Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl-N-dimethyl phosphoramidocyanidate) is approximatelt one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon (p-nitrophenyl diethylphosphate), and a phosphinate PNMPP (p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn²⁺ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.     

pH-dependent changes of ganglioside biosynthesis in neuronal cell culture

Ganglioside biosynthesis was studied in primary cultured murine cerebellar cells after labeling with [14C]galactose. A shift in biosynthesis from "a"-series to "b"-series gangliosides was observed after lowering the pH of the culture medium from 7.4 to 6.2; this effect was fully reversible on changing back to pH 7.4. The observed regulatory effect of pH is in accordance with a recent model of ganglioside biosynthesis. Sialyltransferase II (ST II), the first enzyme for biosynthesis of "b"-series gangliosides, is more active at pH 6.2 than Gal-NAc-transferase, the first enzyme for synthesis of "a"-series gangliosides, which is more active than sialyltransferase II at pH 7.4.     

Neuronal and glial enzyme studies in cell culture

Summary Newborn BALB/c mouse brain was cultured as disaggregated cells after serial trypsin dissociations. The ontogeny of the cultures was followed by assays of cell number, deoxyribonucleic acid, and protein content and by the activities of three enzymes considered to be markers of neuronal differentiation. Aliquots of the freshly dissociated cells were assayed for choline acetylase, acetylcholinesterase, and glutamic acid decarboxylase activities and compared with intact brain. The percentages of recovery of activities, expressed as¹⁴C product formed per mg of protein per 10 min, at pH 6.8 and 37°C, were 37% for choline acetylase, 54% for acetylcholinesterase, and 24% for glutamic acid decarboxylase. The remainder of the freshly dissociated cells were placed into culture; enzyme assays were performed as the cells multiplied and then when the cultures became static. Choline acetylase activity increased as the cells rapidly divided, and glutamic acid decarboxylase activity increased only after the cultures became confluent. Under the culture conditions, acetylcholinesterase was not induced, despite active synthesis of acetylcholine. Neuroblastoma clone N18, C1300 cell line, was grown in cell culture, and the activity of acetylcholinesterase was measured as the cells multiplied and came to confluency. The specific activity of mouse neuroblastoma acetylcholinesterase increased 25-fold when the rate of cell division was restricted. The rate of cell division could be regulated by adjusting the serum concentration. By removing fetal calf serum during the growth period, cell division ceased, and acetylcholinesterase activity was significantly and rapidly induced. Choline-O-acetyltransferase specific activity was measured in rapidly dividing and in static cultures. Its specific activity was highest in nondividing cultures, compared to cultures containing actively dividing cells (6-fold), and the specific activity of thymidylate synthetase was increased 2.5-fold in actively dividing cultures, compared to static cultures. Glioblastoma cells obtained from the rat astrocytoma, clone C6, were grown in culture, and glucose metabolism was measured in control cultures, and in cultures containing norepinephrine (0.017 mg per ml). Norepinephrine produced a 50% inhibition in the incorporation ofd-[¹⁴C]glucose. Cells incubated for 2 hr in the presence ofd-[¹⁴C]glucose, washed and then incubated in control medium or in medium containing norepinephrine, resulted in the release of greater than 50% of radioactive metabolites in the norepinephrine treated plates. Norepinephrine caused a 50% increase in¹⁴CO₂ production in glioblastoma cells incubated withd-[1-¹⁴C]glucose. Norepinephrine, under similar conditions, did not affect the metabolism of glucose in clone C46, C1300 mouse neuroblastoma cells. Portions of this work were supported by a research grant (6-444946-58605) from the American Cancer Society.     

Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides

An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture.     

