Systematic Asymmetric Synthesis of All Diastereomers of (-)-Talaumidin and Their Neurotrophic Activity.

(-)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan isolated from Aristolochia arcuata Masters, shows significant neurite-outgrowth promotion and neuroprotection in primary cultured rat cortical neurons and in NGF-differentiated PC12 cells. The four stereogenic centers on the tetrahydrofuran moiety in 1 result in the presence of seven diastereomers except for their enantiomers. In order to investigate the stereochemistry-activity relationships of the stereoisomers, the systematic synthesis of all stereoisomers of 1 was accomplished by employing Evans aldol, diastereoselective hydroboration, reductive deoxygenation, and Mitsunobu reactions as key steps. The ability of all of the synthesized stereoisomers to promote neurite-outgrowth in PC12 and neuronal cells was evaluated. All stereoisomers exhibited moderate to potent neurotrophic activities in NGF-differentiated PC12 cells at 30 µM and in primary cultured rat cortical neuronal cells at 0.01 µM. In particular, 1e bearing all cis substituents resulted in the most potent neurite-outgrowth promotion.

Probing the biocompatibility of MoS2 nanosheets by cytotoxicity assay and electrical impedance spectroscopy.

Transition metal dichalgogenides such as MoS2 have recently emerged as hot two-dimensional (2D) materials due to their superior electronic and catalytic properties. Recently, we have reported the usefulness of MoS2 nanosheets toward the electrochemical detection of neurotransmitters and glucose (Narayanan et al 2014 Nanotechnology 25 335702). Furthermore, there are reports available in the literature that demonstrate the usefulness of MoS2 nanosheets for biosensing and energy storage applications (Zhu et al 2013 J. Am. Chem. Soc. 135 5998-6001; Pumera and Loo 2014 Trends Anal. Chem. 61 49-53; Lee et al 2014 Sci. Rep. 4 7352; Stephenson et al 2014 Energy Environ. Sci. 7 209-31). Understanding the cytotoxic effect of any material is very important prior to employing them for any in vivo biological applications such as implantable sensors, chips, or carriers for drug delivery and cell imaging purposes. Herein, we report the cytotoxicity of the MoS2 nanosheets based on the cytotoxic assay results and electrical impedance analysis using rat pheochromocytoma cells (PC12) and rat adrenal medulla endothelial cells (RAMEC). Our results indicated that the MoS2 nanosheets synthesized in our work are safe 2D nanosheets for futuristic biomedical applications.

Is the PentaBDE replacement, tris (1,3-dichloro-2-propyl) phosphate (TDCPP), a developmental neurotoxicant? Studies in PC12 cells.

Organophosphate flame retardants (OPFRs) are used as replacements for the commercial PentaBDE mixture that was phased out in 2004. OPFRs are ubiquitous in the environment and detected at high concentrations in residential dust, suggesting widespread human exposure. OPFRs are structurally similar to neurotoxic organophosphate pesticides, raising concerns about exposure and toxicity to humans. This study evaluated the neurotoxicity of tris (1,3-dichloro-2-propyl) phosphate (TDCPP) compared to the organophosphate pesticide, chlorpyrifos (CPF), a known developmental neurotoxicant. We also tested the neurotoxicity of three structurally similar OPFRs, tris (2-chloroethyl) phosphate (TCEP), tris (1-chloropropyl) phosphate (TCPP), and tris (2,3-dibromopropyl) phosphate (TDBPP), and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a major component of PentaBDE. Using undifferentiated and differentiating PC12 cells, changes in DNA synthesis, oxidative stress, differentiation into dopaminergic or cholinergic neurophenotypes, cell number, cell growth and neurite growth were assessed. TDCPP displayed concentration-dependent neurotoxicity, often with effects equivalent to or greater than equimolar concentrations of CPF. TDCPP inhibited DNA synthesis, and all OPFRs decreased cell number and altered neurodifferentiation. Although TDCPP elevated oxidative stress, there was no adverse effect on cell viability or growth. TDCPP and TDBPP promoted differentiation into both neuronal phenotypes, while TCEP and TCPP promoted only the cholinergic phenotype. BDE-47 had no effect on cell number, cell growth or neurite growth. Our results demonstrate that different OPFRs show divergent effects on neurodifferentiation, suggesting the participation of multiple mechanisms of toxicity. Additionally, these data suggest that OPFRs may affect neurodevelopment with similar or greater potency compared to known and suspected neurotoxicants.

Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque.

Using antibodies and recombinant DNA techniques, we have identified plakophilin 2, a novel desmosomal plaque protein of M(r) 100,000 (estimated from SDS-PAGE), which is a member of the arm-repeat family of proteins and can occur in two splice forms (2a and 2b) because of the insertion of a 44 amino acid (aa)-encoding exon. In its aa sequence (837 and 881 aa, calculated pIs: 9.33 and 9.38, mol wts 92,750 and 97,410 kD), it is conspicuously related to the 80-kD plakophilin 1, with which it shares a central region of 9 repeats of the arm-motif, preceeded by a long head region and followed by a very short (11 aa) carboxy-terminal sequence. Plakophilin 2 and its mRNA have been detected in a wide range of tissues and cell types, including cells devoid of desmosomes. By light and electron microscopical immunolocalization, plakophilin 2 has been localized to plaques of desmosomes of one-layered ("simple") and complex epithelia, carcinomas, diverse epithelium-derived cell culture lines, as well as cardiac tissue and the dendritic reticulum cells of lymphatic germinal centers, i.e., desmosomes in which plakophilin 1 is not detected. However, plakophilin 2 has also been localized in the desmosomes of certain but not all stratified epithelia where it coexists with plakophilin 1. Remarkably, plakophilin 2 is also enriched in the karyoplasm of a wide range of cell types, including many that lack desmosomes and in which, therefore, the nuclear state is the only locally enriched form of plakophilin 2 present. We conclude that plakophilins 2a and 2b are basic nuclear proteins that in certain cell types additionally assemble with other proteins to form the desmosomal plaque and serve general nuclear functions as well as a function specific to many but not all desmosomes.

Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex.

Cadherins are calcium-dependent cell adhesion molecules that play fundamental roles in embryonic development, tissue morphogenesis, and cancer. A prerequisite for their function is association with the actin cytoskeleton via the catenins. Tyrosine phosphorylation of beta-catenin, which correlates with a reduction in cadherin-dependent cell adhesion, may provide cells with a mechanism to regulate cadherin activity. Here we report that beta-catenin immune precipitates from PC12 cells contain tyrosine phosphatase activity which dephosphorylates beta-catenin in vitro. In addition, we show that a member of the leukocyte antigen-related protein (LAR)-related transmembrane tyrosine phosphatase family (LAR-PTP) associates with the cadherin-catenin complex. This association required the amino-terminal domain of beta-catenin but does not require the armadillo repeats, which mediate association with cadherins. The interaction also is detected in PC9 cells, which lack alpha-catenin. Thus, the association is not mediated by alpha-catenin or by cadherins. Interestingly, LAR-PTPs are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. We propose that changes in tyrosine phosphorylation of beta-catenin mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth.

Inhibition of medulloblastoma tumorigenesis by the antiproliferative and pro-differentiative gene PC3.

Medulloblastoma, the most common brain tumor in childhood, appears to originate from cerebellar granule cell precursors (GCPs), located in the external granular layer (EGL) of the cerebellum. The antiproliferative gene PC3 (Tis21/BTG2) promotes cerebellar neurogenesis by inducing GCPs to shift from proliferation to differentiation. To assess whether PC3 can prevent the neoplastic transformation of GCPs and medulloblastoma development, we crossed transgenic mice conditionally expressing PC3 (TgPC3) in GCPs with Patched1 heterozygous mice (Ptc(+/-)), a model of medulloblastoma pathogenesis characterized by hyperactivation of the Sonic Hedgehog pathway. Perinatal up-regulation of PC3 in Ptc(+/-)/TgPC3 mice results in a decrease of medulloblastoma incidence of approximately 40% and in a marked reduction of preneoplastic abnormalities, such as hyperplastic EGL areas and lesions. Moreover, overexpression of cyclin D1, hyperproliferation, and defective differentiation--observed in Ptc(+/-) GCPs--are restored to normality in Ptc(+/-)/TgPC3 mice. The PC3-mediated inhibition of cyclin D1 expression correlates with recruitment of PC3 to the cyclin D1 promoter, which is accompanied by histone deacetylation. Remarkably, down-regulation of PC3 is observed in preneoplastic lesions, as well as in human and murine medulloblastomas. As a whole, this indicates that PC3 may prevent medulloblastoma development by controlling cell cycle and promoting differentiation of GCPs.

