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Biomedical subjects

E Bock

Publications and source records attributed to E Bock.

At least 73 records · Page 4Linked to original sources
PubMed1997

NCAM-fibronectin-type-III-domain substrata with and without a six-amino-acid-long proline-rich insert increase the dendritic and axonal arborization of spinal motoneurons.

The neural cell adhesion molecule (NCAM) is a modulator of neurite outgrowth in vitro and in vivo. To see if single or tandem extracellular NCAM domains can influence neurite outgrowth, motoneurons from embryonic rat spinal cord were cultured on several NCAM fusion protein substrata. Motoneurons growing on either of two fusion proteins comprising the combined two fibronectin type III homology domains of NCAM with or without a six-amino-acid-long, proline-rich insert (F3I,II+ and F3I,II, respectively) usually developed three or more neurites per cell. Motoneurons grown on NCAM-immunoglobulin domain I (IgI), by contrast, developed many unipolar and bipolar cells, a situation also seen when motoneurons were cultured on control substrata. The neuritic trees of motoneurons grown on F3I,II and F3I,II+ appeared broader and rounder than motoneurons cultured on either control or IgI substrata, and the spreading indices of motoneurons grown on F3I,II and F3I,II+ were significantly lower than when the other substrata were used. Neither of the NCAM-F3 fusion proteins stimulated the outgrowth of single neurites. By contrast, IgI substratum was able to stimulate neurite outgrowth over control substrata. Both NCAM-F3 substrata induced branches in axons and dendrites, whereas IgI substratum did not affect neurite branching significantly. These data indicated that neurite outgrowth and neurite branching on the chosen substrata were not closely linked to each other. Furthermore, the branching characteristics of motoneuron neurites potentially depend on their differentiation states and, possibly, on the conformation of the two NCAM-F3 domains.

Amino Acid Sequence↗
PubMed1997

The first immunoglobulin-like neural cell adhesion molecule (NCAM) domain is involved in double-reciprocal interaction with the second immunoglobulin-like NCAM domain and in heparin binding.

To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM domains were also produced as fusion proteins. By means of surface plasmon resonance analysis, it was shown that the first Ig-like NCAM domain binds the second Ig-like NCAM domain with a dissociation constant 5.5 +/- 1.6 x 10(-5) M. Furthermore, it was found that the first Ig-like domain binds heparin. It was also demonstrated that the second Ig-like NCAM domain binds heparin and that both domains bind collagen type I via heparin but not collagen type I directly.

Animals↗
PubMed1997

Evaluation of cell morphology by video recording and computer-assisted image analysis.

The aim of this study was to evaluate the effects of various cell culture conditions on cell morphology. Cell morphology was estimated by means of video recording and computer-assisted image analysis. Cell contours from the stored images of either live cells or fixed and stained cells were determined automatically, and cellular area, form factor, and average cell brightness were calculated. Using the mouse fibroblastoid L 929 cell line (L-cells) and the rat glioma BT4C cell line, it was found that a number of methodological parameters strongly affected cell morphology. These included confluency of cells before dissociation, dissociation procedure, cell seeding density, cultivation time, and culture substratum. The substratum, particularly collagen type I and fibronectin, profoundly affected cell morphology. Using drugs affecting cytoskeletal organization or cell substratum interactions, it was shown that average cell brightness was a valuable parameter for estimation of cellular attachment. Cytochalasin D, which impairs actin filaments, caused a dramatic increase in the average cell brightness in both cell lines. Nocodazole, which depolymerizes microtubules, mainly affected the L-cells, whereas the BT4C-cells were largely unaffected, indicating that microtubules were morphological determinants for the former cell line but not for the latter. When cells were grown on fibronectin, an RGD-peptide only affected L-cell attachment, indicating that BT4C-cells only expressed low (if any) amounts of RGD recognizing integrins. The interassay precision of the employed procedure depended on culture substratum; the coefficients of variation ranged from 7-24%. Lowest variations in area determination were found for cells grown on fibronectin. The coefficient of variation of form factor determinations was generally around 20%, independent of substrata and culture time.

Animals↗
PubMed1997

Prediction of teratogenic potency of valproate analogues using cerebellar aggregation cultures.

