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J Gehrmann

Publications and source records attributed to J Gehrmann.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: a model for the role of disulfide bonds in structural stability.

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13)<GI(2-13;3-7)<GI(2-3;7-13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3;7-13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2-7;3-13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7;3-13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.

Amino Acid Sequence↗
PubMed1997

Crystal structure at 1.1 A resolution of alpha-conotoxin PnIB: comparison with alpha-conotoxins PnIA and GI.

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 A and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P212121 with the following unit cell dimensions: a = 14.6 A, b = 26.1 A, and c = 29.2 A. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

Amino Acid Sequence↗
PubMed1997

Morphologic characterization and assessment of mitral regurgitation after repair of atrioventricular defects in children.

Severe postoperative mitral regurgitation renders information on the underlying mechanism before reoperation very important, as a potential for mitral valve reconstruction may facilitate the decision whether to reoperate, especially in the very young. This study compares the efficacy of transthoracic echo-cardiography (TTE) and left-ventricular angiography with that of transesophageal echocardiography (TEE) for detection of the mechanism underlying mitral regurgitation and its quantitative assessment in children after repair of common atrioventricular septal defect. Five children aged 1.5 to 16 years were evaluated by TTE, TEE, and angiography for postoperative mitral regurgitation 1 to 21 months after initial repair. TEE showed septal detachment of the mitral leaflet in four patients and reopening of the mitral cleft in one patient as the cause of mitral regurgitation whereas TTE failed in four and angiography in all patients. TEE allows definite identification of morphologic characteristics of mitral regurgitation and reliable assessment of its severity. Thus redo surgery may be safety performed on the bases of TEE findings alone without confirmation by cardiac catheterization.

Adolescent↗
PubMed1996

The 1.1 A crystal structure of the neuronal acetylcholine receptor antagonist, alpha-conotoxin PnIA from Conus pennaceus.

BACKGROUND: alpha-Conotoxins are peptide toxins, isolated from Conus snails, that block the nicotinic acetylcholine receptor (nAChR). The 16-residue peptides PnIA and PnIB from Conus pennaceus incorporate the same disulfide framework as other alpha-conotoxins but differ in function from most alpha-conotoxins by blocking the neuronal nAChR, rather than the skeletal muscle subtype. The crystal structure determination of PnIA was undertaken to identify structural and surface features that might be important for biological activity. RESULTS: The 1.1 A crystal structure of synthetic PnIA was determined by direct methods using the Shake-and-Bake program. The three-dimensional structure incorporates a beta turn followed by two alpha-helical turns. The conformation is stabilised by two disulfide bridges that form the interior of the molecule, with all other side chains oriented outwards. CONCLUSIONS: The compact architecture of the PnIA toxin provides a rigid framework for presentation of chemical groups that are required for activity. The structure is characterized by distinct hydrophobic and polar surfaces; a 16 A separation of the sole positive and negative charges (these two charged residues being located at opposite ends of the molecule); a hydrophobic region and a protruding tyrosine side chain. These features may be important for the specific interaction of PnIA with neuronal nAChR.

Amino Acid Sequence↗
PubMed1996

Differential expression of fibroblast growth factor-2 and receptor by glial cells in experimental autoimmune encephalomyelitis (EAE)

To assess the expression pattern of basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR-1/flg) during autoimmune inflammation of the CNS, FGF-2, and FGFR1/flg peptide and mRNA levels were examined by immunocytochemistry, by in situ hybridisation and by Northern blot analysis in T cell-mediated EAE of the Lewis rat. In naive control animals as well as in animals injected with non-encephalitogenic, PPD-reactive T lymphocytes, FGF-2 immunoreactivity was low and confined to blood vessels and to a few spinal cord neurons. In rats injected with encephalitogenic, MBP-reactive T lymphocytes, however, FGF-2-immunoreactive cells were detected from day 4 after T cell transfer onward, i.e., from the onset of clinical symptoms. The number of FGF-2 immunoreactive cells was highest between days 6 and 10 after T cell transfer. Increased FGF-2 peptide expression was paralleled by increased FGF-2 mRNA expression on macrophages/microglia in the spinal cord. By 21 days after T cell transfer, i.e. after complete recovery, FGF-2 peptide and mRNA expression had fully subsided. Based on morphological criteria and on double labeling with the macrophage/microglia-binding lectin GSI-B4 two cell types expressed FGF-2: 1) round macrophages within the core, and 2) activated microglia at the edges of white and grey matter perivascular lesions. Paralleling the temporal and spatial expression pattern of FGF-2, FGFR-1/flg immunoreactivity was induced on activated macrophages/microglia but also on reactive astrocytes bordering perivascular inflammatory lesions. In situ hybridisation analysis furthermore showed that macrophages/microglia expressed the FGFR-1/flg mRNA, and that receptor mRNA expression paralleled ligand mRNA expression. Macrophage/microglia-derived FGF-2 could serve two main functions in EAE: 1) regulate microglial activation in an autocrine fashion, and 2) help to target astrocyte-derived insulin-like growth factor-I (IGF-I) to potentially injured oligodendrocytes in demyelination.

