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H W Müller

Publications and source records attributed to H W Müller.

At least 91 records · Page 5Linked to original sources
PubMed1995

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22.

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.

Chromosome Mapping↗
PubMed1995

Restricted appearance of tenascin and chondroitin sulphate proteoglycans after transection and sprouting of adult rat postcommissural fornix.

Transected fibres of the adult rat postcommissural fornix sprout over short distances but fail to traverse the lesion site and terminate in close vicinity to the wound. As a step in defining the molecular environment responsible for regeneration failure at the lesion site, we have used immunocytochemistry to analyse the spatio-temporal expression pattern of two putative growth-inhibitory extracellular matrix components, tenascin and chondroitin sulphate proteoglycans and their topographical relationship to the sprouting axons. Both tenascin and chondroitin sulphate proteoglycan labelling appeared after fornix transection and were confined to the immediate vicinity of the lesion site. While tenascin-labelling was associated with astrocytes and microglia/macrophages, which accumulate preferentially at the tract borders, chondroitin sulphate proteoglycan labelling appeared as a homogeneous meshwork around the wound. Tenascin-like immunoreactivity disappeared between 17 days and 4 weeks, but chondroitin sulphate proteoglycan staining persisted at least up to 14 months after transection. Regrowing fornix fibres invaded and elongated within the chondroitin sulphate proteoglycan-immunopositive region up to the lesion site, where they terminated. This zone of axonal growth inhibition was neither characterized by an increase of chondroitin sulphate proteoglycan immunoreactivity nor by the presence of tenascin-immunopositive structures. The spatio-temporal distribution patterns of tenascin and chondroitin sulphate proteoglycan and the permeability of the chondroitin sulphate proteoglycan-immunopositive region for sprouting axons do not support the hypothesis that chondroitin sulphate proteoglycan alone and/or tenascin inhibit the advance of sprouting fornix fibres.

Animals↗
PubMed1995

Astroglial neurotrophic and neurite-promoting factors.

Astroglial cells express neurotrophic and/or neurite growth-promoting factors, including (a) peptide growth factors (e.g. nerve growth factors, ciliary neuronotrophic factor, fibroblast growth factor), (b) neurotransmitters (such as glutamate, neuropeptide Y), (c) cell adhesion molecules (e.g. N-CAM) and (d) extracellular matrix proteins (including laminin, fibronectin and proteoglycans). Expression of these molecules is regulated during development and/or after CNS lesions. Some of the astroglial peptide growth factors, including nerve growth factor, basic fibroblast growth factor and ciliary neuronotrophic factor, have been shown to exert protective or even regenerative effects on neurons following traumatic, chemical or ischemic lesions. These observations illustrate the enormous therapeutic potential of astroglia-derived neurotrophic molecules.

Animals↗
PubMed1995

Facilitation of learning following injection of the chondroitin sulfate proteoglycan biglycan into the vicinity of the nucleus basalis magnocellularis.

The aim of this study was to examine the effects of biglycan, a small chondroitin sulfate proteoglycan with neurotrophic activity, on memory and reinforcement upon unilateral injection into the region of the nucleus basalis magnocellularis (NBM). In experiment 1, rats with chronically implanted cannulas were injected with biglycan and tested on the uphill avoidance task, which involves punishment of a high-probability turning response on a tilted platform (negative geotaxis). Immediately after the training trial, that is, after a tail-shock was administered upon performing the response, rats received one microinjection (0.5 microliter) of substance P (SP) in a reference dosage of 0.74 pmol or biglycan (doses ranging from 1.3 to 1300.0 nmol) into the NBM region. When tested 24 h later, rats treated with SP (0.74 pmol) or biglycan (2.1 and 2.6 nmol) had significantly longer uphill latencies than vehicle (PBS) controls, indicative of superior learning of the avoidance response. In experiment 2, a test for possible proactive effects of post-trial biglycan on performance during the retention trial was performed. Furthermore, the uphill avoidance task was combined with a conditioned place preference task to assess possible reinforcing effects of biglycan. Rats were injected with either 2.6 or 130.0 nmol biglycan immediately after the training trial of the uphill task. One control group received 2.6 nmol biglycan 5 h after the trial, a second group was sham-operated. Additional groups were included which received biglycan (2.6 or 130.0 nmol), SP (0.74 pmol) or PBS after the training trial but no tail-shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Generation and characterization of a human chromosome 6-specific hncDNA library from a somatic cell hybrid.

