Biomedical subjects

A Schober

Publications and source records attributed to A Schober.

At least 19 recordsLinked to original sources

PubMed1999

Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7-T10 that project to the adrenal medulla. In situ hybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked alpha receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-beta (TGF-beta) receptor II. Administration of GDNF (recombinant human, 1 microg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochrome c applied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-beta isoforms but not GDNF. This suggests that the presence of endogenous TGF-beta was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-beta for permitting trophic actions of GDNF in vitro (Kreiglstein et al., 1998) also applies to the in vivo situation.

Adrenal Glands↗

PubMed1998

On-line polymerase chain reaction (PCR) monitoring.

In this short note, we present the results of a case study for monitoring the whole polymerase chain reaction (PCR) process (all steps) with a glass fiber fluorometer that was described in a former publication. To utilize this fluorometer, which was originally constructed for a PCR machine with three thermostating devices, a new thermostating device has been developed: the glass fiber matrix is integrated into the thermostating device, while the PCR samples are heated and cooled. The device is able to monitor all samples throughout all stages of PCR with the help of an intercalating dye. This approach also permits one to choose arbitrarily different cooling and heating rates.

Fluorometry↗

PubMed1998

TrkB and neurotrophin-4 are important for development and maintenance of sympathetic preganglionic neurons innervating the adrenal medulla.

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7-T10, disappear. Administration of NT-4 in gelfoams (6 microgram) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7-T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.

Adrenal Medulla↗

PubMed1998

Distinct populations of macrophages in the adult rat adrenal gland: a subpopulation with neurotrophin-4-like immunoreactivity.

Macrophages are widely distributed in lymphohaemopoietic and many other mammalian tissues, where they are mainly involved in host defence mechanisms, phagocytosis, wound repair, and secretion of growth factors. Increasing evidence suggests that secretory products of macrophages can influence adrenal gland functions. In the present study, we have used specific antibodies to ED1 (cytoplasmic antigen), ED2 (membrane antigen), ED8 (membrane antigen), and OX-6 (MHC class II/membrane antigen) as markers for macrophages to examine their distribution within the adult rat adrenal gland. ED2 and OX-6 recognize distinct subpopulations of adrenal gland macrophages, whereas macrophages immunoreactive (-ir) for ED1 and ED8 could not be detected. OX-6-ir macrophages were most numerous in the cortical reticularis and glomerulosa zones, while only few cells were found in the zona fasciculata and in the adrenal medulla. Macrophages immunoreactive for ED2 were restricted to the adrenal medulla. The majority of these macrophages were associated with vascular sinuses or chromaffin cells. By double-immunolabelling we found that most of ED2-ir medullary macrophages contain neurotrophin-4 (NT-4)-like ir. Attempts to clarify whether macrophages take up NT-4 from NT-4-ir chromaffin cells indicated that medullary macrophages are immunonegative for chromogranin A and neuropeptide Y, two major secretory products of chromaffin cells. In situ hybridizations and immunofluorescence showed expression of the neurotrophin receptor TrkA, but not TrkB in the adrenal medulla. In vitro studies indicated that NT-4, similar to nerve growth factor, can induce c-fos-ir in chromaffin cells. We conclude that chromaffin cells are putative targets for adrenal medullary NT-4, whose functions remain to be clarified.

Adrenal Cortex↗

PubMed1998

Cationic amino acid transporter mRNA expression in rat kidney and liver.

Expression of rat cationic amino acid transporter 2 (r-CAT-2) mRNA was studied in kidney and liver using Northern blot analysis and nonradioactive in situ hybridization with a probe identifying both the r-CAT-2alpha and -2beta splice variants. Expression of r-CAT-2 mRNA was higher in the liver than in the kidney. Within the kidney, r-CAT-2 mRNA was more abundant in the outer and inner medulla than in the cortex. In the liver lobule, the intensity of the hybridization signal in hepatocytes decreased between the portal area and the central vein. In the kidney, hybridization signals were detected in parietal cells of Bowman's capsule, various tubule cells of outer and inner medulla, in endothelial and interstitial cells of inner medulla, and in papillary epithelial cells.

Amino Acid Transport Systems, Basic↗

PubMed1998

Influence of osmotic stress on heat shock proteins 25 and 72 in mouse mesangial cells.

