iTRAQ-based proteomics analysis on insomnia rats treated with Mongolian medical warm acupuncture.

OBJECTIVE: To explore the proteomic changes in the hypothalamus of rats treated with Mongolian medical warm acupuncture for insomnia therapy based proteomics. METHOD: We used an iTRAQ-based quantitative proteomic approach to identify proteins that potential molecular mechanisms involved in the treatment of insomnia by Mongolian medical warm acupuncture. RESULT: In total, 7477 proteins were identified, of which 36 proteins showed increased levels and 45 proteins showed decreased levels in insomnia model group (M) compared with healthy control group (C), 72 proteins showed increased levels and 44 proteins showed decreased levels from the warm acupuncture treated insomnia group (W) compared with healthy controls (C), 28 proteins showed increased levels and 17 proteins showed decreased levels from the warm acupuncture-treated insomnia group (W) compared with insomnia model group (M). Compared with healthy control groups, warm acupuncture-treated insomnia group showed obvious recovered. Bioinformatics analysis indicated that up-regulation of neuroactive ligand-receptor interaction and oxytocin signaling was the most significantly elevated regulate process of Mongolian medical warm acupuncture treatment for insomnia. Proteins showed that increased/decreased expression in the warm acupuncture-treated insomnia group included Prolargin (PRELP), NMDA receptor synaptonuclear-signaling and neuronal migration factor (NSMF), Transmembrane protein 41B (TMEM41B) and Microtubule-associated protein 1B (MAP1B) to adjust insomnia. CONCLUSION: A combination of findings in the present study suggest that warm acupuncture treatment is efficacious in improving sleep by regulating the protein expression process in an experimental rat model and may be of potential benefit in treating insomnia patients with the added advantage with no adverse effects.

Dioscin relieves endotoxemia induced acute neuro-inflammation and protect neurogenesis via improving 5-HT metabolism.

Sepsis, in addition to causing fatality, is an independent risk factor for cognitive impairment among sepsis survivors. The pathologic mechanism of endotoxemia induced acute neuro-inflammation still has not been fully understood. For the first time, we found the disruption of neurotransmitters 5-HT, impaired neurogenesis and activation of astrocytes coupled with concomitant neuro-inflammation were the potential pathogenesis of endotoxemia induced acute neuro-inflammation in sepsis survivors. In addition, dioscin a natural steroidal saponin isolated from Chinese medicinal herbs, enhanced the serotonergic system and produced anti-depressant effect by enhancing 5-HT levels in hippocampus. What is more, this finding was verified by metabolic analyses of hippocampus, indicating 5-HT related metabolic pathway was involved in the pathogenesis of endotoxemia induced acute neuro-inflammation. Moreover, neuro-inflammation and neurogenesis within hippocampus were indexed using quantitative immunofluorescence analysis of GFAP DCX and Ki67, as well as real-time RT-PCR analysis of some gene expression levels in hippocampus. Our in vivo and in vitro studies show dioscin protects hippocampus from endotoxemia induced cascade neuro-inflammation through neurotransmitter 5-HT and HMGB-1/TLR4 signaling pathway, which accounts for the dioscin therapeutic effect in behavioral tests. Therefore, the current findings suggest that dioscin could be a potential approach for the therapy of endotoxemia induced acute neuro-inflammation.

Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting of Endogenous MAP1LC3B.

Assays that monitor autophagic flux, or degradative completion of autophagy, are crucial for the assessment of the dynamic autophagy process in a variety of systems. Such assays help to distinguish between an increase in autophagosomes resulting from induced autophagic activity versus an increase in autophagosomes due to reduced lysosomal turnover. The majority of flux assays use autophagy protein MAP1LC3B (microtubule-associated proteins 1A/1B light chain 3B, here referred to as LC3B) as a marker for autophagy, and most are based on the use of reporters. Here, we describe a method, suitable for monitoring flux in primary cells and/or when reporters are not available or desirable, that uses lysosomal inhibitors and the analysis of endogenous LC3B-II (the lipidated form of LC3B that is associated with autophagosomes) by western blotting. A common application of this method, detailed here, is to test whether a treatment of interest (e.g., chemotherapy drug) induces autophagic flux in the cells of interest. If it is found that there is no difference in LC3B-II levels between treatment with lysosomal inhibitor alone versus drug plus lysosomal inhibitor, then this suggests that the drug is not inducing autophagic flux. Elevated levels of LC3B-II in treatments with drug plus lysosomal inhibitor, compared with drug treatment alone and inhibitor treatment alone, indicate that the drug is probably leading to an increase in autophagic flux.

