Biomedical subjects

A Hildebrandt

Publications and source records attributed to A Hildebrandt.

At least 19 recordsLinked to original sources

PubMed2006

Optical immunosensor for fast and sensitive detection of DDT and related compounds in river water samples.

A surface plasmon resonance (SPR) based immunosensor has been developed for the monitoring of environmentally persistent pollutants like DDT, its metabolites and analogues in real water samples. A reusable immunosurface is provided via the covalent attachment of the analyte derivative to a self-assembled alkanethiol monolayer formed onto the SPR gold-thin layer. The regeneration of the sensor surface allowed the performance of 270 assay cycles within an analysis time of 20 min for each assay cycle. Immunoassays based on a binding inhibition format were performed by using two monoclonal antibodies (MAbs) with different selectivity. Low limits of detection (LODs), in the sub-nanogram per litre range, were attained for DDT-selective (15 ng L-1) and DDT group-selective immunoassays (31 ng L-1). Both assays were carried out in spiked river water samples without significant effect of the matrix. SPR measurements were validated using gas-chromatography-mass spectrometry. The comparison between methods was in good agreement showing an excellent correlation coefficient (r2=0.995). The SPR analysis of DDT proved to be three times more sensitive than colorimetric ELISAs without the need of labelling and a much lower time of response. Our SPR biosensor portable platform (beta-SPR) is already commercialised by the company SENSIA, S.L. (Spain).

Antibodies↗

PubMed2006

Public knowledge and attitudes on organ donation do not differ in Germany and Spain.

BACKGROUND: The background of this study was the inadequate supply of donor organs in Germany. In Spain, by contrast, a strong increase of organ donors over the past years has created a satisfactory supply situation. Because both countries have similar legal situations, the causes for the drastic differences in organ donation rates remain unclear. The main issue of our study was to investigate the intellectual attitudes toward various aspects of postmortem donations in the populations of both countries as a causative factor for the observed differences. METHODS: We studied 726 persons by questionnaire. Probands, matched for age and gender, were recruited among medical students, in a public library and in a general medical practice in both Germany and Spain. RESULTS: We found no differences in the attitudes toward postmortem organ donation between the two countries. Differences among the social groups within the countries were apparent in the expected direction. CONCLUSION: A higher level of knowledge or a difference in attitudes toward organ donation is probably not the reason for the higher donation rate in Spain. The cause appears to be rather at the organizational level.

Attitude↗

PubMed2005

Determination of carbaryl in natural water samples by a surface plasmon resonance flow-through immunosensor.

The analysis of carbaryl in natural water samples was accomplished using a portable immunosensor based on surface plasmon resonance (SPR) technology. The assay was based on a binding inhibition immunoassay format with the analyte derivative covalently immobilized on the sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed onto the gold-coated sensor surface to allow the reusability of the same sensing surface during 220 regeneration cycles. Reproducibility was evaluated by performing three independent assays in triplicate on 3 different days. The batch-assay variability was also calculated using three different gold-coated sensor surfaces. The intra- and inter-day relative standard deviation were 8.6 and 15.3%, respectively, whilst a variation of 7.4% in assay sensitivity was obtained by employing different sensor chips. The lowest detection limit, calculated as the concentration providing a 10% decrease of the blank signal, was of 1.38 microg L(-1). Matrix effects were also evaluated in different water types, showing I50 values (carbaryl concentrations that produced a 50% decrease of the blank signal) within the range of carbaryl standard curves in distilled water (2.78-3.55 microg L(-1)). The carbaryl immunoassay performance was validated with respect to conventional high-performance liquid chromatography-mass spectrometry (HPLC-MS). The correlation between methods was in good agreement (r2 = 0.998, 0.999 and 0.999) for the three types of natural water samples tested. A complete assay cycle, including regeneration, is accomplished in 20 min. All measurements were carried out with the SPR sensor system (beta-SPR) commercialised by the company SENSIA, SL (Spain). The small size and low-time of response of the beta-SPR platform would allow its utilization in real contaminated locations.

Biosensing Techniques↗

PubMed2005

A novel transcriptome subtraction method for the detection of differentially expressed genes in highly complex eukaryotes.