Previous studies have shown intense staining for heat shock protein 25 (HSP25) in the extraglomerular mesangium (EGM). Because relationships are believed to exist between osmotic stress, expression of HSP25, and protection against stress and because the EGM may be exposed to high local tonicity, we examined the expression of HSP25 and the major stress-inducible and cytoprotective HSP72 in mouse mesangial cells and embryonic lung fibroblasts (3T3) after exposure to hypertonic stress (addition of 150 mM NaCl to the medium for two to seven days). Mesangial, but not 3T3, cells expressed high levels of HSP25 already under control conditions, whereas neither cell line contained HSP72. Hypertonic treatment neither enhanced (mesangial cells) or induced (3T3 cells) HSP25 expression. HSP72, however, was induced strongly in 3T3 cells, but only minimally in mesangial cells. The high level of HSP25 in mesangial cells thus seems not to be a consequence of high tonicity in the EGM because cultured mesangial cells express HSP25 already under control conditions, and osmotic stress did not induce HSP25 in either cell line. Furthermore, high amounts of HSP25 seem to reduce the requirement for HSP72 after stress exposure, suggesting that, in mesangial cells, HSP25 might assume some functions of HSP72.

3T3 Cells↗

PubMed1998

Effect of ischemia on localization of heat shock protein 25 in kidney.

The effects of renal ischemia on the intracellular distribution of the low-molecular weight heat shock protein (HSP)25 were examined using immunofluorescence microscopy. In all kidney zones, ischemia decreased HSP25 in the supernatant of the tissue homogenates and increased it in the pellet fraction (containing mainly nuclei and cytoskeletal components). This was associated with disappearance of HSP25 staining from the brush border of proximal convoluted tubule (PCT) cells. Because no nuclear staining of cortical tubule cells was apparent either in control or ischemic kidneys, ischemia seems to cause a closer association of HSP25 with cytoskeletal components. HSP25 probably participates in the postischemic restructuring of the cytoskeleton of PCT cells.

Animals↗

PubMed1997

Nitric oxide synthase in learning-relevant nuclei of the chick brain: morphology, distribution, and relation to transmitter phenotypes.

Nitric oxide (NO) has been implicated in learning in the hatchling chicken. To examine morphological and neurochemical properties of neurons that contain NO synthase (NOS) in brain regions known to be involved in learning and memory, the NADPH-diaphorase technique was used in conjunction with immunocytochemistry and tract tracing. A distinct cell type was NOS-labeled in the lobus parolfactorius (LPO) in the telencephalon, and neurons were labeled in the area ventralis of Tsai (AVT), the substantia nigra (nucleus tegmenti pedunculo-pontinus, pars compacta, TPc), and the locus coeruleus in the brainstem. Thus, NO may influence processes of learning and memory in the forebrain after release from intrinsic neurons and/or from extrinsic NOS-projections originating from the brainstem. DiI-tracing revealed that most of the NOS-positive neurons in the AVT/TPc project to the basal forebrain. The majority of tyrosine hydroxylase-positive (presumptive dopaminergic) neurons in the AVT and TPc expressed NOS. Double-labeling with antibodies to tyrosine hydroxylase, choline acetyltransferase, somatostatin, and the neurotrophin receptor as a marker for noradrenergic coeruleus neurons showed that NOS was not colocalized with noradrenergic or somatostatinergic neurons, and that less than a third of the cholinergic neurons were double-labeled for NOS. Injections of 6-hydroxydopamine into the brainstem did not reduce the density of NOS-labeled fibers in the LPO, indicating that most of the NO in the LPO originates from intrinsic neurons in the basal forebrain. Thus, NOS-containing presumptive local circuit neurons in the LPO are the most likely source of NO involved in learning of passive avoidance tasks in hatchling chicks.

Animals↗

PubMed1997

Reduced acetylcholinesterase (AChE) activity in adrenal medulla and loss of sympathetic preganglionic neurons in TrkA-deficient, but not TrkB-deficient, mice.

