Biomedical subjects

C Schwarz

Publications and source records attributed to C Schwarz.

At least 19 recordsLinked to original sources

PubMed2000

Serotonin suppresses subthreshold and suprathreshold oscillatory activity of rat inferior olivary neurones in vitro.

The effect of serotonin on membrane potential oscillations of inferior olivary neurones was studied in brainstem slices from 10- to 19-day-old rats. Serotonin at 50 and 5 microM induced a mean depolarization of 9.4 and 7.7 mV, respectively, that was preceded by a reversible suppression of subthreshold membrane potential oscillations. These effects were not changed by 1 microM tetrodotoxin and the suppression of subthreshold oscillations persisted after current-mediated restoration of resting potential. In spontaneously active neurones, serotonin abolished the rhythmicity of action potential firing without affecting spike frequency. Serotonin reduced the slope of the calcium-mediated rebound spike and both the duration and amplitude of the subsequent afterhyperpolarization. Serotonin also shifted the voltage dependence of the rebound spike to more negative values. Hyperpolarizing current pulses (200 ms) revealed that serotonin increased the pre-rectification and steady-state components of membrane resistance by 37 and 38 %, respectively, in 66 % of neurones, but decreased these parameters by 14 and 20% in the remaining cells. The serotonin effects were antagonized by 5 microM methysergide or 1-5 microM ketanserin and were mimicked by 10-20 microM dimethoxy-4-iodoamphetamine but not 10 microM 8-hydroxy-2-(di-N-propylamino)-tetralin. The data indicate that serotonin suppresses the rhythmic activity of olivary neurones via 5-HT2 receptors by inhibition of the T-type calcium current in combination with membrane depolarization due to activation of a cation current (Ih) and block of a resting K+ current (fast IK(ir)). This modulatory action of serotonin may account for the differential propensity of olivary neurones to fire rhythmically during different behavioural states in vivo.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2000

Receptor recognition by a hepatitis B virus reveals a novel mode of high affinity virus-receptor interaction.

The duck hepatitis B virus model system was used to elucidate the characteristics of receptor (carboxypeptidase D, gp180) interaction with polypeptides representing the receptor binding site in the preS part of the large viral surface protein. We demonstrate the pivotal role of carboxypeptidase D for virus entry and show its C-domain represents the virus attachment site, which binds preS with extraordinary affinity. Combining results from surface plasmon resonance spectroscopy and two-dimensional NMR analysis we resolved the contribution of preS sequence elements to complex stability and show that receptor binding potentially occurs in two steps. Initially, a short alpha-helix in the C-terminus of the receptor binding domain facilitates formation of a primary complex. This complex is stabilized sequentially, involving approximately 60 most randomly structured amino acids preceding the helix. Thus, hepadnaviruses exhibit a novel mechanism of high affinity receptor interaction by conserving the potential to adapt structure during binding rather than to preserve it per se. We propose that this process represents an alternative strategy to escape immune surveillance and the evolutionary pressure inherent in the compact hepadnaviral genome organization.

Amino Acid Sequence↗

PubMed2000

[Pathophysiology of acute renal failure at the cellular level].

The influence of inflammation on post-ischemic acute renal failure (ARF) has only recently be appreciated. In this review we therefore discuss the cellular events occurring in ARF with special emphasis on the impact of inflammatory processes on the pathogenesis of ARF. Furthermore, the spectrum of injury leading to sublethal or lethal cell damage and the time course, occurrence and regulation of the two distinct forms of cell death, necrosis and apoptosis will be described extensively. Especially apoptosis and its regulation has been studied only marginally in the setting of ischemic ARF. This overview is mainly focused on tubular cell injury since tubular epithelial cells are the major victims of ischemia whereas cells inside the glomerular tuft show only little pathology. The models of tubular injury described in this paper are ranging from primary cultures of isolated human tubular epithelial cells to experimental ischemic renal failure in rats, and to clinical settings of human ischemic ARF. The cellular events highlighted in this review are the influence of the expression of cellular adhesion molecules on the pathophysiology of ARF, and the regulation and time course of apoptosis. Examples of these processes are being illustrated by figures exhibiting morphology and immunohistochemistry of cell proliferation and cell death regulatory proteins.

Acute Kidney Injury↗

PubMed2000

Reply

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Journal Article↗

PubMed1999

Glutamine deprivation induces the expression of GADD45 and GADD153 primarily by mRNA stabilization.

