The cytoplasmic creatine kinase (CKB) enzyme has a central role in the regeneration of ATP in the brain. We have shown previously that CKB mRNA levels in cultured primary rat brain astrocytes and oligodendrocytes are much higher than in primary neurons. It has been suggested that high CKB expression is essential for the energy-demanding functions of glial cells. Conversely, CKB may be repressed in most neuronal cells; however, CKB protein has previously been detected by immunohistochemistry in several distinct groups of neurons in the adult rodent brain. Presently, little is known of the factors responsible for the high CKB expression in glia and possible repression in neurons. In this report, we investigated if low CKB mRNA was characteristic of some established neuronal cell lines. CKB mRNA was found to be extremely low in mouse CI300 neuroblastomas NS20Y and N1E-115 but 10-fold higher in NG108-15, a hybrid cell composed of a Cl300 neuroblastoma and a rat C6 glioma. Since we showed NG108-15 contained only rat CKB mRNA transcribed from the C6 glioma CKB gene, expression of CKB mRNA may be a manifestation of a glial property in NG108-15 cells. However, CKB mRNA expression in NG108-15 appeared not to be fully activated since it was still 5-fold lower than in (parental) C6 glioma and 10-fold lower than in cellular RNA from either total rat brain or cultured primary astrocytes. When neuronal differentiation was increased in NS20Y and N1E-115 by treating cells with prostaglandin E1 and theophylline, the extremely low CKB mRNA level was not significantly changed. In a comparative study, the CKB mRNA levels in NS20Y, N1E-115 and neuronal RT4-B8 and RT4-E5 cells (from the rat RT4 peripheral neurotumor) were at least 50-fold lower than that in C6 glioma and 100-fold lower than in cultured primary astrocytes. These cell lines may provide a system for the identification of factors involved in the possible repression of CKB in many neuronal cells.
Acetylcholinesterase (AChE), which contains two subfamilies, ace1 and ace2 in insects, was identified to be the target of organophosphorous and carbamate insecticides. To research the sequences and tissues expressions of two aces, full length cDNAs encoding two ace genes were cloned, designated as Bmm-ace1 and Bmm-ace2 from larvae of the Bombyx mandarina. The amino acid sequence of Bmm-ace1 shared 99.71 % homology with its homolog, Bm-ace1, in silkworm, Bombyx mori, with two mutations (G664S and S307P), and the amino acid sequence of Bmm-ace2 shared 99.37 % homology with Bm-ace2, in B. mori , with four mutations (M18I, N233S, I310V and G621S). Tissue expression analysis showed that ace1 gene expressed only in the brains and fat bodies of B. mandarina, while ace2 genes expressed in all the tissues tested. ace1 and ace2 expressed highly in brains and fat bodies. The present results are significant to the study of resistance evolution of Lepidorptera as well as the understanding of the mechanism of pesticide resistance of insects.
Abstract The primordial follicle assembly, activation and the subsequent development are critical processes for female reproduction. A limited number of primordial follicles are activated to enter the growing follicle pool each wave, and the primordial follicle pool progressively diminishes over a woman's life‐time. The number of remaining primordial follicles represents the ovarian reserve. Identification and functional investigation of the factors involved in follicular initial recruitment will be of great significance to the understanding of the female reproduction process and ovarian ageing. In this study, we aimed to study whether and how semaphorin 6C ( Sema6c ) regulated the primordial follicle activation in the neonatal mouse ovary. The attenuation of SEMA 6C expression by Si RNA accelerated the primordial follicle activation in the in vitro ovary culture system. PI 3K‐ AKT ‐rpS6 pathway was activated when SEMA 6C expression was down‐regulated. And the LY 294002 could reverse the effect of low SEMA 6C expression on primordial follicle activation. Our findings revealed that Sema6c was involved in the activation of primordial follicles, and the down‐regulation of SEMA 6C led to massive primordial follicle activation by interacting with the PI 3K‐ AKT ‐rpS6 pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian ageing.
