Ultrastructure of human neural stem/progenitor cells and neurospheres

BACKGROUND： Biological and morphological characteristics of neural stem/progenitor cells （NSPCs） have been widely investigated. OBJECTIVE： To explore the ultrastructure of human embryo-derived NSPCs and neurospheres cultivated in vitro using electron microscopy. DESIGN, TIME AND SETTING： A cell biology experiment was performed at the Brain Tumor Laboratory of Soochow University, and Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University between August 2007 and April 2008. MATERIALS： Human fetal brain tissue was obtained from an 8-week-old aborted fetus; serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium was provided by Gibco, USA; scanning electron microscope was provided by Hitachi Instruments, Japan; transmission electron microscope was provided by JEOL, Japan. METHODS： NSPCs were isolated from human fetal brain tissue and cultivated in serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium. Cells were passaged every 5-7 days. After three passages, NSPCs were harvested and used for ultrastructural examination. MAIN OUTCOME MEASURES： Ultrastructural examination of human NSPCs and adjacent cells in neurospheres. RESULTS： Individual NSPCs were visible as spherical morphologies with rough surfaces under scanning electron microscope. Generally, they had large nuclei and little cytoplasm. Nuclei were frequently globular with large amounts of euchromatin and a small quantity of heterochromatin, and most NSPCs had only one nucleolus. The Golgi apparatus and endoplasmic reticulum were underdeveloped; however, autophagosomes were clearly visible. The neurospheres were made up of NSPCs and non-fixiform material inside. Between adjacent cells and at the cytoplasmic surface of apposed plasma membranes, there were vesicle-like structures. Some membrane boundaries with high permeabilities were observed between some contiguous NSPCs in neurospheres, possibly attributable to plasmalemmal fusion between adjacent cells. CONCLUSION： A large number of autophagosomes were observed in NSPCs and gap junctions were visible between adjacent NSPCs.

Zinc transporter-3 expression and long-term cognitive impairments in a rat model of neonatal concurrent seizure

BACKGROUND： Developmental seizures, which are pathologically characterized by regenerative sprouting of hippocampal mossy fibers, cause long-term damaging effects to synaptic plasticity. Zn^2＋ metabolism has been shown to contribute to the regenerative sprouting of hippocampal mossy fibers Furthermore, zinc transporter-3 （ZnT3） is responsible for Zn^2＋ transport in the hippocampal mossy fiber pathway. OBJECTIVE： To investigate the effects of long-term recurrent neonatal seizures on learning, memory formation and hippocampal ZnT3 expression in rats. DESIGN, TIME AND SETTING： Based on molecular biological research and behavioral examination a randomized, controlled, animal experiment was performed at the Laboratory Animal Center, Peking University Health Science Center, between October 2004 and July 2005. MATERIALS： Flurothyl was purchased from Aldrich Chemical Co., USA. ZnT3 mRNA in situ hybridization kits were provided by Tianjin Haoyang Biological Manufacture Co., Ltd., China. Morris water maze was produced by Shanghai Jiliang Science and Technology Co., Ltd., China. METHODS： Sixty, 6-day old, Wistar rats were randomly divided into three groups： single seizure （n = 21）, recurrent seizure （n = 21, one seizure daily for 6 consecutive days）, and control （n = 18）. Seizures were induced by flurothyl gas inhalation, in the single seizure and recurrent seizure groups. MAIN OUTCOME MEASURES： At postnatal days 12, 46 and 90, rat hippocampal ZnT3 mRNA expression was detected by RT-PCR; at postnatal days 46 and 90, ZnT3 mRNA expression was determined by in situ hybridization; and at postnatal days 41-46 and 85 90, rat spatial learning and memory formation were examined by the Morris water maze test. RESULTS： RT-PCR results revealed that at postnatal day 12, ZnT3 expression was significantly greater in the recurrent seizure group than in the control and single seizure groups, and at day 46, it was also significantly greater in the recurrent seizure group compared with the control group （P 〈 0.05）. In situ hybridization results showed that at postnatal day 46, the recurrent seizure group exhibited increased hippocampal ZnT3 expression over the control and single seizure groups （P〈0.05）. Morris water maze test results displayed that, in the first place navigation test at postnatal day 44, and the second test at days 87-88, the recurrent seizure group exhibited significantly higher value of average escape latency compared with the control group （P 〈 0.05）. In the two spatial probe tests, the search strategies were significantly inferior in the recurrent seizure group than in the control and single seizure groups （P 〈 0.05）. CONCLUSION： Neonatal concurrent seizures produce long-term damaging effects on hippocampal ZnT3 expression and cognitive function, while both of which have no parallel correlation.

