Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

Antioxidant Effect of Phenelzine on MPP+-Induced Cell Viability Loss in Differentiated PC12 Cells

Phenelzine, deprenyl, and antioxidants (SOD, catalase, ascorbate, or rutin) reduced the loss of cell viability in differentiated PC12 cells treated with 250 M MPP⁺, whereas N-acetylcysteine and dithiothreitol did not inhibit cell death. Phenelzine reduced the condensation and fragmentation of nuclei caused by MPP⁺ in PC12 cells. Phenelzine and deprenyl prevented the MPP⁺-induced decrease in mitochondrial membrane potential, cytochrome c release, formation of reactive oxygen species, and depletion of GSH in PC12 cells. Phenelzine revealed a scavenging action on hydrogen peroxide and reduced the hydrogen peroxide–induced cell death in PC12 cells, whereas deprenyl did not depress the cytotoxic effect of hydrogen peroxide. Both compounds reduced the iron and EDTA-mediated degradation of 2-deoxy-d-ribose degradation. The results suggest that phenelzine attenuates the MPP⁺-induced viability loss in PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.     

Botulinum toxin a inhibits acetylcholine release from cultured neurons in vitro

Summary Clostridium botulinum type toxin A (BoTx) blocks stimulus-induced acetylcholine (ACh) release from presynaptic nerve terminals at peripheral neuromuscular junctions. However, the detailed mechanism of this effect remains elusive. One obstacle in solving this problem is the lack of a suitable in vitro homogenous cholinergic neuronal model system. We studied the clonal pheochromocytoma PC12 cell line to establish such a model. PC12 cells were differentiated in culture by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release properties. Stimulation of these cells with high K⁺ (80 mM) in the perfusion medium markedly increased calcium-dependent [³H]ACh release compared to undifferentiated cells. Stimulated [³H]ACh release was totally inhibited by pretreatment of cells with 2 nM BoTx for 2 h. BoTx inhibition of [³H]ACh release was time- and concentration-dependent. A 50% inhibition was obtained after 2 h incubation with a low (0.02 nM) toxin concentration. The time required for 2 nM BoTx to cause a measurable inhibition (18%) of stimulated [³H]ACh release was 30 min. Botulinum toxin inhibition of stimulated ACh release was prevented by toxin antiserum and heat treatment, suggesting the specificity of the toxin effect. Our results show that by differentiation with NGF, PC12 cells can be shifted from an insensitive to a sensitive state with respect to BoTx inhibition of stimulated ACh release. This cell line, therefore, may serve as a valuable in vitro cholinergic model system to study the mechanism of action of BoTx.     

Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells

Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC₅₀ of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC₅₀, 11 μM) and dehydroascorbate (EC₅₀, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote-olysis of the purified enzyme as determined by sodium do-decyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions.     

Differentiation-dependent sensitivity to apoptogenic factors in PC12 cells

We have investigated the role of the mitochondrial pathway during cell death following serum and nerve growth factor (NGF)/dibutyryl cyclic AMP (Bt(2)cAMP) withdrawal in undifferentiated or NGF/Bt(2)cAMP-differentiated PC12 cells, respectively. Holocytochrome c, Smac/DIABLO, and Omi/HtrA2 are released rapidly following trophic factor deprivation in PC12 cells. Bcl-2 and Akt inhibited this release. The protection, however, persisted longer in differentiated PC12 cells. In differentiated, but not undifferentiated cells, Bcl-2 and Akt also inhibited apoptosis downstream of holocytochrome c release. Thus, undifferentiated PC12 cells showed marked sensitivity to induction of apoptosis by microinjected cytochrome c even in the presence of NGF, Bcl-2, or Akt. In contrast, in differentiated cells these factors suppressed cell death. Consistent with these observations, in vitro processing of procaspase 9 in response to cytochrome c was observed in extracts from undifferentiated but not differentiated cells expressing Akt or Bcl-2. Endogenous caspase 9 was cleaved during cell death, whereas dominant negative caspase 9 inhibited cell death. The results from determining the role of inhibitors of apoptosis (IAPs) suggest that acquisition of inhibition by IAPs is part of the differentiation program. Ubiquitin-DeltaN-AVPI Smac/DIABLO induced cell death in differentiated cells only. c-IAP-2 is unregulated in differentiated cells, whereas X-linked IAP levels decreased in these cells coincident with cell death. Moreover, expressing X-linked IAP rendered undifferentiated cells resistant to microinjected cytochrome c. Overall, the inhibitory regulation, of cell death at the level of release of mitochondrial apoptogenic factors and at post-mitochondrial activation of caspase 9 observed in differentiated PC12 cells, is reduced or absent in the undifferentiated counterparts.     

