Isolation and Characterization of NP-POL Nonapeptide for Possible Therapeutic Use in Parkinson's Disease.

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinson's disease.

Investigation of amyloid formation inhibition of chemically and biogenically from Citrus aurantium L. blossoms and Rose damascena oils of gold nanoparticles: Toxicity evaluation in rat pheochromocytoma PC12 cells.

Fibrillation inhibition effects of chemically and biogenically gold nanoparticles (GNPs) were investigated in vitro using human insulin as a model for fibrillation of protein. This inspection was followed using the Congo red assay, thioflavin T fluorescence measurements, transmission electron microscopy, and evaluation of cytotoxicity effects on rat pheochromocytoma PC12 cells. Biogenic GNPs were synthesized using oil extracts of Citrus aurantium L. blossoms and Rose damascena blossoms as reducing and concomitant agents. Congo red assay showed development of fibril formation of insulin at acidic media at 60°C over a period of 48h. In these circumstances, transmission electron micrographs confirmed the progress of fibril state from globular chains to amyloid. However, the results of ThT fluorescence measurements indicated a concentration-dependent inhibiting effect of chemically synthesized GNPs on insulin fibrillation in vitro, simultaneously by conversion of the formed fibrils into amorphous aggregates. Furthermore, biogenic GNPs were found to more effectively inhibit the fibril formation, compared to chemically synthesized GNPs. Accordingly, just 0.05nmolL-1 of the biogenic GNPs showed similar inhibition property of chemically synthesized GNPs with a concentration of 10nmolL-1. Both types of GNPs diminished toxicity of insulin fibrils in rat pheochromocytoma PC12 cells viability.

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation.

Estrogen receptors (ERs) α and ß are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as a model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases.

Identification of flavonoids from Flammulina velutipes and its neuroprotective effect on pheochromocytoma-12 cells.

The flavonoids from Flammulina velutipes (FVF) were isolated and the chemical composition of FVF was identified by high-performance liquid chromatography/mass spectrometry (HPLC/MS). The antioxidant activity and the neuroprotective effect against H2O2-induced pheochromocytoma-12 cells (PC12) cytotoxicity of FVF was investigated. Six flavonoids from FVF were identified as arbutin, epicatechin, phillyrin, apigenin, kaempferol and formononetin. Obtained results suggested that FVF possesses strong antioxidant properties in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities as well as reducing power. Pretreatment of PC12 cells with FVF significantly increased the survival rate of cells, glutathione level and superoxide dismutase activity. Conversely, the pretreatment reduced the release of lactate dehydrogenase and the accumulation of both intracellular reactive oxygen species and malondialdehyde. These findings collectively suggested that the protective effects of FVF against H2O2-induced neurotoxicity in PC12 cells as well as the potential for neuroprotection of FVF could be due to its antioxidant action.

Pleurotus giganteus (Berk.) Karunarathna & K.D. Hyde: Nutritional value and in vitro neurite outgrowth activity in rat pheochromocytoma cells.

