Quantitative IR studies of acyl chain conformational order in fatty acid homogeneous membranes of live cells of Acholeplasma laidlawii B.

Acholeplasma laidlawii B has been grown highly enriched in myristic, pentadecanoic, and palmitic acids. The conformational order in the acyl chains of living cell membranes has been compared with that in the membranes of lysed cells and the lipids extracted therefrom, using Fourier-transform infrared (FT-IR) spectroscopic techniques. A gel-liquid-crystal-phase transition (25-45 degrees C) was noted for both the live cells and the membranes. Surprisingly, CH2 wagging progressions, characteristic of coupled oscillators from acyl chains in the all-trans conformation, were detected in the live cells and in the cell membrane. A simple model suggested the presence of 1.7 gauche bonds/chain at the growth temperature (37 degrees C) in each case. Conformational order in the live cells and in the membranes was virtually identical over the range of cell viability (5-40 degrees C), as measured by the thermotropic responses of the k = 1 and k = 2 components of the wagging mode progression. This result was confirmed by studies of the thermotropic response of the symmetric CH2 stretching vibrations, a qualitative index of acyl chain order. In contrast, the membrane lipid extracts (i) show much more conformational disorder from 5 to 25 degrees C than either the live cells or the membranes, (ii) undergo a gel-liquid transition over a broader temperature range and with a reduced magnitude of change in the symmetric CH2 stretching frequency, and (iii) demonstrate a second transition centered at 50 degrees C, which is detected by a large increase in the localized CH2 wagging mode (1368 cm-1) that arises from conformationally disordered kink+gtg states.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement of the cell body away from the base of a process. In differentiated cells, PMA induced the rapid extension of small lamellas and subsequent formation of short-lived elongated processes from the lateral edges of neurites. Additionally, growth cones exhibited enhanced modulation in shape after PMA treatment. These reversible reorganizations were similar to the actinoplast-tubuloplast transformations exhibited by PMA-treated fibroblasts. We suggest that actinoplast-tubuloplast reorganizations play essential roles in morphogenesis where stable cytoplasmic extensions are induced by external stimuli. In particular, PMA-induced reorganizations of neural cells in culture may be a model for morphological modulations that occur in nerve tissue.

Inositol metabolism during neuroblastoma B50 cell differentiation: effects of differentiating agents on inositol uptake.

Inositol uptake was studied in the rat CNS neuroblastoma B50 cell line. Eadie-Hofstee analysis of the uptake pattern reveals two defined modes of inositol entry into the cell. The high-affinity uptake component requires the presence of extracellular sodium and is inhibited by phloridzin. Analysis of the uptake velocities of the high-affinity uptake component provided the following apparent kinetic parameters: Km = 13.7 microM and Vmax = 14.7 pmol/mg of protein/min (without correcting for residual diffusion) and Km = 12.9 microM and Vmax = 12.3 pmol/mg of protein/min (with correction). At physiological concentrations, the high-affinity transport process contributes approximately 70% to total uptake; the remainder is due to a low-affinity diffusion-like process. Uptake inhibition studies reveal that the uptake process is sensitive to ouabain, amiloride, and dichlorobenzamil inhibition but relatively insensitive to cytochalasin B or phloretin. When neuroblastoma B50 cells are induced to differentiate morphologically with high extracellular calcium or with dibutyryl cyclic AMP, a significant decrease in inositol uptake is observed. The dibutyryl cyclic AMP-mediated inhibition of uptake affects only the high-affinity uptake component and is noncompetitive in nature. The high extracellular calcium-mediated inhibition is less specific; it involves "disappearance" of the high-affinity process, some inhibition of the low-affinity process, and an increase of inositol efflux. The significance of these observations is discussed in the context of neuroblastoma B50 cell differentiation.

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei.

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.

Multiple types of neurotransmitter binding sites in the rat neuroblastoma B 50 cell line. I. Characterization and binding affinity changes during various differentiation paradigms.

In this study, it is shown that the neuroblastoma B 50 cell line of rat central nervous system origin possesses both adrenergic and cholinergic binding sites. The adrenergic binding site population comprises a major alpha 1-adrenergic component (using [3H]prazosin as ligand; KD = 0.55 nM; 19.3 fmol/mg protein), and a minor beta-adrenergic species (KD = 0.45 nM; 3.4 fmol/mg protein using [125I]cyanopindolol as ligand). The cholinergic binding site is of a muscarinic type. Binding studies with quinuclidinyl benzylate (QNB) indicate the presence of two binding sites with different affinities. This is supported by competition studies with agonists. However, in view of this unusual binding behavior of the antagonist, the possibility of a one-site model for QNB is also considered and affords the following parameters: KD = 0.247 nM, Bmax = 29.6 fmoles/mg protein. When the cells are induced to differentiate with dibutyryl cyclic AMP (db-cAMP) or high extracellular calcium, all of these binding sites undergo a variety of changes in their affinities for their respective ligands. Db-cAMP increases the affinity of the alpha 1-site for prazosin while high extracellular calcium lowers it. In both cases the number of binding sites remains unchanged. The affinity of the beta-adrenergic component is markedly enhanced upon induction of differentiation with either extracellular calcium or/and db-cAMP. Finally, the muscarinic cholinergic binding sites exhibit a decrease in receptor number upon db-cAMP treatment and an apparent loss of one of the binding sites upon calcium treatment. The significance of these affinity changes is discussed for each type of binding site.