Depletion and recovery of ATP in V79 cells with exposure to inhibitors of glycolysis and oxidative phosphorylation

Summary Cells of the cultured hamster cell line V79 were labeled with tritiated adenosine and incubated for up to 30 min in the presence of inhibitors of glycolysis and oxidative phosphorylation. These inhibitors were (a) 5 mM KCN plus 5 mM iodoacetate, (b) 5 mM KCN plus 5 mM KF, and (c) 15 mM KCN plus 15 mM KF. The fate of the tritium label was examined during incubation with inhibitors and also during subsequent incubation in growth medium in the absence of inhibitors. The tritiated ATP pool was found to decrease in cells incubated in the presence of any of the inhibitor combinations, but only in the presence of 15 mM KCN plus 15 mM KF was this pool decreased below the level of detection. After cells were incubated with KCN plus KF, a high level of ATP was recovered when the inhibitors were removed. Cells incubated with KCN plus iodoacetate retained depletion levels of ATP. Plating efficiency and trypan blue staining showed that KCN-KF treated cells retained viability, whereas KCN-iodoacetate treated cells did not. Cells were examined for ability to take up tritiated uridine before, during, and after depletion of ATP by incubation in the presence of 15 mM KCN plus 15 mM KF. These cells were found to have a variation in uridine uptake that was related directly to intracellular ATP level. Cells in which the ATP was very low exhibited little or no uridine uptake, whereas cells in which the ATP level was near normal exhibited normal uridine uptake. This work was supported in part by Grant GM24271 from the National Institutes of Health, Bethesda, Maryland.     

Induction of peroxisome proliferation and increase of catalase activity in yeast,Candida albicans,by cadmium

The effects of cadmium on the growth rate, catalase activity, and peroxisome proliferation in yeast,Candida albicans, were evaluated. The yeast growth was markedly inhibited by 1 mM cadmium at the initial hours. The toxic effect of cadmium on the cell growth persisted. The catalase activity of the cells treated with 1 mM Cd²⁺ first decreased, and then rose at 24 h to about 2.6 times that of the controls. The average number of peroxisomes per cell in the yeast treated with 1 mM Cd²⁺ was about sixfold higher than the control groups. The proliferation of peroxisomes and the increase of catalase activity following cadmium toxicity gives credence to the hypothesis that cadmium toxicity is related to its potential to induce oxidative stress in cells.     

Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells

Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.     

In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process

The fate of alcohol oxidase (AO) in chemostatgrown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the release of flavin adenine dinucleotide (FAD) from the holo enzyme. However, dissociation of octameric AO into subunits was not observed. Subsequent growth of intact cyanide-treated cells in fresh methanol media was paralelled by proteolytic degradation of part of the peroxisomes present in the cells. The recovery of AO activity, concurrently observed in these cultures, was accounted for by synthesis of new enzyme protein. Reactivation of previously inactivated AO was not observed, even in the presence of FAD in such cultures. Newly synthesized AO protein was incorporated in only few of the peroxisomes present in the cells. ³¹P nuclear magnetic resonance (NMR) studies showed that cyanide-treatment of the cells led to a dissipation of the pH gradient across the peroxisomal membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.Abbreviations AO alcohol oxidase - FAD flavin adenine dinucleotide - CHI cycloheximide - NMR nuclear magnetic resonance - FPLC fast protein liquid chromatography - RIE rocket immuno electrophoresis     