Animals↗
PubMed1995

Encephalitogenic, myelin basic protein-specific T cells from naive rat thymus: preferential use of the T cell receptor gene V beta 8.2 and expression of the CD4-CD8- phenotype.

Using a primary limiting dilution approach to generate T cell lines, we compared myelin basic protein (MBP)-specific T cell clones from naive unprimed Lewis rat thymuses with the corresponding T cell repertoire of primed rats. We found that in the native thymus repertoire MBP-specific, encephalitogenic T cell clones preferentially use T cell receptor V beta 8.2 genes, along with CDR3 sequences typical for the primed Lewis anti-MBP response. In contrast to T cells from primed immune organs, which all display the CD4+CD8- phenotype, the majority of naive thymus-derived T cell clones expressed reduced levels of the CD4 co-receptor. Some clones were completely CD4-CD8-, while others included CD4-CD8- subpopulations along with CD4+CD8- T cells. In the one mixed population examined in detail, the CD4-CD8- and CD4+CD8-T cell subpopulations used a T cell receptor with identical beta chain sequence. The data suggest that in the Lewis rat the biased T cell receptor gene usage by encephalitogenic T cells is a property of the natural thymic T cell repertoire, possibly as a consequence of positive selection. The unusually low expression of CD4 in the major histocompatibility complex class II-restricted autoreactive T cells could be related to their escape from negative selection within the thymus.

Animals↗
PubMed1995

Inflammatory reaction in experimental autoimmune encephalomyelitis (EAE) is accompanied by a microglial expression of the beta A4-amyloid precursor protein (APP).

Neuropathological studies of the amyloid depositions and senile plaques in the brains of elderly patients or patients diagnosed with Alzheimer's disease reveal the conspicuous presence of numerous proteins which are usually expressed during reactions of the immune system. This has led to speculations that the pathomechanism of neurodegenerative diseases might involve inflammatory processes. These considerations constitute the theoretical basis for therapeutic intervention with antiinflammatory drugs in neurodegenerative diseases. Here, we show that the beta A4-amyloid precursor (APP) is rapidly induced in microglia in a model of experimental autoimmune encephalomyelitis (EAE). Using specific monoclonal antibodies against APP, the first glial cells newly expressing APP immunoreactivity were found at an early preclinical stage, i.e., 24 h after T-cell transfer. At the peak of clinical disease (6 days after T-cell transfer), numerous characteristically ramified cells were strongly positive for APP. Based on morphology and double-labeling, most of the de novo APP-expressing cells were identified as microglia. Additionally, APP-immunoreactive round cells were detected in and around perivascular infiltrates. Reflecting the course of the clinical disease, the induction of APP immunoreactivity terminated in the postclinical stage, i.e., 14 days after T-cell transfer. These results support earlier work demonstrating that microglia can rapidly de novo synthesise APP not only in response to direct nerve injury (Banati et al: Glia 9:199, 1993a) but also in immune-mediated disease. Apart from its possible therapeutic relevance, such a production of APP--reminiscent of an acute phase protein-suggests a role of APP in immune and repair mechanisms of the central nervous system.

Amyloid beta-Protein Precursor↗
PubMed1995

Amyloid precursor protein (APP) expression in multiple sclerosis lesions.