Multiple types of neurotransmitter binding sites in the rat neuroblastoma B 50 cell line. II. Response of second messenger systems to physiological stimuli in proliferating and differentiated cells.

The functionality of the alpha 1-, beta-adrenergic and muscarinic cholinergic binding sites of neuroblastoma B 50 is investigated under proliferating and differentiating conditions. In proliferating cells, the stimulation of the alpha 1-adrenergic and muscarinic cholinergic binding sites by their respective agonists causes an increase in both extracellular calcium association with the cells and phosphatidylinositol (PI) turnover; effects usually associated with functional receptors. When the cells are induced to differentiate morphologically with dibutyryl cyclic AMP (db-cAMP), extracellular calcium or a combination of both, the activity of the muscarinic receptor-coupled PI turnover is strictly correlated with the binding affinity of the receptor. This is not the case for the alpha 1-adrenergic receptor stimulation of PI turnover. The latter result, however, may be explained in terms of the intrinsic properties of the inducing agents used to cause neurite extension. The stimulation of the beta-adrenergic binding site with isoproterenol in proliferating cells, both with and without a phosphodiesterase inhibitor present, does not result in cellular cAMP accumulation. In morphologically differentiated cells, only the db-cAMP-induced state exhibits an increase in [3H]adenosine incorporation into cellular cAMP upon isoproterenol stimulation. This happens only in the presence of a phosphodiesterase inhibitor. The data presented in this study are discussed in terms of the affinity of the receptors for their respective ligands and in terms of the intrinsic properties of the inducing agents.

Extracellular calcium-induced neuroblastoma cell differentiation: involvement of phosphatidylinositol turnover.

The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions; this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.

Chemical inducers of differentiation cause conformational changes in the chromatin and deoxyribonucleic acid of murine erythroleukemia cells.

The chemical inducers of murine erythroleukemia cell differentiation, dimethyl sulfoxide, sodium butyrate, and ethionine, elicited conformational changes in the DNA and chromatin of treated cells. The chromatin from dimethyl sulfoxide treated and butyrate-treated cells exhibited circular dichroic spectra different from that of the noninduced control. The molar ellipticity [theta]282.5 in isotonic saline decreased from 4900 deg X cm2 X dmol-1 for control chromatin to 3800 and 3600 deg X cm2 X dmol-1 for dimethyl sulfoxide treated and butyrate-treated chromatin, respectively, while that from ethionine-treated chromatin remained virtually unchanged (5400 deg X cm2 X dmol-1). Increasing the ionic strength to 2.5 or 5 M with NaCl resulted in a substantial, uniform, decrease in molar ellipticity. Thermal denaturation profiles of high molecular weight DNA prepared from each of the inducer-treated cells showed a pronounced hyperchromic shift but no change in Tm when compared to control DNA. Circular dichroic spectra of the DNA indicated a decrease in ellipticity [theta]277 from 9600 deg X cm2 X dmol-1 to 8900, 8300, and 8800 deg X cm2 X dmol-1 for ethionine, dimethyl sulfoxide, and butyrate treated cells, respectively. Treatment of the DNA with 3 M NaCl canceled the UV and CD differences. These measurements indicate an increased stacking of bases or an increased compactness of the DNA from induced cells. Concomitant with specific modifications such as hypomethylation of DNA, the data can be interpreted in terms of conformational changes in chromatin resulting from core histone acetylation.

Maintenance of DNA integrity during murine erythroleukemia cell differentiation induced by ethionine and other hypomethylation agents.

The extent of single-strand nicks in DNA from murine erythroleukemia cells induced to differentiate to hemoglobin synthesis in the presence of the hypomethylating agent ethionine was estimated and compared to those levels in uninduced cells and from cells induced to differentiate upon exposure to dimethylsulfoxide or butyrate ion. Although ethionine has been shown to cause more extensive hypomethylation in the DNA of induced cells than that caused by dimethylsulfoxide or butyrate ion, the frequency of detected single-strand breaks in the DNA of uninduced, control cells was not significantly different from that of cells exposed to any of these inducing chemicals. This data indicates that no correlation exists between DNA hypomethylation and DNA single-strand breaks and that unmethylated CpG loci likely do not operate as specific endonuclease recognition sites or as potential origins of transcription in these mammalian cells.