SHORT-TERM REGULATION OF CATECHOLAMINE BIOSYNTHESIS IN A NERVE GROWTH FACTOR RESPONSIVE CLONAL LINE OF RAT PHEOCHROMOCYTOMA CELLS

Abstract— A clonal cell line (designated PC12) has been previously established from a transplantable rat adrenal medullary pheochromocytoma. Tissue cultures of PC12 cells synthesize, store, release and take up catecholamines. PC12 cells also respond to nerve growth factor (NGF) protein by cessation of mitosis and extension of neurites. The present studies concern the comparison of several aspects of catecholamine metabolism in PC12 cultures with that in normal noradrenergic tissues. One question was why the ratio of dopamine to norepinephrine in PC12 cultures (in contrast to that in normal noradrenergic tissue) is considerably more than one. The presence of exogenous reduced ascorbate (a cofactor for dopamine-β-monooxygenase) enhanced by 5–10-fold the rate at which PC12 cultures converted [³H]tyrosine to [³H]norepinephrine. Under such conditions, the rate of synthesis of [³H]do-pamine was unchanged. It was also found that the ratio of norepinephrine to dopamine increased by 10-fold when the cells were grown in vivo as tumors. Since tissue culture medium is essentially free of reduced ascorbate, it is likely that the absence of this cofactor is responsible for the low norepinephrine to dopamine ratio in PC12 cultures. Experiments were also carried out on short-term regulation of catecholamine synthesis in PC12 cultures. These studies revealed the following: (1) The rate of conversion of [³H]tyrosine to [³H]catechols was increased 2–3-fold (as compared with controls) in the presence of depolarizing levels of K⁺ (51.5 mM), and by 2-fold in the presence of 0.5–2 mM-dibutyryl cyclic adenosine 3′, 5’monophosphoric acid (db-cAMP). (2) Similar increases occurred in cultures which had been treated with (and had responded to) nerve growth factor. (3) The stimulatory effects of 51.5 mM-K⁺ rapidly returned toward control levels when the cultures were returned to control medium and (4) required the presence of Ca²⁺ in the extracellular medium. (5) Stimulation of catechol synthesis by 51.5 mM-K⁺ and db-cAMP also occurred in the presence of an inhibitor of DOPA decar-boxylase. Thus, the ultimate effects of these agents were probably at the level of conversion of tyrosine to dopa by tyrosine 3-monooxygenase. (6) Simultaneous exposure of cultures to 51.5 mM-K⁺ and mM-db-cAMP gave additive levels of stimulation. Such findings demonstrate that catecholamine synthesis in cultures of PC12 cells undergoes short-term regulation which is similar to that previously demonstrated in normal monoaminergic tissues. As a homogeneous tissue culture line, the PC12 bears certain advantages for studying the primary mechanisms of such effects.     

Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity

Past studies have shown that serum-free cultures of PC12 cells are a useful model system for studying the neuronal cell death which occurs after neurotrophic factor deprivation. In this experimental paradigm, nerve growth factor (NGF) rescues the cells from death. It is reported here that serum-deprived PC12 cells manifest an endonuclease activity that leads to internucleosomal cleavage of their cellular DNA. This activity is detected within 3 h of serum withdrawal and several hours before any morphological sign of cell degeneration or death. NGF and serum, which promote survival of the cells, inhibit the DNA fragmentation. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases in vitro, suppresses the endonuclease activity and promotes long-term survival of PC12 cells in serum-free cultures. This effect appears to be independent of macromolecular synthesis. In addition, ATA promotes long-term survival of cultured sympathetic neurons after NGF withdrawal. ATA neither promotes nor maintains neurite outgrowth. It is hypothesized that the activation of an endogenous endonuclease could be responsible for neuronal cell death after neurotrophic factor deprivation and that growth factors could promote survival by leading to inhibition of constitutively present endonucleases.     

