the molecule''s substituents. Approximately 56% of the APIs currently in use are chiral compounds, and 88% of these chiral APIs are administered therapeutically as the racemate [i.e., an equal mixture of the two enantiomers, indicated by (±)]. The chirality of environmental contaminants, such as APIs, must be taken into consideration in order to fully understand the environmental fate and effects of these compounds. The enantiomers of a chiral API are able to interact differently with other chiral compounds, such as enzymes, and therefore potentially have different effects when they are released into the environment (5, 14, 33). It is widely known that the enantiomers of a chiral API may have different toxicological and biological effects than each other and than the racemate (an equal mixture of the two enantiomers) (25, 54). It has been shown that the (S) enantiomers of the β-blocking agents atenolol and propranolol are more potent in humans than the corresponding antipodes (3, 11, 35, 45) and that a number of the biotransformation pathways for β-blockers are stereoselective in humans (30). The mode of action of the drugs and their enantiomers in prokaryotic systems is not known. It is therefore necessary to increase our understanding of the fate and biological effects of chiral pharmaceuticals on typical microflora in the aquatic environment in order to fully appreciate the risks (19). Of particular interest is the group of APIs termed β-blockers as they all contain at least one chiral center and are generally administered therapeutically as the racemate (30). In addition, they are widely used, and for example, approximately 29 and 12 tonnes of atenolol and propranolol, respectively, are consumed each year in the United Kingdom (2, 6, 7).In order to explore the effects of the APIs on biological systems, we employed Fourier-transform infrared (FT-IR) spectroscopy; this is a phenotypic typing technique which has previously been used to generate metabolic fingerprints of bacteria (22, 53). Previous studies have successfully discriminated bacteria to the subspecies level (31, 50, 53) through detection of subtle changes in the biochemical phenotypes of the bacteria. We recently demonstrated use of FT-IR spectroscopy coupled with suitable chemometrics to physiologically assess bioprocesses (unpublished data). In addition, a combination of FT-IR spectroscopy and trajectory analysis has been used for identification of metabolic changes in fermentations (22). FT-IR spectroscopy is an automated high-throughput technique (10 to 60 s per sample is typical) that requires minimal sample preparation, and this makes it relatively inexpensive. It is therefore an ideal screening method to explore the effects of APIs on a number of bacterial systems.In this study the chirality-specific metabolism of the β₁-selective adrenergic blocking agent atenolol and the nonselective β-adrenergic blocking agent propranolol by a range of environmental microorganisms was investigated (14, 41, 48). FT-IR spectroscopy was employed to monitor biochemical changes in the spectral fingerprints of whole bacterial cells during growth of the microorganisms in the presence of the selected APIs. In addition, we monitored the fate of the APIs with chiral high-performance liquid chromatography (HPLC), which allowed quantification of the enantiomers.