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Alexander Rapp

Publications and source records attributed to Alexander Rapp.

16 recordsLinked to original sources
PubMed2025

Dynamic association of H3K36me3 with pericentromeric heterochromatin regulates its replication time.

The flexibility of the spatio-temporal genome replication program during development and disease highlights the regulatory role of plastic epigenetic mechanisms over genetic determinants. Histone post-translational modifications are broadly implicated in replication timing control, yet the specific mechanisms through which individual histone marks influence replication dynamics, particularly in heterochromatin, remain unclear. Here, we demonstrate that H3K36me3 dynamically enriches at pericentromeric heterochromatin, composed of major satellite DNA repeats, prior to replication during mid S phase in mouse embryonic stem cells. By knocking down lysine 36-specific methyltransferases or by targeting the H3K36M oncohistone to pericentromeric heterochromatin, we reduce global or local H3K36me3 levels, respectively, revealing its essential role in preserving the replication timing of constitutive heterochromatin. Loss of H3K36me3 accompanies increased RNA polymerase II serine-5 phosphorylation and lowered major satellite RNA levels, indicating transcriptional dysregulation. Notably, we identify a strand-specific contribution of major satellite forward transcripts in regulating the replication timing of constitutive heterochromatin and maintaining chromatin stability, highlighting the importance of non-coding RNAs as critical regulators of replication timing.

Heterochromatin↗
PubMed2006

Neural correlates of working memory dysfunction in first-episode schizophrenia patients: an fMRI multi-center study.

Working memory dysfunction is a prominent impairment in patients with schizophrenia. Our aim was to determine cerebral dysfunctions by means of functional magnetic resonance imaging (fMRI) in a large sample of first-episode schizophrenia patients during a working memory task. 75 first-episode schizophrenia patients and 81 control subjects, recruited within a multi-center study, performed 2- and 0-back tasks while brain activation was measured with fMRI. In order to guarantee comparability between data quality from different scanners, we developed and adopted a standardized, fully automated quality assurance of scanner hard- and software as well as a measure for in vivo data quality. After these quality-control measures had been implemented, 48 patients and 57 controls were included in the final analysis. During attention-related processes, even when the performance between patients and controls was comparable, there was a recognizable emergence of cerebral dysfunctions with hypoactivations in the ventrolateral prefrontal cortex (VLPFC), in the superior temporal cortex and in the thalamus. During working memory performance, parietal hypoactivations, especially in the precuneus, were prominent and were accompanied by poorer performance in patients. A hyperfrontality emerged in the ventrolateral prefrontal cortex. Hence, results point to a dysfunctional ventrolateral prefrontal-parietal network during working memory in patients, suggesting impairments in basic functions such as retrieval, storage and maintenance. The brain activation pattern of this large and significant sample of first-episode schizophrenia patients indicates an imbalanced system failing to adjust the amount of brain activity required in the cerebral network involved in attention and working memory.

Adolescent↗
PubMed2006

[Does garlic influence rheologic properties and blood flow in progressive systemic sclerosis?].

BACKGROUND: According to traditional European naturopathy garlic is an agent that increases perfusion. In studies with healthy subjects and in-vitro research garlic has shown influences on erythrocyte and thrombocyte aggregation as well as on vasoregulation. However, data on its effects in clinical populations are still lacking. Garlic may be useful for systemic sclerosis which is characterised by impaired perfusion that often cannot sufficiently be influenced by standard treatment. We investigated if dried garlic powder can influence rheologic properties and vasomotor function in systemic sclerosis. SUBJECTS AND METHODS: During a randomised, double blind pilot study, 20 female inpatients with systemic sclerosis received a 7 day add-on therapy with either 900 mg dried garlic powder or placebo. Rheologic properties (erythrocyte aggregation, ADP-induced thromboycyte aggregation, plasma viscosity, fibrinogenous plasma level, blood sedimentation rate) were assessed initially as well as after 1 and 7 days of treatment. Vasomotor function was evaluated using near-infrared red photoplethysmography, a new diagnostic tool to assess microcirculation. Furthermore, acral skin temperature was measured. RESULTS: After 7 days, only the verum treatment had induced a significant reduction of ADP-induced thrombocyte aggregation and a decrease in erythrocyte aggregation. Results showed no significant effects on vasomotor function, but an immediate effect of garlic on acral skin temperature. DISCUSSION: According to the 'Qualitatenlehre' of traditional European naturopathy, garlic is classified as a 'heating agent'. Our results suggest that the improvement of rheologic properties could be a possible biological correlate for this. Although further research is required, we conclude garlic could be a rational add-on therapy in the 'Kaltekrankheit' ('cold disease') of systemic sclerosis.