Chromosome specific cDNA libraries are a useful source of candidate genes for disorders which have been linked to particular chromosomes. Here, we report the generation of a cDNA library made from a somatic cell hybrid retaining chromosome 6 as its only human component. In order to ascertain the chromosomal location of cDNAs the library was amplified by inter-Alu-PCR and used as probe for competitive in situ suppression (CISS). To identify human specific cDNA clones the library was screened with PD39, a highly human specific Alu consensus probe. Out of 350,000 clones 360 were found to hybridize with PD39. Nucleotide sequences were determined for 40 clones with inserts larger than 500 basepairs (bp) and a sequence comparison was performed at the National Center for Biotechnology Information using BLASTN. One clone was shown to be identical to Manganese Superoxide Dismutase (MnSOD/SOD2) which has previously been assigned to chromosome 6q25. Localization of 11 clones was determined using PCR and clone-specific primer pairs on a hybrid mapping panel DNA set. Two PCR-localized clones and five additional clones were localized by fluorescence in situ hybridization. Transcripts for five clones were identified by RT-PCR. The generation of chromosome 6-specific hncDNAs from a somatic cell hybrid should aid in the identification of disease-associated genes localized on this chromosome.

Animals↗
PubMed1995

Adhesive properties of proteolipid protein are responsible for the compaction of CNS myelin sheaths.

We have studied the molecular function of proteolipid protein (PLP), the main integral membrane protein of CNS myelin, by generating mice lacking PLP expression. Here, we demonstrate that these PLP-minus mice show no pleiotropism as mice carrying point mutations within the PLP gene. The expression of other myelin genes (myelin basic protein, MBP; myelin associated glycoprotein, MAG; UDP-galactose-ceramide galactosyl transferase, CGT) is unimpaired on the RNA level. Protein level immunofluorescence analysis by confocal microscopy reveals that in PLP-minus mice there is a complete absence of PLP, a scattered appearance of MBP, and MAG expressed more widely in regions lacking MBP staining, which may be a compensatory mechanism. In electron microscopy the myelin lamellae of the ensheathed CNS axons are loosely wrapped with wide extracellular spaces between turning loops. Intraperiod dense lines are missing. The lateral loops of the paranode form regular axoglial junctions. In PLP-minus mice axons form regular axoglial junctions. In PLP-minus mice axons with large diameters are loosely myelinated, whereas small axons remain unmyelinated. Functionally, the mutant mice show deficits in their locomotor activity. We propose that adhesion properties of the extracellular domains of PLP are responsible for the tight apposition of the plasma membrane processes of oligodendrocytes wrapping axons to form the compact myelin sheath.

Adhesiveness↗
PubMed1994

Peripheral myelin protein-22 expression in Charcot-Marie-Tooth disease type 1a sural nerve biopsies.

Peripheral myelin protein-22 (PMP22) is expressed in myelinating Schwann cells and shows significant homology to murine growth arrest-specific gene gas3. Charcot-Marie-Tooth disease type 1a (CMT1a) is a common hereditary demyelinating neuropathy. Recently it was demonstrated that the gene for PMP22 is duplicated in CMT1a patients. A gene dosage mechanism has been postulated to cause CMT1a. According to this hypothesis, the increase in copy number of PMP22 gene would lead to an elevated expression of PMP22 and thereby cause the demyelinating phenotype of CMT1a. In the present communication we analyzed PMP22 mRNA and protein expression in sural nerve biopsies from CMT1a patients and normal controls. We show that PMP22 mRNA expression in CMT1a is not uniform. We found both elevated as well as normal PMP22 mRNA levels in patients. Interestingly, the highest PMP22 mRNA level was found in the least affected patient. In contrast to the mRNA levels, PMP22 was clearly reduced in all CMT1a patients as shown by immunohistochemistry. Thus the CMT1a phenotype may not be strictly correlated with increased PMP22 mRNA and protein expression. Possible roles of PMP22 in the pathogenesis of CMT1a are discussed.

Biopsy↗
PubMed1994

Differential expression of two mRNA species indicates a dual function of peripheral myelin protein PMP22 in cell growth and myelination.