The expression of the growth arrest- and DNA damage-inducible genes, GADD45 and GADD153/CHOP (C/EBP-homologous protein), as well as GRP78 (glucose-regulated protein of 78 kDa) was examined in several human breast cell lines subjected to acute glutamine (GLN) deprivation. GLN deprivation caused rapid elevation of GADD45 and GADD153/CHOP mRNA levels in cells that were highly dependent upon GLN for growth and viability. Both GADD mRNAs were rapidly elevated up to several hundred-fold. In contrast, GRP78 expression was elevated by no more than 4-fold by GLN deprivation. The magnitude of GADD up-regulation roughly correlated with the extent of GLN dependence of each cell line. The levels of all three mRNAs were responsive to alterations of ambient GLN content in a physiologically relevant concentration range that corresponded to the affinities of cellular GLN transporters. Provision of GLN-derived metabolites partially inhibited the induction of GADD expression in GLN-deprived cells. Nuclear run-on assays and mRNA decay studies suggested that the primary mechanism leading to increased GADD mRNA levels was not transcriptional, but rather that GADD45 and GADD153/CHOP expression were up-regulated in response to GLN deprivation via marked stabilization of these mRNAs. These results suggest that the expression of GADD genes contributes to growth arrest and/or protection from metabolic damage during GLN-poor conditions.

Breast Neoplasms↗

PubMed1999

Two cases with interstitial deletions of chromosome 2 and sex reversal in one.

We present two children with de novo interstitial deletions of the long arm of chromosome 2 (karyotypes 46,XY, del(2)(q31.1q31.3) and 46,XY, del(2)(q24.3q31.3), respectively). The first child had severe learning difficulties, growth retardation, unilateral ptosis, small palpebral fissures, a cleft uvula, and bilateral cutaneous syndactyly of the second and third toes. Despite her male karyotype, she had female external genitalia with hypoplasia of the clitoris and labia minora. This is the first reported case of feminization of the external genitalia in a genotypic male with an interstitial deletion of chromosome 2q31 and adds to the growing amount of evidence for a gene involved in sex determination in this chromosome region. The second child had severe mental and growth retardation, ptosis, down-slanting palpebral fissures, low-set ears, micrognathia, finger camptodactyly, and brachysyndactyly of the second to fifth toes. The clinical manifestations associated with deletions of 2q31 to 2q33 are similar to those found with proximal deletions at 2q24 to 2q31 and of band 2q24, suggesting that the phenotype may result from haploinsufficiency for one or more genes located at 2q31. Microsatellite marker studies showed that both children had paternally derived deletions that included the HOXD gene cluster and the EVX2, DLX1, and DLX2 genes known to be important in limb development.

Abnormalities, Multiple↗

PubMed1999

GABAergic inhibition in the rat pontine nuclei is exclusively extrinsic: evidence from an in situ hybridization study for GAD67 mRNA.

As clearly indicated by our electrophysiological work, GABAergic inhibition plays a powerful role in the pontine nuclei (PN), the major link between cerebral cortex and the cerebellum. Using the technique of in situ hybridization for the mRNA encoding for the gamma-aminobutyric acid (GABA)-synthesizing isoenzyme glutamic acid decarboxylase67 (GAD67), we demonstrate here the total absence of potentially GABAergic neurons from the rat PN. This negative finding supports the notion that GABAergic inhibition in the PN of rats, unlike that of higher mammals, is exclusively based on extrapontine GABAergic afferents.

Animals↗

PubMed1999

Binding of signals relevant for action: towards a hypothesis of the functional role of the pontine nuclei.

If numbers matter, the projection that connects the cerebral cortex to the cerebellum is probably one of the most-important pathways through the CNS. Its extensive development as one ascends the phylogenetic scale parallels that of the cerebral hemispheres and the cerebellum, and it accompanies improvements in motor skills, suggesting that this system might have a decisive role in the generation of skilled movement. This article focuses on the pontine nuclei (PN), which are intercalated in the cerebro-cerebellar pathway, a large nuclear complex in the ventral brainstem of mammals, whose raison d'être has as yet not been examined. By considering recent morphological and electrophysiological findings, this article argues that the PN are an interface that is needed to accommodate the grossly different computational principles governing the cerebral cortex and the cerebellum.

Animals↗

PubMed1999

Sequence determinants of modified cobra venom neurotoxin which induce immune resistance to experimental allergic encephalomyelitis: molecular mechanisms for immunologic action.

A nontoxic, iodoacetamide-modified cobratoxin derivative (CAM-NTX) induced resistance to experimental allergic encephalomyelitis (EAE) in guinea pigs. Resistance was retained after trypsin digestion and shown to reside in N-terminal and central peptides of CAM-NTX. A similarly modified protein cardiotoxin (CAM-CTX), representative of proteins homologous with cobratoxin, was not immunosuppressive. Depressed clinical symptoms in EAE-resistant animals correlated with reduced lymphocytic infiltration of the brain. Antibody to myelin basic protein (MBP) was reduced in immunosuppressed animals. The immunoinhibitory determinants in CAM-NTX may mimic immune response suppressor proteins (SIRS-alpha 7) and the EAE-resistance region of MBP.

