响应面法优化紫山药花青苷提取工艺

以紫山药为实验材料,以酸性乙醇为提取溶剂通过Box-Behnken响应面法及Design-Expert 8.0.6分析软件建立二次多项式数学模型优化紫山药花青苷的提取工艺及参数。研究表明,五种单因素对花青苷得率影响大小的顺序为X5(盐酸质量分数)X2(时间)X4(乙醇体积分数)X3(料液比)X1(温度)。并且温度和料液比、温度和乙醇体积分数、提取时间和料液比、提取时间和盐酸体积分数之间存在交互作用。得到紫山药花青苷最佳提取工艺参数为温度:81℃、时间:3.5 h、料液比:1∶25、乙醇体积分数:70%、盐酸质量分数:18‰。在此条件下花青苷平均得率达到4.928 mg/g,相对标准偏差为0.31%,与数学模型理论得率间的相对误差小于1.0%。     

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.     

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.     

A new sequence representation as applied in better specificity elucidation for human immunodeficiency virus type 1 protease

Factor analysis scales of generalized amino acid information (FASGAI) involving hydrophobicity, alpha and turn propensities, bulky properties, compositional characteristics, local flexibility, and electronic properties were derived from 516 property parameters of 20-coded amino acids, and was then employed to represent sequence structures of 746 peptides with 8 amino acid residues. Cleavage site prediction models for human immunodeficiency virus type 1 protease by linear discriminant analysis and support vector machine with radial basis function kernel were constructed to identify if they could be cleaved or not, and were further utilized to investigate the cleavage specificity. These diversified properties, including the bulky properties, secondary conformation characteristics, electronic properties, and hydrophobicity at the first, the second, the fourth, the fifth, and the sixth residue, are possibly important factors in determining HIV PR cleavage or not. Particularly, maximal positive and negative influences result from the bulky properties of different sites. Further results from analysis of variance also likely reflect that the HIV PR recognizes diversified key properties of various sites in the octameric sequences. Satisfactory results show that FASGAI can not only be used to represent sequence structures of various functional peptides, but alsoprovide a potential feasible measure for exploring relationship between protein motif sequences and their functions.     

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.     

Prediction of atherosclerotic plaque ruptures with high-frequency ultrasound imaging and serum inflammatory markers

Atherosclerotic plaque rupture and thrombosis are the main causes of acute coronary syndrome. In the present study, we investigated whether ultrasound imaging and inflammatory parameters are predictive of plaque rupture in a newly established animal model. We developed a rabbit model for plaque rupture by locally delivering recombinant p53 adenovirus to plaques in rabbits fed a high-cholesterol diet for 10 wk, and plaque rupture was triggered using Chinese Russell's viper venom and histamine. We found that 81.1% of rabbits transfected with p53 (n = 37) had the ruptured plaques, which was significantly higher than results in rabbits transfected with the control vector (26.3%, n = 38; P < 0.001). Among measured biomarkers, high-sensitive C-reactive protein, soluble intercellular adhesion molecule-1, and soluble vascular cell adhesion molecule-1 were significantly different between rabbits with and without ruptured plaques. Using high-frequency duplex and intravascular ultrasound imaging techniques, we obtained a list of parameters. With the multivariate logistic regression model, we identified that plaque eccentric index, plaque area, high-sensitive C-reactive protein, and corrected integrated backscatter intensity were significant predictors of plaque rupture, with odds ratios of 7.056 [95% confidence interval (CI): 1.958, approximately 25.430], 1.942 (95% CI: 1.058, approximately 3.564), 1.025 (95% CI: 1.007, approximately 1.043), and 0.856 (95% CI: 0.775, approximately 0.946), respectively. Localized p53 overexpression technique induces plaque rupture, and the combined measurement of ultrasound and biochemical markers is a valuable tool in predicting plaque rupture.     

Endoreduplication of human smooth muscle cells induced by 2-methoxyestradiol: a role for cyclin-dependent kinase 2

Endoreduplication has been suggested to contribute to the development of hypertrophy of smooth muscle cells (SMCs) in hypertension. However, endoreduplication in vascular SMCs and the underlying molecular mechanisms are not clear. Treatment of human SMCs with 10 microM 2-methoxyestradiol (2-ME) for 24 h induces accumulation of cells with > or =4N DNA content, and some polyploid/aneuploid cells actively synthesize their DNA, suggesting the occurrence of endoreduplication. In addition, 2-ME treatment upregulates the expression of cyclin-dependent kinase 2 (Cdk2). The present study was designed to characterize endoreduplication of human SMCs and explore the potential roles of Cdk2 in endoreduplication induced by 2-ME. Treatment with 2-ME (10 microM) for 2-4 days not only caused increases in >4N cells and their reentry into S phase but also induced overduplication of chromosomes. Furthermore, 2-ME increased the kinase activity of Cdk2 and its interaction with cyclin E. Inducible overexpression of dominant-negative Cdk2 in human SMCs inhibited both DNA synthesis of >4N cells and the accumulation of >4N cells induced by 2-ME. We conclude that 2-ME induces endoreduplication of human SMCs and Cdk2 plays an important role in endoreduplication in response to 2-ME.