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Biomedical subjects

E Bock

Publications and source records attributed to E Bock.

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

High rate of aerobic nitrification and denitrification by Nitrosomonas eutropha grown in a fermentor with complete biomass retention in the presence of gaseous NO2 or NO.

A pure culture of the obligately lithoautotrophic ammonia-oxidizer Nitrosomonas eutropha was grown in a laboratory-scale bioreactor with complete biomass retention. The air supply was supplemented with nitrogen dioxide (NO2; 25 or 50 ppm) or nitric oxide (NO; 25 or 50 ppm). Compared to cultures grown without these nitrogenous oxides, the addition of NO2 or NO to the culture resulted in a significant increase of the nitrification rate, specific activity of ammonia oxidation, growth rate, and maximum cell densities. In contrast, the growth yield slightly decreased in the presence of NO or NO2. Maximum cell densities of about 2 x 10(10) cells ml-1 and a maximum nitrification rate of about 221 mmol NH4+ l-1 day-1 were obtained after 3 weeks in the presence of 50 ppm NO2. Furthermore, in the stationary phase about 50% of the nitrite produced was aerobically denitrified to dinitrogen (N2) and traces of nitrous oxide (N2O). When cells were supplemented with NO, a high rate of aerobic denitrification occurred only during the first days of the exponential growth phase.

Aerobiosis↗
PubMed1998

Anaerobic ammonia oxidation by cell-free extracts of Nitrosomonas eutropha.

Cell-free extracts of Nitrosomonas eutropha oxidized ammonia to nitrite with NO2 (N2O4) as electron acceptor. The ammonia oxidation activity was shown to be sensitive against oxygen. In the absence of oxygen ammonia and NO2 were consumed in a ratio of approximately 1:2 and hydroxylamine occurred as an intermediate. NO was released in amounts equimolar to the consumption of NO2. After passing the cell suspension through a French pressure cell and fractionating it by density gradient centrifugation using a linear sucrose gradient, two soluble and two membrane fractions were detectable. Highest ammonia oxidation activity was measured in the membrane fractions and highest hydroxylamine oxidation activity in the soluble fractions. The KS values of the ammonia oxidizing system in cell-free extracts was about 20 microns NH3 and remained unchanged between pH 7.25 to 8.25.

Ammonia↗
PubMed1998

Biodeterioration of natural stone with special reference to nitrifying bacteria.

An evaluation of field data from historical buildings in Germany showed that chemoorganotrophic bacteria are the most numerous microorganisms in building stones, followed by fungi and nitrifying bacteria. Chemoorganotrophic bacteria and fungi were present in almost every sample. Ammonia and nitrite oxidizers were found in 55 and 62% of the samples, respectively. Within months, natural stone was colonized by chemoorganotrophic microorganisms. The highest cell numbers were usually found near the surface. The colonization of natural stone by nitrifying bacteria took several years. The highest cell numbers were in some cases found underneath the surface. Nitrifying bacteria showed a preference for calcareous material with a medium pore radius between 1 and 10 microns. Cell numbers of nitrifying bacteria did not correlate to the nitrate content of the stone material. We demonstrated that the stone inhabiting microflora can cause significant loss of nitrate by denitrification. Our data strongly suggested that microbial colonization of historical buildings was enhanced by anthropogenic air pollution. Samples taken from stone material with a pore radius < or = 1 micron had significantly higher cell numbers when they were covered with black crusts. A comparison of samples taken between 1990-1995 from buildings throughout Germany showed that in Eastern Germany a significantly stronger colonization with facultatively methylotrophic bacteria and nitrifying bacteria existed. The same was true for natural stone from an urban exposure site when compared to material from a rural exposure site. Data from outdoor exposure and laboratory simulation experiments indicated that the colonization of calcareous stone by nitrifying bacteria was enhanced by chemical weathering.

Biodegradation, Environmental↗
PubMed1998

Methanotrophs and methanogens in masonry

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH4 g of stone-1 day-1. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH4 g of stone-1 day-1 was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called "mini-methane"-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.

Journal Article↗
PubMed1998

Fra-1 induces morphological transformation and increases in vitro invasiveness and motility of epithelioid adenocarcinoma cells.