We have designed a novel transcriptome subtraction method for the genome-scale analysis of differential gene expression in highly complex eukaryotes, in which suppression subtractive hybridization (SSH) is performed first to enrich the target and, after exchange of adapters, negative subtraction chain (NSC) is then used to eliminate the remaining background. NSC evolved from differential subtraction chain (DSC). We designed novel adapters which make the subtraction system more robust. SSH and NSC were then combined to successfully detect differentially expressed genes in Solanum. The combined technique improves qualitatively upon SSH, the only commercially available transcriptome subtraction system, by detecting target genes in the middle abundance class, to which most differentially expressed genes in highly complex eukaryotes are expected to belong. The main advantage of the combined technique with SSH/NSC is its ability to isolate differentially expressed genes quickly and cost-efficiently from non-standard models, for those microarrays are unavailable.

Gene Expression Profiling↗

PubMed2004

Novel formulation of nonlocal electrostatics.

The accurate modeling of the dielectric properties of water is crucial for many applications in physics, computational chemistry, and molecular biology. This becomes possible in the framework of nonlocal electrostatics, for which we propose a novel formulation allowing for numerical solutions for the nontrivial molecular geometries arising in the applications mentioned before. Our approach is based on the introduction of a secondary field psi, which acts as the potential for the rotation free part of the dielectric displacement field D. For many relevant models, the dielectric function of the medium can be expressed as the Green's function of a local differential operator. In this case, the resulting coupled Poisson (-Boltzmann) equations for psi and the electrostatic potential phi reduce to a system of coupled partial differential equations. The approach is illustrated by its application to simple geometries.

Biopolymers↗

PubMed2003

Prevalence of four species of Borrelia burgdorferi sensu lato and coinfection with Anaplasma phagocytophila in Ixodes ricinus ticks in central Germany.

A total of 305 Ixodes ricinus ticks collected from three areas of Thuringia in central Germany were investigated for infection with Borrelia burgdorferi sensu lato species and Anaplasma phagocytophila. Overall, 11.1% were infected with Borrelia burgdorferi and 2.3% with Anaplasma phagocytophila. Adult ticks showed a significantly higher rate of infection with both borreliae and Anaplasma phagocytophila. Borrelia garinii (55.9%) was detected most frequently, followed by Borrelia burgdorferi sensu stricto (32.4%), Borrelia afzelii (17.6%), and Borrelia valaisiana (5.9%). Four ticks had dual infection with Borrelia garinii and Borrelia burgdorferi sensu stricto. Two of the Borrelia-positive ticks were coinfected with Anaplasma phagocytophila.

Adult↗

PubMed2002

Downregulation of beta2-microglobulin in human cord blood somatic stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells?

Adherently growing, non-hematopoietic somatic stem cells isolated from human cord blood were stained with the fluorescent dye PKH26 and transplanted into livers of SCID-mice to examine a possible cell fate transition. Already 7 days after transplantation stem cells were well integrated into the liver tissue. Human albumin that was not expressed by the stem cells before transplantation was detectable in the host's livers after injection of cord blood stem cells. Human alpha1-antitrypsin was detectable in stem cells already before transplantation and remained positive in the mouse liver. The most interesting observation in this study was the downregulation of human beta2-microglobulin (beta2M) in the stem cells after transplantation: beta2M is expressed constitutively in our cord blood stem cells. However, beta2M was no longer detectable by RT-PCR in all tissues where human albumin and alpha1-antitrypsin were expressed after stem cell transplantation. beta2M is known to participate as an integral part of the major histocompatibility complex. Absence of beta2M makes the residual heavy chain inactive as an antigen. Thus, downregulation of beta2M may represent an escape mechanism from killer-T cells and may be a molecular mechanism explaining the recently described "immunological blindness" [37] of stem cells. In contrast to the results obtained after direct injection of stem cells as a suspension, no consistent downregulation of beta2M was observed after transplantation of stem cells encapsulated in alginate beads to generate a compartment where stem cells are protected from the host's natural killer cells. No expression of human genes was observed after transplantation of human cord blood derived mononuclear cells (MNC) that were used as a negative control. In conclusion, we have shown that human cord blood somatic stem cells survive and are reprogrammed after transplantation into mouse livers, although a complete transdifferentiation to hepatocytes did not occur within 7 days, since some marker genes (GATA4 and alpha-fetoprotein) were still negative. Switching off expression of beta2M may be part of an intriguing and novel mechanism explaining why stem cells escape the host's immune system.

