Biomedical subjects
E Bock
Publications and source records attributed to E Bock.
Nitrification at Low pH by Aggregated Chemolithotrophic Bacteria.
A study was performed to gain insight into the mechanism of acid-tolerant, chemolithotrophic nitrification. Microorganisms that nitrified at pH 4 were enriched from two Dutch acid soils. Nitrate production in the enrichment cultures was indicated to be of a chemolithoautotrophic nature as it was (i) completely inhibited by acetylene at a concentration as low as 1 mumol/liter and (ii) strongly retarded under conditions of carbon dioxide limitation. Electron microscopy of the enrichment cultures showed the presence of bacteria that were morphologically similar to strains of known chemolithotrophic nitrifying genera. Many of the enriched bacteria, in particular those that were identified as ammonium oxidizers, were aggregated. Filtration experiments indicated that aggregated cells were able to nitrify at low pH, whereas single cells were not. It is hypothesized that cells inside the aggregates are protected against the toxicity of nitrous acid. Nitrification by aggregated chemolithoautotrophic bacteria may be the dominating process of nitrate formation in many acid soils as it does not appear to depend on the existence of microsites of high pH (acid-sensitive autotrophic nitrification) or on the availability of organic carbon (heterotrophic nitrification).
[Cell adhesion--during morphogenesis and in the immune system].
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The cell adhesion molecule Cell-CAM 105 is an ecto-ATPase and a member of the immunoglobulin superfamily.
Cell-CAM 105 (C-CAM), a cell adhesion molecule in rat hepatocytes, was digested with trypsin, and peptides were isolated and sequenced by Edman degradation. The sequences of 4 peptides agreed with different regions of rat liver ecto-ATPase. Detailed biochemical analyses confirmed the identity between C-CAM and the ecto-ATPase. C-CAM/ecto-ATPase is a transmembrane protein having 4 immunoglobulin-like domains in the extracellular portion, demonstrating membership of the immunoglobulin superfamily. The ATPase activity suggests that ATP might influence cell adhesion, which would explain the inhibitory effect of exogenously added ATP on adhesion of several cell types.
Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes.
A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized the five NCAM mRNAs in rat brain.
Characterization of N-cadherin mRNA in chicken brain and heart by means of oligonucleotide probes.
It has previously been reported, that there is only one mRNA of 4.3 kb for chicken N-cadherin in brain and heart and one gene [(1988) J. Cell Biol. 106, 873-881]. Using three synthetic oligonucleotide probes derived from the published chicken N-cadherin cDNA sequence we found hybridization to at least four mRNAs in chicken brain and heart. The measured sizes were 8.0, 4.7, 3.8, and 3.3 kb. The 8.0 kb mRNA is only seen in brain, and only when a cytoplasmic probe is employed. The 3.8 kb mRNA is only observed in heart.
Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP-212.
Monoclonal antibodies (MAbs) 123C3 and 123A8 generated against a membrane preparation of a small cell lung carcinoma (SCLC) specimen recognize not only SCLC and bronchial carcinoids but also a significant portion of non-small cell lung carcinomas (non-SCLC) of various histological types. Together with 13 other monoclonal antibodies, which show preference for SCLC, they have been ranked as SCLC cluster 1 (SC-1) Mabs. In this study we show that SC-1 MAbs are directed against a restricted number of epitopes, and that SC-1 MAbs and a polyclonal antiserum directed against the neural cell adhesion molecule (NCAM) recognize identical glycoproteins, indicating that SC-1 antigens are closely related to or identical with NCAM. Long polysialic acid units composed of alpha-(2,8)-linked N-acetylneuraminic acid units, which in mammals are found exclusively on NCAM, were present on SC-1 antigens in SCLC. This provides further evidence that SC-1 MAbs recognize NCAM. The SC-1 antigens in the SCLC cell line H69 were present in two forms, NCAM-containing alpha-(2,8)-polysialic acid units identified by antiserum 735, the NCAM-H form, and the less sialylated NCAM-L form. The NCAM-H form consisted of diffusely migrating sialoglycoproteins with a molecular weight of 200,000-250,000, which resolved after neuraminidase treatment into two proteins with molecular weights of 140,000 and 180,000. Since the NCAM-H form is expressed in the lung tumor type with a poor prognosis, our results suggest that NCAM might be implicated in the invasive behavior of these NCAM-positive lung tumors.
Glial fibrillary acidic protein and beta A4 protein deposits in temporal lobe of aging brain and senile dementia of the Alzheimer type: relation with the cognitive state and with quantitative studies of senile plaques and neurofibrillary tangles.