BACKGROUND: Drugs dedicated to alleviate neurodegenerative diseases like Parkinson's and Alzheimer's have always been associated with debilitating side effects. Medicinal mushrooms which harness neuropharmacological compounds offer a potential possibility for protection against such diseases. Pleurotus giganteus (formerly known as Panus giganteus) has been consumed by the indigenous people in Peninsular Malaysia for many years. Domestication of this wild mushroom is gaining popularity but to our knowledge, medicinal properties reported for this culinary mushroom are minimal. METHODS: The fruiting bodies P. giganteus were analysed for its nutritional values. Cytotoxicity of the mushroom's aqueous and ethanolic extracts towards PC12, a rat pheochromocytoma cell line was assessed by using 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Neurite outgrowth stimulation assay was carried out with nerve growth factor (NGF) as control. To elucidate signaling mechanisms involved by mushroom extract-induced neurite outgrowth, treatment of specific inhibitor for MEK/ERK and PI3K signalling pathway was carried out. RESULTS: The fruiting bodies of P. giganteus were found to have high carbohydrate, dietary fibre, potassium, phenolic compounds and triterpenoids. Both aqueous and ethanolic extracts induced neurite outgrowth of PC12 cells in a dose- and time-dependant manner with no detectable cytotoxic effect. At day 3, 25 µg/ml of aqueous extract and 15 µg/ml of ethanolic extract showed the highest percentage of neurite-bearing cells, i.e. 31.7 ± 1.1% and 33.3 ± 0.9%; respectively. Inhibition treatment results suggested that MEK/ERK and PI3K/Akt are responsible for neurite outgrowth of PC12 cells stimulated by P. giganteus extract. The high potassium content (1345.7 mg/100 g) may be responsible for promoting neurite extension, too. CONCLUSIONS: P. giganteus contains bioactive compounds that mimic NGF and are responsible for neurite stimulation. Hence, this mushroom may be developed as a nutraceutical for the mitigation of neurodegenerative diseases.

[Study on protective effect of salvianolic acid B on glutamate-induced excitotoxicity in pheochromocytoma PC12 cells].

OBJECTIVE: To study the protective effect and mechanism of salvianolic acid B (Sal B) on glutamate-induced excito-toxicity. METHOD: Glutamate-induced PC12 cell injury model was established to detect the cell survival rate by MTT, the leakage rate of lactic dehydrogenases using LDH, and the cell apoptosis by using AO/EB double staining for fluorescence microscope and PI single staining flow cytometry which was also used to detect the content of intracellular reactive oxygen species. The expression of Caspase-3 protein was also detected by the Western blotting method. RESULT: Sal B is proved to inhibit glutamate-induced PC12 cells from injury and prevent them from releasing LDH within the range from 50 micromol x L(-1) to 200 micromol x L(-1). Meanwhile, Sal B has an effect on significantly reducing the expression of inhibit glutamate-induced active Caspase-3 protein, inhibiting accumulated glutamate-induced ROS and decreasing PC12 cell apoptosis rate within the range from 50 micromol x L(-1) to 200 micromol x L(-1). CONCLUSION: The study proves that Sal B prevented against glutamate-induced cell injury via inhibiting ROS formation and Caspase-3 pathway-dependent apoptosis in PC12 cells.

Identification of coffee components that stimulate dopamine release from pheochromocytoma cells (PC-12).

Coffee and caffeine are known to affect the limbic system, but data on the influence of coffee and coffee constituents on neurotransmitter release is limited. We investigated dopamine release and Ca(2+)-mobilization in pheochromocytoma cells (PC-12 cells) after stimulation with two lyophilized coffee beverages prepared from either Coffea arabica (AR) or Coffea canephora var. robusta (RB) beans and constituents thereof. Both coffee lyophilizates showed effects in dilutions between 1:100 and 1:10,000. To identify the active coffee compound, coffee constituents were tested in beverage and plasma representative concentrations. Caffeine, trigonelline, N-methylpyridinium, chlorogenic acid, catechol, pyrogallol and 5-hydroxytryptamides increased calcium signaling and dopamine release, although with different efficacies. While N-methylpyridinium stimulated the Ca(2+)-mobilization most potently (EC(200): 0.14±0.29µM), treatment of the cells with pyrogallol (EC(200): 48±14nM) or 5-hydroxytryptamides (EC(200): 10±3nM) lead to the most pronounced effect on dopamine release. In contrast, no effect was seen for the reconstituted biomimetic mixture. We therefore conclude that each of the coffee constituents tested stimulated the dopamine release in PC-12 cells. Since no effect was found for their biomimetic mixture, we hypothesize other coffee constituents being responsible for the dopamine release demonstrated for AR and RB coffee brews.

Increased uptake of [¹²³I]meta-iodobenzylguanidine, [¹8F]fluorodopamine, and [³H]norepinephrine in mouse pheochromocytoma cells and tumors after treatment with the histone deacetylase inhibitors.