Effect of lower chlorinated hydroxylated-polychlorobiphenyls on development of PC12 cells.

Hydroxylated polychlorobiphenyls (OH-PCBs) are major metabolites of PCBs that are widely distributed in the environment. While the effects of penta- to hepta-chlorinated OH-PCBs on neuronal differentiation have been widely reported, those of lower chlorinated OH-PCBs have not been extensively studied. To investigate the effects of lower chlorinated OH-PCBs on neuronal development, we studied the effects of mono- to hexa-chlorinated OH-PCBs on PC12 cells. Morphological changes were examined using an automatic system IN Cell Analyzer. Seventeen of the 20 OH-PCBs investigated promoted neuronal elongation in an OH-PCB concentration-dependent manner, while three OH-PCB congeners suppressed neuronal elongation based on Dunnett's analysis. In particular, the top five OH-PCBs (4OH-PCB2, 4'OH-PCB3, 4'OH-PCB25, 4'OH-PCB68, and 4'OH-PCB159), which have hydroxyl groups at the para-position and chlorine substitutions at the 2, 4, or 3' positions, significantly promoted neuronal elongation. Moreover, these neuronal elongations were suppressed by U0126, and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was observed in PC12 cells treated with 4OH-PCB2, 4'OH-PCB25, and 4'OH-PCB159. Taken together, our results indicate that the effect of OH-PCB on neuronal development is not dependent on the number of chlorine groups but on the chemical structure, and the mitogen-activated kinase kinase (MEK)-ERK1/2 signaling pathway is involved in this process.

Re-evaluation of Culture Condition of PC12 and SH-SY5Y Cells Based on Growth Rate and Amino Acid Consumption.

BACKGROUND/AIM: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively). MATERIALS AND METHODS: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization. RESULTS: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aß1-42 and Aß25-35 suppressed their growth nearly by 50%. CONCLUSION: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity.

Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein.

OBJECTIVE: Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein-coupled receptor transport from the endoplasmic reticulum to the cell surface. METHODS AND RESULTS: Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum-export signal. Using a yeast two-hybrid system, we identified a Golgi membrane-associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor. CONCLUSIONS: Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.

Distinct signalling particles containing ERK/MEK and B-Raf in PC12 cells.

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein-protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated 'signalling particles' with an estimated size of 60-75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.

Enhanced levels of annexins in pancreatic carcinoma cells of Syrian hamsters and their intrapancreatic allografts.

Annexins are a family of calcium- and phospholipid-binding proteins related by amino acid sequence homology. Annexins I and II are substrates for protein tyrosine kinases. Recent investigations have revealed a possible involvement of annexins I and II in mitogenic signal transduction and cell proliferation. To investigate further the involvement of annexins in cell proliferation, we measured the levels of annexins I and II and the enzyme 3-phosphoglycerate kinase (PGK) (annexin II and PGK are components of the primer recognition protein complex) in normal Syrian hamster pancreas, three hamster pancreatic ductal carcinoma cell lines, and allografts of the three cell lines into hamster pancreas. All three carcinoma cell lines had 5-8-fold higher levels of annexin II compared to normal pancreas. An inverse relationship was seen between level of annexin II and the doubling time of the cell culture. In intrapancreatic allografts, annexin II levels were 3-6-fold higher than in normal pancreas. Annexin I levels were 2-3-fold higher in the allografts. Significant increases (5-6-fold) in specific activity of PGK were seen in all allografts examined. However, the level of PGK, as measured by immunoblotting, was not significantly altered. Immunohistochemical staining revealed heterogeneity in the reactivity of the antiannexin and anti-PGK antibodies with tumor cells. Strikingly, the reactivity and staining intensity were greater in the proliferating regions of the primary tumors and in the metastatic foci. Mitotic cells were either unstained or very weakly stained. We conclude from these findings that annexin II and PGK, as primer recognition proteins, may have a role in cell proliferation.

Kinetic studies on the hydrogen peroxide elimination by cultured PC12 cells: rate limitation by glucose-6-phosphate dehydrogenase.

Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and brain ischemia, and hydrogen peroxide (H(2)O(2)) plays a central role in the stress. In this study, we have examined the kinetics of H(2)O(2) elimination by PC12 cells as a neuronal model in connection with the enzyme activities supporting the reaction. Similarly to other cell lines previously studied, H(2)O(2) removal kinetics could be divided into two reactions: one apparently following the Michaelis-Menten kinetics (GSH-dependent reaction) and the other following the first-order kinetics (mainly catalyzed by catalase). Based on the enzyme activities in the cell homogenate, it was inferred that glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the GSH- and NADPH-dependent H(2)O(2) elimination by PC12 cells. This is in contrast with fibroblasts and endothelial cells previously examined, in which glutathione reductase (GR) is rate-limiting in the reaction sequence. Treatment of PC12 cells with nerve growth factor increased G6PD activity in the cell homogenate and H(2)O(2) removal activity of the whole cells, with a concomitant increase in the resistance against H(2)O(2) toxicity. These results suggest the importance of G6PD in the antioxidant function of brain and pathogenesis of the oxidative stress-related diseases.

Synaptotagmin I-DeltaC2B. A novel synaptotagmin isoform with a single C2 domain in the bovine adrenal medulla.

Synaptotagmin I is a 65 kDa type 1 membrane glycoprotein found in secretory organelles that plays a key role in regulated exocytosis. We have characterised two forms (long and short) of synaptotagmin I that are present in the bovine adrenal medulla. The long form is a type I integral membrane protein which has two cytoplasmic C2 domains and corresponds to the previously characterised full-length synaptotagmin I isoform. The short-form synaptotagmin I-DeltaC2B has the same structure in the lumenal and transmembrane sequences, but synaptotagmin I-DeltaC2B is truncated such that it only has a single cytoplasmic C2 domain. Analysis of synaptotagmin I-DeltaC2B expression indicates that synaptotagmin I-DeltaC2B is preferentially expressed in the bovine adrenal medulla. However, it is absent from the dense core chromaffin granules. Furthermore, when expressed in the rat pheochromocytoma cell line PC12 bovine synaptotagmin I-DeltaC2B is largely absent from dense core granules and synaptic-like microvesicles. Instead, indirect immunofluorescence microscopy reveals the intracellular location of synaptotagmin I-DeltaC2B to be the plasma membrane.

A 56,000 Mr phosphoseryl protein in PC12 cell lysates strongly associates with protein-A sepharose beads and was observed in immune complex kinase assays for PP60c-src.

Using an immune complex kinase assay to measure pp60c-src kinase activity, we have identified a 56,000 Mr protein (p56) from PC12 cell lysates that co-purified with pp60c-src by strong association with protein-A sepharose beads. The p56 protein was strongly phosphorylated on serine but no tyrosine or threonine phosphorylation was evident. However, pp60c-src was strongly phosphorylated on tyrosine, weakly phosphorylated on serine with no observed threonine phosphorylation. P56 was not a proteolytic breakdown product of pp60c-src, since it was neither tyrosine phosphorylated nor was it recognized by anti-src antibody. P56 was also not recognised by other antibodies to 56kD signalling molecules such as p56lck. The identify of p56 awaits further investigation but its appearance in immunoprecipitates of pp60c-src using protein-A sepharose beads is of interest but complicates the the interpretation of results from immune complex kinase assays in PC12 cells.

The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1.

Retinoic acid-receptor-related orphan receptor (ROR) alpha is a nuclear receptor involved in many pathophysiological processes such as cerebellar ataxia, inflammation, atherosclerosis and angiogenesis. In the present study we first demonstrate that hypoxia increases the amount of Rora transcripts in a wide panel of cell lines derived from diverse tissues. In addition, we identified a functional promoter sequence upstream of the first exon of the human Rora gene, spanning -487 and -45 from the translation initiation site of RORalpha1. When cloned in a luciferase reporter vector, this sequence allowed the efficient transcription of the luciferase gene in several cell lines. Interestingly, the activity of the Rora promoter was enhanced by hypoxia in HepG2 human hepatoma cells, and this effect was dependent on an HRE (hypoxia response element) spanning from -229 to -225. Using electrophoretic-mobility-shift assays, we showed that HIF-1 (hypoxia-inducible factor 1), which plays a key role in the transcriptional response to hypoxia, bound to this HRE. Overexpression of HIF-1alpha increased the activity of the Rora promoter through the HRE. Overexpression of a dominant-negative form of HIF-1alpha producing transcriptionally inactive HIF-1alpha/HIF-1beta dimers abolished hypoxic activation of the Rora promoter. This indicated that HIF-1 is involved in the response of RORalpha to hypoxia. Taken together, our data reveal Rora as a new HIF-1 target gene. This illustrates, at the molecular level, the existence of cross-talk between signalling pathways mediated by HIF-1 and those mediated by nuclear receptors.

