Showing papers by "Michael Meyer published in 2002"

Use of hybridization kinetics for differentiating specific from non‐specific binding to oligonucleotide microarrays

Abstract: Hybridization kinetics were found to be significantly different for specific and non-specific binding of labeled cRNA to surface-bound oligonucleotides on microarrays. We show direct evidence that in a complex sample specific binding takes longer to reach hybridization equilibrium than the nonspecific binding. We find that this property can be used to estimate and to correct for the hybridization contributed by non-specific binding. Useful applications are illustrated including the selection of superior oligonucleotides, and the reduction of false positives in exon identification.

Stable and efficient cassette exchange under non‐selectable conditions by combined use of two site‐specific recombinases

Abstract: Work of the last decade has proven the ‘one gene– one product–one function’ hypothesis an oversimplification. To further unravel the emerging ‘one gene–multiple products–even more functions’ concept, new methods (such as subtle knock-in and tightly regulated conditional mutations) for the analysis of gene function in health and disease are required. Another class of improvements (such as tetraploid fusion and cassette exchange) addresses the efficiency with which targeted mutant strains can be generated. Recombinase-mediated cassette exchange (RMCE), which in theory is well suited for the rapid generation of multiple alleles of a given locus, is hampered by its low efficiency in the absence of selection and, especially in vivo, by the promiscuity of the participating recombinase recognition sites. Here we present a novel approach which circumvents this problem by the use of two independent recombinase systems. The strategy, which uses loxP on one and FRT on the other side of the cassette together with a Cre/Flpe expression vector, prevents excisive events and results in higher rates of cassette integration without selection than previously described. This method has a huge potential for the generation of allelic series in embryonic stem cells and, importantly, in pre-implantation embryos in vivo.

A disrupted circumstellar torus inside eta Carinae's Homunculus Nebula

Abstract: We present thermal infrared images of the bipolar nebula surrounding eta Carinae at six wavelengths from 4.8 to 24.5 microns. These were obtained with the MIRAC3 camera system at the Magellan Observatory. Our images reveal new intricate structure in the bright core of the nebula, allowing us to re-evaluate interpretations of morphology seen in images with lower resolution. Complex structures in the core might not arise from a pair of overlapping rings or a cool (110 K) and very massive dust torus, as has been suggested recently. Instead, it seems more likely that the arcs and compact knots comprise a warm (350 K) disrupted torus at the intersection of the larger polar lobes. Some of the arcs appear to break out of the inner core region, and may be associated with equatorial features seen in optical images. The torus could have been disrupted by a post-eruption stellar wind, or by ejecta from the Great Eruption itself if the torus existed before that event. Kinematic data are required to rule out either possibility.

Conditional inactivation of the calbindin D-28k (Calb1) gene by Cre/loxP-mediated recombination.

Abstract: Calbindin D-28k (calbindin), the product of the Calb1 gene on mouse chromosome 4, belongs to a large family of proteins that use EF hand motives to bind calcium (Persechini et al., 1989; Heizmann and Hunziker, 1991). It is expressed in various populations of neurons (Celio 1990; Baimbridge et al., 1992) and a limited number of extraneural tissues (Christakos et al., 1989). Recent studies on calbindin null mutant or antisense mouse strains demonstrated that endogenous calbindin is required for motor coordination, shaping of calcium transients (Airaksinen et al., 1997a), and synaptic plasticity (Klapstein et al., 1998; Jouvenceau et al., 1999). Its role as a neuroprotective calcium buffer, however, could not be confirmed (Airaksinen et al., 1997b; Klapstein et al., 1998). In order to study the function of calbindin in defined neuronal populations, we generated a conditional allele (in the following referred to as Calb to distinguish it from the general null mutation allele Calb; Airaksinen et al., 1997a). A strategy (Gu et al., 1994) based on the Cre/loxP system (Sternberg and Hamilton, 1981; Le and Sauer, 2000) was chosen. The targeting vector was obtained by inserting a single loxP site upstream and a floxed neo-TK cassette downstream of the first exon (Fig. 1a). After electroporation and G418 selection of R1 embryonic stem cells (Airaksinen et al., 1997a), homologous recombinants were identified by Southern blotting (Fig. 1b) using an outside probe (see Fig. 1a). Homologous recombination occurred in 1 in 50 clones. Two recombined clones were selected for electroporation with a Cre expression plasmid. Gancyclovir-resistant clones were screened for the presence of the first exon and the absence of the selection cassette. Eleven clones fulfilling both criteria were obtained and one of these was used for injection into C57BL/6 (B6) blastocysts. Resulting chimeras were crossed to B6 and offspring from intercrosses of heterozygous F1 mice was used for most of the following analysis. Currently, the strain is maintained by backcrossing to B6. Homozygous Calb mice were checked for possible effects of the loxP sites (their location is detailed in Fig. 1c) on calbindin expression. Western blotting and immunostaining with calbindin antibodies (Fig. 1d, e) and RT-PCR (not shown; conditions as in Fig. 2) did not reveal differences between Calb/Calb and wildtype littermates. Importantly, both groups were also indistinguishable in the open field (horizontal activity, Fig. 1f; vertical activity, not shown) (Barski et al., 2000) and runway (Fig. 1g) (Airaksinen et al., 1997a) behavioral tests. Conditional recombination of the Calb allele was tested by mating of Calb/Calb animals with pcp2 mice that express Cre specifically in cerebellar Purkinje cells (Barski et al., 2000). Mice heterozygous for both mutations were intercrossed, and offspring of all possible genotypes was obtained in mendelian ratios. Efficiency and specificity of recombination were checked in animals harboring two Calb alleles and at least one pcp2 allele (in the following referred to as recombined). RT-PCR on RNA from cerebellum of recombined animals revealed almost complete absence of calbindin-specific transcripts (Fig 2b). In contrast, expression in hippocampus, cortex, and kidney of the same animals was not different from wild type. Similar observations were made in Western blots (Fig. 2c). Residual calbindin gene product is derived from the low number (about 2%) of not-recombined Purkinje cells and from calbindin protein present in climbing fibers, a major Purkinje cell afferent. General deletion of the calbindin gene results in remarkably subtle histological changes (Airaksinen et al., 1997a). Using immunostaining for various cerebellar marker proteins, we extend this observation now to the Purkinje cell-specific null mutant (Fig. 2d). From this experiment, there is also no indication of toxic effects of prolonged Cre expression (Schmidt et al., 2000).