The aim of this study was to develop a novel in vitro system suitable for preclinical testing for developmental toxicity of drugs. An assay system consisting of primary cultures of dissociated cerebella from 6-day-old mice was chosen, since it allowed quantification of neuronal aggregation and fasciculated neurites. A human teratogen, the antiepileptic drug valproic acid (VPA), as well as its structural analogues, ( +/- )-4-en-VPA and E-2-en-VPA, with varying teratogenic activities, were tested and found to affect aggregation and fiber formation of cerebellar neurons. Based on a dose-response study, the concentrations of compounds causing 50%, inhibition (IC50) of formation of thick and thin fibers were determined. The lowest IC50 values were found for VPA (52 +/- 7 and 86 +/- 11 microM for thick and thin fibers, respectively), which in vivo caused the highest rate of exencephaly among the three compounds tested, ( +/- )-4-en-VPA exhibited intermediate values (150 +/- 30 and 300 +/- 40 microM), whereas the highest IC50 values were found for E-2-en-VPA (260 +/- 42 and 430 +/- 40 microM). The latter compound does not induce neural tube defects, but has been shown to have neurobehavioral effects in prenatally exposed animals. Subsequently, the purified S- and R-enantiomers of 4-yn-VPA (teratogenic and non-teratogenic, respectively) were tested for their effects on aggregation and fiber formation of the cerebellar neurons. Treatment with S-4-yn-VPA resulted in pronounced changes in numbers of aggregates and fasciculated processes compared to the cultures treated with R-4-yn-VPA, indicating that the intrinsic stereoselective potency of the enantiomers may be correlated to the difference in their effects on cerebellar neurons in vitro. Thus, the teratogenic potency of VPA and its analogues correlated with their effects on aggregation of neural cells and formation of fasciculated neurites in primary cultures of dissociated cerebella, indicating that the in vitro assay system employed may be used as a pre-screening test for prediction of teratogenic potency of drugs.

Animals↗
PubMed1997

Characterization of microwell cultures of dissociated brain tissue for studies of cell-cell interactions.

Microwell cultures of dissociated tissue from prenatal rat hippocampus and cerebral cortex as well as from early postnatal cerebellum were used for quantification of neuronal aggregation, process extension, and fasciculation. It was shown that the cells in culture from these different brain regions developed differently with regard to both architecture and rate of differentiation. The effect of a polyclonal antibody against the neural cell adhesion molecule (NCAM), the excitatory amino acid receptor agonist N-methyl-D-aspartate (NMDA), and the neurotoxin acrylamide on aggregation and fiber formation was investigated. Exposure to the NCAM antibody led to formation of fewer but larger aggregates and stimulated the morphological development of the cultures. Acrylamide affected aggregate formation, leading to smaller but more numerous aggregates, and it inhibited process extension and fasciculation. Treatment with NMDA affected process formation and led to formation of more numerous but smaller aggregates. Some of these effects were strongly tissue-dependent. Thus, large differences were seen regarding the effect of the NCAM antibody on aggregation and process extension in cultures from the different brain areas. The culture systems appear to represent convenient and reliable screening tools to study the influence of putative morphoregulatory substances on cell-cell interactions during early neuronal development.

Animals↗
PubMed1997

Effect of NCAM-transfection on growth and invasion of a human cancer cell line.

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.

Cell Cycle↗
PubMed1996

Functional characterization of NCAM fibronectin type III domains: demonstration of modulatory effects of the proline-rich sequence encoded by alternatively spliced exons a and AAG.

In order to characterize the functions of the two fibronectin type III (F3) homology domains of the neural cell adhesion molecule (NCAM), we investigated the effects of two variants, expressed as fusion proteins, of the NCAM-F3 domains on attachment and spreading of NCAM-expressing fibroblasts, cerebellar cell aggregation and fiber formation, and on growth cones. The two fusion proteins were different with regard to a short proline-rich insert of six amino acids between the two F3 domains. Immobilized NCAM-F3 fusion proteins were found to mediate attachment of both transmembrane and lipid-anchored NCAM expressing fibroblasts. Also NCAM-negative cells adhered to the NCAM-F3 substratum, although to a lesser extent, implying the possibility of a heterophilic ligand to NCAM-F3 domains on the surface of fibroblasts. Cellular spreading on NCAM-F3 substratum was selectively increased in fibroblasts expressing transmembrane NCAM, and only the NCAM-F3 fusion protein lacking the proline-rich insert was able to elicit this effect. Primary cultures of mouse cerebellum were strongly inhibited with regard to formation of cellular aggregates and fibers, when incubated in the presence of either of the two NCAM-F3 fusion proteins, the fusion protein with the proline-rich insert being the more effective one. Finally, the morphology of growth cones from rat cerebellar granule cells changed significantly when grown on NCAM-F3 substrata as revealed by computer-assisted image analysis. Thus, our data indicate that the NCAM-F3 domain are involved in cell-cell adhesion, and that insertion of the proline-rich sequence has a modulatory effect on NCAM-F3 domain functions.