ß-Asarone prevents autophagy and synaptic loss by reducing ROCK expression in asenescence-accelerated prone 8 mice.

ß-Asarone is an active component of the Acori graminei rhizome that is a traditional Chinese medicine clinically used in treating dementia in China. However, the cognitive effect of ß-asarone and its mechanism has remained elusive. Here, we used asenescence-accelerated prone 8 (SAMP8) mice, which mimic many of the salient features of Alzheimer׳s disease (AD), to further investigate whether modulation of the ROCK signaling pathway and/or autophagy, synaptic loss is involved in the effects of ß-asarone on learning and memory. SAMP8 mice at the age of 6 months were intragastrically administered by ß-asarone or a vehicle daily for 2 months. Senescence-accelerated-resistant (SAMR1) mice were used as the control. Our results demonstrate that autophagy and ROCK expression were increased significantly in 8 months SAMP8 mice, which were concomitant with that SAMP8 mice at the same age displayed a significant synaptic loss and cognitive deficits. The up-regulation of ROCK expression and autophage in the hippocampus of SAMP8 were significantly reduced by ß-asarone, and prevents synaptic loss and improved cognitive function of the SAMP8 mice. ß-asarone decreased neuronophagia and lipofuscin in the hippocampus of SAMP8 mice, but did not reduce Aß42 levels and malondialdehyde levels and superoxide dismutase activities. Moreover, suppression of ROCK2 by siRNA significantly reduced the effects of ß-asarone on the autophage and synaptic proteins expression in PC12 cells damage induced by Aß1-40. Taken together, ß-asarone prevents autophagy and synaptic loss by reducing ROCK expression in SAMP8 mice.

Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity.

BACKGROUND: We previously demonstrated the autophagy-inducing activity in the crude extract of areca nut (ANE) and its 30-100 kDa fraction (ANE 30-100 K). This study aimed to analyze whether chronic ANE and ANE 30-100 K stimulations lead to higher stress resistance and autophagic activity in oral cells, and whether the resulting autophagic status in stimulated cells correlates with stress resistance. MATERIALS AND METHODS: Malignant cells from the mouth oral epidermoid carcinoma Meng-1 (OECM-1) and blood (Jurkat T) origins were stimulated with non-cytotoxic ANE and ANE 30-100 K for 3 months. Sensitivity to anticancer drugs of and autophagy status in stimulated cells, analyzed respectively by XTT assay and calculating microtubule-associated protein 1 light chain 3-II LC3-II/ß-actin ratios from Western blot, were compared to non-treated cells. Autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), were used to assess whether autophagy inhibition interferes the altered chemoresistance. RESULTS: Areca nut extract-stimulated (ANE-s) and ANE 30-100 K-stimulated (30-100 K-s) OECM-1 and Jurkat T cells generally exhibited higher cisplatin and 5-fluorouracil (5-FU) resistances, compared to non-stimulated cells. Most stimulated cells expressed significantly higher levels of LC3-II and Atg4B proteins. Interestingly, these cells also showed stronger tolerances against hypoxia environment and expressed higher LC3-II levels under glucose-deprived and hypoxia conditions. Finally, both 3-MA and CQ alleviated, albeit to different degrees, the increased chemoresistance in ANE-s and/or 30-100 K-s cells. CONCLUSIONS: Chronic stimulations of ANE or ANE 30-100 K may increase tolerance of oral cancer and leukemia T cells to anticancer drugs, as well as to glucose deprivation and hypoxia conditions, and cause an elevation of autophagy activity responsible for increased drug resistance.

Pyrithiamine-induced thiamine deficiency alters proliferation and neurogenesis in both neurogenic and vulnerable areas of the rat brain.