Application of neuronal cell culture in human degenerative brain disorders

Neuronal cell culture system has been used for the study of pathochemical evaluations in human degenerative brain disorders, particularly for Krabbe's disease and neuronal ceroid lipofuscinosis. To understand the pathochemistry of Krabbe's disease, we added psychosine into neuronal cell cultures and psychosine treated cells showed the destruction of cytoskeleton and pathy intracellular changes. Electron microscopic finding showed the swelling of the mitochondria. Oligodendrocytes and Schwann cells were isolated from the brains and sciatic nerve of twitcher mouse as an authentic murine model of globoid cell leukodystrophy. Oligodendroglial cells cultured for 22 days were stained by anti-galactocerebroside antibodies. In twitcher oligodendrocyte processes were wirelike and progressively degenerated and there were few membranous expansion. Schwann cells from twitcher could not elongated their processes. These data suggest that psychosine might be important factor to result in these pathological conditions. Furthermore, we studied the effect of protease inhibitors, E-64 on dissociated primary cultures from fetal rat brain. After treated with E-64 in a concentration from 0.1-50 micrograms/ml, numerous cytoplasmic accumulations appeared in neuronal cells. These morphological pictures resemble with those of neuronal ceroid lipofuscinosis, Batten disease. We will discuss the relationship between the deficiency of catepsin H in Batten disease and inclusion bodies found in E-64.     

Wnt proteins promote neuronal differentiation in neural stem cell culture

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from studies of mouse and chick models, but its action at the cellular level has been poorly understand. In this study, we examine the in vitro function of Wnt signaling in embryonic neural stem cells, dissociated from neurospheres derived from E11.5 mouse telencephalon. Conditioned media containing active Wnt-3a proteins are added to the neural stem cells and its effect on regeneration of neurospheres and differentiation into neuronal and glial cells was examined. Wnt-3a proteins inhibit regeneration of neurospheres, but promote differentiation into MAP2-positive neuronal cells. Wnt-3a proteins also increase the number of GFAP-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4. These results indicate that Wnt-3a signaling can inhibit the maintenance of neural stem cells, but rather promote the differentiation of neural stem cells into several cell lineages.     

Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture

In situ zymography is a unique technique for detection and localization of enzyme?Csubstrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the ??in position?? activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.     

Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe

We have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects their neurites. The hybridization is detected only in neuronal structures. Consequently, detection of the biotinylated neurofilament DNA probe by silver-intensified streptavidin-gold can be specifically used to identify neuronal cell bodies.     

The retention of differentiated properties by lens epithelial cells in clonal cell culture

A small number of cells of lens epithelium from newly hatched chickens were cultured at clonal density to investigate the retention of differentiated properties during cellular growth in vitro. Singly plated cells proliferated to produce colonies, at least some of which were considered to be true clones of single cell origin. The differentiation of lens fibers occurring in many colonies was identified through observations by electron microscopy as well as immunofluorescence utilizing specific antiserum against lens fibers. Primary or secondary mass cultures of cells of lens epithelium contained cells which produce differentiated colonies when cultured at clonal density. Colony-producing cells can be differentially dissociated from monolayers by EDTA treatment without using tyrpsin. For successful culture of cells of lens epithelium at clonal density, the use of conditioned medium is necessary.     

Proteins secreted by clonal cell lines : Changes in metabolism with culture growth

Clonal cell lines release glycoproteins into their culture medium, some of which appear to be derived from the outer cell surface. These proteins do not originate from lysed cells, nor do they comigrate on SDS acrylamide gels with the proteins of substrate attached material. When the proteins released from exponentially dividing cells are compared with those from stationary phase cells, marked differences are found. In addition, the proteins released from normal stationary cells differ from those precociously growth arrested with db-cAMP or by serum deprivation. The spectrum of proteins released by the serum-deprived cells is more like that of normal stationary phase cells than db-cAMP-inhibited cells.     

Superoxide dismutase activity in nerve cell culture

Superoxide dismutase (EC 1.15.1.1) activity was assayed in preparations obtained from several clonal lines of nerve cell culture, by enzymatic and nonenzymatic assays. The enzyme was found in dialyzed homogenates of washed cells and in partially purified fractions. The enzyme was also found in cells which had been grown in media containing 5-bromodeoxyuridine, where cell differentiation was observed.     

