Calmodulin-Binding Proteins in Human Y-79 Retinoblastoma and HTB-14 Glioma Cell Lines

The Y-79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial-like (glial fibrillary acidic protein positive) or neuron-like (neuron-specific enolase-positive) cells. To determine whether Y-79 retinoblastoma cells express neuronotypic calmodulin-binding proteins, Y-79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum-containing medium or were maintained in serum-free media. Using a biotinylated calmodulin blot overlay technique, we found that Y-79 cells treated with dbcAMP or butyrate expressed low levels of membrane-bound calmodulin-binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate-treated cells also expressed a calmodulin-binding peptide of 135 kDa. Since butyrate treatment of Y-79 cells induces the expression and the secretion of interphotoreceptor retinoid-binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin-binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135-kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial-like characteristics in Y-79 cells, we compared the calmodulin-binding protein pattern in these cells with that seen in human HTB-14 glioma cells. The HTB-14 line did not express calmodulin-binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y-79 cells express membrane-bound calmodulin-binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB-14 glioma cells.     

RNA synthesis and coding capacity of polyadenylated and nonpolyadenylated mRNA from cultures of differentiating Drosophila melanogaster myoblasts

We have investigated the synthesis and coding capacity of RNA isolated from cultures of differentiating Drosophila embryonic muscle cells. We find that following muscle cell fusion, the sedimentation profile of newly synthesized polyadenylated RNA becomes somewhat lighter. In vitro translation products analyzed by two-dimensional gel electrophoresis indicate that the coding capacity of translatable myogenic mRNA changes during differentiation. A group of several muscle-specific proteins (including the contractile proteins) is translated only from mRNA isolated after the initiation of fusion. This pattern coincides with proteins synthesized in vivo during differentiation. Additionally, we find that polyadenylated and nonpolyadenylated myogenic mRNA from a given developmental stage in culture have extremely similar coding potentials.     

Effects of Dietary n-3 Fatty Acids on the Phospholipid Molecular Species of Monkey Brain

Y-79 human retinoblastoma cells grown in serum-free medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y-79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y-79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of approximately 3 fmol/10(6) cells (approximately 1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly-D-lysine and fibronectin and continued culture in serum-free medium. After 8 days in serum-free culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/10(6) cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15-17 days (7-9 days of Bt2cAMP treatment) expressing receptor levels of 15-20 fmol/10(6) cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y-79 (dY-79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM-09151-2 approximately spiperone greater than domperidone approximately (+)-butaclamol approximately fluphenazine greater than chlorpromazine greater than (-)-sulpiride greater than (+)-sulpiride greater than promethazine greater than (+)-SCH 23390 much greater than (-)-butaclamol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

Recoverin in Cultured Human Retinoblastoma Cells: Enhanced Expression During Morphological Differentiation

Abstract: Recoverin is a calcium-binding protein expressed in retinal photoreceptors. It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin. The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina. A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein. Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate. Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents. In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 m M butyrate to the culture medium. Butyrate treatment also caused an increase in the development of neurite-like cellular processes. Addition of 4 m M dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes. Western and northern blots of butyrate and dbcAMP-treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry. These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.     

Changes in translatable mRNAs during the larval-pupal transformation of the epidermis of the tobacco hornworm