Blood Viscosity↗
PubMed2006

Near-field scanning optical microscopy in cell biology and cytogenetics.

Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented.

Animals↗
PubMed2005

Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy.

Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labeled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c-myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.

Biophysical Phenomena↗
PubMed2005

Assessment of DNA damage in target tumor cells after thermoablation in mice.

PURPOSE: To determine the effects of temperature on cell death for cells in culture and to compare these effects with the results of in vivo experiments in which heating is induced in mice with implanted human adenocarcinoma by using magnetic methods. MATERIALS AND METHODS: Experimentation was approved by the regional animal care committee. Human adenocarcinoma cells (MX-1) and human fibroblasts (HTB-125) were exposed to defined temperatures of 45 degrees -90 degrees C for 4 minutes. Single- and double-strand DNA breaks (expressed as a percentage of the total DNA in tail) were identified by using the alkaline comet assay, and cell survival was determined by using the cloning assay and trypan blue exclusion. For in vivo experiments, MX-1 tumors were implanted into 14 mice. Magnetic heating at temperatures of 59 degrees -96 degrees C was subsequently performed by injecting magnetic material into the tumor (7 mg +/- 3 [+/- standard deviation] magnetite per tumor) and applying an alternating magnetic field (8.8 kA/m; 400 kHz) for 4 minutes. The efficiency of the temperature-dependent induction of DNA damage in isolated tumor cells was quantified and compared with that in cultured cells. RESULTS: Results of experiments with cell cultures revealed a strong correlation between DNA damage, cell survival, and temperature, as determined with the cloning assay and trypan blue exclusion. The threshold thermoablasive temperature for tumor cell elimination was found to be 55 degrees-60 degrees C. Moreover, a strong impairment in cell survival was found when damaged DNA accounted for more than 50% of the total DNA. The heating sensitivities of malignant and nonmalignant cells did not differ. After the magnetic heating of tumors in vivo to temperatures of up to 96 degrees C (rectal temperatures between 27 degrees C +/- 2 and 29 degrees C +/- 2), isolated tumor cells showed a mean of 71.9% +/- 24.5 of total DNA in the tail per cell compared with nontreated tumors, which showed 8.0% +/- 3.1. CONCLUSION: There appears to be a threshold temperature for the induction of irreversible DNA damage. This is reflected by the results of in vivo experiments in tumor-bearing mice after high temperatures were applied to tumors by using magnetic heating.

Adenocarcinoma↗
PubMed2005

The comet-FISH technique: a tool for detection of specific DNA damage and repair.

The comet-FISH technique described in this protocol is a tool to detect genome region-specific DNA damage and repair. It is a combination of two established techniques, the comet assay (or single-cell gel electrophoresis, or the single-cell gel test), to separate highly fragmented from moderately or nonfragmented DNA and to measure it, and fluorescence in situ hybridization (FISH), to specifically label DNA sequences of interest. Comet-FISH exists in two versions, based on the neutral and the alkaline comet assays. A detailed description of the comet assay is given in Chapter 9, so readers who are not familiar with this technique can work directly with the protocol described here, without referring to additional protocols reported elsewhere. The neutral version of the comet assay detects double-strand breaks, while the alkaline version detects both double- and single-strand breaks as well as abasic sites or sites of incomplete repair. This chapter also details cell preparation and production of the hybridization probes adapted to the comet-FISH technique. Finally, microscopic analysis of comet-FISH results is described, and possible procedures of quantification of the specific DNA damage are presented.