Thiamine deficiency (TD) leads to Wernicke's encephalopathy (WE), in which focal histological lesions occur in periventricular areas of the brain. Recently, impaired neurogenesis has been reported in the hippocampus during the dietary form of TD, and in pyrithiamine-induced TD (PTD), a well-characterized model of WE. To further characterize the consequences of PTD on neural stem/progenitor cell (NSPC) activity, we have examined the effect of this treatment in the rat on both the subventricular zone (SVZ) of the rostral lateral ventricle and subgranular layer (SGL) of the hippocampus, and in the thalamus and inferior colliculus, two vulnerable brain regions in this disorder. In both the SVZ and SGL, PTD led to a decrease in the numbers of bromodeoxyuridine-stained cells, indicating that proliferation of NSPCs destined for neurogenesis in these areas was reduced. Doublecortin (DCX) immunostaining in the SGL was decreased, indicating a reduction in neuroblast formation, consistent with impaired NSPC activity. DCX labeling was not apparent in focal areas of vulnerability. In the thalamus, proliferation of cells was absent while in the inferior colliculus, numerous actively dividing cells were apparent, indicative of a differential response between these two brain regions. Exposure of cultured neurospheres to PTD resulted in decreased proliferation of NSPCs, consistent with our in vivo findings. Together, these results indicate that PTD considerably affects cell proliferation and neurogenesis activity in both neurogenic areas and parts of the brain known to display structural and functional vulnerability, confirming and extending recent findings on the effects of TD on neurogenesis. Future use of NSPCs in vitro may allow a closer and more detailed examination of the mechanism(s) underlying inhibition of these cells during TD.

Inhibition of autophagy augments chemotherapy in human salivary adenoid cystic carcinoma.

Although cisplatin (DDP)-based adjuvant chemotherapy is widely used in the treatment of salivary adenoid cystic carcinoma (SACC), SACCs have developed resistance to cisplatin, resulting in chemotherapy failure. Autophagy serves as a critical adaptive response, which was increased in tumor cells in chemotherapy. However, the function of autophagy is not clear in SACC. In this study, apoptosis induced by DDP in SACC high metastatic cell line (ACC-M) was revealed using MTT assay, flow cytometry, and caspase-3 immunoblotting. The autophagy activation induced by DDP treatment was measured by transmission electron microscopy, green fluorescent protein-light chain 3 plasmid transfection LC3 immunoblotting and p62 immunoblotting. 3-methyladenine (3-MA) or small interference RNA targeting beclin 1 (beclin 1 siRNA) inhibited autophagy and significantly enhanced DDP-induced apoptosis. ACC-M xenografts in nude mice further verified the synergistic effect of DDP and 3-MA. In conclusion, autophagy activation was caused to protect cancer cells from DDP-induced apoptosis and autophagy inhibition could be a promising strategy for adjuvant chemotherapy in SACC.

Annexin and survivin in locally advanced rectal cancer: indicators of resistance to preoperative chemoradiotherapy?

BACKGROUND: The aim of preoperative chemoradiotherapy is improvement of local control in patients with locally advanced rectal cancer (LARC). Recent studies have shown that annexin and survivin are involved in the resistance capability of tumours. We sought to determine whether survivin, annexin A4 or annexin A5 expression predict resistance to preoperative chemoradiotherapy. MATERIAL AND METHODS: Biopsies of tumour and normal rectal tissue were taken from 38 patients with LARC (cT3/4Nx or Tx/N+) before the start of chemoradiotherapy and during surgery. mRNA expression of annexin A4/A5 and survivin was measured by real-time polymerase chain reaction (RT-PCR) and correlated with down-staging and progression-free survival (PFS). RESULTS: Significantly higher mRNA levels of survivin, and annexin A4/A5 were detected in untreated tumour compared with normal mucosa. After chemoradiotherapy, this difference disappeared for survivin and annexin A4. Annexin A5 expression in the tumour increased during chemoradiotherapy. No correlation between the mRNA levels of survivin, annexin A4/A5 and tumour down-staging or PFS was noticed. CONCLUSIONS: In the present analysis of 38 patients with LARC undergoing neoadjuvant chemoradiotherapy, the expression levels of survivin and annexin A4 and A5 did not correlate with down-staging. Moreover, with regard to PFS, none of these markers was found to be prognostically relevant.

Brazilein inhibits survivin protein and mRNA expression and induces apoptosis in hepatocellular carcinoma HepG2 cells.