At the initiation of metamorphosis when exposed to ecdysteroid in the absence of juvenile hormone (JH), the lepidopteran epidermis changes its commitment from one for larval differentiation to one for pupal differentiation. Changes in mRNA populations during this change both in vivo and in vitro were followed by a one-dimensional SDS-gel electrophoretic analysis of translation products made in a mRNA-dependent rabbit reticulocyte lysate system. The larval epidermal cell was found to lose its translatable mRNAs for larval cuticular proteins and the larval-specific pigment insecticyanin during the change in commitment; these never reappeared. For Class I cuticular proteins and for insecticyanin, this loss occurred during the exposure to ecdysteroid, each with a differing time course. By contrast, Class II cuticular mRNAs first increased during this time, then also disappeared by the time the cells were pupally committed. In vitro these mRNAs appeared in only trace amounts in response to 20-hydroxyecdysone (20-HE). The pupally committed cell (late in the wandering stage) contained mRNAs for three low-molecular-weight proteins which were precipitable with the pupal cuticular antiserum. The remainder of the pupal cuticular mRNAs were not translatable until the third day after wandering, a time when pupal cuticle is being deposited in response to a molting surge of ecdysteroid. The pupally committed cell also had at least one new noncuticular mRNA which coded for a 34K protein and which was absent from both larval and pupal epidermal cells making cuticle. Since its appearance in response to 20-HE in vitro is repressed by JH, it is called a pupal commitment-specific protein. Thus, during the change of commitment 20-HE inactivates larval-specific genes irreversibly in a sequential cascade of events. The activation of most pupal-specific genes then requires a subsequent exposure to more ecdysteroid.     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

Laminin: An initiating role in retinoblastoma cell attachment and differentiation

Summary The effect of laminin (LN) on the attachment and differentiation of a human retinoblastoma cell line (Y-79) was investigated. We found that 10 μg/ml LN in the serum-free culture medium for 3 to 4 d induces 20 to 30% of the cells to firmly attach to a plastic substratum. This effect seems to be complex because the short-term effect of LN is inhibition of cell attachment. The specificity of LN may be indicated because antilaminin antibody counteracted this effect. The attached cells form small processes immediately after attachment and continue to proliferate, forming small colonies. Treatment of these cells with 4 mM dibutyryl-cyclic AMP (db-cAMP) or 2 mM sodium butyrate starting on the 3rd or 4th d of culture results in extensive differentiation of all the attached cells. Db-cAMP provokes the formation of long ramifying neuritelike processes whereas butyrate induces the appearance of epithelial-like cells with a flat morphology. Thus, laminin seems to act in concert with other agents promoting attachment and potentiating differentiation.     

Butyrate inhibits the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.     

Messenger RNA coding for glutamine synthetase in cerebral hemispheres and astroglial cultures from mouse brain: A developmental study

Messenger RNAs from mouse brain hemispheres and from an enriched astroglial population of the same area were isolated, characterized and used to study Glutamine Synthetase (GS) processing during postnatal development using a m-RNA stimulated reticulocyte lysate system.The RNA preparations yielded distinct polypeptide products upon translation including high molecular weight species. Polypeptides in the range of 43–55 kDa appeared developmentally regulated in brain hemispheres but not in astroglia. After immunoprecipitation of the translation products with a GS antibody a major monomeric polypeptide was isolated on SDS/PAGE which migrated at the same position as the purified brain GS antigen (43 kDa).The translatable mRNA were optimal in the perinatal period and decreased until 300 days while GS-mRNA increased during the same period of time and closely paralleled the previously described GS activity profile in this brain area reaching an optimum at 15 days. Astroglial mRNAs were optimal at 18 days in vitro and decreased thereafter. The GS-mRNA was much lower in control astroglial cultures than in brain tissue, but in the presence of 10⁻⁶M hydrocortisone increased all over the growth period. The highest stimulation of GS-mRNA was observed at 18 days whereas the global mRNA decreased in the presence of the hormone.The GS-mRNAs from either 15-day-old brain hemispheres or 18-day in vitro hydrocortisone stimulated cultures were partially purified on a 5–30% linear sucrose gradient. Two GS-mRNAs, which sedimented respectively at 17S and 23S, were characterized. In addition, based on the profile of total proteins translated in vitro, we estimated that GS-mRNA constituted 0.01% of the brain hemisphere fraction and 0.3% in the astroglial hormone stimulated cells.     