Animals↗
PubMed1996

99m-Tc-HMPAO-SPECT in diagnosis of early cerebral vasculitis.

The objective of this study was to evaluate whether mild neurological symptoms suggestive of neuropsychiatric involvement may be associated with cerebral perfusion defects as detected by functional brain imaging with 99m-Tc-HMPAO-SPECT (single photon emission computed tomography). SPECT analysis for the early detection of central nervous system (CNS) involvement was evaluated in 40 consecutive patients with systemic vasculitis or with Sneddon's syndrome. Of these, 18 patients showed overt neuropsychiatric symptoms, so-called major symptoms (e.g. motoric or sensible defects); 6 had mild symptoms like headache or cognitive disorders, so-called minor symptoms: 16 patients did not present with any of these symptoms. SPECT abnormalities were detected in 16 of the 18 patients with overt neuropsychiatric symptoms (89%). Five of the 6 patients with minor symptoms (83%) and 5 of the 16 patients without neurological symptoms (31%) also had SPECT abnormalities. There was no relation to disease activity or duration. We concluded that the high sensitivity of SPECT (87.5%) in detecting perfusion abnormalities among the evaluated group of patients indicates its suitability for early diagnosis of vasculitic CNS involvement.

Adult↗
PubMed1996

Two types of amyloid precursor protein (APP) mRNA in rat glioma cell lines: upregulation via a cyclic AMP-dependent pathway.

APP is a multifunctional transmembrane glycoprotein and the only known natural source of beta A4 peptide-the major constituent of senile plaques in Alzheimer's disease (AD). The expression and cAMP-dependent regulation of the APP gene were investigated in primary cultures of rat astrocytes and two related glioma cell lines, BT4C and BT4Cn, which exhibit distinct invasive phenotypes. Besides the well-characterized 3.5 kb APP mRNA class, a robust expression of an unusual 2.8 kb APP mRNA class was revealed by Northern blotting in both glioma cell lines, but not in the astrocytes. Low amounts of the 2.8 kb APP mRNA species were also observed in rat liver and occasionally in aged rat brain. The 2.8 kb APP mRNA contained exons 1-18 and may thus be generated by truncation of the 3' untranslated region. For the first time, regulation of the APP gene via a cAMP-dependent mechanism was shown. Exposure to dBcAMP dramatically upregulated the 3.5 and 2.8 kb transcripts in BT4C cells, and, to a lesser extent, in BT4Cn cells where the constitutive expression of the APP gene was much higher. Elucidation of the factors involved in cAMP-dependent induction of APP mRNA in these cells may shed more light on the molecular mechanisms of APP overexpression.

Amyloid beta-Protein Precursor↗
PubMed1996

Quantitative whole body scintigraphy--a simplified approach.

In this paper we present investigations on a simplified method of quantitative whole body scintigraphy by using a dual head LFOV-gamma camera and a calibration algorithm without the need of additional attenuation or scatter correction. Validation of this approach to the anthropomorphic phantom as well as in patient studies showed a high accuracy concerning quantification of whole body activity (102.8% and 97.72%, resp.), by contrast organ activities were recovered with an error range up to 12%. The described method can be easily performed using commercially available software packages and is recommendable especially for quantitative whole body scintigraphy in a clinical setting.

Gamma Cameras↗
PubMed1995

NCAM-antibodies modulate induction of long-term potentiation in rat hippocampal CA1.

The neural cell adhesion molecule (NCAM) probably plays a role in neural plasticity in the adult vertebrate brain. We here present evidence that NCAM may be involved in long-term potentiation (LTP) in the CA1-region of rat hippocampal slices. It is shown that local application of antibodies against NCAM inhibits subsequent LTP-induction. Thus NCAM may be directly involved in the initial phase of LTP-induction. These results have important implications for the possible involvement of NCAM in learning and memory.

2-Amino-5-phosphonovalerate↗
PubMed1995

A new obligately chemolithoautotrophic, nitrite-oxidizing bacterium, Nitrospira moscoviensis sp. nov. and its phylogenetic relationship.