The aim of this study was to compare brain glial fibrillary acidic protein (GFAP) levels to the modifications of cognitive functions (Blessed test score [BTS]), the density of the main neuropathological lesions (senile plaques [SP] and neurofibrillary tangles [NFT]), and the density of the two main subtypes of beta A4 deposits (classic plaques and diffuse deposits) in a series of patients with normal aging and senile dementia of the Alzheimer type of various degrees of severity. GFAP levels (enzyme-linked immunosorbent assay [ELISA] technique) and the densities of changes were measured in the temporal lobe of 12 women over 75 years of age. Under these conditions, the ELISA assay could determine GFAP in brain homogenates (aqueous-Triton buffer soluble extract) in a range from 2.5 ng to 600 ng per assay. Least affected patients (with a BTS of 19 and over) all ranged below 60 micrograms/mg protein. Most affected patients (with a BTS under 6) ranged above 150 micrograms/mg protein. However, interindividual variations were wide. A significant correlation between the BTS and the amount of GFAP could be found only when using the non parametric test of Spearman. There was a significant positive correlation between the amount of GFAP and the density of 1) SP, 2) NFT both revealed by Bodian's silver stain, and 3) classic beta A4 plaques shown by immunocytochemistry. On the contrary, no correlation was observed with diffuse beta A4 deposits. One case with very large amounts of diffuse beta A4 deposits without SP or NFT showed no associated GFAP reactivity. This suggests that GFAP production is a critical event in the formation of classic SP.
Structure and function of the neural cell adhesion molecules NCAM and L1.
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A developmental study of soluble L1.
L1 is a neural cell adhesion molecule expressed by neurons and it is involved in cell interactions during axon elongation and fasciculation. L1 from rat brain consists of a membrane-inserted Mr 200,000 polypeptide from which two polypeptides of Mr 180,000 and Mr 140,000 can be derived. These latter polypeptides appear both as membrane-associated and as soluble molecules. In this report, both total and soluble L1 in rat brain have been quantified by an enzyme-linked immunosorbent assay. The amount of total L1 per gram brain varies with postnatal age showing a peak value at postnatal day 7. The variation in soluble L1 coincides with the changes in total L1. Thus, soluble L1 constitutes ca 2% of total L1 at all ages investigated. The soluble Mr 140,000 and 180,000 L1 polypeptides are also present in cerebrospinal fluid. Studies of membrane L1 catabolism in cultured fetal rat brain neurons show that the half-life of membrane L1 is less than 24 hr. As a part of membrane L1 catabolism, small amounts of soluble L1 polypeptides are released to include cell surroundings.
Cell adhesion molecules in brain.
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[Plasma level studies in volunteers after intramuscular injection of various doses of etofenamate in an oily solution].
Plasma level Studies on Volunteers after Intramuscular Application of Different Doses of Etofenamate in Oily Solution. After i.m. injections of etofenamate (active substance of Rheumon i.m.) in oily solution to 12 volunteers, courses of plasma levels of etofenamate, flufenamic acid and fenamate (sum of etofenamate and flufenamic acid) were measured by HPTLC. Maximum levels of etofenamate, flufenamic acid and fenamate, as well as areas under the plasma level time curve (AUC) after 250, 500 and 1000 mg etofenamate respectively are proportional to dose. Maxima of fenamate plasma levels are reached after 6.3, 6.2 and 5.4 h respectively, half maximal levels are present already after 2 h. The mean residence time is 21.8, 18.8 and 15.7 h. These values obtained from different doses are not statistically different from each other. Pharmacokinetics are therefore linear and dose independent. The courses of fenamate levels can be described by a two compartment model. The elimination half lives after 250, 500 and 1000 mg are 2.1, 2.3 and 1.9 h, the invasion half-lives (dominant half-life) 8.8, 7.8 and 6.8 h. Terminal half-lives are 50.3, 63.7 and 35.4 h. Since plasma levels have decreased to 2% of the maximum level after one terminal half-life, they have no practical importance for the duration of activity or for accumulation. No sex related differences are found for dose dependent and independent parameters. From the data it can be derived that after i.m. injection of etofenamate in oily solution a prolongation of the dominant half-life occurs by a factor of 4-5 (as compared to oral data) which is caused by prolonged liberation from the oily depot. This long lasting liberation of etofenamate leads to a prolonged residence time after a fast increase, at the same time avoiding unnecessary high peak levels. Therefore it is guaranteed that even after i.m. administration of 1000 mg etofenamate in oily solution plasma levels of fenamate do not exceed those after 300 mg given orally. According to pharmacokinetic data a fast onset of action, good tolerability and a therapeutic action over a period of 24 h can be expected.
Dystrophin in electric organ of Torpedo californica homologous to that in human muscle.
We have found that dystrophin is highly concentrated at neuromuscular junctions and innervated membranes of the electric organ of Torpedo californica. In acetylcholine receptor-rich Torpedo membrane preparations dystrophin represents approximately 0.4% of total protein and can be extracted from these membranes by alkaline treatment in the absence of detergent, indicating that it is a peripheral membrane protein. Polyclonal antibodies raised against electrophoretically isolated Torpedo dystrophin cross-react with dystrophin in human muscle and unequivocally discriminate between normal and Duchenne muscular dystrophy patient's muscle. These results indicate that dystrophin is phylogenetically a highly conserved protein and that the relatively abundant dystrophin in electric organ would facilitate further investigations of its structure and function.
Differential expression of cell adhesion molecules in variants of K1735 melanoma cells differing in metastatic capacity.