Creatine kinases (CK) catalyze the reversible transfer of a high energy phosphate group between creatine phosphate and ADP to regenerate ATP in cell types where the requirements for ATP are extensive and/or sudden. Previously, we have shown in primary rat brain cell cultures that brain CK (CKB) mRNA levels are highest in astrocytes and oligodendrocytes and much lower in neuronal cells. However, little is known of the factors which regulate CKB expression in the central nervous system and peripheral nervous system. To begin to investigate these factors, we asked in this report (1) if this pattern of CKB expression was also characteristic of some established glial and neuronal cell lines derived from the PNS; (2) whether CKB expression could be rapidly modulated by culture conditions, and (3) if CKB is expressed in cells with characteristics of glial cell progenitors. In subconfluent cells, CKB mRNA and enzyme activity were found to be high in both the rat RT4 peripheral neurotumor stem cell RT4-AC36A and its glial cell derivative RT4-D6. Conversely, CKB mRNA and activity were 5- and 8-fold lower, respectively, in the neuronal derivative RT4-E5 and, more dramatically, CKB was undetectable in neuronal RT4-B8 cells. Maintaining RT4-D6 glial cells at confluence rapidly increased CKB enzyme activity by 7-fold, such that D6 cells contained about 25% of the CKB level in lysates prepared from either whole adult rat brain or primary cultures of rat brain astrocytes. The levels of CKB mRNA and immunoreactive protein were also correspondingly increased in confluent D6 cells. These confluence-mediated increases in CKB appeared to be due to cell-cell contact and not the depletion of serum growth factors or an increase in intra-cellular cAMP. This study indicates that CKB expression is highest in cells displaying glial properties and can be rapidly modulated by appropriate culture conditions. The results are discussed in relation to the factors which may regulate CKB expression in vivo.
Major histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells.Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR.MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05).Our data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.
Objective To study the activity of macroautophagy and chaperone-medicated antophagy (CMA) in dopaminergic cells under oxidative stress and their possible roles in neurodegeneration.Methods PC 12 cells were treated with various concentrations of rotenone (0,20,100 and 500 nmol/L).The morphological characteristics of autophagosomes were observed by transmission electron microscopy (TEM),and macroautophagic activity was determined by detecting light chain (LC3) expression of microtubule-associated protein by Western blotting and monodansylcadaverine (MDC) immunofluorescence.Molecular chaperone mediated autophagy was estimated by the expressions of lysosomal-associated membrane protein 2α (Lamp-2α),heat shock cognate 70 (Hsc70) and heat shock protein 90 (Hsp90),which were detected by Western blotting.Results In control group (0 nmol/L rotenone),TEM showed that the cells presented a low autophagic activity with less autophagic vacuoles; in rotenone treatment groups,the autophagosomes with different forms were significantly increased,and the phenomenon ofautophagic mitochondria was observed.A large of vacuole-like bodies were found in the cytoplasm of the cells treated with high concentration of rotenone.The MDC immunofluorescence showed that after the treatment of rotenone,the red fluorescence showed punctate distribution,which was also significantly enhanced with the increasing of the concentrations of rotenone.Western blotting showed dose-dependent expressions of LC3-Ⅰ and LC3-Ⅱ.As compared with control cells,the expressions ofLamp-2α,Hsc70 and Hsp90 in rotenone treatment groups were also increased significantly (P＜0.05),but didn't show statistical difference between each two rotenone treatment groups (P＞0.05).Conclusions The macroautophagy and autophagy mediated by molecular chaperone are both activated in PC12 cells under oxidative stress conditions.And the increased oxidative stress results in an enhanced activity of macroautophagy,while the activity of CMA is saturated.
Key words:
Oxidative stress; Autophagy; PC12 cells; Rotenone
Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.
Mammalian oocytes go through a long and complex developmental process, while acquiring the competencies that are required for fertilization and embryogenesis. Recent studies revealed that the communication between oocytes and granulosa cells (GCs) is a critical process for female follicle development. In the current study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the cell junctions between oocytes and GCs in mice preantral follicles. The attenuation of SEMA6C expression by siRNA decreased the cell-cell junctions and accelerated follicle atresia in vitro. PI3K-AKT pathway was activated when SEMA6C expression was downregulated. And the LY294002, a PI3K inhibitor, could reverse the effect of low SEMA6C expression on cell junctions in preantral follicles. Our findings revealed that Sema6c was involved in follicle development, and the suppression of SEMA6C led to cell junction defection by activating the PI3K/AKT pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian aging.
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