TrkA high-affinity receptors are essential for the normal development of sympathetic paravertebral neurons and subpopulations of sensory neurons. Paravertebral sympathetic neurons and chromaffin cells of the adrenal medulla share an ontogenetic origin, responsiveness to NGF, and expression of TrkA. Which aspects of development of the adrenal medulla might be regulated via TrkA are unknown. In the present study we demonstrate that mice deficient for TrkA, but not the neurotrophin receptor TrkB, show an early postnatal progressive reduction of acetylcholinesterase (AChE) enzymatic activity in the adrenal medulla and in preganglionic sympathetic neurons within the thoracic spinal cord, which are also significantly reduced in number. Quantitative determinations of specific AChE activity revealed a massive decrease (-62%) in the adrenal gland and a lesser, but still pronounced, reduction in the thoracic spinal cord (-40%). Other markers of the adrenal medulla and its innervation, including various neuropeptides, chromogranin B, secretogranin II, amine transporters, the catecholamine-synthesizing enzymes tyrosine hydroxylase and PNMT, synaptophysin, and L1, essentially were unchanged. Interestingly, AChE immunoreactivity appeared unaltered, too. Preganglionic sympathetic neurons, in contrast to adrenal medullary cells, do not express TrkA. They must, therefore, be affected indirectly by the TrkA knock-out, possibly via a retrograde signal from chromaffin cells. Our results suggest that signaling via TrkA, but not TrkB, may be involved in the postnatal regulation of AChE activity in the adrenal medulla and its preganglionic nerves.

Acetylcholinesterase↗

PubMed1997

The response of heat shock proteins 25 and 72 to ischaemia in different kidney zones.

Induction of heat shock proteins (HSPs) following cell injury contributes to the protection of vital cell functions. It was, therefore, of interest to study the effects of transient renal ischaemia on the abundance and distribution of two HSPs, HSP25 and HSP72, in renal tissue using Western-blot techniques. Analyses were performed on the supernatant (HSP25, HSP72) and pellet (HSP25) of homogenates obtained from cortex (CX) and outer (OM) and inner (IM) medulla of the rat kidney immediately after 60 min of ischaemia followed by varying periods of reperfusion. Ischaemia of the left kidney caused HSP25 contents to decrease in CX, OM and IM by 73, 89 and 54% respectively, compared with the corresponding zones of the contralateral control kidney. This initial decrease in supernatant HSP25 was accompanied by an increased abundance of HSP25 in the pellet. Following reperfusion, HSP25 contents in the supernatant gradually increased in CX and OM, reaching, after 24 h, values that were 5.4- and 2.5-fold higher, respectively, than those in the control kidneys. After 7 or 14 days of reperfusion, HSP25 contents had not completely normalised in CX, but had reached control levels in OM. In IM, the HSP25 content remained below control throughout the entire reperfusion period. HSP72 (supernatant) was below the detection limit in the CX of the control kidney. Similar to the level of HSP25, that of HSP72 was also markedly lower in OM and IM immediately after ischaemia. The intrarenal distribution of HSP72 and the sequence of zonal changes in HSP72 contents were similar to those observed for HSP25. These results are compatible with the view that, during ischaemia and the initial reperfusion period, HSP25 migrates from the cytoplasmic compartment (supernatant) into the nucleus and/or associates with cytoskeletal structures. The observation that both HSP25 and HSP72 are transiently induced in CX and OM, but not in IM, may be explained by the fact that, while all kidney cells are exposed to ischaemic stress, only inner medullary cells experience a major postischaemic attenuation of osmotic stress.

Animals↗

PubMed1997

Optimizing doped libraries by using genetic algorithms.

The insertion of random sequences into protein-encoding genes in combination with biological selection techniques has become a valuable tool in the design of molecules that have useful and possibly novel properties. By employing highly effective screening protocols, a functional and unique structure that had not been anticipated can be distinguished among a huge collection of inactive molecules that together represent all possible amino acid combinations. This technique is severely limited by its restriction to a library of manageable size. One approach for limiting the size of a mutant library relies on 'doping schemes', where subsets of amino acids are generated that reveal only certain combinations of amino acids in a protein sequence. Three mononucleotide mixtures for each codon concerned must be designed, such that the resulting codons that are assembled during chemical gene synthesis represent the desired amino acid mixture on the level of the translated protein. In this paper we present a doping algorithm that "reverse translates' a desired mixture of certain amino acids into three mixtures of mononucleotides. The algorithm is designed to optimally bias these mixtures towards the codons of choice. This approach combines a genetic algorithm with local optimization strategies based on the downhill simplex method. Disparate relative representations of all amino acids (and stop codons) within a target set can be generated. Optional weighing factors are employed to emphasize the frequencies of certain amino acids and their codon usage, and to compensate for reaction rates of different mononucleotide building blocks (synthons) during chemical DNA synthesis. The effect of statistical errors that accompany an experimental realization of calculated nucleotide mixtures on the generated mixtures of amino acids is simulated. These simulations show that the robustness of different optima with respect to small deviations from calculated values depends on their concomitant fitness. Furthermore, the calculations probe the fitness landscape locally and allow a preliminary assessment of its structure.