Steady-state kinetics of the photosystem I reaction in chloroplasts of Dunaliella which contain variable concentrations of plastocyanin

The endogenous plastocyanin (PC) concentrations of Dunaliella cultures were varied from 0.3 to 3.1 molecules per pigment 700 (P700) by decreasing the Cu⁺ supply of the nutrient. With these cultures the amount of PC which is sufficient for maximum photosynthesis in intact cells was determined to be about 1 to 1.5 PC/P700. Chloroplasts were also prepared from these cells and were employed in enzyme kinetic measurements of the PSI reaction from ascorbate reduced diaminodurene (DAD) to methylviologen/O₂. The k _m value for DAD in this reaction was 106 M. A decrease of the endogenous PC concentration caused no change of the k _m value but affected the V _max in the DAD-dependent reaction. A similar interference of the PC concentration on the maximum reaction rate could also be observed when the light intensity was varied.     

Effects of Fetal Bovine Serum on Ferrous Ion-Induced Oxidative Stress in Pheochromocytoma (PC12) Cells

Ferrous ion (Fe²⁺) has been considered to be a cause of neuronal oxidative injury. Since body fluids contain protein and serum is an essential component of tissue culture medium, we have examined the role of serum protein on Fe²⁺-mediated oxidative stress using PC12 cells and rat cerebral cortices. Fe²⁺ or the combination of ascorbate and Fe²⁺ increased concentrations of thiobarbituric acid reactive substances (TBARS) in PC12 cells and cerebrocortical homogenates in medium (RPMI 1640), but did not increase TBARS when the medium was supplemented with 10% fetal bovine serum. Treatment with ascorbate/Fe²⁺ in serum-free medium reduced endogenous glutathione (GSH) concentration in PC12 cells. However, the medium supplemented with serum did not reduce GSH concentrations. PC12 cell death induced by ascorbate/Fe²⁺ was alleviated by increasing serum or bovine albumin concentrations in the medium. These observations indicated that oxidative injury caused by the transition metal ion could be lessened by adding fetal bovine serum to culture medium.     

Isolation and characterization of rat primary lung cells

Summary Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×10⁸ cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC⁵⁰=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC₅₀=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC₅₀=0.1 mM). Paraquat was more toxic to lung cells (EC₅₀=0.03 mM) than to rat (EC₅₀=2.8 mM) and mouse (EC₅₀=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC₅₀=2.6 mM) compared to rat (EC₅₀=0.2 mM) and mouse (EC₅₀=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC₅₀=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC₅₀=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants. Parts of the study had been presented orally at the meeting of the German Society of Toxicology and Pharmacology in Mainz (FRG), March 15–17, 1994.     

Purification and characterization of two ascorbate peroxidases of rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.) expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g^–1 dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min^–1 mg^–1 protein, respectively. The K_m values for ascorbate were 4 and 1 mM, respectively, and those for H₂O₂ were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.Revisions requested 27 September 2004; Revisions received 12 November 2004     

CEP-1347 (KT7515), an Inhibitor of JNK Activation, Rescues Sympathetic Neurons and Neuronally Differentiated PC12 Cells from Death Evoked by three Distinct Insults