[¹³¹I]meta-iodobenzylguanidine ([¹³¹I]MIBG) is the most commonly used treatment for metastatic pheochromocytoma and paraganglioma. It enters the chromaffin cells via the membrane norepinephrine transporter; however, its success has been modest. We studied the ability of histone deacetylase (HDAC) inhibitors to enhance [¹²³I]MIBG uptake by tumors in a mouse metastatic pheochromocytoma model. HDAC inhibitors are known to arrest growth, induce differentiation and apoptosis in various cancer cells, and further inhibit tumor growth. We report the in vitro and in vivo effects of two HDAC inhibitors, romidepsin and trichostatin A, on the uptake of [(3)H]norepinephrine, [¹²³I]MIBG, and [(18)F]fluorodopamine in a mouse model of metastatic pheochromocytoma. The effects of both inhibitors on norepinephrine transporter activity were assessed in mouse pheochromocytoma (MPC) cells by using the transporter-blocking agent desipramine and the vesicular-blocking agent reserpine. HDAC inhibitors increased [(3)H]norepinephrine, [¹²³I]MIBG, and [(18)F]fluorodopamine uptake through the norepinephrine transporter in MPC cells. In vivo, inhibitor treatment resulted in significantly increased uptake of [(18)F]fluorodopamine positron emission tomography (PET) in pheochromocytoma liver metastases (19.1 ± 3.2% injected dose per gram of tumor (%ID/g) compared to liver metastases in pretreatment scans 5.9 ± 0.6%; P<0.001). Biodistribution analysis after inhibitors treatment confirmed the PET results. The uptake of [(123)I]MIBG was significantly increased in liver metastases 9.5 ± 1.1% compared to 3.19 ± 0.4% in untreated control liver metastases (P<0.05). We found that HDAC inhibitors caused an increase in the amount of norepinephrine transporter expressed in tumors. HDAC inhibitors may enhance the therapeutic efficacy of [(131)I]MIBG treatment in patients with advanced malignant pheochromocytoma and paraganglioma.

[Protective effects and machanism of muskone on pheochromocytoma cell injure induced by glutamate].

OBJECTIVE: To explore the mechanism of glutamate (Glu)-induced PC12 cell apoptosis and the protective effect of muscone. METHOD: PC12 cells were randomly divided into six groups: normal group (Normal), model group injured by glutamate (Glu), nimodipine group (Nim) and muskone groups (Mus) of high, middle and low doses. The PC12 cells were pretreated with or without different concentrations of muskone for 30 min and then exposed to glutamate. MTT assay for cell survival, flow cytometric detection of apoptotic cells, DCF assay for reactive oxygen species (ROS) and flow cytometric assay was performed to determine the mitochondrial membrane potential in PC12 cell. RESULT: PC12 cell damage, the concentrations of [Ca2+], and apoptosis induced by Glu were decreased after being administrated with paeoniflorin. CONCLUSION: Muskone inhibited Glu-induced apoptosis in PC12 cells. The mechanism is related to inhibiting intracellular Ca2+ overload and maintaining mitochondral membrance potential.

Radiation dosimetry, pharmacokinetics, and safety of ultratrace Iobenguane I-131 in patients with malignant pheochromocytoma/paraganglioma or metastatic carcinoid.