A gram-negative, non-motile, non-marine, nitrite-oxidizing bacterium was isolated from an enrichment culture initiated with a sample from a partially corroded area of an iron pipe of a heating system in Moscow, Russia. The cells were 0.9-2.2 microns x 0.2-0.4 microns in size. They were helical- to vibroid-shaped and often formed spirals with up to three turns 0.8-1.0 micron in width. The organism possessed an enlarged periplasmic space and lacked intracytoplasmic membranes and carboxysomes. The cells tended to excrete extracellular polymers, forming aggregates. The bacterium grew optimally at 39 degrees C and pH 7.6-8.0 in a mineral medium with nitrite as sole energy source and carbon dioxide as sole carbon source. The optimal nitrite concentration was 0.35 mM. Nitrite was oxidized to nitrate stoichiometrically. The doubling time was 12 h in a mineral medium with 7.5 mM nitrite. The cell yield was low; only 0.9 mg protein/l was formed during oxidation of 7.5 mM nitrite. Under anoxic conditions, hydrogen was used as electron donor with nitrate as electron acceptor. Organic matter (yeast extract, meat extract, peptone) supported neither mixotrophic nor heterotrophic growth. At concentrations as low as 0.75 g organic matter/l or higher, growth of nitrite-oxidizing cells was inhibited. The cells contained cytochromes of the b- and c-type. The G+C content of DNA was 56.9 +/- 0.4 mol%. The chemolithoautotrophic nitrite-oxidizer differed from the terrestrial members of the genus Nitrobacter with regard to morphology and substrate range and equaled Nitrospira marina in both characteristics. The isolated bacterium is designated as a new species of the genus Nitrospira.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

The ability to re-express polysialylated NCAM in soleus muscle after denervation is reduced in aged rats compared to young adult rats.

The neural cell-adhesion molecule, NCAM, contains an unusual homopolymer of sialic acid units, polysialic acid. This carbohydrate seems to be involved in neurite outgrowth, bundling and branching, processes which are important during reinnervation. In aged rats, reinnervation of denervated muscle fibres is incomplete. In this study, age-related changes in the degree of polysialylation of NCAM re-expressed after denervation were examined using a monoclonal antibody recognizing polysialic acid and a polyclonal antibody recognizing NCAM. The results show that, after denervation, the degree of polysialylation on NCAM was clearly reduced in rat soleus muscle of aged, compared to young, adult rats. This age-related change in expression of polysialic acid probably influences the reinnervation process in aged muscle.

Animals↗
PubMed1995

Localization of NCAM on NCAM-B-expressing cells with inhibited migration in collagen.

The extracellular matrix is a key element in neuronal development and tumour invasion, providing a substratum which sustains the adhesion and migration of cells. In order to study interactions between the neural cell adhesion molecule (NCAM) and collagen, we transfected mouse L cells with cDNA encoding the human transmembrane NCAM isoform of 140 kDa (NCAM-B). An L-cell/collagen type I system was used to study the influence of NCAM expression on in vitro invasion. We here report that migration of NCAM-expressing cells in collagen was inhibited compared to that of NCAM-negative cells transfected with the empty vector. Immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold electron microscopy using anti-human NCAM antibodies demonstrated a heterogeneous distribution of NCAM on the plasma membrane of transfected L cells grown on collagen. NCAM was preferentially located at the surface of broad cytoplasmic protrusions and slender extensions, some of which were facing the collagen. This was in contrast to the homogeneous surface distribution of NCAM on cells grown on plastic. These data suggest that NCAM and collagen type I interact, and that this might lead to the migration inhibition of NCAM-expressing cells.

Animals↗
PubMed1995

High-resolution SPECT of the temporomandibular joint in chronic craniofacial pain disorders: a pilot study.

Chronic craniofacial pain disorders commonly cause physicians diagnostic difficulties. The purpose of this study was, on one hand, to detect pathological focuses of the craniofacial skeleton using a new system of high-resolution single photon emission computertomography (SPECT), and on the other hand, to compare the results with those obtained via high-field magnetic resonance imaging (MRI) as far as temporomandibular joint affections are concerned. SPECT can be regarded as a supplementary diagnostic mean for patients displaying the symptoms of chronic craniofacial pain disorders, especially in cases where clinical and paraclinical investigations do not coïncide or which are refractory to treatment, not least to differentiate between somatic and psychogenic causes, respectively.

Adult↗