Aged↗

PubMed2002

Characterization of minute differences between genomes of strains of Penicillium nalgiovense using subtractive suppression hybridization without cloning.

AIMS: Characterization of integrated DNA fragments of unknown base sequences from foreign species using the genetically modified mould Penicillium nalgiovense. METHODS AND RESULTS: A modified suppression subtractive hybridization method was deployed in order to detect and characterize foreign genes. Combined with a special two-dimensional polyacrylamide gel electrophoretic technique the usual background was eliminated from the prospective foreign polymerase chain reaction fragments according to their base composition (Müller et al. 1981). CONCLUSIONS AND SIGNIFICANCE AND IMPACT OF THE STUDY: DNA fragments obtained from the procedure were suitable for direct DNA sequencing. Each DNA fragment derived from the tester genomic DNA was found to be completely absent in the driver organism.

Cloning, Molecular↗

PubMed2001

Structure prediction of protein complexes by an NMR-based protein docking algorithm.

Protein docking algorithms can be used to study the driving forces and reaction mechanisms of docking processes. They are also able to speed up the lengthy process of experimental structure elucidation of protein complexes by proposing potential structures. In this paper, we are discussing a variant of the protein-protein docking problem, where the input consists of the tertiary structures of proteins A and B plus an unassigned one-dimensional 1H-NMR spectrum of the complex AB. We present a new scoring function for evaluating and ranking potential complex structures produced by a docking algorithm. The scoring function computes a 'theoretical' 1H-NMR spectrum for each tentative complex structure and subtracts the calculated spectrum from the experimental one. The absolute areas of the difference spectra are then used to rank the potential complex structures. In contrast to formerly published approaches (e.g. [Morelli et al. (2000) Biochemistry, 39, 2530-2537]) we do not use distance constraints (intermolecular NOE constraints). We have tested the approach with four protein complexes whose three-dimensional structures are stored in the PDB data bank (Bernstein et al., 1977) and whose 1H-NMR shift assignments are available from the BMRB database. The best result was obtained for an example, where all standard scoring functions failed completely. Here, our new scoring function achieved an almost perfect separation between good approximations of the true complex structure and false positives.

Algorithms↗

PubMed2000

Two-dimensional agarose gel electrophoresis as a tool to isolate genus- and species-specific repetitive DNA sequences.

Two-dimensional electrophoresis in agarose gels separates DNA-restriction fragments not only by molecular weight but also according to their AT-cluster content. The method produced genus-specific spot patterns of multicopy DNA fragments of grains as well as spot patterns of highly repetitive DNA fragments of ciliates, demonstrated for barley, spelt, and Tetrahymena. Further investigations in regard to their specificity by hybridization with three other grain species (wheat, oat, and rye) and three ciliate species (Tetrahymena thermophila, Tetrahymena pigmentosa, and Tetrahymena borealis) were performed. The DNA samples from spelt and Tetrahymena were demonstrated to be genus specific for Triticum and species specific for Tetrahymena pyriformis, respectively.

Animals↗

PubMed2000

Calcium and malate are sporulation-promoting factors of Physarum polycephalum.

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.

Animals↗

PubMed1999

Detection of polycyclic aromatic hydrocarbon degradation genes in different soil bacteria by polymerase chain reaction and DNA hybridization.

Twenty different strains of Pseudomonas, Mycobacterium, Gordona, Sphingomonas, Rhodococcus and Xanthomonas which degrade polycyclic aromatic hydrocarbons (PAH) were characterized in respect to genes encoding degradation enzymes for PAH. Genomic DNA from these strains was hybridized with a fragment of ndoB, coding for the large iron sulfur protein (ISP alpha) of the naphthalene dioxygenase from Pseudomonas putida PaW736 (NCIB 9816). A group of seven naphthalene-degrading Pseudomonas strains showed strong hybridization with the ndoB probe, and five Gordona, Mycobacterium, Rhodococcus and Pseudomonas strains able to degrade higher molecular weight PAH showed weaker hybridization signals. Using a polymerase chain reaction (PCR) approach, seven naphthalene-degrading Pseudomonas strains showed a PCR fragment of the expected size with ndoB-specific primers and additionally ten strains of Gordona, Mycobacterium, Pseudomonas, Sphingomonas and Xanthomonas able to degrade higher molecular weight PAH were detected with degenerate primer-pools specific for the ISP alpha [2Fe-2S]-Rieske center of diverse aromatic hydrocarbon dioxygenases. This suggests a molecular relationship between genes coding for PAH catabolism in various PAH-degrading bacterial taxa, which could be used to evaluate the PAH-degradation potential of mixed populations.