Animals↗
PubMed2005

Fragility of telomeres after bleomycin and cisplatin combined treatment measured in human leukocytes with the Comet-FISH technique.

UNLABELLED: THE AIM of the present study was the comparative investigation of action of widely applied anticancer preparations: cisplatin (cis-DDP) and bleomycin (BLM) on total DNA and telomeres damage in human blood cells. METHODS: The "Comet-FISH technique" -- single cell gel electrophoresis ("comet assay") in combination with fluorescent in situ hybridization (FISH) was used for this purpose. This newly applied combined approach permits to detect on the same specimen the total DNA damage in individual cells and evaluate specific DNA sequences as well. Telomere -- specific -- PNA (peptide nucleic acid) probes were used for the localization of telomeres in the comet's head and their migration to the tail. THE RESULTS obtained indicate that in control variants, due to DNA metabolism and handling, approximately 7% of the DNA and 17% of the telomeres were found in the tail. In cells treated with BLM alone, telomeres leak out with equal probability as total DNA. In turn, the combination of cis-DDP with BLM reduces telomere migration more than the migration of total DNA due to cis-DDP crosslinking effect. Thus, preferentially telomeric action of the cis-DDP can be concluded. CONCLUSION: The Comet-FISH approach permitted us to reveal the induction of DNA breaks with BLM and its modification due to platinum-crosslink formation, using telomeric PNA probes.

Adult↗
PubMed2004

Spatial association of homologous pericentric regions in human lymphocyte nuclei during repair.

Spatial positioning of pericentric chromosome regions in human lymphocyte cell nuclei was investigated during repair after H(2)O(2)/L-histidine treatment. Fifteen to three-hundred minutes after treatment, these regions of chromosomes 1, 15, and X were labeled by fluorescence in situ hybridization. The relative locus distances (LL-distances), the relative distances to the nuclear center (LC-distances), and the locus-nuclear center-locus angles (LCL-angles) were measured in approximately 5000 nuclei after two-dimensional microscopy. Experimental frequency histograms were compared to control data from untreated stimulated and quiescent (G(0)) nuclei and to a theoretical two-dimensional projection from random points. Based on the frequency distributions of the LL-distances and the LCL-angles, an increase of closely associated labeled regions was found shortly after repair activation. For longer repair times this effect decreased. After 300 min the frequency distribution of the LL-distances was found to be compatible with the random distance distribution again. The LL-distance frequency histograms for quiescent nuclei did not significantly differ from the theoretical random distribution, although this was the case for the stimulated control of chromosomes 15 and X. It may be inferred that, concerning the distances, homologous pericentric regions appear not to be randomly distributed during S-phase, and are subjected to dynamic processes during replication and repair.

Cell Nucleus↗
PubMed2004

After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available.

After induction of DNA double-strand breaks (DSB) two repair systems, the error-prone 'nonhomologous end joining' (NHEJ) and the more accurate 'homologous recombination repair' (HRR) can compete for the same individual DSB site. In the human keratinocyte cell line, HaCaT, we have tested the spatial co-localisation and the temporal sequence of events. We used UV-A (365 nm) as a damaging agent, which can be applied in clearly defined doses and can lead to rare DSBs via propagation of clustered single-strand breaks (SSBs). DNA fragmentation and repair was measured by the Comet assay and persisting DSBs were quantified by the micronucleus assay. Direct DSB detection was performed by immunohistochemical labelling of gamma-H2AX, a phosphorylated histone that is assumed to form one foci per DSB. Intra- and inter-pathway interactions were quantified by co-localisation, FRET imaging and by co-immunoprecipitation (Co-IP) of XRCC4, DNA-PK and Ku70 as representatives of NHEJ, Rad51 and Rad52 for HRR and gamma-H2AX, Mre11 and Rad50 as representatives of both pathways. In G2 cells, where both systems are available, the temporal sequence after irradiation is: (1) gamma-H2AX (2) Mre11 (3) DNA-PK Rad51 (4) XRCC4. That is, the first two proteins involved in both pathways 'label' the damaged site and initiate repair, followed by the NHEJ, which is temporally overlapping with HRR activity. Taking all these observations together we suggest that a cell tries to repair DSBs with a combination of both HRR and NHEJ, if available.