The amyloid precursor protein (APP) is rapidly induced in reactive glial cells in response to several pathological stimuli including inflammation. In the present study, observations previously made in animal models of autoimmune central nervous system inflammation have been extended to the analysis of multiple sclerosis (MS) lesions. A total of thirty fresh-frozen tissue blocks from six histopathologically normal control and six MS cases have been examined immunocytochemically with monoclonal antibodies directed against either C- or N-terminal epitopes of APP. Histopathological evaluation of disease progression was based on hematoxylin-eosin and oil red O staining and immunocytochemistry for T cells, macrophages/microglia, astrocytes, and oligodendrocytes. In control cases, APP immunoreactivity was generally low and confined to blood vessel walls, oligodendrocytes in white, and neurons in grey matter. In actively demyelinating plaques, however, levels of APP immunoreactivity were high, localised on T lymphocytes, foamy macrophages, activated microglia, and reactive astrocytes including astrocytic processes. In more chronic lesions, levels of APP immunoreactivity were generally lower than in acute lesions, mainly found on reactive astrocytes, their processes and a few macrophages/microglia depending on the stage of plaque development. In addition, a few 14E-positive oligodendrocytes and, moreover, numerous axons exhibited APP immunoreactivity, which was particularly pronounced with anti-C-terminal antibodies. These results demonstrate that APP is induced on reactive glial cells but also on T lymphocytes during demyelination. The extent of APP expression appears to be correlated to histopathological lesion development and thus suggests that APP detection serves as a sensitive marker for disease progression in MS.

Adult↗
PubMed1995

Rapid appearance of beta-amyloid precursor protein immunoreactivity in glial cells following excitotoxic brain injury.

Clinical and experimental data have indicated an up-regulation of amyloid precursor protein (APP) after various types of CNS injury. In the present study the cellular source of lesion-induced APP has been investigated in a neurotoxic CNS model. Quinolinic acid injection into the striatum results in neuronal degeneration, while glial cells survive. APP immunoreactivity was detected in glial cells starting at postoperative day 3 and persisted until day 21, the last time point studied. Double immunocytochemistry identified the majority of APP-immunoreactive cells as glial fibrillary acidic protein-immunoreactive astrocytes. There was no evidence of amyloid fibril deposition during this time. It is concluded that following excitotoxic neuronal degneration APP is mainly produced by reactive astrocytes in the lesioned area.

Amyloid beta-Protein Precursor↗
PubMed1995

Microglia: intrinsic immuneffector cell of the brain.

Microglia form a regularly spaced network of resident glial cells throughout the central nervous system (CNS). They are morphologically, immunophenotypically and functionally related to cells of the monocyte/macrophage lineage. In the ultimate vicinity of the blood-brain barrier two specialized subsets of macrophages/microglia can be distinguished: firstly, perivascular cells which are enclosed within the basal lamina and secondly juxtavascular microglia which make direct contact with the parenchymal side of the CNS vascular basal lamina but represent true intraparenchymal resident microglia. Bone marrow chimera experiments indicates that a high percentage of the perivascular cells undergoes replacement with bone marrow-derived cells. In contrast, juxtavascular microglia like other resident microglia form a highly stable pool of CNS cells with extremely little turnover with the bone marrow compartment. Both the perivascular cells and the juxtavascular microglia play an important role in initiating and maintaining CNS autoimmune injury due to their strategic localization at a site close to the blood-brain barrier, their rapid inducibility for MHC class II antigens and their potential scavenger role as phagocytic cells. The constantly replaced pool of perivascular cells probably represents an entry route by which HIV gets access to the brain. Microglia are the first cell type to respond to several types of CNS injury. Microglial activation involves a stereotypic pattern of cellular responses, such as proliferation, increased or de-novo expression of immunomolecules, recruitment to the site of injury and functional changes, e.g., the release of cytotoxic and/or inflammatory mediators. In addition, microglia have a strong antigen presenting function and a pronounced cytotoxic function. Microglial activation is a graded response, i.e., microglia only transform into intrinsic brain phagocytes under conditions of neuronal and or synaptic/terminal degeneration. In T-cell-mediated autoimmune injury of the nervous system, microglial activation follows these lines and occurs at an early stage of disease development. In experimental autoimmune encephalomyelitis (EAE), microglia proliferate vigorously, show a strong expression of MHC class I and II antigens, cell adhesion molecules, release of reactive oxygen intermediates and inflammatory cytokines and transform into phagocytic cells. Due to their pronounced antigen presenting function in vitro, activated microglia rather than astrocytes or endothelial cells are the candidates as intrinsic antigen presenting cel of the brain. In contrast to microglia, astrocytes react with a delay, appear to encase morphologically the inflammatory lesion and may be instrumental in downregulating the T-cell-mediated immune injury by inducing T-cell apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain↗
PubMed1995

Changes in the expression pattern of blood-brain barrier-associated pericytic aminopeptidase N (pAP N) in the course of acute experimental autoimmune encephalomyelitis.