Leaf cell water and enzyme activity

This work supports further the thesis that under conditions of water stress, cell water content may supersede hormonal regulation in effecting enzyme activity, thus becoming a regulatory factor in cellular metabolism. Addition of NaCl to the root medium of barley plants (Hordeum vulgare L.) markedly increased leaf RNase activity parallel to an increase of leaf water saturation deficit (WSD). Kinetin and abscisic acid, applied to the salinated plants, also modified RNase activity, as well as leaf-WSD. The familiar pattern of effects of these hormones on leaf RNase as well as leaf chlorophyll content was inverted, kinetin effected a relative increase in RNase activity and a decrease in leaf chlorophyll, whereas abscisic acid effected a relative decrease in RNase activity and maintained chlorophyll content. A close relationship between enzyme activity and leaf WSD became evident when leaf RNase and protease activities in the salinated plants were plotted against leaf WSD. This close relationship was maintained irrespective of the hormonal treatments, which in themselves markedly modified leaf WSD. As predicted, high relative humidity which relived the leaves from salt-induced water stress prevented the salt-induced rise in RNase activity.     

Some factors controlling cell polarity in chick retinal pigment epithelial cells in clonal culture

In clonal culture differentiated chick retinal pigmented epithelial (RPE) cells form a monolayer which shows little or no cellular division. The cells usually rest on a basal and reticular lamina and are polarized with their apical surface towards the medium. The apical surface is characterized by apical protrusions, an extensive apical web of microfilaments and junctional complexes which join the apical-lateral borders. A PA/S positive material with a felt-like appearance from the serum component of the medium coats the surfaces of the tissue culture plates. A similar material is found on any membrane filter which has been exposed to medium containing serum. When such a filter brought in contact with the upper surfaces of the RPE cells, the apical surface characteristics are lost, the cells often accumulate Alcian Blue positive material between the cells and the filter and secrete a reticular and a basal lamina, i.e. they establish a second basal surface. Once this has occurred, the cells appear to either detach from the plate and reverse their polarity, or undergo division forming two cell layers. In the latter case new apical surfaces are created between the cell layers but the cells appear to join to form circular structures rather than sheets. These results suggest that contact with this felt-like material initiates formation of a basal surface. They further suggest that where the apical surface has been converted to a basal one the cell attempts to restore the apical surface either by separating from the plate and reversing its polarity or by creating circular structures and developing new apices oriented toward the center of the circle.     

Estimating the life-span of oligodendrocytes from clonal data on their development in cell culture

This paper presents a new method to analyze clonal data on oligodendrocyte development in cell culture. The process of oligodendrocyte generation from precursor cells is modelled as a multi-type Bellman-Harris branching process as suggested in an earlier paper [K. Boucher, A. Zorin, A.Y. Yakovlev, M. Mayer-Proschel, M. Noble, An alternative stochastic model of generation of oligodendrocytes in cell culture, J. Math. Biol. 43 (2001) 22]. This model has been extended to allow for death of oligodendrocytes as well as a dissimilar distribution of the first mitotic cycle duration as compared to the subsequent cycles of precursor cells, which lengths are assumed to be independent and identically distributed random variables. Since the time-span of oligodendrocytes is not directly observable in clonal data, plausible parametric assumptions are invoked to make estimation problems tractable. In particular, the time to cell death follows a two-parameter gamma distribution, while the lapse of time between the event of cell death and the event of cell disintegration is assumed to be exponentially distributed. A simulated pseudo maximum likelihood method for estimation of model parameters has been developed using simulation-based approximations of the expected numbers and variance-covariance matrices for different types of cells. Finite sample properties of the estimation procedure are studied by computer simulations. The proposed method is illustrated with an analysis of the clonal development of O-2A progenitor cells isolated from the rat optic nerve and the corpus callosum.     

Phase II enzyme induction by a carotenoid,lutein, in a PC12D neuronal cell line

The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.     

A combined Bodian-Nissl stain for improved network analysis in neuronal cell culture

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.