Antigens, Nuclear↗
PubMed2004

Comet-FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage.

For the optimal use of anticancer drugs a knowledge of the whole spectrum of side-effects is required. A potential hazard, so far only scarcely investigated, is uncontrolled effects of drugs such as bleomycin (BLM) and mitomycin C (MMC) on telomere shortening in non-cancerous tissues of the treated person. For the first time, directly labelled telomere-specific peptide nucleic acid (PNA) hybridization probes were applied in comet-FISH to detect DNA fragmentation on an intermediate scale. The effects of BLM and MMC were measured in peripheral blood cells of three human volunteers, following ex vivo incubation. Fragmentation of telomeres and subtelomeric regions was highly specifically detected by the comet-FISH assay, a combination of the comet assay and fluorescence in situ hybridization. As a technical detail, the effects of the hybridization procedure have been studied on the level of single comets. Image analysis before and after the hybridization process reveals a small decrease in the detected fragmented DNA, probably due to diffusion of small fragments. It could not only be shown that both drugs actually induce breaks in telomere-associated DNA, but also that the comet-FISH technique, as a quantitative approach, is a useful tool for the detection and evaluation of the role of sequence-specific DNA damage after mutagenic action. The breakage frequency for DNA of or adjacent to telomeric repeats was found to be proportional to that of the total DNA, which hints at random induction of DNA breaks by BLM and MMC. In terms of therapy, the results indicate that no over- or under-proportional effects on telomeres of BLM or MMC need be expected.

Antibiotics, Antineoplastic↗
PubMed2004

Mismatch negativity responses in schizophrenia: a combined fMRI and whole-head MEG study.

OBJECTIVE: Mismatch negativity is an event-related brain response sensitive to deviations within a sequence of repetitive auditory stimuli. It is thought to reflect short-term sensory memory and is independent of higher-level cognitive processes. Mismatch negativity response is diminished in patients with schizophrenia. Little is known about the mechanisms of this decreased response, the contribution of the different hemispheres, and its locus of generation. METHOD: Patients with schizophrenia (N=12) and matched comparison subjects (N=12) were studied. A novel design to measure mismatch negativity responses to deviant auditory stimuli was generated by using the switching noises from the functional magnetic resonance imaging (fMRI) scanner, thus avoiding any interfering background sound. Stimuli included deviants of amplitude (9 dB lower) and duration (76 msec shorter) presented in a random sequence. The scanner noise was recorded and applied to the same subjects in a whole-head magnetoencephalography (MEG) device. Neuromagnetic and hemodynamic responses to the identical stimuli were compared between the patients and comparison subjects. RESULTS: As expected, neuromagnetic mismatch fields were smaller in the patient group. More specifically, a lateralization to the right for duration deviance was only found in comparison subjects. For the relative amplitude of the blood-oxygen-level-dependent signal (measured with fMRI), differences emerged in the secondary (planum temporale), but not primary (Heschl's gyrus), auditory cortex. Duration deviants achieved a right hemispheric advantage only in the comparison group. A significantly stronger lateralization to the left was found for the deviant amplitude stimuli in the patients. CONCLUSIONS: The data support the view of altered hemispheric interactions in the formation of the short-term memory traces necessary for the integration of auditory stimuli. This process is predominantly mediated by the planum temporale (secondary auditory cortex). Altered interaction of regions within the superior temporal plane and across hemispheres could be in part responsible for language-mediated cognitive (e.g., verbal memory) and psychopathological (hallucinations, formal thought disorder) symptoms in schizophrenia.