Fractal dynamics of heart beat interval fluctuations in corticotropin-releasing factor receptor subtype 2 deficient mice.

Abstract: Non-linear fractal analysis of cardiac interbeat time series was performed in corticotropin-releasing factor receptor subtype 2 (CRFR2) deficient mice. Heart rate dynamics in mice constitutes a self-similar, scale-invariant, random fractal process with persistent intrinsic long-range correlations and inverse power-law properties. We hypothesized that the sustained tachycardic response elicited by intraperitoneal (ip) injection of human/rat CRF (h/rCRF) is mediated by CRFR2. In wildtype control animals, heart rate was increased to about maximum levels (~ 750 bpm) while in CRFR2-deficient animals baseline values were retained (~ 580 bpm). The tachycardic response elicited by ip-application is mediated by CRFR2 and is interpreted to result from sympathetic stimulation. However, the functional integrity of CRFR2 would not present a prerequisite to maintaining the responsiveness and resiliency of cardiac control to external environmental perturbations experimentally induced by extrinsic ip-application of h/rCRF or under physiological conditions that may be associated with an increased peripheral release of CRF. Under stressful physiological conditions achieved by novelty exposure, CRFR2 is not involved in the cardiodynamic regulation to external short-term stress. While the hypothesis of involvement of CRFR2 in cardiac regulation upon pharmacological stimulation cannot be rejected, the present findings suggest that the mechanism of action is by sympathetic stimulation, but would not unambiguously allow to draw any conclusions as to the physiological role of CRFR2 in the control of cardiac dynamics.

Fractal Scaling of Heartrate Dynamics in Health and Disease

Abstract: The dynamics of heartbeat interval time series were studied by a modified random walk analysis recently introduced as Detrended Fluctuation Analysis In this analysis, the intrinsic fractal long-range power-law correlations of beat-to-beat fluctuations generated by the dynamical system, after decomposition from extrinsic uncorrelated sources, can be quantified by the scaling exponent which, in healthy subjects, is - 10 The finding of a scaling coefficient of 10, indicating scale-invariant long-range power-law correlations (1/f noise) of heartbeat fluctuations, would reflect a genuinely self-similar fractal process that typically generates fluctuations on a wide range of time scales The 1/f dynamics of heartbeat interval fluctuations are unaffected by exposure to chronic hypoxia at high altitude (> 5000 m) suggesting that the neuroautonomic cardiac control system is preadapted to hypoxia Functional (hypothermia, cardiac disease) and/or structural (cardiac transplantation, early cardiac development) inactivation of neuroautonomic control is associated with the breakdown of fractal complexity reflected by anticorrelated random walk-like dynamics, indicating that in these conditions the heart is unadapted to its environment

ALIGNMENT OF Ar + IONS PRODUCED AFTER RESONANT AUGER DECAY OF Ar * 2p 5 3d RESONANCES

Abstract: Fluorescence spectra have been recorded in the wavelength region 300 nm ≤ λ(fluo) ≤ 500 nm after resonant 2p → 3d excitation of atomic Ar using monochromatic synchrotron light The radiative emission is characterized by strong transitions from Ar+3p4 (1S, 1D, 3P) nl ionic states It is mainly assigned to processes of the type 3p44d → 3p44p and 3p44p → 3p44s In addition, we have analyzed the degree of polarization of the fluorescence lines and we have determined the alignment of the Ar+ ions, which are produced after the resonant Auger decay In comparison to investigations based on photoelectron spectroscopy, the spectral analysis of the fluorescence provides complementary information about the electronic relaxation during the resonant Auger process and, in particular, it allows one to examine directly the remaining ion

Information States in Cardiac Rhythm

Abstract: The cardiac rhythm of a healthy person is shown to be characterized by a negative exponential probability distribution in heart rate fluctuations for sufficient time delay between beats. This distribution, the hallmark of a statistical mechanical formulation, allows for the characterization ofthe textural and structural information. Properties of the joint variation of these forms of information show evidence of the existence of two states separated by a critical value of heart rate variability. For low variability the heart beats regularly, but as the variability increases the heart rate becomes increasingly intermittent. The implication of the information states is that the heart is a self adjusting mechanism over the span of metabolic demands. The method allows for a comparison of how some hearts (congestive heart failure and children) differ from adult normal hearts, and others (normal mice) resemble those ofhumans. it is shown that the information treatment of non-linear cardiodynamics described here incorporates the multifractal property of chaos in the cardiac rhythm.