This is a first of many phase 1 study of Ultratrace Iobenguane I-131 (Ultratrace 131I-MIBG; Molecular Insight Pharmaceuticals, Inc., Cambridge, MA). High-specific-activity Ultratrace 131I-MIBG may provide improved efficacy and tolerability over carrier-added 131I-MIBG. We investigated the pharmacokinetics (PK), radiation dosimetry, and clinical safety in 11 patients with confirmed pheochromocytoma/paraganglioma (Pheo) or carcinoid tumors. A single 5.0-mCi (185 MBq) injection of Ultratrace 131I-MIBG, supplemented with 185 microg of unlabeled MIBG to simulate the amount of MIBG anticipated in a therapeutic dose, was administered. Over 120 hours postdose, blood and urine were collected for PK, and sequential whole-body planar imaging was performed. Patients were followed for adverse events for 2 weeks. Ultratrace 131I-MIBG is rapidly cleared from the blood and excreted in urine (80.3% +/- 2.8% of dose at 120 hours). For a therapeutic administration of 500 mCi (18.5 GBq), our estimate of the projected dose is 1.4 Gy for marrow and 10.4 Gy for kidneys. Safety results showed 12 mild adverse events, all considered unrelated to study drug, in 8 of 11 patients. These findings support the further development of Ultratrace 131I-MIBG for the treatment of neuroendocrine tumors, such as metastatic Pheo and carcinoid.

Functional analysis of cultured neural cells for evaluating cold/cool- and hot/warm-natured Chinese herbs.

Recently, modern scientific research has been required to understand pharmacological basis of traditional Chinese medicine (TCM) theory based on the ancient clinical experience, and to investigate the molecular mechanisms of action of Chinese herbs. Here, 20 Chinese herbs, classified into 4 properties (hot, warm, cold and cool) of TCM, were analyzed for their ability to exhibit antioxidant action, to enhance glucose uptake by murine microglia N9 cells, and to influence neurotransmitter norepinephrine (NE) release from rat pheochromocytoma PC12 cells. We found a generally protective effect of both hot/warm-natured and cold/cool-natured herbs against H(2)O(2)-induced N9 cell death, partially by elevating superoxide dismutase (SOD) activity. Glucose uptake was elevated after treatment with some hot/warm-natured herbs. In addition, most herbs with hot/warm nature tended to stimulate NE release, while such stimulatory effect was not observed in the herbs with cold/cool nature. Two cold/cool-natured herbs, Rhizoma coptidis and Radix scutellariae, even significantly suppressed the release. These results suggest that the distinct abilities of Chinese herbs to regulate neural cell functions appear to be correlated with their natures identified in traditional TCM theory, and may be a useful guide for their utility in neural degenerative diseases.

A case of pheochromocytoma presenting as low back pain.

OBJECTIVE: This case report describes and discusses a patient with a pheochromocytoma who presented to a chiropractic office with low back pain. CLINICAL FEATURES: The patient is a 51-year-old woman who was self-referred to our chiropractic service with low back pain that appeared to be musculoskeletal in nature. Four days after chiropractic consultation, she collapsed in cardiogenic shock with signs of congestive heart failure, left ventricular dysfunction, and hypotension. Computed tomographic image of the abdomen revealed a right-sided adrenal mass that was confirmed via laboratory analysis to be a pheochromocytoma. INTERVENTION AND OUTCOME: The patient underwent laparoscopic adrenalectomy and made a full recovery. Her initial back symptoms resolved with tumor excision. CONCLUSION: Pheochromocytomas are rare catecholamine-producing tumors of the adrenal glands that can mimic musculoskeletal conditions such as low back pain. Chiropractic physicians should be aware of the various clinical presentations and, when pheochromocytoma is suspected, make prompt referral to medical providers for diagnostic evaluation and treatment.

Royal jelly-induced neurite outgrowth from rat pheochromocytoma PC12 cells requires integrin signal independent of activation of extracellular signal-regulated kinases.