Hepatocellular carcinomas represent the third leading cause of cancer-related deaths worldwide. Survivin, a structurally unique member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in a wide range of malignancies, including hepatocellular carcinoma. Due to its involvement in cancer progression and treatment resistance, survivin is currently undergoing extensive investigation as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Brazilein, a compound obtained in a large amount from the dried heartwood of Caesalpinia sappan Linn., which has long been used in traditional medicine in China, has some pharmacological activities. Human hepatocellular carcinoma HepG2 cells were treated with brazilein and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Brazilein treatment of cells for 48 h at 5 and 10 microg/ml doses resulted in significantly decrease in survivin protein expression. We also observed that brazilein caused a strong decrease in survivin mRNA expression. In other studies, down-regulation of survivin by brazilein was associated with a strong and prominent caspases-9 and -3 activation as well as PARP cleavage. It was also shown that brazilein induced a strong apoptotic cell death, as shown by DNA ladder assay, and growth inhibition of HepG2 cells. Further studies are needed to investigate in vivo effect of brazilein on survivin expression and associated biological effects in hepatocellular carcinoma that could provide useful information for brazilein efficacy in the prevention/intervention of human hepatocellular carcinoma.

Tanshinone IIA inhibits leukemia THP-1 cell growth by induction of apoptosis.

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, has been reported to have anti-tumor effects on a large variety of cancer cells. The present study was undertaken to investigate the in vitro antiproliferation and apoptosis inducing effects of Tanshinone IIA on leukemia THP-1 cell lines and its mechanisms of action. MTT assay was used to detect the cell growth inhibitory rate; cell apoptotic rate and the mitochondrial membrane potential (Deltapsim) were investigated by flow cytometry (FCM), apoptotic morphology was observed by Hoechst 33258 staining and DNA fragmentation analysis. The expression of caspase-3 and different apoptosis modulators were analyzed by Western blotting. The results revealed that Tanshinone IIA inhibited the growth of THP-1 cells and caused significant apoptosis, the suppression was both in time- and dose-dependent manner. After treatment by Tanshinone IIA for 48 h, the percentage of disruption of Deltapsim gradually increased in a dose-dependent manner along with marked changes of cell apoptosis. Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit and a dose-dependent cleavage of PARP, with the appearance of 89-kDa fragment; The expression of Bcl-2 and survivin was down-regulated remarkably while Bax expression was up-regulated concurrently after the cells were treated with Tanshinone IIA for 48 h. We therefore conclude that Tanshinone IIA has significant growth inhibition effects on THP-1 cells by induction of apoptosis, and that Tanshinone IIA-induced apoptosis on THP-1 cells is mainly related to the disruption of Deltapsim and activation of caspase-3 as well as down-regulation of anti-apoptotic protein Bcl-2, survivin and up-regulation of pro-apoptotic protein Bax. The results indicate that Tanshinone IIA may serve as a potential anti-leukemia reagent.

Edaravone (MCI-186) scavenges reactive oxygen species and ameliorates tissue damage in the murine spinal cord injury model.

The present study evaluated the effect of the free radical scavenger edaravone on lesion volume and neurological dysfunction after spinal cord injury (SCI) in mice, and investigated its protective effects on superoxide generation. Female C57BL/6 mice were subjected to SCI using a pneumatic impact device and were treated with 3 mg/kg of edaravone or vehicle 30 minutes before the insult. Motor functions were quantitatively evaluated. Lesion volume was assessed by Dohrmann's two-cone method after one week. In situ detection of superoxide in the injured cord was carried out using the superoxide-sensitive dye dihydroethidium (DHE) staining technique. Pretreatment with edaravone significantly improved motor dysfunction and reduced the lesion volume to about 63% of the control (p < 0.05). Semi-quantitative measurements of red fluorescence emitted from DHE revealed that the superoxide concentration increased in the lesion periphery at 1 and 3 hours after the insult, and that pretreatment with edaravone significantly inhibited the increase of superoxide concentration in the lesion periphery at both time points (p < 0.0001). Double staining with DHE and monoclonal antibody against MAP2 showed that most cells positive for DHE were also positive for MAP2. These findings suggest that edaravone ameliorates tissue damage by scavenging reactive oxygen species, especially in the neurons, after SCI.

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells.

BACKGROUND: Although extensive research has been performed to control differentiation of neural stem cells - still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation - allowing for an increase in percentage yield of neuronal cells. RESULTS: Uncommitted GFAP and SOX2 positive neural progenitors - so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100beta-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells--i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. CONCLUSION: We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells - such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

Survivin, p53, and Ki-67 as predictors of histopathologic response in locally advanced rectal cancer treated with preoperative chemoradiotherapy.