Two peripheral myelin protein PMP22 transcripts, CD25 and SR13, have been identified by Northern blot and RNA-polymerase chain reaction (PCR) methods in rat. The CD25 and SR13 mRNA species (each approximately 1.8 kb in size) differ significantly in their 5'-untranslated region (5'-UTR) sequences but encode the same protein. While CD25 mRNA is largely confined to the peripheral nervous system, the SR13 transcript is more ubiquitously expressed in rat tissues. Both transcripts are differentially expressed during postnatal sciatic nerve development. While CD25 expression steadily increases from low levels in neonates up to a maximum at postnatal day 14, SR13 mRNA levels are elevated at birth but decrease throughout adulthood. CD25 and SR13 transcripts are expressed at very low constant levels in developing and adult brain. In degenerating and regenerating segments of injured peripheral nerve changes in CD25 mRNA levels clearly resemble the expression pattern of other myelin genes, whereas expression of SR13 is inversely correlated with the time course of Schwann cell proliferation. In cultured rat meningeal fibroblasts SR13 mRNA expression is strictly growth arrest-specific and independent of forskolin. On the other hand, regulation of CD25 mRNA levels in these cells is more complex with respect to interfering effects of serum and forskolin. In cultured Schwann cells neither CD25 nor SR13 expression is growth arrest-specific. However, both transcript levels are consistently enhanced by forskolin under all conditions of cell growth tested. Expression of CD25 (but not SR13) depends on high Schwann cell density. Our results substantiate the hypothesis that PMP22 serves two biological functions, one related to cell growth (SR13) and another to myelination (CD25).

Animals↗
PubMed1994

Transplants of immature astrocytes promote axonal regeneration in the adult rat brain.

To study beneficial effects of immature astrocytes on axonal regeneration in the injured adult mammalian brain, we have stereotactically implanted cultured astrocytes from embryonic (E 14-16) rat cerebral cortex into the lesion site following transection of the postcommissural fornix. The spatio-temporal pattern of axonal degeneration and regrowth in the proximal fornix stump was investigated using wheat germ agglutinin-horseradish peroxidase tracing techniques and quantitative analysis of myelinated axon profiles. Transection of the postcommissural fornix tract caused disintegration of the axons in the distal stump as well as rapid and pronounced retrograde axonal degeneration up to 800-1,200 microns proximal to the lesion site. While a small bundle of subicular fibers spontaneously extended to the lesion site within 4 weeks after injury, axonal regeneration was markedly stimulated in those animals that had received an astroglial implant. Following the former pathway, regenerating axons sprouted towards the implant but did not penetrate the graft. Instead, the axons elongated over the surface of the transplant, avoiding growth into the surrounding neuropil or into the distal fornix segment. In grafted animals we further observed a substantial increase in the number of myelinated axons of approximately 31.5% (at the level of 800 microns) and approximately 40% (at the 400 microns level) compared with the injured tract lacking a transplant. Our results indicate the capacity of juvenile astrocytes to stimulate axonal regeneration after injury of the post-commissural fornix tract in the adult rat brain. We further demonstrate myelination of the regenerated axons.

Animals↗
PubMed1994

Extensive and long-lasting changes of glial cells following transection of the postcommissural fornix in the adult rat.

Injury to the adult brain results in the formation of a glial scar that involves both activated astroglia and microglia/macrophages. Although reactive gliosis has been linked to failure of axonal regeneration in the adult mammalian CNS, the spatio-temporal pattern of the postlesion responses in morphology and distribution of the major cellular constituents of the gliosis has attracted little attention. In an attempt to define these relationships, we have undertaken a systematic study of astrocytic and microglial/macrophagic responses after stereotactic transection of the postcommissural fornix in rat. To visualize astrocytes, microglia, and macrophages, antibodies against glial fibrillary acidic protein (GFAP), vimentin (VIM), complement receptor type 3 (OX42), and ED1 were used. The cellular responses occurring between 2 h and 1 year postlesion (PL) at various distances distal and proximal to the lesion site were studied. Transection of the postcommissural fornix leads to: 1) a very early microglial reaction both distal and proximal to the lesion, as indicated by OX42-immunoreactivity, followed by a massive astrocytic response; 2) a transient GFAP(-) but VIM(+) region around the lesion center; 3) the appearance of numerous ED1(+) macrophages at PL3d; and 4) long-lasting (at least 1 year) persistence of both astrocytes and microglia/macrophages. The timing and extension of the sequential glial reactions after postcommissural fornix transection are discussed in relation to the myelin degradation and spontaneous sprouting of injured axons that have previously been observed in this lesion model (Wunderlich et al: Glia 10:49-58, 1994).

