Dissimilarity in the folding of human cytosolic creatine kinase isoenzymes

Creatine kinase (CK, EC 2.7.3.2) plays a key role in the energy homeostasis of excitable cells. The cytosolic human CK isoenzymes exist as homodimers (HMCK and HBCK) or a heterodimer (MBCK) formed by the muscle CK subunit (M) and/or brain CK subunit (B) with highly conserved three-dimensional structures composed of a small N-terminal domain (NTD) and a large C-terminal domain (CTD). The isoforms of CK provide a novel system to investigate the sequence/structural determinants of multimeric/multidomain protein folding. In this research, the role of NTD and CTD as well as the domain interactions in CK folding was investigated by comparing the equilibrium and kinetic folding parameters of HMCK, HBCK, MBCK and two domain-swapped chimeric forms (BnMc and MnBc). Spectroscopic results indicated that the five proteins had distinct structural features depending on the domain organizations. MBCK BnMc had the smallest CD signals and the lowest stability against guanidine chloride-induced denaturation. During the biphasic kinetic refolding, three proteins (HMCK, BnMc and MnBc), which contained either the NTD or CTD of the M subunit and similar microenvironments of the Trp fluorophores, refolded about 10-fold faster than HBCK for both the fast and slow phase. The fast folding of these three proteins led to an accumulation of the aggregation-prone intermediate and slowed down the reactivation rate thereby during the kinetic refolding. Our results suggested that the intra- and inter-subunit domain interactions modified the behavior of kinetic refolding. The alternation of domain interactions based on isoenzymes also provides a valuable strategy to improve the properties of multidomain enzymes in biotechnology.     

Self-renewing Pten-/- TP53-/- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.     

mTOR and the differentiation of mesenchymal stem cells

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase, belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells. mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.     

苦瓜多糖的提取、分离纯化及组成性质

正交实验确定提取工艺后,用热水提取法得到苦瓜多糖(MCP).对MCP进行DEAE-32离子交换层析分离,得到3个多糖组分MCP1、MCP2和MCP3. 进一步采用Sephacryl S-400凝胶层析进行分离,经凝胶层析和高效液相色谱检测表明,MCP1、MCP2为均一性多糖组分.通过高效液相凝胶色谱法测定了两者的相对分子质量分别为1.16×106和7.45×105.用PMP衍生化法测定其单糖,结果表明: MCP1系由Man、Rham、GlcUA、GalUA、Glu、Gal、Xyl、Ara等单糖组成的杂多糖,摩尔比为1.03:2.93:1.00:14.95:2.16:30.70:2.85:4.50.MCP2系由Rham、GalUA、Gal、Xyl、Ara等单糖组成的杂多糖,对应的摩尔比为1.63:21.88:4.66:1.00:1.29.紫外光谱表明该多糖不含蛋白质和核酸.     

消融右肺动脉神经节丛对肾上腺素能刺激和胆碱 能刺激诱发房颤的影响

目的:探讨右肺动脉神经节丛(RPVGP)消融对胆碱能及儿茶酚胺诱发房颤的影响。方法:20只犬麻醉开胸后,暴露RPVGP,分别在消融RPVGP前后,经股静脉静滴乙酰胆碱(ACh)及儿茶酚胺。测量房颤诱发率及两类递质诱发房颤的阈浓度。结果:RPVGP消融前,静滴Ach和异丙基肾上腺素(IPA)及肾上腺素(EPI)(1～100μmol/l)均可诱发AF,诱发率100%。Ach、IPA和EPI的诱发阈浓度分别为2.6±0.3μmol/l,3.3±0.2μmol/l,5.6±0.2μmol/l。RPVGP消融后,Ach及儿茶酚胺的AF诱发率分别降至10%及35%,且三种递质的诱发阈浓度分别提高至2.6±0.3μmol/l、22.5±2.4μmol/l和26.±2.6μmol/(lP<0.05)。结论:消融RPVGP使乙酰胆碱和儿茶酚胺诱发房颤的阈浓度增高,并降低此二类介质的房颤诱发率。     

Intrauterine growth restriction alters the metabonome of the serum and jejunum in piglets

Intrauterine growth restriction (IUGR) is not only an underlying factor for stunted postnatal growth and newborn deaths, but also associated with disease prevalence, such as hypertension and diabetes, in both adult humans and animals. To investigate the metabolic status of IUGR, the differences in serum and jejunal tissue metabonome were examined in IUGR and normal weight 21 day old piglets. IUGR piglets had a significantly lower birth weight (785 ± 42 g vs. 1451 ± 124 g), weaned weight (3053 ± 375 g vs. 6489 ± 545 g) and average daily gain (108 ± 16 g vs. 240 ± 21 g) than normal weight piglets (p < 0.05). IUGR piglets also had a shorter villus height and smaller villus height to crypt depth ratio (p < 0.05) in jejunum. An NMR-based metabonomic study found that serum levels of glycoprotein, albumin and threonine were higher in IUGR than in normal weight piglets, while serum levels of HDL, lipids, unsaturated lipids, glycerophosphorylcholine, myo-inositol, citrate, glutamine and tyrosine were lower in IUGR piglets (p < 0.05). In addition, marked changes in jejunal metabolites, including elevated levels of lipids and unsaturated lipids, and decreased levels of valine, alanine, glutamine, glutamate, choline, glycerophosphorylcholine, trimethylamine-N-oxide, scyllo-inositol, lactate, creatine, glucose, galactose, phenylalanine, tyrosine, glutathione, inosine and taurine were observed in IUGR piglets (p < 0.05). These novel findings indicate that IUGR piglets have a distinctive metabolic status compared to normal weight piglets, including changes in lipogenesis, lipid oxidation, energy supply and utilization, amino acid and protein metabolism, and antioxidant ability; these changes could contribute to impaired growth and jejunal function.