PURPOSE: The ability to predict response to chemoradiotherapy before the treatment may allow protecting poorly responding patients from the side effects of neoadjuvant treatment. Several molecular markers have been proposed to radio and chemosensitivity of rectal cancer. In this study, from pre-irradiation tumor biopsies, a novel and promising candidate factor survivin, and p53 and Ki-67 were assessed as predictors of response to preoperative chemoradiotherapy. MATERIALS AND METHODS: Expression of each marker was evaluated by immunohistochemistry on pretreatment biopsies from 37 patients having rectal cancer treated with preoperative chemoradiotherapy and curative surgery. Treatment response was assessed histopathologically in the resected surgical specimen. RESULTS: There was no correlation between expression of p53, Ki-67, and survivin with response to preoperative chemoradiotherapy and prognosis. CONCLUSIONS: Our data suggest that these molecular markers are not helpful to identify patients who would have benefit from neoadjuvant treatment of rectal cancer. Further investigations are necessary to select patients for preoperative treatment based on analysis of the preoperative biopsies.

Small molecule regulators of autophagy identified by an image-based high-throughput screen.

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.

Suppressive effects of dehydroepiandrosterone and the nuclear factor-kappaB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo.

Dehydroepiandrosterone (DHEA) is believed to have an anti-tumor effect, as well as anti-inflammatory, antioxidant, and anti-aging effects. To clarify the possible inhibitory action of DHEA on pituitary tumor cells, we tested the effects of DHEA, alone or in combination with the nuclear factor-kappaB (NF-kappaB) inhibitor parthenolide (PRT), on AtT20 corticotroph cell growth and function both in vitro and in vivo. We found that, in vitro, DHEA and PRT had potent inhibitory effects on pro-opiomelanocortin and NF-kappaB-dependent gene expression. They also suppressed the transcription activity of survivin, a representative anti-apoptotic factor, and induced apoptosis in this cell line. Furthermore, using BALB/C nude mice with xenografts of AtT20 cells in vivo, we found that the combined administration of DHEA and PRT significantly attenuated tumor growth and survivin expression. The treatment also decreased the elevated plasma corticosterone levels and ameliorated the malnutrition induced by tumor growth. Altogether, these results suggested that combined treatments of DHEA and PRT potently inhibit the growth and function of corticotroph tumor cells both in vitro and in vivo. This effect may, at least partly, be caused by the suppressive effects of these compounds, such as survivin and other inhibitor of apoptosis proteins, on NF-kappaB-mediated gene transcription.

Quantitative neuropathologic correlates of changes in ratio of N-acetylaspartate to creatine in macaque brain.

PURPOSE: To elucidate the neuropathologic basis of transient changes in the ratio of N-acetylaspartate (NAA) to creatine (Cr) in the primate brain by using a simian immunodeficiency virus (SIV)-infected macaque model of the neurologic manifestation of acquired immune deficiency syndrome. MATERIALS AND METHODS: This study was approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care and the Institutional Animal Care and Use Committee of Harvard University. Rhesus macaques infected with SIV were evaluated during the 1st month of infection. A total of 11 animals were studied, including four control animals, three animals sacrificed 12 days after infection, three animals sacrificed 14 days after infection, and one animal sacrificed 28 days after infection. All animals underwent in vivo proton ((1)H) magnetic resonance (MR) spectroscopy, and postmortem frontal lobe tissue was investigated by using high-spectral-resolution (1)H MR spectroscopy of brain extracts. In addition, quantitative neuropathologic analyses were performed. Stereologic analysis was performed to determine neuronal counts, and immunohistochemical analysis was performed to analyze three neuronal markers: synaptophysin, microtubule-associated protein 2 (MAP2), and calbindin. Analysis of variance (ANOVA) was used to determine substantial changes in neuropathologic and MR spectroscopic markers. Spearman rank correlations were calculated between plasma viral load and neuropathologic and spectroscopic markers. RESULTS: During acute infection with SIV, the macaque brain exhibited significant changes in NAA/Cr (P < .02, ANOVA) and synaptophysin (P < .013, ANOVA). There was no significant change in the concentration of Cr. No significant changes were found in neuronal counts or other immunohistochemical neuronal markers. With the Spearman rank test, a significant direct correlation was detected between synaptophysin and ex vivo NAA/Cr (r(s) = 0.72, P < .013). No correlation between NAA/Cr and neuronal counts, calbindin, or MAP2 was found. CONCLUSION: NAA/Cr is a sensitive marker of neuronal injury, not necessarily neuronal loss, and best correlates with synaptophysin, a marker of synaptodendritic dysfunction.

[Cloning and subcellular localization of apr-1--a new gene of tumor specific antigen family].