We showed earlier that neurite outgrowth of rat pheochromocytoma PC12 cells was stimulated by royal jelly extract (PERJ) or its unique component, AMP N(1)-oxide, via adenosine A2a receptors. In this study, we found that stimulated neurite outgrowth occurred in medium supplemented with serum, but not in serum-free medium. The pentapeptide GRGDS, which includes the RGD sequence commonly shared by extracellular matrix (ECM) components, could attenuate the effect of serum, suggesting that integrin receptor signaling was essential for the neurite outgrowth induced by PERJ or AMP N(1)-oxide. PERJ or AMP N(1)-oxide also activated extracellular signal-regulated kinases 1 or 2 (ERK1/2); however, this activation was not associated with the neurite outgrowth. As it is known that Mn(2+) induces neurite outgrowth from PC12 cells and activates ERK1/2 through integrin signals and that activation of ERK1/2 is essential for Mn2+-induced neurite outgrowth, a difference in the mechanism between Mn(2+)-induced and PERJ- or AMP N(1)-oxide-induced neurite outgrowth is suggested. Furthermore, we demonstrated that PERJ contained no ECM component-like substances. These results demonstrate that AMP N(1)-oxide and its analogues were the only entities in PERJ with neurite outgrowth-inducing activity and that they required integrin signaling in addition to activation of A2a receptors to induce neurite outgrowth.

Malignant pheochromocytomas and paragangliomas: a phase II study of therapy with high-dose 131I-metaiodobenzylguanidine (131I-MIBG).

Thirty patients with malignant pheochromocytoma (PHEO) or paraganglioma (PGL) were treated with high-dose 131I-MIBG. Patients were 11-62 (mean 39) years old: 19 patients males and 11 females. Nineteen patients had PGL, three of which were multifocal. Six PGLs were nonsecretory. Eleven patients had PHEO. All 30 patients had prior surgery. Fourteen patients were refractory to prior radiation or chemotherapy before 131I-MIBG. Peripheral blood stem cells (PBSCs) were collected and cryopreserved. 131I-MIBG was synthesized on-site, by exchange-labeling 131I with 127I-MIBG in a solid-phase Cu2+-catalyzed exchange reaction. 131I-MIBG was infused over 2 h via a peripheral IV. Doses ranged from 557 mCi to 1185 mCi (7.4 mCi/kg to 18.75 mCi/kg). Median dose was 833 mCi (12.55 mCi/kg). Marrow hypoplasia commenced 3 weeks after 131I-MIBG therapy. After the first 131I-MIBG therapy, 19 patients required platelet transfusions; 19 received GCSF; 12 received epoeitin or RBCs. Four patients received a PBSC infusion. High-dose 131I-MIBG resulted in the following overall tumor responses in 30 patients: 4 sustained complete remissions (CRs); 15 sustained partial remissions (PRs); 1 sustained stable disease (SD); 5 progressive disease (PD); 5 initial PRs or SD but relapsed to PD. Twenty-three of the 30 patients remain alive; deaths were from PD (5), myelodysplasia (1), and unrelated cause (1). Overall predicted survival at 5 years is 75% (Kaplan Meier estimate). For patients with metastatic PHEO or PGL, who have good *I-MIBG uptake on diagnostic scanning, high-dose 131I-MIBG therapy was effective in producing a sustained CR, PR, or SD in 67% of patients, with tolerable toxicity.

Differentiation of pheochromocytoma PC12 cells induced by human urine extract and the involvement of the extracellular signal-regulated kinase signaling pathway.

OBJECTIVE: The aim of this study was to investigate the effects of a human urine preparation on the differentiation of tumor cells. DESIGN: The pheochromocytoma PC12 cells were used to examine the effects of a human urine preparation, CDA-2 on the induction of differentiation markers, neurofilaments, and compared with that induced by nerve growth factor (NGF). The MAPK/ERK kinase (MEK) inhibitor U0126 was used to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathway in this differentiation inducing effect. RESULTS: We find that CDA-2 could induce differentiation of pheochromocytoma PC12 cells, as evidenced by the markedly increased expression of neurofilaments to a level comparable to those induced by NGF. This phenomenon was accompanied by the phosphorylation of extracellular-signal-regulated kinase (ERK) and could be inhibited by the MEK inhibitor, U0126. CONCLUSIONS: Our results demonstrate the presence of active components in the human urine extract that can induce differentiation of PC12 cells and may involve the ERK signaling pathway. This may provide new insights for seeking novel differentiation agents and offer hope for cancer patients.

Differential activity of lipoic acid enantiomers in cell culture.

