Biomedical subjects

Holger Schulze

Publications and source records attributed to Holger Schulze.

14 recordsLinked to original sources

PubMed2006

Cortical and subcortical sides of auditory rhythms and pitches.

It is commonly assumed that different perceptual qualities arising from sensory stimuli depend on their physical nature being transformed by specific peripheral receptors, for example, colour, vibration or heat. A notable unexplained exception is the low and high repetition rates of any sound perceived as rhythm or pitch, respectively. Using auditory discrimination learning in bilaterally auditory cortex ablated animals, we demonstrate that the perceptual quality of sounds depends on the way the brain processes stimuli rather than on their physical nature. In this context, cortical and subcortical processing steps have different roles in analysing different aspects of sounds with the complete analysis accomplished not before information converges in the auditory cortex.

Acoustic Stimulation↗

PubMed2006

A new format of electrodes for the electrochemical reduction of cytochromes P450.

New approach to the electrochemical reduction of cytochromes P450 (P450s, CYPs) at electrodes chemically modified with appropriate substrates for P450s ("reverse" electrodes) was proposed. The method is based on the analysis of cyclic voltammograms, square-wave voltammograms and amperograms with subsequent determination of electrochemical characteristics such as catalytic current and redox potential. The sensitivity of proposed method is 0.2-1 nmol P450/electrode. The changes of maximal current and of redox potentials in square-wave voltammograms as well as the changes of catalytic current in amperometric experiments proved to be informative and reliable. Planar regime of screen-printed electrodes (strip-type sensors) enabled to utilise 20-60 microl of electrolyte volume. The enzyme-substrate pairs P450 2B4/benzphetamine and P450scc/cholesterol were investigated. Electrochemical parameters of electrodes with unspecific P450 substrates differed considerably from electrodes with appropriate substrates.

Biosensing Techniques↗

PubMed2006

High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification.

The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.

Acetylcholinesterase↗

PubMed2006

Comparison of estimates for volumes of brain ablations derived from structural MRI and classical histology.

Estimates of auditory cortex ablation sizes in a rodent model as derived from classical histology (volume reconstructions from Nissl-stained brain sections) and structural magnetic resonance imaging (MRI) (T1-weighted whole-brain scans from a 4.7 T animal scanner) were compared in the same specimens (Mongolian gerbils). Estimates of lesion volumes obtained with the two methods were very similar, robust, highly correlated and not significantly different from each other. Hence, the general usefulness of structural MRI for the determination of brain lesion size in small animal models is demonstrated. MRI therefore seems to be well suited to determine proper size and location of an experimental brain ablation prior to (potentially extensive) behavioral testing.

Animals↗

PubMed2006

Differential effects of iontophoretic in vivo application of the GABA(A)-antagonists bicuculline and gabazine in sensory cortex.

We have compared the effects of microiontophoretic application of the GABA(A)-receptor antagonists bicuculline (BIC) and gabazine (SR95531) on responses to pure tones and to sinusoidally amplitude-modulated (AM) tones in cells recorded extracellularly from primary auditory cortex (AI) of Mongolian gerbils. Besides similar effects in increasing spontaneous and stimulus-evoked activity and their duration, both drugs elicited differential effects on spectral tuning and synchronized responses to AM tones. In contrast to gabazine, iontophoresis of the less potent GABA(A)-antagonist BIC often resulted in substantial broadening of frequency tuning for pure tones and an elimination of synchronized responses to AM tones, particularly with high ejecting currents. BIC-induced effects which could not be replicated by application of gabazine were presumably due to the well-documented, non-GABAergic side-effects of BIC on calcium-dependent potassium channels. Our results thus provide strong evidence that GABA(A)-mediated inhibition in AI does not sharpen frequency tuning for pure tones, but rather contributes to the processing of fast temporal modulations of sound envelopes. They also demonstrate that BIC can have effects on neuronal response selectivity which are not due to blockade of GABAergic inhibition. The results have profound implications for microiontophoretic studies of the role of intracortical inhibition in sensory cortex.