The c-Jun N-terminal kinase signaling cascade appears to play a role in some cases of cell death, including neuronal apoptosis. CEP-1347 (KT7515), an indolocarbazole of the K252a family, blocks this stress signaling cascade and promotes survival. Here, we used CEP-1347 to probe whether neuronal death pathways activated by distinct insults also possess elements in common. Cultured rat sympathetic neurons and neuronally differentiated PC12 cells were induced to die by withdrawal of nerve growth factor, exposure to ultraviolet irradiation, or subjection to oxidative stress. In each case, death was prevented by 100-200 nM CEP-1347. Moreover, in each of these death paradigms, c-Jun N-terminal kinase 1 activity in neuronally differentiated PC12 cells was elevated by two- or threefold, and this increase was totally blocked by CEP-1347 at concentrations that promoted survival. In contrast, 200 nM CEP-1347 did not block death due to serum withdrawal from undifferentiated PC12 cells or to activation of Fas in Jurkat T cell cultures, even though in each case c-Jun N-terminal kinase 1 activation occurred and was inhibited by CEP-1347. These observations suggest that some but not all death pathways triggered by different insults can include a common mechanistic component, a likely candidate for which is activation of the c-Jun N-terminal kinase signaling cascade.     

Dp71Δ78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ_78‐79 dystrophin mutant is stably expressed in PC12‐C11 cells. To investigate the effect of Dp71Δ_78‐79 overexpression on the protein profile of PC12‐C11 cells, we compared the expression profiles of undifferentiated and NGF‐differentiated PC12‐C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ_78‐79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12‐C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ_78‐79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.     

The "Point of no Return" Concept in Cell Division. The Effects of Some Metabolic Inhibitors on Synchronized Chlorella pyrenoidosa

The coarse of growth and cell division in synchronized cultures of Chlorella pyrenoidosa was studied after the addition of metabolic inhibitors at differing times during the cell cycle (14 h light - 10 h darkness with nitrate as nitrogen source. 12 h light: 12 h darkness with urea as nitrogen source). Dinitrophenol (DNP) added to a final concentration of 0.3 mM at any time in the synchronization cycle, the compound remaining in the suspension from the time of addition to the end of the dark period, inhibited spore formation completely. Growth measured as increase in cell volume was less sensitive to the action of the inhibitor. Chloramphenicol (CAP) added dining the 0–5 h interval to a final concentration of 0.1 mM resulted in 80 per cent inhibition of cell division. Similar treatment started at successive times thereafter resulted in a gradual decrease of the inhibition. Treatment at the 14th hour and during the dark period did not affect the sporulation. Similar experiments with 0.9 mM puromycin added at various times during the illumination period gave almost complete inhibition of cell division, while the growth was reduced by only 25 per cent. para-Fluorophenylalanine (p-FPhe) at 3.3 × 10^?2 mM stopped cell division nearly completely irrespective of addition time in the light period. Addition during the dark period also prevented an increase in the number of tree cells. In this case about half of the cells produced spores which were not released. It is concluded that DNP inhibits all stages of preparation for cell division, as well as the division process itself. With CAP a genuine transition point of preparation for cell division was observed, although its interpretation as related to protein synthesis is somewhat uncertain. With puromycin and p-FPhe no transitions were observed.     

Immunocytochemical localization of calmodulin in PC12 cells and its possible interaction with histones

The subcellular localization of calmodulin, a multi-functional calcium-binding regulatory protein, was examined immunocytochemically in undifferentiated PC12 rat pheochromocytoma cells and cells differentiated with nerve growth factor (NGF) and dibutyryl cyclic AMP. In undifferentiated PC12 cells, diffuse immunostaining for calmodulin was observed in the cytoplasm, and weak, patch-like staining was found in the nucleus. In differentiated cells, intense immunostaining for calmodulin was observed in the cytoplasm, while nuclear immunostaining was still evident. Immunoreactivity for calmodulin was also observed along newly-formed neuritic processes, with strong staining in varicosity-like structures and growth cones. Using double-label immunochemistry, the relative intensity of immunostaining for calmodulin among the nuclei was found to correlate with the relative intensity of immunostaining for histones in the same nuclei. A comparison of a profile of ¹²⁵I-calmodulin binding in the nuclear fraction from PC12 cells to that of immunoblotting for histones in the same fraction indicated that some of the histones are calmodulin-binding proteins in PC 12 cells. These results show that the level and subcellular distribution of calmodulin are altered during the course of nerve cell differentiation and suggest the possibility that histones may function as major nuclear binding proteins for calmodulin.