Cytochrome P450 bio-affinity detection coupled to gradient HPLC: on-line screening of affinities to cytochrome P4501A2 and 2D6

Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column.     

An Anti-Ubiquitin Antibody Response in Transitional Cell Carcinoma of the Urinary Bladder

Background

To use combinatorial epitope mapping (“fingerprinting”) of the antibody response to identify targets of the humoral immune response in patients with transitional cell carcinoma (TCC) of the bladder.

Methods

A combinatorial random peptide library was screened on the circulating pool of immunoglobulins purified from an index patient with a high risk TCC (pTa high grade plus carcinoma in situ) to identify corresponding target antigens. A patient cohort was investigated for antibody titers against ubiquitin.

Results

We selected, isolated, and validated an immunogenic peptide motif from ubiquitin as a dominant epitope of the humoral response. Patients with TCC had significantly higher antibody titers against ubiquitin than healthy donors (p<0.007), prostate cancer patients (p<0.0007), and all patients without TCC taken together (p<0.0001). Titers from superficial tumors were not significantly different from muscle invasive tumors (p = 0.0929). For antibody response against ubiquitin, sensitivity for detection of TCC was 0.44, specificity 0.96, positive predictive value 0.96 and negative predictive value 0.41. No significant titer changes were observed during the standard BCG induction immunotherapy.

Conclusions

This is the first report to demonstrate an anti-ubiquitin antibody response in patients with TCC. Although sensitivity of antibody production was low, a high specificity and positive predictive value make ubiquitin an interesting candidate for further diagnostic and possibly immune modulating studies.     

Experiences of Community-Living Older Adults Receiving Integrated Care Based on the Chronic Care Model: A Qualitative Study

BackgroundIntegrated care models aim to solve the problem of fragmented and poorly coordinated care in current healthcare systems. These models aim to be patient-centered by providing continuous and coordinated care and by considering the needs and preferences of patients. The objective of this study was to evaluate the opinions and experiences of community-living older adults with regard to integrated care and support, along with the extent to which it meets their health and social needs.MethodsSemi-structured interviews were conducted with 23 older adults receiving integrated care and support through “Embrace,” an integrated care model for community-living older adults that is based on the Chronic Care Model and a population health management model. Embrace is currently fully operational in the northern region of the Netherlands. Data analysis was based on the grounded theory approach.ResultsResponses of participants concerned two focus areas: 1) Experiences with aging, with the themes “Struggling with health,” “Increasing dependency,” “Decreasing social interaction,” “Loss of control,” and “Fears;” and 2) Experiences with Embrace, with the themes “Relationship with the case manager,” “Interactions,” and “Feeling in control, safe, and secure”. The prospect of becoming dependent and losing control was a key concept in the lives of the older adults interviewed. Embrace reinforced the participants’ ability to stay in control, even if they were dependent on others. Furthermore, participants felt safe and secure, in contrast to the fears of increasing dependency within the standard care system.ConclusionThe results indicate that integrated care and support provided through Embrace met the health and social needs of older adults, who were coping with the consequences of aging.     

Butler-Volmer-Monod model for describing bio-anode polarization curves

A kinetic model of the bio-anode was developed based on a simple representation of the underlying biochemical conversions as described by enzyme kinetics, and electron transfer reactions as described by the Butler-Volmer electron transfer kinetics. This Butler-Volmer-Monod model was well able to describe the measured bio-anode polarization curves. The Butler-Volmer-Monod model was compared to the Nernst-Monod model described the experimental data significantly better. The Butler-Volmer-Monod model has the Nernst-Monod model as its full electrochemically reversible limit. Contrary to the Nernst-Monod model, the Butler-Volmer-Monod model predicts zero current at equilibrium potential. Besides, the Butler-Volmer-Monod model predicts that the apparent Monod constant is dependent on anode potential, which was supported by experimental results.     

High-resolution bioactivity profiling of mixtures toward the acetylcholine binding protein using a nanofractionation spotter technology

This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC(50)s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantage of this "atline" technology using postcolumn bioaffinity analysis when compared to continuous flow online postcolumn bioaffinity profiling is the possibility to choose postcolumn incubation times freely without compromising resolution due to diffusion effects.     

Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry

This paper describes the establishment of flow cytometric methods for recombinant Pichia pastoris strains, and their application to a lab scale fed batch fermentation. Using a strain which secretes human trypsinogen, the viability and the product which remained associated to the cell were measured with propidium iodide and immunofluorescent staining, respectively. Viability decreases significantly below 70% during the methanol fed batch phase, indicating a stress situation triggered by the fermentation conditions. Cell associated product is accumulated earlier after methanol induction than secreted product. These data demonstrate that flow cytometry is a powerful tool for the analysis and optimization of recombinant protein production processes, and they indicate the need to further improve a widely used fermentation protocol for P. pastoris.     

A circadian oscillator in Aspergillus spp. regulates daily development and gene expression

We have established the presence of a circadian clock in Aspergillus flavus and Aspergillus nidulans by morphological and molecular assays, respectively. In A. flavus, the clock regulates an easily assayable rhythm in the development of sclerotia, which are large survival structures produced by many fungi. This developmental rhythm exhibits all of the principal clock properties. The rhythm is maintained in constant environmental conditions with a period of 33 h at 30°C, it can be entrained by environmental signals, and it is temperature compensated. This endogenous 33-h period is one of the longest natural circadian rhythms reported for any organism, and this likely contributes to some unique responses of the clock to environmental signals. In A. nidulans, no obvious rhythms in development are apparent. However, a free running and entrainable rhythm in the accumulation of gpdA mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) is observed, suggesting the presence of a circadian clock in this species. We are unable to identify an Aspergillus ortholog of frequency, a gene required for normal circadian rhythmicity in Neurospora crassa. Together, our data indicate the existence of an Aspergillus circadian clock, which has properties that differ from that of the well-described clock of N. crassa.     

Determining acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow assay

A fluorometric assay for acetylcholinesterase inhibitory activity was developed in a flow system using the fluorogenic substrate 7-acetoxy-1-methyl quinolinium iodide which is hydrolysed to the highly fluorescent 7-hydroxy-1-methyl quinolinium iodide. The detection limit of galanthamine is 0.5 microM, which is about 20 times more sensitive than in the colorimetric flow assay. In the presence of 30% methanol or of 5% acetonitrile, about 70% of the enzyme activity could still be detected. Various plant extracts have been screened using the described system including bulbs of Galanthus nivalis, Eucharis amazonica (E. x grandiflora), Crinum powelli and Nerine bowdenii (all members of the Amaryllidaceae), which showed strong AchE inhibitory activity.