The role of cerebral pericytes in blood-brain barrier (BBB) mechanisms is still a matter of controversy. Because acute experimental autoimmune encephalomyelitis (EAE) is characterized by a transient and focal perturbation of the BBB, we have utilized the model of adoptive transfer EAE to correlate the expression of the pericytic aminopeptidase N (pAP N) with the acute functional state of the BBB. We demonstrate that a significant downregulation of microvascular pAP N expression occurs, and the observed perturbation of the enzymatic BBB complement seems to be a sustained effect which persists even after recovery from clinical disease. At the peak of clinical disease, numerous pAP N expressing invasive cells were detected in white matter of the lumbar spinal cord. Through the use of a panel of different immunocytochemical markers these pAP N-immunopositive cells were characterized as ED 1-positive, most likely hematogenous macrophages. Activated resident microglial cells participate in the EAE-induced inflammatory process to only a minor extent.

Acute Disease↗
PubMed1995

Colony-stimulating factors regulate programmed cell death of rat microglia/brain macrophages in vitro.

Programmed cell death of activated microglia appears to be one mechanism how steady state of microglia is achieved in vivo. Programmed cell death of microglia might result either from the downregulation of microglial mitogens/survival factors or from signals which directly induce microglial cell death. To further elucidate the mechanisms regulating programmed cell death in microglia, growth factor and cytokine dependence of microglial proliferation and cell death have been examined in vitro in microglia/brain macrophage cultures established from neonatal rat brain. Microglial proliferation was assessed by PCNA labelling and DNA fragmentation by the TUNEL technique in the presence or absence of several cytokines including IL-1, IL-6, TGF beta 1, TNF alpha, M-CSF and GM-CSF. Results of TUNEL labellings were supplemented by gel electrophoretic analysis of DNA extracted from cultured microglia which showed laddering of DNA fragments. Of all cytokines/growth factors tested, GM-CSF and M-CSF were not only the strongest microglial mitogens but, moreover, withdrawal of M-CSF or GM-CSF significantly enhanced rates of microglial cell death by DNA fragmentation. Expression of microglial growth factors, in particular colony-stimulating factors, may thus be instrumental in controlling steady states of microglia in the injured nervous system.

Animals↗
PubMed1995

Glial expression of the beta-amyloid precursor protein (APP) in global ischemia.

The beta-amyloid precursor protein (APP) bears characteristics of an acute-phase protein and therefore is likely to be involved in the glial response to brain injury. In the brain, APP is rapidly synthesized by activated glial cells in response to comparatively mild neuronal lesions, e.g., a remote peripheral nerve injury. Perfusion deficits in the brain result largely in neuronal necrosis and are a common condition in elderly patients. This neuronal necrosis is accompanied by a pronounced reaction of astrocytes and microglia, which can also be observed in animal models. We have therefore studied in the rat, immunocytochemically, the induction of APP after 30 min of global ischemia caused by four-vessel occlusion. The postischemic brain injuries were examined at survival times from 12 h to 7 days. From day 3 onward, APP immunoreactivity was strongly induced in the CA1 and CA4 regions of the rat dorsal hippocampus as well as in the dorsolateral striatum. In these areas, the majority of APP-immunoreactive cells were reactive glial fibrillary acidic protein (GFAP)-positive astrocytes, as shown by double-immunofluorescence labeling for GFAP and APP. Additionally, small ramified cells, most likely activated microglia, expressed APP immunoreactivity. In contrast, in the parietal cortex, APP immunoreactivity occurred focally in clusters of activated microglia rather than in astrocytes, as demonstrated by double-immunofluorescence labeling for APP and the microglia-binding lectin Griffonia simplicifolia isolectin B4. In conclusion, following global ischemia, APP is induced in reactive glial cells with spatial differences in the distribution pattern of APP induction in astrocytes and microglia.

Amyloid beta-Protein Precursor↗
PubMed1995

Gene transfer through the blood-nerve barrier: NGF-engineered neuritogenic T lymphocytes attenuate experimental autoimmune neuritis.

Nerve-specific autoimmune T lymphocytes were used as vehicles to deliver therapeutically useful neurotrophic factors across the endothelial blood-nerve barrier. P2 protein-reactive T-lymphocyte lines from Lewis rats were transduced with a recombinant retrovirus containing the mouse nerve growth factor (NGF) gene. The engineered T cells released high amounts of NGF dependent on antigenic stimulation in vitro. After intravenous injection, the T cells infiltrated the rat peripheral nervous system and persisted there for at least two weeks. Local release of NGF from engineered T cells was demonstrable by immunocytochemistry and by an anti-inflammatory effect on infiltrating macrophages.

Animals↗