It is unclear whether the two enantiomeric forms (R & S) of lipoic acid (LA) share similar pharmacological activity and the exact cellular targets of LA are not well identified. We oxidatively stressed 3 cell culture systems representing different cell types. Mitochondrial metabolism was the primary endpoint. When C6 glioma was damaged by hydrogen peroxide (H2O2), all forms of LA protected. Racemic and S-LA were less effective than the R-isomer that was also protective in tertiary butyl hydroperoxide (TBHP)-damaged C6 glioma. In PC12 cells, little damage was produced by TBHP; R-LA increased mitochondrial metabolism above the level of non-damaged control. In H2O2 damaged PC12 cells, R-LA and racemic LA (but not S-LA) not only protected against damage, but increased mitochondrial metabolism above the non-damaged control level. When BAE cells were damaged with H2O2, R- and racemic LA protected while S-LA was ineffective.

Chromaffin cell catecholamine secretion: bisindolylmaleimide compounds exhibit novel and potent antagonist effects at the nicotinic cholinergic receptor in pheochromocytoma cells.

Activation of protein kinase C (PKC) stimulates nicotine-induced catecholamine secretion. PKC down-regulation by prolonged pretreatment with phorbol 12-myristate 13-acetate diminished nicotine-induced catecholamine secretion only slightly (approximately 16%), suggesting substantial PKC independence of nicotinic receptor activation. However, we found that bisindolylmaleimide compounds (which are also putative PKC chemical inhibitors) dramatically inhibited nicotine-induced catecholamine secretion (IC(50) values of approximately 24-37 nM). This inhibition was specific for the nicotinic cholinergic receptor. Catecholamine secretion induced by other nicotinic agonists (such as epibatidine, anatoxin, or cytisine) was also powerfully antagonized by bisindolylmaleimide II (IC(50) values of approximately 60-90 nM). Even high-dose nicotinic agonists failed to overcome the inhibition by bisindolylmaleimide II, suggesting noncompetitive nicotinic antagonism by this class of compounds. Nicotinic inhibition by bisindolylmaleimide seemed not to be readily reversible. Structure-activity studies of bisindolylmaleimide compounds revealed that bisindolylmaleimides I through III are the most potent nicotinic antagonists at the nicotinic cholinergic receptor in PC-12 cells (IC(50) < or =37 nM), whereas bisindolylmaleimide IV and V have far less nicotinic antagonist activity (IC(50) >1 microM); the active compounds I through III have cationic tails at an indole nitrogen, whereas the least potent compounds IV and V do not. By contrast, a free NH within the maleimide ring is crucial for PKC inhibition by this class of compounds. We conclude that bisindolylmaleimides I through III are some of the most potent noncompetitive neuronal nicotinic antagonists, indeed the most potent such antagonists we have observed in PC-12 cells. Nicotinic antagonism of these compounds seems to be independent of PKC inhibition.

NID67, a small putative membrane protein, is preferentially induced by NGF in PC12 pheochromocytoma cells.

In an effort to identify genes involved in neuronal differentiation, we have used representational difference analysis (RDA) to clone cDNAs that are preferentially induced by nerve growth factor (NGF) vs. epidermal growth factor (EGF) in PC12 pheochromocytoma cells. We now report the cloning of a previously unknown primary response gene, NID67. In addition to a robust induction by NGF and FGF, both of which cause PC12 cells to differentiate, NID67 is strongly induced by forskolin, A23187 and ATP. EGF, TPA and KCl induce NID67 only weakly. NID67 mRNA is most abundant in heart, ovary and adrenal. Modest levels are present in most brain regions, testis, thyroid, thymus, pituitary, kidney and intestine; little NID67 is present in skeletal muscle and cerebellum. The NID67 cDNA contains a 180 bp open reading frame (ORF) that encodes a 60 amino acid protein. The central 29 amino acids are very hydrophobic and very likely comprise a transmembrane domain. Mouse and human NID67 cDNAs contain an ORF similar to NID67; the rat and human protein sequences are 85% identical whereas the rat and mouse sequences are 92% identical. In vitro transcription and translation reactions confirmed that the ORF we identified produces a 6000 Da protein product. Several small membrane proteins are similar to NID67; they contain a transmembrane domain and little more. All of these proteins participate in forming or regulating ion channels. NID67 may play a similar role in cellular physiology.