Effects of cadmium on gene expression in cadmium-tolerant and cadmium-sensitiveDatura innoxia cells

The effect of Cd on gene expression in suspension cultures of twoDatura innoxia cell lines with differing Cd tolerance was studied.In vivo labeling experiments using [³H] leucine showed that Cd induced the synthesis of a similar range of proteins in both cell lines at a concentration which will kill the sensitive but not the tolerant cells. Corresponding changes in levels of translatable mRNA were also observed. The induction of the synthesis of proteins by Cd was transient since Cd-tolerant cells growing continuously in 250 M CdCl₂ contained a similar set ofin vitro translation products to cells growing in the absence of Cd. Although Cd had a similar effect on gene expression in both cell lines, Cd-tolerant cells possess two abundant mRNAs which are constitutively produced. These mRNAs encode proteins of low molecular weight (about 11 kDa) and are either absent or present at a low level in Cd-sensitive cells. The functions of these proteins are not known but they may be involved in the tolerance mechanism. Two-dimensional gel electrophoresis ofin vitro translation products showed that many of the Cd-induced proteins are also induced by heat shock. A 42°C heat shock resulted in agreater range and more intense induction of translatable mRNAs than 4 h exposure to 250 M CdCl₂. However a subset of mRNAs were induced specifically by Cd while other mRNAs were heat shock-specific. There was no difference in the ability of the two cell lines to tolerate heat shock. This was also reflected by the same pattern of major proteins induced by heat shock in the two cell lines.     

Neurochemical and morphological studies on differentiation of NG108-15 cells by phorbol ester and forskolin

12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin or dibutyryl cAMP induced neurite outgrowth and inhibition of cell growth in NG108-15 cells. TPA, forskolin and dibutyryl cAMP significantly increased specific activity of choline acetyltransferase. Forskolin markedly stimulated cAMP accumulation, but not TPA, suggesting that forskolin could induce differentiation by increasing the cAMP content via adenylate cyclase activation, but TPA-induced differentiation seems not to be due to the raise of the cAMP level. Incubation of the cells with TPA, forskolin or dibutyryl cAMP for 24 h resulted in enhancement of 50 mM K⁺-evoked Ca²⁺ influx and neurite elongation, although incubation with these agents for 1 h didn't affect these events. From these results, it is suggested that TPA and forskolin induce differentiation of NG108-15 cells to acetylcholine neurons via different mechanisms: protein kinase C activation by TPA and cAMP-dependent protein kinase activation by forskolin. In addition, it is likely that Ca²⁺ channels in cells differentiated by TPA, forskolin or dibutyryl cAMP become sensitive to depolarization.     

Calmodulin mRNA in Barley (Hordeum vulgare L.) : Apparent Regulation by Cell Proliferation and Light

Calmodulin is encoded by a 650-nucleotide mRNA in higher plants. This messenger was identified in barley and pea by a combination of in vitro translation and blot hybridization experiments using anti-sense RNA produced from an eel calmodulin cDNA probe. In all plant tissues tested, calmodulin mRNA represents between 0.01 and 0.1% of the total translatable mRNA population. Calmodulin mRNA levels are three- to fourfold higher in the meristematic zone of the first leaf of barley. At all other stages of leaf cell differentiation, calmodulin mRNA levels are nearly identical. During light-induced development in barley leaves, the relative proportion of translatable calmodulin mRNA declines about twofold. Cytoplasmic mRNAs that may encode calmodulin-like proteins were also detected. The levels of several of these putative Ca²⁺-binding protein mRNAs are modulated during the course of light-induced barley leaf cell development.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis

The rate of protein synthesis in unfertilized sea urchin eggs is very low, although all components for protein synthesis are present. To determine whether egg messenger RNAs are unavailable for translation because of “masking” by phenol-soluble inhibitors, crude and purified nonpolyribosomal messenger ribonucleoprotein complexes (mRNPs) from eggs of Strongylocentrotus purpuratus were translated in vitro in a wheat germ cell-free system. Crude and purified egg mRNPs were nearly as translatable as the mRNAs extracted from the mRNPs, suggesting that the mRNPs were not masked. No difference in the relative translational activities of mRNPs and their constituent mRNAs was revealed by isolating the mRNPs in buffers of different ionic strength or in the presence of protease and ribonuclease inhibitors. Furthermore, kinetic analysis of the in vitro translation and translation of the mRNPs and mRNAs at several concentrations of K⁺, Mg²⁺, and template all indicate that mRNPs are efficient templates for directing protein synthesis. Separation on polyacrylamide gels of the products of in vitro and in vivo translation demonstrated that both mRNPs and mRNAs extracted from the mRNPs synthesized in vitro high-molecular-weight polypeptides, some of which were also synthesized in vivo. Although sea urchin egg mRNPs may not be masked, there are several alternative mechanisms for regulating translation in the egg.     

Induction of neural-like cells and acetylcholinesterase activity in cultures of F9 teratocarcinoma treated with retinoic acid and dibutyryl cyclic adenosine monophosphate

Cultures of F9 embryonal carcinoma cells treated with retinoic acid showed partial differentiation to endoderm cells as previously reported [Strickland, S., and Mahdavi, V. (1978).Cell15, 393–403]. Addition of dibutyryl cAMP to cultures pretreated with retinoic acid led to a second distinctive change in the cell population, with the formation of many neural-like cells. The appearance of these cells coincided with large increases in specific acetylcholinesterase activity of the cultures. Provided the cultures had been exposed to retinoic acid for at least 48 hr beforehand, the morphological and enzymatic changes became apparent between 24 and 48 hr after the addition of dibutyryl cAMP. The changes proceeded more abruptly and extensively when cells were grown in nongelatinized culture dishes. On gelatin-coated surfaces, the differentiated cells occasionally showed local areas of ordered arrangements. It is suggested that this system may be useful in analyzing early events in neural differentiation.     

Effect of sodium butyrate on mammalian cells in culture: a review.

Sodium butyrate produces reversible changes in morphology, growth rate, and enzyme activities of several mammalian cell types in culture. Some of these changes are similar to those produced by agents which increase the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) or by analogs of cAMP. Sodium butyrate increases the intracellular level of cAMP by about two fold in neuroblastoma cells; therefore, some of the effects of sodium butyrate on these cells may in part be mediated by cAMP. Sodium butyrate appears to have properties of a good chemotherapeutic agent for neuroblastoma tumors because the treatment of neuroblastoma cells in culture causes cell death and "differentiation"; however, it is either innocuous or produces reversible morphological and biochemical alterations in other cell types.     

Biosynthesis of nonspecific lipid transfer proteins in germinating castor bean seeds

The biosynthesis of nonspecific lipid transfer proteins (ns-LTPs) in germinating castor bean (Ricinus communis L.) seeds were investigated. Lipid transfer activities of ns-LTPs in the cotyledons, axis, and endosperm increased with growth after germination. The activity increases were accompanied by increased amounts of ns-LTPs in each tissue, as measured by immunoblot using anti-ns-LTP serum. These results suggest that the ns-LTPs are synthesized de novo in each tissue after germination and not activated from inactive proteins synthesized before germination. Comparison of the immunoblot products in each tissue from 4-day-old seedlings indicate the occurrence of tissue-specific isoforms of ns-LTPs; 9 kilodaltons (major) and 7 kilodaltons (minor) in the cotyledons, and 7 kilodaltons (major) and 9 kilodaltons (minor) in the axis, whereas only the 8-kilodalton ns-LTP is present in the endosperm. In vitro translation from poly(A)⁺ RNAs from three tissues of castor bean seedlings and the detection of immunoprecipitated products indicate that translatable mRNAs for ns-LTPs exist in the three tissues a day before the synthesis of ns-LTPs; the translation products, which are 3.5 to 4.0 kilodaltons larger than ns-LTPs, were processed to the mature ns-LTPs. The production of mature ns-LTPs from translatable mRNAs without any delay suggests that gene expression of ns-LTPs in castor bean seedlings is controlled at a step before the formation of translatable mRNAs.