Bacteria↗

PubMed1999

cDNA cloning and cadmium-induced expression of metallothionein mRNA in the zebra mussel Dreissena polymorpha.

Using pooled degenerate oligonucleotides inferred from the N-terminal amino acid sequence of Dreissena polymorpha metallothionein and a Cys-X-Cys motif characteristic for known metallothioneins, a 150-bp metallothionein-specific reverse transcription PCR product was generated. The PCR product was used to screen a Dreissena polymorpha cDNA library, and a complete metallothionein cDNA sequence from Dreissena was identified. Four clones with the identical sequence were detected, supporting the idea of a single metallothionein gene in Dreissena. The sequence contains a 141-bp 5' untranslated region and a 572-bp 3' untranslated region with two polyadenylation signals. The coding region spans 219 bp. The deduced amino acid sequence shows 21 cysteine residues present in the metallothionein-typical motifs. Induction studies were performed with 50 microg Cd2+/L for up to 16 days. The exposed mussels show a sevenfold higher metallothionein mRNA level compared with uninduced control mussels.

Amino Acid Sequence↗

PubMed1997

Long-term decline of atmospheric and marine pollution by polycyclic aromatic hydrocarbons (PAHS) in Germany.

Concentrations of benzo[a]pyrene and benzo[e]pyrene in the atmosphere as markers for the class of PAHs decreased by about 70% within one decade in clean air as well as in industrially polluted areas of Germany when measured with passive samplers (spruce sprouts, poplar and beech leaves). The same trend has been found for East-Germany during 1991-1995. Mussels (Mytilus edulis) were found to accumulate PAHs from the aquatic environment and exhibited a seasonal periodicity of the PAH concentration. After an initial decline from 1985 to 1990, the PAH concentration remained constant until 1995 in the North Sea area investigated.

Air Pollutants↗

PubMed1997

Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion.

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.

Artifacts↗

PubMed1996

Isolation and characterisation of human cardiac fibroblasts from explanted adult hearts.

Fibrosis makes an important contribution to the pathophysiological events leading to the development of heart failure in ischemic and hypertensive heart disease. Since cardiac fibroblasts are mainly responsible for the synthesis and deposition of the extracellular matrix, we have established a method for isolating and cultivating human cardiac fibroblasts from explanted human hearts. The cell yield was 2.14+/-0.25x10(6 )cells in five independent isolations and the cell purity was 95-97%, contaminating cells being vascular smooth muscle cells and pericytes. Cultured cells were studied with respect to growth properties, morphology and deposition of components of the extracellular matrix. Isolated cells displayed a differentiated phenotype, including the second passage in culture; they synthesised collagen I, III, IV, fibronectin, vitronectin, tenascin and chondroitin sulphate and expressed an atypical angiotensin receptor. This atypical angiotensin receptor internalised angiotensins II and III but not angiotensin IV in a time-dependent manner. Stimulation of the cells with angiotensins II and III but not with angiotensin IV resulted in a dose-dependent stimulation of DNA synthesis. Co-incubation with the subtype-specific receptor antagonists Losartan and PD 123317 did not prevent the stimulation of DNA synthesis. The further characterisation of this receptor should provide insights into the pathobiochemical events leading to heart failure in hypertension and ischemic heart disease.

Adult↗

PubMed1996

Aortic valve debridement by ultrasonic surgical aspirator in degenerative, aortic valve stenosis: follow-up with Doppler echocardiography.