Mutant alpha-synuclein and autophagy in PC12 cells

Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively. Results demonstrated that mutant a-synuclein resulted in cell death via autophagy and involved a-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutant a-synuclein （A30P） also mediated toxicity of 1-methyl-4-phenylpyridinium ion. Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted α-synuclein aggregation and cell death. To further determine the role of autophagy due to mutant α-synuclein, the present study measured expression of microtubule-associated protein light chain 3. Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3 while rapamycin promoted its expression. These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

Trehalose ameliorates autophagy dysregulation in aged cortex and acts as an exercise mimetic to delay brain aging in elderly mice

Exercise is recognized as an effective strategy to delay brain aging, which is related to the activation of autophagy. Trehalose is a natural compound that can activate autophagy and exert beneficial effects in delaying brain aging. In this study, we investigated whether trehalose may exert neuroprotection similar to those of exercise in delaying age-related cognitive decline. Fifteen-month-old male C57BL/6 mice underwent swim exercise and/or were treated with 2% trehalose for 12 weeks. Trehalose, exercise and the combination of exercise and trehalose intervention improved the learning and memory of aged mice. They also improved the ratio of LC3-II/LC3-I, the protein level of LC3-II, Bnip3L, and Parkin respectively. Additionally, both exercise and trehalose increased the phosphorylation of AMPK. Exercise decreased cortical phosphorylation of m TOR and S6k, whereas trehalose did not change these cortical levels. These data indicated that exercise and trehalose might modulate autophagy through m TOR-dependent or m TOR-independent pathways, respectively. However, a combination of exercise and trehalose did not play a synergistic role in improving cognitive function and modulation of autophagy. Taken together, our findings suggest that trehalose exerts similar effects to those of exercise in delaying age-related cognitive decline and that it may thus represent an exercise mimetic to delay brain aging.

Inhibition of Differentiation by Transforming Growth Factor β1 in Rhabdomyosarcoma Cells

OBJECTIVE To study the effect of transforming growth factor?1 (TGF-β1)on differentiation of rhabdomyosarcoma(RMS)cells METHODS RD(human embryonal RMS cell line)cells,cultured in differentiation medium containing 9-cis retinoic acid(9CRA),were treated with TGF-β1.Proliferation of the cells was examined by the MTT assay.The differentiation specific proteins(sarcomeric actin and MyHC)and myogenic transcription factors(MyoD1 and myogenin)in the RD cel s were assessed by immunofluorescence staining. RESULTS Compared to control cel s,treatment with lower concentrations of TGF-?1(0.1 and 0.2 ng/ml)induced an increase in OD values after 4 d(P<0.01),whereas higher concentrations of TGF-?1(2 and 5 ng/ml)led to a reduction of cell viability(P<0.01).After exposure to 9CRA,the viability of the cells decreased significantly(P<0.01),while sarcomeric actin,MyHC and myogenin were induced.These changes were antagonized by TGF-β1(0.1 ng/ml).No changes were observed in expression of MyoD1. CONCLUSION The RMS cells,derived from myogenic progenitors are committed to a myogenic fate,but are arrested in the differentiation course by the addition of TGF-?1 which represses some of the myogenic transcription factors.

A Salmonella enterica serovar Typhi plasmid induces rapid and massive apoptosis in infected macrophages

pRST98 is a chimeric plasmid isolated from Salmonella enterica serovar Typhi(S.typhi)that mediates the functions of drug resistance and virulence.Previously,we reported that Salmonella plasmid virulence(spv)genes were present in S.typhi.In our current study,we investigated whether plasmid pRST98 exhibits significant cytotoxicity in macrophages.pRST98 was transferred into the avirulent Salmonella enterica serovar Typhimurium(S.typhimurium)strain RIA to create the transconjugant pRST98/RIA.The standard S.typhimurium virulent strain SR-11,which carries a 100-kb virulence plasmid,was used as a positive control.The bacterial strains were incubated with a murine macrophage-like cell line(J774A.1)in vitro.Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling,and the survival of Salmonella strains in J774A.1 cells was determined.Results showed that macrophages infected with strain pRST98/RIA displayed greater levels of apoptosis than those infected with RIA and that pRST98 may increase bacterial survival in macrophages.Further studies showed that the pRST98-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pRST98 may activate caspase-9 and then caspase-3.The research data indicate that the virulence of bacteria that contain the pRST98 plasmid is enhanced;the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.