Acoustic Stimulation↗

PubMed2006

Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylusbrasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant.

Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.

Acetylcholinesterase↗

PubMed2005

Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.

The sensitivity of acetylcholinesterase (AChE) biosensors for insecticide detection could be increased substantially by engineering AChE B of Nippostrongylus brasiliensis. The introduction of 10 single and 4 double mutations into the AChE peptide chain led to an increase in sensitivity to 10 of the 11 insecticides tested. The combination of three mutants with the wild-type enzyme in a multienzyme biosensor array enabled the detection of 11 out of the 14 most important organophosphates and carbamates at concentrations below 10 microg/kg, the maximum residue limit of infant food. The detection limit for pirimiphos methyl could be reduced from 10 microg/L to a value as low as 1 ng/L (3.5 x 10(-)(12) mol/L). The newly created biosensors exhibited an extraordinary high storage stability. There was no loss of sensitivity of N. brasiliensis AChE B, immobilized on screen-printed, disposable electrodes, even after 17-month storage at room temperature.

Acetylcholinesterase↗

PubMed2005

Disposable biosensor test for organophosphate and carbamate insecticides in milk.

A highly sensitive and rapid biosensor test based on disposable screen-printed thick-film electrodes was developed, which is suitable for monitoring organophosphate and carbamate residues in foods of animal origin with increased fat contents such as milk. The wild-type enzyme was combined with three engineered variants of Nippostrongylus brasiliensis acetylcholinesterase (NbAChE), to obtain enhanced sensitivity. The sample pretreatment could be reduced to a minimum. There was no extraction or fat removal necessary. With the biosensor test paraoxon concentrations down to 1 microg/L could be detected in milk. The detection limit for carbaryl was 20 microg/L. Recovery rates for paraoxon and carbaryl in milk samples lay between 89 and 107%. Ten milk samples from local markets were tested both with the biosensor test and with standard chromatographic multiresidue methods. Two milk samples caused AChE inhibition rates of >50%. Accordingly, 4 microg/L tebufenpyrad, 4 microg/L tetraconazole, and 2 microg/L bifenthrin were detected in one of these milk samples. The other milk sample contained 2 microg/L tebufenpyrad.

Acetylcholinesterase↗

PubMed2004

Activation of phosphorothionate pesticides based on a cytochrome P450 BM-3 (CYP102 A1) mutant for expanded neurotoxin detection in food using acetylcholinesterase biosensors.

A novel enzymatic in vitro activation method for phosphorothionates has been developed to allow their detection with acetylcholinesterase (AChE) biosensors. Activation is necessary because this group of insecticides shows nearly no inhibitory effect toward AChE in their pure nonmetabolized form. In contrast, they exert a strong inhibitory effect on AChE after oxidation as it takes place by metabolic activation in higher organisms. Standard chemical methods to oxidize phosphorothionates showed inherent disadvantages that impede their direct use in food analysis. In contrast, a genetically engineered triple mutant of P450 BM-3 (CYP102 A1) could convert the two frequently used insecticides parathion and chlorpyrifos into their oxo variants as was confirmed by GC/MS measurements. The wild-type protein was unable to do so. In the case of chlorpyrifos, the enzymatic activation was as good as the chemical oxidation. In the case of parathion, the P450 activation was more efficient than the oxidation by NBS but neither activation method yielded an AChE inhibition that was as high as with paraoxon. The application of the method to infant food in combination with a disposable AChE biosensor enabled detection of chlorpyrifos and parathion at concentrations down to 20 microg/kg within an overall assay time of 95 min.

Acetylcholinesterase↗

PubMed2003

Design of acetylcholinesterases for biosensor applications.

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.

Acetylcholinesterase↗

PubMed2002

Auditory sensory memory for random waveforms in the Mongolian gerbil.