OBJECTIVE: Recently there has been an emphasis on reconstructing diseases native valves as an alternative to prosthetic valve replacement. Whereas; the surgical repair of aortic valve stenosis has been always problematic. This study was performed to estimate the clinical results after aortic valve debridement using ultrasonic energy. METHODS: Between 1990-1994 26 patients underwent ultrasonic aortic valve decalcification. There were 15 females and 11 males, the age was in average 74 years. As a concomitant diagnosis 88% patients (23) had mostly mild, aortic valve insufficiency, 16 (61%) had coronary artery disease and 11 (42%) had mild mitral valve incompetence. All of the patients were operated with cardiopulmonary bypass using moderate hypothermia, cardioplegical arrest and topical cooling for myocardial protection. The calcifications were removed tangentially using Cavitron Ultrasonic Surgical Aspirator. (CUSA), CAVITRON, USA. Leaflet perforation and/or unsatisfactory valve closure have been indications for aortic valve replacement. RESULTS: Two operative death (8%) have occurred and six patients have died in the further course (follow-up mean 17 months ranging from 4 to 61 months). Postoperative Doppler-Echocardiography results taken directly after surgery and then again 17 months later (n = 18) showed a decrease of peak and mean gradients across the aortic valve three and two times respectively (p < 0.001). 17 months after debridement we observed a mild rise in both gradients (by peak gradient p < 0.05). Directly postoperative, the aortic valve area increased doubly and decreased 17 months later slight, but it was still statistically significant in comparison with our preoperative data (p < 0.001). Follow-up echocardiography demonstrated late onset of moderate aortic valve insufficiency in 6 patients. The classification of New York Heart Association was improved in 13 (72%) survivors after 17 months. CONCLUSIONS: Ultrasonic debridement of aortic valve stenosis allows precise and energy-controlled removal of calcium, increased doubly the valve area and decreased of peak and mean gradients statistically significant. The advantages of preserving the native aortic valve in elderly patients are relative good arguments; although a longer follow-up is necessary to establish this procedure.

Aged↗

PubMed1995

Regulation, chamber localization, and subtype distribution of angiotensin II receptors in human hearts.

BACKGROUND: To assess the chamber localization, subtype distribution, and regulation of human myocardial angiotensin II receptors in heart failure, we determined the binding of angiotensin II, Sar1Ile8-angiotensin II, and the subtype-specific antagonists Dup 753 (AT1-specific) and PD 123319 (AT2-specific) in atria from patients with normal (left ventricular ejection fraction > 55%) or moderately impaired (left ventricular ejection fraction 30% to 55%) cardiac function and in atria and ventricles from explanted end-stage failing hearts. Sarcolemmal and combined fractions, the latter including internalized receptors, were studied. In addition, AT1 mRNA content was analyzed by polymerase chain reaction after reverse transcription. METHODS AND RESULTS: The number of angiotensin II binding sites (Bmax) in sarcolemmal fractions was significantly reduced in explanted end-stage failing hearts in comparison with control subjects and moderate heart failure (Bmax 3.9 +/- 0.8 versus 11.2 +/- 1.7 and 9.6 +/- 0.8 fmol/mg protein, respectively). A comparable 65% reduction in receptor numbers was found in combined fractions from end-stage failing hearts, indicating that the loss of binding sites was not due to their internalization. The dissociation constants were comparable in sarcolemmal and combined fractions and in nonfailing and failing hearts, ranging from 0.5 +/- 0.2 to 1.2 +/- 0.5 nmol/L. In nonfailing hearts, 69 +/- 4% of binding sites were blocked by the subtype-2-specific inhibitor PD 123319 and were therefore classified as AT2; 33 +/- 5% were blocked by the subtype-1-specific inhibitor DUP 753 and thus classified as subtype 1. In explanted hearts, comparable ratios of 66 +/- 5% AT2 sites and 34 +/- 5% AT1 sites were found. AT1 cDNA amplification signals by polymerase chain reaction were reduced to about one third of the level in control subjects in end-stage failing hearts. CONCLUSIONS: Angiotensin II receptors in human myocardium are present in relatively low numbers, and AT2 is the predominant subtype. A significant loss of angiotensin II receptors occurs in end stage but not in moderate heart failure. The loss of receptors affects both subtypes to a comparable degree. The data suggest that the decrease in receptor density is due to a decrease in steady-state mRNA abundance.

Binding Sites↗