[(123)I]metaiodobenzylguanidine and [(111)In]octreotide uptake in begnign and malignant pheochromocytomas.

Selecting the appropriate approach for resection and follow-up of pheochromocytomas (PCCs) is highly dependent upon reliable localization and exclusion of multifocal, bilateral, or metastatic disease. Metaiodobenzylguanidine (MIBG) scintigraphy was developed for functional localization of catecholamine-secreting tissues. Somatostatin receptor imaging (SRI) has a high sensitivity for localizing head and neck paragangliomas, but studies of intraabdominal PCCs are rare. In this study we review our experience of [(123)I]MIBG and SRI, performed since 1983 and 1989, respectively, in the work-up of primary and recurrent PCCs. Scintigraphic results were correlated with catecholamine secretion, size and site, malignancy, associated tumor syndromes, and morphological features. [(123)I]MIBG scans were performed in a total of 75 patients, in 70 cases before resection of primary PCCs and in 5 cases because of recurrent disease. Ninety-one PCCs were resected. The overall detection rates were 83.3% and 89.8% for PCCs larger than 1.0 cm. Multifocal disease was detected in 4 patients with [(123)I]MIBG. [(123)I]MIBG uptake correlated with greater size of PCC (r = 0.33; P = 0.008) and greater concentration of plasma epinephrine (r = 0.32; P = 0.006). [(123)I]MIBG-negative PCCs (n = 14) had significantly (P = 0.01) smaller diameters than [(123I)]MIBG-positive tumors. Furthermore, [(123)I]MIBG uptake was significantly higher in unilateral (P = 0.02), benign (P = 0.02), sporadic (P = 0.02), intraadrenal (P = 0.02), and capsular invasive (P = 0.03) PCCs than in bilateral, malignant, MEN2A/2B-related, extraadrenal, and noninvasive PCCs, respectively. The detection rate of SRI was only 25% (8 of 32) for primary benign PCCs. In 14 patients metastases occurred, which were effectively visualized with [(123)I]MIBG in 8 of 14 cases. SRI was able to detect metastases in 7 of 8 cases, including 3 [(123)I]MIBG-negative metastatic cases. In addition, [(123)I]MIBG and SRI detected 2 recurrences. In conclusion, [(123)I]MIBG uptake is correlated with the size, epinephrine production, and site of PCCs. Its role in bilateral and MEN2A/2B-related PCCs seems limited. In cases of recurrent elevation of catecholamines, localization of metastases and/or recurrence should be attempted with [(123)I]MIBG scintigraphy. In suspicious metastatic PCCs, SRI might be considered to supplement [(123)I]MIBG scintigraphy.

Ganoderma extract activates MAP kinases and induces the neuronal differentiation of rat pheochromocytoma PC12 cells.

The pharmacology and clinical application of traditional Chinese medicine has been extensively documented. We have used an in vitro model system, PC12 cells, to demonstrate the presence of neuroactive compounds in Ganoderma lucidum (lingzhi). Ganoderma extract induced the neuronal differentiation of PC12 cells and prevented nerve growth factor-dependent PC12 neurons from apoptosis. Moreover, these effects of ganoderma might be mediated via the ras/extracellular signal-regulated kinase (Erk) and cAMP-response element binding protein (CREB) signaling pathways, as demonstrated by the phosphorylation of Erk1, Erk2 and CREB. Thus, our data not only present the first evidence of the presence of neuroactive compounds that mediate the neuronal differentiation and neuroprotection of the PC12 cells, but also reveal the potential signaling molecules involved in its action.