Mongolian gerbils (N = 21) were trained to discriminate between continuous and repeated auditory white noise. While for periods up to 40 ms of the repeated noise spectral effects make this a perceptual task, longer periods require auditory sensory memory to solve the task. Short periods (20 ms) could easily be discriminated by naive gerbils (discrimination performance, i.e. hit rate minus false alarm rate >80% after 8 days of training). Discrimination was more difficult for longer periods (100 ms: discrimination performance approximately 50% after 18 days of training). By long-term training (156 days) using an optimized training paradigm two further gerbils learned to discriminate up to a period length of 360 ms but could not proceed at 400 ms. While this falls short of human performance, it demonstrates for the first time sensory memory for random waveforms in animals.

Acoustic Stimulation↗

PubMed2002

Rapid detection of neurotoxic insecticides in food using disposable acetyicholinesterase-biosensors and simple solvent extraction.

The extensive use of pesticides to protect agricultural crops necessitates reliable tools for the detection of residues in food and water, thus ensuring environmental protection and consumer safety. Neuroinhibitors such as organophosphates and carbamates in particular, represent a potential hazard to human health. These compounds are frequently found in food, but conventional methods of analysis are limited as they are either time consuming or not sufficiently sensitive. As a result, a rapid and sensitive biosensor test based on AChE-inhibition was developed. The disposable AChE-biosensor was directly applied in solvent extracts of food samples using isooctane as extraction solvent. A complete assay could be performed in less than 2 h. Recovery rates of 84% were obtained in tests with spiked orange juice samples. Tests in food samples with a lower water content resulted in reduced recovery rates (44% for peach pap baby food). Phosphorothionate insecticides in food could be detected after direct oxidation with N-bromosuccinimide and solvent extraction. The assay displayed a detection limit of 2 microg/kg paraoxon, which was sufficient for the monitoring of maximum residue limits in food according to EU regulations.

Acetylcholinesterase↗

PubMed2002

Development, validation, and application of an acetylcholinesterase-biosensor test for the direct detection of insecticide residues in infant food.

A highly sensitive and rapid food-screening test based on disposable screen-printed biosensors was developed, which is suitable for monitoring infant food. The exposure of infants and children to neurotoxic organophosphates and carbamates is of particular concern because of their higher susceptibility to adverse effects. The European Union has, therefore, set a very low limit for pesticides in infant food, which must not contain concentrations exceeding 10 microg/kg for any given pesticide. The maximum residue limit (MRL) has been set to be near the determination threshold that is typically achieved for pesticides with traditional analytical methods. The biosensor method could detect levels lower than 5 microg/kg and thus clearly fulfills the demands of the EU. To substantiate these measurements, recovery rates were determined and amounted on average to 104% in food. Matrix effects were eliminated by the introduction of a special electrode treatment. The test was compared with two traditional pesticide multiresidue analysis methods (GC-MS, LC-MS) using 26 fruit and vegetable samples from local markets and 23 samples of processed infant food from Germany, Spain, Poland and USA. Three infant food samples exceeded the MRL of 10 microg/kg when analyzed by either biosensor test or multiresidue methods.

Acetylcholinesterase↗

PubMed2002

Superposition of horseshoe-like periodicity and linear tonotopic maps in auditory cortex of the Mongolian gerbil.

The segregation of an individual sound from a mixture of concurrent sounds, the so-called cocktail-party phenomenon, is a fundamental and largely unexplained capability of the auditory system. Speaker recognition involves grouping of the various spectral (frequency) components of an individual's voice and segregating them from other competing voices. The important parameter for grouping may be the periodicity of sound waves because the spectral components of a given voice have one periodicity, viz. fundamental frequency, as their common denominator. To determine the relationship between the representations of spectral content and periodicity in the primary auditory cortex (AI), we used optical recording of intrinsic signals and electrophysiological mapping in Mongolian gerbils (Meriones unguiculatus). We found that periodicity maps as an almost circular gradient superimposed on the linear tonotopic gradient in the low frequency part of AI. This geometry of the periodicity map may imply competitive signal processing in support of the theory of "winner-takes-all".

Acoustic Stimulation↗