BACKGROUND & OBJECTIVE: apr-1 was cloned by improved polymerase chain reaction (PCR)-based subtractive hybridization from all-trans retinoic acid (ATRA)-induced apoptotic leukemia HL-60 cells in 1999. Preliminary results showed that apr-1 might be an apoptosis-related gene (GenBank ID: NM_014061). This study was to explore the background of apr-1 through gene cloning, bioinformatic analysis, and subcellular locating. METHODS: The cDNA encoding Apr-1 was amplified by reverse transcription-PCR (RT-PCR), and sequenced. Open reading frame (ORF) of apr-1 was analyzed with ORF finder software. Chromosome locus was defined by genome blast software. Conserved domains of amino acids were analyzed by protein blast software. Align (Cluster W) software in Vector NTI software package was used to analyze homogeneous genes (or proteins), and to draw the Phylogenetic Tree. Subcellular localization of apr-1 was performed. RESULTS: apr-1 was mapped to chromosome Xp11.22 with the ORF locating in 1 exon. Two MAGE conserved domains were found in Apr-1. Apr-1 shared homology with MAGE-A1, MAGE-B1, MAGE-C1, MAGE-D1, and Necdin. Phylogenetic analysis showed that Apr-1 was more closely related to MAGE-D1 and Necdin. Gene products of apr-1 were located in the nuclei of eukaryocytes. CONCLUSIONS: apr-1 is a member of MAGE family, and might belong to type II MAGE genes.

Absence of histological lesions in primate models of ECT and magnetic seizure therapy.

OBJECTIVE: The authors present preliminary findings from the first nonhuman primate neuropathological study of ECT to use perfusion fixation and adequate controls and the first to compare ECT with magnetic seizure therapy, to their knowledge. METHOD: Twelve Macaca mullata received 6 weeks of daily ECT, magnetic seizure therapy, or anesthesia alone. After perfusion fixation, their brains were examined while masked to intervention. RESULTS: No identified lesions were attributable to the interventions. Cortical and hippocampal immunoreactivity for glial fibrillary acidic protein (an astrocytic marker) was most intense in the group that received ECT. CONCLUSIONS: This small but rigorous primate study supports the view that ECT does not produce histological lesions in the brain and provides the first comparable safety data on magnetic seizure therapy.

Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem.

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker alpha-internexin, while the other stained with the astrocyte marker GFAP. The alpha-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These alpha-internexin positive cells coimmunostained for the neuronal markers MAP2, type III beta-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these alpha-internexin positive cells revealed small Ca(2+) currents with a peak current of -0.5 +/- 0.1 pA/pF at a membrane potential of -20 mV (n = 5) and half-maximal activation at -30 mV (n = 5). Na(+) currents with a peak current density of -154.5 +/- 49.8 pA/pF at a membrane potential of -15 mV (n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.

The effects of dietary sulfur amino acid deficiency on rat brain glutathione concentration and neural damage in global hemispheric hypoxia-ischemia.

Primary brain injury in stroke is followed by an excitotoxic cascade, oxidative stress and further neural damage. Glutathione is critical and depleted in oxidative stress. Since cysteine is limiting in glutathione synthesis, this study investigated the effect of dietary sulfur amino acid (SAA) deficiency on neural damage in a rat model of global hemispheric hypoxia-ischemia (GHHI). Animals were fed with SAA deficient ("deficient") or control diet for 3 days, subjected to right common carotid artery ligation and hypoxia, and diet continued for 3 more days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in "deficient" rats, shown by mean (+/- SEM) global and hippocampal grid scores of 2.5 +/- 0.7 and 34.9 +/- 9.3%, respectively, vs. controls' scores of 0.1 +/- 0.1 and 0.1 +/- 0.1%, respectively. Mean brain (+/- SEM) reduced glutathione was not different between groups at 6h post hypoxia-ischemia, but was decreased in "deficient" animals 3 days later in neocortex (1.46 micromoles/g wet weight +/- 0.05 vs. 1.67 +/- 0.04 in controls) and thalamus (1.60 micromoles/g wet weight +/- 0.05 vs. 1.78 +/- 0.03 in controls). Administration of a cysteine precursor to "deficient" animals did not ameliorate neural damage. These findings suggest that well-nourished but not "deficient" animals tolerate a mild brain insult. The decline in brain glutathione in the "deficient" animals may be one of several contributing mechanisms.