Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

马尾松雌球果的发生和早期发育研究

采用常规石蜡制片技术对马尾松雌球果的发生和早期发育进行了研究。结果表明：雌球果原基发生时间为10月中旬,不同的树龄和着生部位,其发生时间不同。雌球果原基与营养茎端在外部形态及内部细胞组织学分区结构有明显差异。营养茎端外形扁平,内部顶端分生组织结构有顶端原始细胞区、中央母细胞区、形成层状过渡区、周围分生组织区及肋状分生组织区5个明显的分区;而雌球果原基外形呈圆锥状,内部结构只有套层和髓区。12月初,最初的苞片原基在雌球果原基的鳞片的叶腋处产生,之后其由基部向顶部连续发生。翌年1月初,在苞片原基的叶腋处,珠鳞原基发生,发生方向亦为向顶发育。2月底,苞片体积不再发生变化,珠鳞膨大端的基部的近轴面分化出2个倒生胚珠。从雌球果原基发生到胚珠分化历时4个多月。亚热带的冬季气候对马尾松雌球果的生长发育没有明显的抑制作用。     

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.     

含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白     

雪莲类PEBP基因在大肠杆菌中的表达及鉴定

本实验旨在构建雪莲类PEBP基因原核表达载体,在大肠杆菌中表达并纯化类PEBP基因所编码的蛋白,为进一步研究奠定基础.将雪莲类PEBP基因开放阅读框序列克隆到原核表达载体pET30( )上,转化感受态表达菌株BL21(DE3),低浓度IPTG低温诱导融合蛋白的表达,纯化产物,Western blotting鉴定目的蛋白.IFTG低诱导PEBP,经SDS-PAGE分析,其相对分子量约为28 kD,与预期相符,表达量约占菌体蛋白的26.8%,并且通过亲和层析纯化了重组融合蛋白,Western blotting鉴定为阳性.成功构建了原核表达载体pET-PEBP,获得了高效表达产物,并为进一步研究雪莲类PEBP基因的抗冻功能打下基础.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997     

Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 371(Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.