Two cell lines originating from a common ancestral tumor, CSML0 and CSML100, were used as a model to study AP-1 transcription factors at different steps of tumor progression. CSML0 cells have an epithelial morphology; they express epithelial but not mesenchymal markers and are invasive neither in vitro nor in vivo. CSML100 possesses all characteristics of a highly progressive carcinoma. These cells do not form tight contacts, are highly invasive in vitro, and are metastatic in vivo. AP-1 activity was considerably higher in CSML100 cells than in CSML0 cells. There was a common predominant Jun component, namely, JunD, detected in both cell lines. We found that the enhanced level of AP-1 in CSML100 cells was due to high expression of Fra-1 and Fra-2 proteins, which were undetectable in CSML0 nuclear extracts. Analysis of the transcription of different AP-1 members in various cell lines derived from tumors of epithelial origin revealed a correlation of fra-1 expression with mesenchymal characteristics of carcinoma cells. Moreover, we show here for the first time that the expression of exogenous Fra-1 in epithelioid cells results in morphological changes that resemble fibroblastoid conversion. Cells acquire an elongated shape and become more motile and invasive in vitro. Morphological alterations were accompanied by transcriptional activation of certain genes whose expression is often induced at late stages of tumor progression. These data suggest a critical role of the Fra-1 protein in the development of epithelial tumors.

Adenocarcinoma↗
PubMed1998

Valproic acid, but not its non-teratogenic analogue 2-isopropylpentanoic acid, affects proliferation, viability and neuronal differentiation of the human teratocarcinoma cell line NTera-2.

The antiepileptic drug valproic acid (VPA) is an established human teratogen that affects neural development in human fetuses exposed to the drug during early pregnancy. The development of simple cell culture models that reflect normal neuronal development will be useful for the screening of neural developmental toxicity of new drugs and for the identification of the specific molecular targets for the teratogenic action. The present study tests a human NT2 cell line for its suitability to serve as such a model. Treatment with VPA, but not with its non-teratogenic-analogue, 2-isopropylpentanoic acid (IPPA), inhibited cell growth, had a cytotoxic effect and blocked retinoic acid-induced neuronal differentiation of NT2 cells. Differentiation was evaluated by immunostaining for neurofilament proteins, microtubule associated proteins-2 (MAP-2), tau and neural cell adhesion molecule, NCAM. However once differentiation had taken place, VPA did not revert this process, although it did affect neuronal aggregation and neurite fasciculation. These results suggest that the human NT2 cell line might be useful for identifying drugs affecting neuronal development in humans.

Anticonvulsants↗
PubMed1997

Demonstration of an extracellular ATP-binding site in NCAM: functional implications of nucleotide binding.

A minor fraction of the total ecto-type (E-type) ATPase activity of rat synaptosomes has been detected in immunoprecipitates of the neural cell adhesion molecule, NCAM, indicating that this either is an intrinsic enzymatic activity of NCAM or of an ATPase tightly associated to NCAM [Dzhandzhugazyan & Bock (1993) FEBS Lett. 336, 279-283]. We here demonstrate ATPase activity in preparations of the lipid-anchored as well as the transmembrane NCAM isoforms immunoisolated from transfected L-cells. A fraction of the E-type ATPase activity is spontaneously released from synaptosomes. Released material was fractionated by various chromatographic procedures and an extracellular fragment of NCAM was shown to co-elute with the major part of the enzymatic activity. Furthermore, it was shown that agarose-coupled NCAM-antibodies retained 85% of the ATPase activity released from synaptosomes after treatment with phosphatidylinositol-specific phospholipase C. These findings restricted the association or expression of the enzymatic activity to the extracellular part of NCAM. An affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine, FSBA, was shown to inhibit ATPase activity of immunoisolated NCAM, and incorporation of FSBA was detected in all three major NCAM isoforms (A, B, and C). An excess of ATP prevented both inactivation of the enzyme and affinity labeling of NCAM. Thus, NCAM contains an ATP-binding site, and this site is localized extracellularly and probably has the catalytic function. Binding of the substrate or FSBA protected a proteolytic cleavage site in NCAM localized close to the membrane presumably by induction of a local conformational change in NCAM, indicating a mechanism by which ATP may regulate NCAM adhesion and adhesion-triggered processes. A possible role of this mechanism in synaptic plasticity and memory consolidation is proposed.

Adenosine↗
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↗