Adult↗
PubMed2003

Brain regions sensitive to the face inversion effect: a functional magnetic resonance imaging study in humans.

Perception of upright faces relies on configural processing. Therefore recognition of inverted, compared to upright faces is impaired. In a functional magnetic resonance imaging experiment we investigated the neural correlate of a face inversion task. Thirteen healthy subjects were presented with a equal number of upright and inverted faces alternating with a low level baseline with an upright and inverted picture of an abstract symbol. Brain activation was calculated for upright minus inverted faces. For this differential contrast, we found a signal change in the right superior temporal sulcus and right insula. Configural properties are processed in a network comprising right superior temporal and insular cortex.

Adult↗
PubMed2003

COMBO-FISH: specific labeling of nondenatured chromatin targets by computer-selected DNA oligonucleotide probe combinations.

Here we present the principle of fluorescence in situ hybridization (FISH) with combinatorial oligonucleotide (COMBO) probes as a new approach for the specific labeling of genomic sites. COMBO-FISH takes advantage of homopurine/homopyrimidine oligonucleotides that form triple helices with intact duplex genomic DNA, without the need for prior denaturation of the target sequence that is usually applied for probe binding in standard FISH protocols. An analysis of human genome databases has shown that homopurine/homopyrimidine sequences longer than 14 bp are nearly homogeneously distributed over the genome, and they represent from 1% to 2% of the entire genome. Because the observation volume in a confocal laser-scanning microscope equipped with a high numerical aperture lens typically corresponds to an approximate 250-kb chromatin domain in a normal mammalian cell nucleus, this volume should contain 150-200 homopurine/homopyrimidine stretches. Using DNA database information, one can configure a set of distinct, uniformly labeled oligonucleotide probes from these stretches that is expected to exclusively co-localize within a 250-kb chromatin domain. Due to the diffraction-limited resolution of a microscope, the fluorescence signals of the configured oligonucleotide probe set merge into a typical, nearly homogenous FISH spot. Using a set of 32 homopyrimidine probes, we performed experiments in the Abelson murine leukemia region of human chromosome 9 as some of the very first proofs-of-principle of COMBO-FISH. Although the experimental protocol currently contains several steps that are incompatible with living cell conditions, the theoretical approach may be the first methodological advance toward the long-term but still elusive goal of carrying out specific FISH in high-resolution fluorescence microscopy of vital cells.

Chromatin↗
PubMed2002

Mismatch responses to randomized gradient switching noise as reflected by fMRI and whole-head magnetoencephalography.

The central auditory system of the human brain uses a variety of mechanisms to analyze auditory scenes, among others, preattentive detection of sudden changes in the sound environment. Electroencephalography (EEG) and magnetoencephalography (MEG) provide a measure to monitor neuronal cortical currents. The mismatch negativity (MMN) or field (MMNm) reflect preattentive activation in response to deviants within a sequence of homogenous auditory stimuli. Functional magnetic resonance imaging (fMRI) allows for a higher spatial resolution as compared to the extracranial electrophysiological techniques. The image encoding gradients of echo planar imaging (EPI) sequences, however, elicit an interfering background noise. To circumvent this shortcoming, the present study applied multi-echo EPI mimicking an auditory oddball design. The gradient trains (SOA = 800 msec, 94.5 dB SPL, stimulus duration = 152 msec) comprised amplitude (-9 dB) and duration (76 msec) deviants in a randomized sequence. Moreover, the scanner noise was recorded and applied in a whole-head MEG device to validate the properties of this specific material. Robust fMRI activation patterns emerged in response to the deviant gradient switching. Changes in amplitude activated the entire auditory cortex, whereas the duration deviants elicited right-lateralized signal increase in secondary areas. The recorded scanner noise evoked reliably right-lateralized mismatch MEG responses. Source localization was in accordance with activation of secondary auditory cortex. The presented paradigm provides a robust and feasible tool to study the functional anatomy of early cognitive auditory processing in clinical populations such as schizophrenia.

Acoustic Stimulation↗