Animals↗
PubMed1994

Relationship between injury-induced astrogliosis, laminin expression and axonal sprouting in the adult rat brain.

Lesion-induced regenerative sprouting of CNS axons is accompanied by structural and metabolic changes of astrocytes. In order to evaluate the effects of these astrocytic changes on axonal regeneration, we investigated the spatio-temporal relationship of gliosis, laminin expression and axonal sprouting in the postcommissural fornix of the adult rat. Using immunocytochemical methods we observed (1) a perilesional area with a transient lack of astrocytes and axons, (2) the reappearance of reactive astrocytes followed by the ingrowth of sprouting fibres and finally an increase in laminin-immunoreactivity, (3) the absence of lesion-induced laminin-expression in reactive astrocytes and (4) the formation and long-lasting (at least 28 months) persistence of a dense plexus of laminin-immunopositive blood vessels at the site of transection and in the proximal and distal stumps. These data indicate that astrogliosis is permeable for regrowing axons and that injury-induced axonal sprouting in the transected postcommissural fornix may be mediated by laminin-independent mechanisms.

Animals↗
PubMed1993

Strychnine-sensitive glycine receptors in cultured primary neurons from rat neocortex.

After 1 day in vitro (DIV) glycine (1 mM) evoked chloride-dependent membrane currents in about 50% of primary cultured rat neocortical neurons and more than 98% of the cells were glycine-sensitive after 2 DIV lasting for at least up to 12 DIV which was similar to GABA chemosensitivity. Strychnine (IC50 40 nM) and picrotoxin (30 microM) but not bicuculline (50 microM) blocked the glycine-evoked currents. The results provide evidence for a very early expression of glycine receptors on cortical neurons leading to a powerful chloride channel-operating capacity during early development.

Animals↗
PubMed1993

Depolarization of cultured astrocytes by leukotriene B4. Evidence for the induction of a K+ conductance inhibitor.

Since astrocytes have been shown to participate in intracerebral immunological processes we investigated the effect of the immune mediators, leukotrienes (LT) B4, LTC4 and LTD4 on membrane properties of cultured astrocytes from neonatal rat brain. When LTB4 was added to the bath solution the membrane potential slowly decreased from -96 mV to -38 mV. While LTB4 at a concentration of 500 nM was ineffective, depolarization occurred when concentrations of 750 nM and above were used. The depolarizing effect was specific for LTB4, since LTC4 and LTD4, other arachidonic acid derivates, failed to depolarize astrocytes at even higher concentrations (1 microM). When the K+ conductance blocker, Ba2+ (2 mM), was added to the bath solution, astrocytes depolarized to the same degree but no further depolarization was achieved when LTB4 was added. Bath application of Co2+ (1 mM), in order to reduce putative Ca2+ inward currents or reduced internal chloride concentration, did not alter the LTB4-induced depolarization, thus arguing against additional Ca(2+)- or Cl(-)-dependent depolarizing effects. The LTB4-induced depolarization could be markedly reduced, however, by preincubation of the cells with cycloheximide (2 microM), which blocks translation and thereby protein synthesis. Cycloheximide alone had no effect on the membrane potential. These data indicate that, in astrocytes, LTB4 stimulates the synthesis of a protein, which, in turn, inhibits K+ conductances. This effect could impair glial, as well as neuronal, functions during CNS diseases accompanied by immunological processes.

Animals↗
PubMed1993

Coexpression of PMP22 gene with MBP and P0 during de novo myelination and nerve repair.