Amino Acid Sequence↗

PubMed1998

NMR investigation of the multidrug transporter EmrE, an integral membrane protein.

EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane protein has been purified and studied in its full-length form by high-resolution NMR spectroscopy in a mixture of chloroform/methanol/water (6:6:1, by vol.). Full activity is maintained after reconstitution of the protein into proteoliposomes from this solvent mixture. A series of heteronuclear (1H-15N) two- and three-dimensional experiments, as well as triple resonance experiments, were applied to the 110-residue protein and led to the assignment of the 1H, 15N and a large part of the 13C backbone resonances as well as many of the sidechain resonances. A preliminary analysis of the secondary structure, based on sequential NOE connectivities, deviation of chemical shifts from random coil values and 3J(NH-H alpha) coupling constants supports a model where the protein forms four alpha-helices between residues 4-26 (TM1), 32-53 (TM2), 58-76 (TM3) and 85-106 (TM4). For the residues of helices TM2 and TM3 a significant line broadening occurs due to slow conformational processes.

Amino Acid Sequence↗

PubMed1998

Dentate gyrus basket cell GABAA receptors are blocked by Zn2+ via changes of their desensitization kinetics: an in situ patch-clamp and single-cell PCR study.

Although GABA type A receptors (GABAARs) in principal cells have been studied in detail, there is only limited information about GABAARs in interneurons. We have used the patch-clamp technique in acute rat hippocampal slices in combination with single-cell PCR to determine kinetic, pharmacological, and structural properties of dentate gyrus basket cell GABAARs. Application of 1 mM GABA (100 msec) to nucleated patches via a piezo-driven fast application device resulted in a current with a fast rise and a marked biexponential decay (time constants 2.4 and 61.8 msec). This decay could be attributed to strong receptor desensitization. Dose-response curves for the peak and the slow component yielded EC50 values of 139 and 24 microM, respectively. Zn2+ caused a marked blocking effect on both the peak and the slow component via a noncompetitive mechanism (IC50 values of 8 and 16 microM). This led to an acceleration of the slow component as well as a prolongation of recovery from desensitization. Zn2+ sensitivity was suggested to depend on the absence of gamma-subunits in GABAARs. To test this hypothesis we performed single-cell reverse transcription PCR that revealed primarily the presence of alpha2-, beta2-, beta3-, gamma1-, and gamma2-subunit mRNAs. In addition, flunitrazepam increased the receptor affinity for its agonist, indicating the presence of functional benzodiazepine binding sites, i.e., gamma-subunits. Thus, additional factors seem to co-determine the Zn2+ sensitivity of native GABAARs. The modulatory effects of Zn2+ on GABAAR desensitization suggest direct influences on synaptic integration via changes in inhibition and shunting at GABAergic synapses.

Animals↗

PubMed1998

Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways.

Huntingtin is a cytoplasmic protein that is found in neurons and somatic cells. In patients with Huntington's disease (HD), the NH2-terminal region of huntingtin has an expanded polyglutamine tract. An abnormal protein interaction by mutant huntingtin has been proposed as a mechanism for HD pathogenesis. Huntingtin associates with vesicle membranes and interacts with proteins involved in vesicle trafficking. It is unclear where along vesicle transport pathways wild-type and mutant huntingtins are found and whether polyglutamine expansion affects this localization. To distinguish wild-type and mutant huntingtin, fibroblasts from normals and HD patients with two mutant alleles (homozygotes) were examined. Immunofluorescence confocal microscopy showed that mutant huntingtin localized with clathrin in membranes of the trans Golgi network and in clathrin-coated and noncoated endosomal vesicles in the cytoplasm and along plasma membranes. Separation of organelles in Nycodenz gradients showed that in normal and HD homozygote patient cells, huntingtin was present in membrane fractions enriched in clathrin. Similar results were obtained in fibroblasts from heterozyote juvenile HD patients who had a highly expanded polyglutamine tract in the HD allele. Western blot analysis of membrane fractions from rat brain showed that wild-type huntingtin was present in fractions that contained purified clathrin-coated membranes or a mixture of clathrin-coated and noncoated membranes. Electron microscopy of huntingtin immunoreactivity in rat brain revealed labeling along dendritic plasma membranes in association with clathrin-coated pits and clusters of noncoated endosomal vesicles 40-60 nm in diameter. These data suggest that wild-type and mutant huntingtin can influence vesicle transport in the secretory and endocytic pathways through associations with clathrin-coated vesicles.

Alleles↗

PubMed1998

Solid lipid nanoparticles (SLN) for controlled drug delivery--drug release and release mechanism.