Algorithms↗

PubMed1996

Is body focus restricted to self-evaluation? Body focus in the evaluation of self and others.

OBJECTIVE: Clinicians have suggested that the core pathology of the eating disorders is an extreme body focus in self-evaluation. This study investigated whether women who focus on their own bodies place a similar focus on body shape when evaluating others and expect others to have a strong body focus in their self-evaluations. METHOD: Eighty-four undergraduate women completed the Eating Attitudes Test (EAT-26), which largely measures body focus in self-evaluation. Each was also shown a series of photographs of women and asked what aspects of the photos they first noticed and how the depicted women felt about themselves. Finally, each responded to scenarios in which they or a hypothetical woman overate or dieted. RESULTS: High EAT-26 scores were associated with an elevated number of "fat" or "thin" feelings attributed to women in the slides and a marginally greater number of body-related observations made about the slides. High EAT-26 scores were also associated with inferred negative feelings of others after overeating and positive feelings of others after dieting. In fact, the relationships between EAT-26 scores and feelings in response to hypothetical overeating and dieting situations were no stronger when applied to the self than when applied to others. However, when presented with a situation in which they overate, participants showed stronger relationships between their EAT-26 scores and their expected weight change and dieting behavior than they predicted would occur for others after an identical period of overeating. DISCUSSION: Extrapolating findings to a clinical population, women with eating disorders may focus on others' body shapes as well as their own. These women may also expect others to be as emotionally invested in their own body shapes as they are themselves, but may not expect others to be as prone to gain weight or to diet as strictly as they do. Treatment for this somewhat recalcitrant belief system is discussed, as are directions for future research.

Adolescent↗

PubMed1996

Hamming chromatography.

Selection of molecules with desired properties from random pools of biopolymers has become a powerful tool in biotechnology. On designing an evolution experiment, a certain knowledge of the concomitant fitness landscape is clearly helpful to set up the optimal experimental conditions. The correlation function is a useful means of characterizing a given landscape, since it can be efficiently measured if one has a method of separating a pool of random sequences according to their Hamming distance from a moderately small number of test sequences. In this paper we describe a special type of hybridization chromatography, where a mixture of oligomers (partially) complementary to a given test sequence is hybridized to the test sequence, covalently bound to a matrix. DNA oligomers are eluted in an 'effective temperature gradient' using conditions that minimize the differences of effects of GC versus AT pairs on the melting temperatures. This method should be a means to quickly separate error classes and thus be the crucial step in characterizing fitness landscapes of biopolymers through an experimental approach. It would also be a useful tool to design sequence pools with a bias towards desired mutant spectra.

Bacteriophage T7↗

PubMed1995

Noradrenergic neurons in the locus coeruleus of birds express TrkA, transport NGF, and respond to NGF.

The chicken locus coeruleus contains a population of noradrenergic neurons which express the neurotrophin receptor p75 (von Bartheld and Bothwell, 1992). To determine which neurotrophin may regulate the development of noradrenergic neurons in the chicken locus coeruleus, expression of trk receptors, retrograde transport of neurotrophins, and responses to NGF were examined. P75-expressing noradrenergic neurons were found to project to the basal forebrain. They transport radio-iodinated NGF after injections into this target. The retrograde transport of NGF is specific to the noradrenergic neuronal population as evidenced by double labeling with antibodies against dopamine-beta-hydroxylase. The same neuronal population expresses trkA receptor mRNA. The size of noradrenergic neurons in the locus coeruleus proper, but not in the nucleus subcoeruleus, is significantly increased after injections of NGF into the telencephalon, consistent with the hypothesis that target-derived NGF provides trophic support. Noradrenergic coeruleus neurons are rescued from toxic effects of 6-hydroxydopamine injected into the telencephalon when NGF is injected into the midbrain. NGF has no rescue effect when it is coinjected with 6-hydroxy-dopamine into the telencephalon. In explant or dissociated cultures, noradrenergic coeruleus neurons do not respond to elevated levels of NGF with increased neurite outgrowth. Taken together, these results suggest that NGF plays a role in the development and maintenance of noradrenergic coeruleus neurons in the chick brain. The data also support our previous conclusion that major species differences exist between birds (chicken) and mammals with regard to trophic regulation of presumptive homologous neuronal populations.

Animals↗

PubMed1995

Multichannel PCR and serial transfer machine as a future tool in evolutionary biotechnology.