RNA interference-mediated silencing of synaptotagmin IX, but not synaptotagmin I, inhibits dense-core vesicle exocytosis in PC12 cells.

Although PC12 cells express three synaptotagmin isoforms (Syts I, IV and IX), all of which have been proposed to regulate dense-core vesicle exocytosis, it remains unknown which of the Sytisoforms acts as the major Ca2+ sensor for dense-core vesicle exocytosis. In the present study, it has been shown by immunoaffinity purification and immunocytochemistry that Syts I and IX, but not Syt IV, are present on the same secretory vesicles in PC12 cells. Silencing of Syt IX with specific small interfering RNA significantly reduced high KCl-dependent neuropeptide Y secretion from PC12 cells, whereas silencing of Syt I with specific small interfering RNA had no significant effect. The results indicate that Syts I and IX are not functionally equivalent and that Syt IX, and not Syt I, is indispensable for the regulation of Ca2+-dependent dense-core vesicle exocytosis in PC12 cells.

Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B).

Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41-49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108-170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B-EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)-Rattus norvegicus IP3K (aa 108-170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.

Colocalization of low- and high-affinity NGF receptors on PC12 cells, C6 glioma cells and dorsal root ganglion neurons.

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types.

Reduced cyclin D1 expression in the cerebella of nutritionally deprived rats correlates with developmental delay and decreased cellular DNA synthesis.

Nutritional deprivation in the early postnatal period severely inhibits cerebellar growth and development, which is related in part to reduced levels of growth factors. Cyclin D1 encodes a growth factor-inducible regulatory subunit of a serine/thereonine kinase that is capable of phosphorylating the tumor suppressor pRB, thereby allowing normal progression through the G1 phase of the cell-cycle. Because the abundance of cyclin D1 is rate limiting in this progression, we examined the regulation of cyclin D1 expression in vivo, using a model of nutritional deprivation. Cyclin D1 expression in cerebella of fed control rats was detected in the external granular layer and was associated with cellular proliferation within this layer. Nutritional deprivation of rats reduced cerebellar weight, as well as the thickness of the molecular layer that largely consists of cells migrating from the external granular layer. Refeeding partially restored cerebellar weight, molecular layer thickness and increased external granular layer cyclin D1 immunostaining. Since nutritional deprivation is accompanied by lower levels of circulating insulin-like growth factor-I (IGF-I), we determined whether IGF-I directly stimulated the cyclin D1 promoter. The human cyclin D1 promoter linked to the luciferase reporter gene was stably integrated into PC12 cells. IGF-I stimulated cyclin D1 promoter activity 4- to 6-fold at 6 hours (h). These findings are consistent with the notion that nutritional deprivation may affect proliferative growth by altering expression of cyclin D1 in the germinal cell layer and that regulation of cyclin D1 expression by growth factors may contribute to normal neonatal cerebellar development. The reduction in cyclin D1 expression as cells differentiate in the cerebellum is consistent with a potential role for cyclin D1 in this process.

Detection of living cells that express AP1 using a fluorolabeled DNA probe.

Activator protein 1 (AP1) is a complex of Fos and Jun, and it regulates the transcription of genes possessing the AP1-binding sequence. The purpose of this study was to detect living cells that express AP1 after stimulation with a tumor promoter. The Fos and Jun components of AP1 were induced rapidly and transiently in PC12 cells following the addition of phorbol ester (phorbol 12-myristate 13-acetate, PMA). The DNA fragment containing the AP1-binding sequence was combined with ethidium bromide, which was used as a fluorescent probe. The probe was transfected into the cells using cationic liposomes. Fluorescence in the transfected cells was observed using a fluorescence microscope. The nuclei of transfected cells emitted strong fluorescence in the presence of PMA, whereas weak fluorescence was retained in the cytoplasm in its absence. The former phenomenon is evidence that AP1 combined with the fluorescent probe was transported into the nuclei. This study suggests that such a fluorolabeling method is feasible to detect living AP1-expressed neurons.