Solid lipid nanoparticles (SLN) are particulate systems for parenteral drug administration with mean particle diameters ranging from 50 up to 1000 nm. The model drugs tetracaine, etomidate and prednisolone were incorporated (1, 5 and 10%) to study the drug load, effect of drug incorporation on the structure of the lipid matrix and the release profiles and mechanism. SLN were produced by high pressure homogenization of aqueous surfactant solutions containing the drug-loaded lipids in the melted or in the solid state (500/1500 bar, 3/10 cycles). In case of tetracaine and etomidate, high drug loadings up to 10% could be achieved when using Compritol 888 ATO and Dynasan 112 as matrix material. The melting behavior of the drug loaded particles revealed that little or no interactions between drug and lipid occurred. A burst drug release (100% release < 1 min) was observed with tetracaine and etomidate SLN, which was attributed to the large surface area of the nanoparticles and drug enrichment in the outer shell of the particles. In contrast, prednisolone loaded SLN showed a distinctly prolonged release over a monitored period of 5 weeks. Depending on the chemical nature of the lipid matrix, 83.8 and 37.1% drug were released (cholesterol and compritol, respectively). These results demonstrate the principle suitability of SLN as a prolonged release formulation for lipophilic drugs.

Chemistry, Pharmaceutical↗

PubMed1998

Automated backbone assignment of labeled proteins using the threshold accepting algorithm.

The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C beta information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A2, a protein with 124 residues.

Algorithms↗

PubMed1998

Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206.

Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.

Blotting, Western↗

PubMed1997

Actin: a target of lipopolysaccharide-induced phosphorylation in human monocytes.

We have previously reported that lipopolysaccharide (LPS) causes altered phosphate labelling of cytosolic proteins of 36 kDa and 38 kDa (p36/38) and that inhibition of phosphorylation is accompanied by a loss of cytokine production. Here we have purified the two phosphorylated proteins via two-dimensional polyacrylamide gel electrophoresis. P 36 was found to consist of two proteins p36a and p36b. The proteins were analysed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and p36b was identified as gamma-actin, p36a as beta/gamma-actin. The ability of LPS to cause altered phosphate labelling of beta/gamma-actin suggests a participation of the microfilament network in LPS-induced monocyte activation.

Actins↗

PubMed1997

Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles (SLN).

Solid lipid nanoparticles (SLN) of a quality acceptable for i.v. administration were freeze-dried. Dynasan 112 and Compritol ATO 888 were used as lipid matrices for the SLN, stabilisers were Lipoid S 75 and poloxamer 188, respectively. To study the protective effect of various types and concentrations of cryoprotectants (e.g. carbohydrates), freeze-thaw cycles were carried out as a pre-test. The sugar trehalose proved to be most effective in preventing particle growth during freezing and thawing and also in the freeze-drying process. Changes in particle size distribution during lyophilisation could be minimised by optimising the parameters of the lyophilisation process, i.e. freezing velocity and redispersion method. Lyophilised drug-free SLN could be reconstituted in a quality considered suitable for i.v. injection with regard to the size distribution. Loading with model drugs (tetracaine, etomidate) impairs the quality of reconstituted SLN. However, the lyophilisate quality is sufficient for formulations less critical to limited particle growth, e.g. freeze-dried SLN for oral administration.

Journal Article↗

PubMed1997

Projection from the cerebellar lateral nucleus to precerebellar nuclei in the mossy fiber pathway is glutamatergic: a study combining anterograde tracing with immunogold labeling in the rat.

The pontine nuclei (PN) and the nucleus reticularis tegmenti pontis (NRTP) are sources of an excitatory projection to the cerebellar cortex via mossy fibers and a direct excitatory projection to the cerebellar nuclei. These precerebellar nuclei, in turn, receive a feedback projection from the cerebellar nuclei, which mostly originate in the lateral nucleus (LN). It has been suggested that the feedback projection from the LN partially uses gamma-aminobutyric acid (GABA) as a transmitter. We tested this hypothesis by using a combination of anterograde tracing (biotinylated dextran amine injection into the LN) and postembedding GABA and glutamate immunogold histochemistry. The pattern of labeling in the PN and the NRTP was compared with that of cerebellonuclear terminals in two other target structures, the parvocellular part of the nucleus ruber (RNp) and the ventromedial and ventrolateral thalamus (VM/VL). The projection to the inferior olive (IO), which is known to be predominantly GABAergic, served as a control. A quantitative analysis of the synaptic terminals labeled by the tracer within the PN, the NRTP, and the VL/VM revealed no GABA immunoreactivity. Only one clearly labeled terminal was found in the RNp. In contrast, 72% of the terminals in the IO were clearly GABA immunoreactive, confirming the reliability of our staining protocol. Correspondingly, glutamate immunohistochemistry labeled the majority of the cerebellonuclear terminals in the PN (88%), the NRTP (90%), the RNp (93%), and the VM/VL (63%) but labeled only 5% in the IO. These data do not support a role for GABAergic inhibition either in the feedback systems from the LN to the PN and the NRTP or within the projections to the RNp and the VM/VL.

Animals↗