As an improved strategy for producing functional macromolecules by in vitro evolutionary optimization, we propose an automated machine that can process up to 960 samples in parallel. It consists of a 960-well PCR machine with special sealed plastic reaction vessels and appropriate handling devices. We show that the heat-sealing technique does not significantly affect the activity of Taq DNA Polymerase or that of the temperature-sensitive Q beta RNA polymerase but avoids cross-contamination and evaporation of the samples. Initial experiments demonstrate the suitability of the apparatus to uniformly process the samples and to perform the thermocycling. Serial transfer of reaction products into fresh reaction solution was used to initiate further rounds of amplification as a typical experimental setup for an evolutionary biotechnology application.

Allolevivirus↗

PubMed1994

Central projections of the nervus terminalis and the nervus praeopticus in the lungfish brain revealed by nitric oxide synthase.

Lungfishes possess two cranial nerves that are associated with the olfactory system: the nervus terminalis enters the telencephalon with the olfactory nerve, and the nervus praeopticus enters the diencephalon at the level of the optic nerve. We investigated the central projections of the nervus terminalis and the nervus praeopticus in the Australian lungfish (Neoceratodus forsteri) and in the African lungfish (Protopterus dolloi) by NADPH-diaphorase histochemistry (nitric oxide synthase; NOS) and compared them with the projections of the nervus terminalis of the frog (Xenopus laevis). In Neoceratodus, NOS-positive fascicles of the nervus terminalis divide and project with a ventral component through the septum and with a dorsal component through the pallium; fibers of both trajectories extend caudally beyond the anterior commissure and join the lateral forebrain bundle. In the nervus praeopticus, about 300 fibers contain NOS; they innervate the preoptic nucleus and continue their course through the diencephalon; many fibers cross in the commissure of the posterior tuberculum. In Protopterus, ganglion cells of the nervus terminalis and of the nervus praeopticus contain NOS. NOS-positive fibers of the nervus terminalis project through the septal region but not through the pallium. Several major fascicles cross in the rostral part of the anterior commissure, where they are joined by a small number of NOS-containing fibers of the nervus praeopticus. Both nerves innervate the preoptic nucleus. The number and pathways of the fascicles of the nervus terminalis are not always symmetric between the two sides. The nervus terminalis fascicles remain in a ventral position, whereas the nervus praeopticus gives rise to the more dorsal fascicles. Many fibers of the two nerves extend throughout the diencephalon and cross in the commissure of the posterior tuberculum. These findings demonstrate many similarities but also significant differences between the contributions of the nervus terminalis and the nervus praeopticus to forebrain projections in the two lungfishes. They support the view that the nervus praeopticus is part of a nervus terminalis system comparable to that in frogs and other nonmammalian vertebrates.

Amino Acid Oxidoreductases↗

PubMed1994

NADPH-diaphorase in the central nervous system of the larval lamprey (Lampetra planeri).

The distribution of nitric oxide synthase (NOS) in the lamprey brain was studied by using reduced nicotinamide-adenine-dinucleotide phosphate (NADPH)-diaphorase histochemistry to further elucidate the evolution of neurons synthesizing nitric oxide. Intense labeling of fibers and/or neurons was found in portions of the lamprey central nervous system, such as the olfactory system, the pineal organ, the habenular region, the nervus stato-acousticus (N. VIII), the brainstem, and the spinal cord, and also in the adenohypophysis. Labeled giant cells located at the floor of the 3rd and 4th ventricle were recognized as reticulospinal neurons. Mauthner and Müller cells were identified according to morphological criteria. Eight pairs of Müller cells and one pair of Mauthner cells were labeled by NADPH histochemistry. None of these cells had, as yet, been described to display NOS activity in any vertebrate. The massive staining of these cells and the apparent lack of labeling, e.g., in teleost fishes, may be a histochemical correlate to already known differences of functions served by these cells in different species. In addition, our results suggest that the nitric oxide (NO) system has appeared early in vertebrate evolution.

Animals↗

PubMed1993

Enzymehistochemical demonstration of nitric oxide synthase in the diencephalon of the rainbow trout (Oncorhynchus mickiss).

Nitric oxide, a free radical, has recently been shown to exert major influences on CNS functions in mammals. It is synthesized by NO-synthase. For the first time, this study reveals this enzyme's existence in the CNS of a teleost fish and describes its distribution in the diencephalon, where the paraventricular organ displays an extraordinarily high activity. The study contributes to an evolutionary perspective of the biological role played by nitric oxide in the vertebrate CNS, and raises questions regarding the significance of this gas in cerebrospinal fluid-contacting neurons.

Amino Acid Oxidoreductases↗