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V Berezin

Publications and source records attributed to V Berezin.

45 records · Page 3Linked to original sources
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↗
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↗
PubMed1983

Enzyme-linked immunosorbent assay of the D2-glycoprotein.

D2 is a glycoprotein enriched in neuronal membranes and probably involved in intercellular adhesion. An immunochemical relationship between D2 and the neuronal cell adhesion molecule from chick has been demonstrated. Changes in D2 concentration in human body fluids correlate to certain neurological diseases. We here report the purification of the D2 membrane proteins from fetal and adult human brain and the demonstration of physicochemical differences between the two proteins. Enrichments of 133 times (fetal D2) and 350 times (adult D2) were found. Specific rabbit antisera against the purified D2 proteins were produced, and this enabled the setting up of an enzyme-linked immunosorbent assay for D2 quantification in human brain extracts, cerebrospinal fluids, sera, and amniotic fluids.

Adult↗
PubMed1983

Quantification of the D2-glycoprotein in amniotic fluid and serum from pregnancies with fetal neural tube defects.

D2 is a glycoprotein existing in both membrane-bound and soluble forms. Employing a specific rabbit antibody against purified human brain D2, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of D2 and applied it to amniotic fluids from 87 normal and 36 pathological pregnancies. With a cut-off point of 150 ng D2/ml, no false positive D2 values were obtained in any of the amniotic fluids from normal fetuses, although the alpha-fetoprotein concentrations were slightly increased in 13 cases. No false negative D2 values were found in any of the 18 investigated amniotic fluids from fetuses with anencephaly. Of 8 amniotic fluids from fetuses with spina bifida, 2 false negative D2 values were found. No false negative alpha-fetoprotein values were found in any of the cases with neural tube defects in this study. In 10 amniotic fluids from fetuses with other malformations, 5 samples showed raised D2 concentrations. The D2 level in sera from 10 women carrying normal fetuses and 16 women carrying malformed fetuses was also determined, but no statistically significant difference in D2 level was found in the pathological sera when compared with normal sera. It was concluded that the determination of D2 concentrations in amniotic fluid by means of the D2-ELISA may be used as an additional test in the screening of fetal malformations in early pregnancy.

Amniotic Fluid↗
PubMedDate not supplied

The neural cell adhesion molecule in synaptic plasticity and ageing.

By mediating cell adhesion and signal transduction, the neural cell adhesion molecule (NCAM) regulates neurite outgrowth, fasciculation and target recognition in the developing nervous system. In addition, a number of studies suggest an important role for the NCAM in regeneration and learning in the adult nervous system. NCAM-deficient mice are impaired in spatial learning. Moreover, by interfering with normal NCAM function by intracranial injections of NCAM-antibodies, long-term potentiation (LTP) in rat hippocampal slices and learning in rats and chicks have been inhibited. In the vertebrate nervous system, NCAM is the dominant carrier of polysialic acid (PSA), an unusual carbohydrate consisting of long homopolymers of sialic acid. The PSA-NCAM expression decreases markedly during development. However, an upregulation of polysialic acid (PSA) in restricted brain areas including the hippocampus has been observed following learning. Moreover, enzymatic removal of PSA results in impaired LTP and learning. In muscle, the PSA-NCAM expression is upregulated following denervation. This response is weakened in aging rats. The expression of NCAM and PSA have been shown to be regulated by neuronal activity suggesting that the NCAM may promote structural remodelling in an activity dependent manner associated with learning and regeneration.

Aging↗