We have investigated the expression of 2 neural-cell adhesion molecules, NCAM and LI, in K1735-C116 and -MI melanoma cells which differ qualitatively in their metastatic potential, i.e., MI cells are metastatic whereas C116 cells are not. We have found that NCAM in C116 cells are expressed as 2 quantitatively major glycosylated polypeptides with Mr of 145,000 and 120,000 and a minor 190,000 Mr polypeptide, whereas MI cells expressed NCAM as 3 glycosylated polypeptides with MR of 200,000, 140,000 and 120,000. The amount of NCAM in MI cells constituted only 60% of the amount observed in C116 cells. In C116 cells, the 145,000 and 120,000 Mr NCAM polypeptides were sulphated whereas NCAM did not appear to be sulphated in MI cells. No phosphorylation of NCAM in the 2 cell lines was observed. LI was expressed as a phosphorylated glycoprotein with Mr of 210,000 in MI cells whereas no LI expression was observed in C116 cells. LI was not sulphated in MI cells.
NCAM and Thy-1 in special sense organs of the developing mouse.
The distribution of the neural cell adhesion molecule (NCAM) and Thy-1 in the olfactory mucosa and olfactory bulb, the eye and the inner ear was examined with immunocytochemistry in mouse embryos from embryonic day 12 (E 12) to embryonic day 19 (E 19). In general, neurons are completely outlined with NCAM, whereas Thy-1 outlines only dendrites and axons. A variable cytoplasmic staining for Thy-1 is present in the perikarya. Neurons directly associated with special sense organs express NCAM and Thy-1 already from the earliest stage and throughout the period investigated, apart from the olfactory neurons in which Thy-1 disappears at E 19. The mitral cells in the olfactory bulb show Thy-1 but no NCAM reactivity. In the eye, lens fibers express Thy-1 and the pigmented layer expresses NCAM; neither of the two molecules can be detected at E 19. In the inner ear, hair cells express NCAM at E 19. Based on the distribution during the developmental period studied and on the cellular localisation of reaction products, it is suggested that the NCAM adhesion function could be of a more general nature by keeping appropriate cell membranes in close contact and thereby allowing more specific molecular interactions to take place. Thy-1, which is located on dendrites and axons, could be such a specific factor and function as recognition molecule in the developing nervous system.
Soluble neural cell adhesion molecule in brain, cerebrospinal fluid and plasma in the developing rat.
Neural cell adhesion molecule (NCAM) is a glycoprotein found in neurons, glial cells and muscle cells. In this report we describe the polypeptide composition and amount of soluble NCAM in brain, cerebrospinal fluid (CSF) and plasma in rats of various ages. One-two percent of total NCAM in rat brain was shown to be buffer-soluble. Soluble NCAM in brain, extracted at pH 7.2, constituted an increasing relative proportion of total NCAM during development, whereas the concentration of soluble NCAM in both CSF and plasma decreased in the same period. By radioiodination of brain extracts, CSF, and plasma, soluble NCAM was found to be composed of up to five polypeptides with molecular weights (Mr) of 200,000, 170,000, 150,000, 115,000 and 80,000. The relative composition did not vary significantly with age. The major forms in brain were the polypeptides with Mr of 200,000 and 115,000, whereas in CSF all forms seemed to appear in equal amounts. In plasma the 200,000 and the 170,000 Mr polypeptides were not observed and here the major forms were the 150,000 and 115,000 Mr polypeptides.
Equilibrium binding analysis of neural cell adhesion molecule binding to heparin.
The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin-Sepharose-based solid-phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 +/- 1.4 X 10(-8) M is observed for binding of newborn rat NCAM to heparin. This is approximately 25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, approximately 5 micrograms/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, approximately 15 micrograms/ml).
Heterogeneity of soluble neural cell adhesion molecule.
Soluble neural cell adhesion molecule (NCAM) from rat brain neuronal cell culture media consists predominantly of a polypeptide of Mr approximately 115,000. Minor amounts of a polypeptide of Mr approximately 180,000 and two inconsistently appearing components of Mr 160,000 and 145,000 are also observed. The Mr 115,000 component is derived from the neuronal membrane NCAM components NCAM-A of Mr 190,000, NCAM-B of Mr 140,000, or both. Thus, as a part of the catabolism of membrane NCAM-A plus -B, a minor fraction is posttranslationally cleaved and recovered in the media as discernible soluble NCAM polypeptides. The half-life of membrane NCAM-A plus -B is less than 24 h. Astrocyte culture media contains a predominant soluble NCAM component of Mr 120,000 derived from membrane-associated NCAM-C. A close comparison of deglycosylated soluble NCAM from astrocyte and neuronal cultures showed a small but consistent difference in Mr, a result suggesting that different NCAM polypeptides are released from the membrane of neurons and astrocytes. In contrast to the Mr 115,000-120,000 NCAM polypeptides, the Mr 180,000 polypeptide from neuronal culture media does not seem to be derived from membrane-attached NCAM and may therefore represent a secreted NCAM isoform.