The recently cloned PMP22 gene, the rat variant of the murine growth arrest-specific gene gas3, encodes a new 22 kD integral membrane glycoprotein of peripheral myelin. By means of in situ hybridization and immunohistochemistry, we have (1) analyzed PMP22 expression in myelinated and nonmyelinated peripheral nerves, and (2) compared the spatio-temporal changes in the expression of PMP22 mRNA with the expression of the myelin genes P0 and MBP (myelin basic protein) in developing as well as degenerating and regenerating sciatic nerve of rat. (3) We further investigated the expression of PMP22 mRNA by Northern blot in cultured Schwann cells maintained under different conditions of cell growth and arrest. Expression of PMP22 mRNA is restricted to Schwann cells of myelinated peripheral nerve. Transection of sciatic nerve in adult rat leads to a simultaneous and rapid decline in both PMP22 and P0 mRNA to nondetectable levels in the degenerating distal stump. When a demyelinated and axon-free distal stump, as indicated by the lack of MBP and neurofilament immunoreactivity, was reanastomosed to its proximal counterpart, the coordinated reexpression of PMP22 and MBP succeeded axonal regeneration through the distal segment with a delay of 1-2 weeks. As in regenerating nerve, a striking synchrony of expression of PMP22 and P0 transcripts, as well as MBP immunoreactivity, could be observed during sciatic nerve development. Further, in vitro evidence suggests that, unlike NIH3T3-fibroblasts, expression of PMP22/gas3 is not strictly growth arrest-specific in Schwann cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Spontaneous activity and recurrent inhibition in cultured hippocampal networks.

As a model for an integrated neuronal network based on the concept of modular units, we have investigated the occurrence of spontaneous activity and the formation of synaptic circuits in primary cultures of dissociated hippocampal neurons from the embryonic rat. Sodium-dependent action potentials (APs) could be elicited after 1 day in vitro (DIV), whereas spontaneous postsynaptic potentials (PSPs), "miniature" PSPs and APs appeared after 3-6 DIV. The number of cells with spontaneous APs and the rate of APs increased during development of the neuritic network. In addition to a stochastic spike interval distribution, pyramid-shaped neurons could be identified after 10-12 DIV, which fired preferentially at interspike intervals between 20-120 ms and 190-400 ms. This distinctive bimodal interspike interval pattern was sensitive to GABA-A antagonists. Simultaneous recordings of pairs of neurons demonstrated recurrent inhibitory, GABA-ergic synaptic circuits. In addition, a subpopulation of GABAergic neurons could be visualized by immunocytochemistry. These results are discussed in relation to the hypothesis that spontaneous firing of connected neurons is network-driven, based on synaptic "noise" and patterned by recurrent inhibition.

Action Potentials↗
PubMed1993

Paroxysmal long-lasting depolarizations in cultured hippocampal neurons are generated by activation of NMDA and non-NMDA receptors.

In primary cultures of hippocampal neurons from the embryonic rat, spontaneous depolarizations lasting up to 6 sec and resembling paroxysmal depolarization shifts (PDSs) appeared after 11 days in vitro. These depolarizations are presumably generated by synaptic events, because: (1) both their appearance and duration are independent of membrane potential, (2) the amplitudes of the underlying currents depend monotonically on membrane potential, and (3) they are reversed at the reversal potential of the excitatory postsynaptic potentials (EPSPs). In addition, PDSs disappeared reversibly when sodium-dependent action potentials were blocked by tetrodotoxin (10 microM) and when synaptic transmission was reduced by elevated Mg2+ (5 mM). Further, the fact that these depolarizations can appear simultaneously in two neurons in paired recordings also points to a synaptic origin. Inhibition of glutaminergic synaptic transmission by kynurenic acid (50 microM) and the NMDA-antagonist D-2-amino-5-phosphonovaleric acid (APV; 50 microM) led to a marked shortening of the depolarizations. This blocking effect of kynurenic acid and APV and comparison with the currents elicited by locally applied glutamate or NMDA provide evidence for an activation of both types of glutamate receptors to induce PDSs. The role of alteration of glutaminergic synaptic transmission in the induction and maintenance of these depolarizations is discussed in the context of results from the literature on the appearance of PDSs in cultures grown under chronic blockade of glutamate receptors.

2-Amino-5-phosphonovalerate↗
PubMed1993

Amplified met gene linked to double minutes in human glioblastoma.

The met proto-oncogene was found to be amplified in a human glioblastoma cell line (T3095) established from a glioblastoma multiform WHO grade IV. Amplification of epidermal growth factor receptor, transforming growth factor alpha and N-myc which have been described previously in glioblastoma were not observed in T3095. There was, however, an 8-fold met amplification. Giemsa-stained metaphases of T3095 cells revealed multiple (> 5) double minutes (dmins) in the majority of cells. Following xenografting in nude mice there was a significant increase in the number and frequency of dmins. The increase in dmins correlates with the level of met amplification (50-fold), suggesting localisation of the amplified met on dmins. Here we report the first case of met amplification in glioblastoma. Correlation between met amplification and extrachromosomal elements (dmins) has not been reported previously.

Animals↗