Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

人轮状病毒NSP4基因变异与功能关系的初步研究

在比较我国人A组轮状病毒一般腹泻患者分离株和重症患者分离株非结构蛋白(NSP)4 cDNA序列时发现,两者在可能与致病性有关的区域(aa131～146)内存在着显著的差异.为进一步探讨这种变异是否与毒力改变有关,利用杆状病毒表达载体在昆虫细胞Sf9中表达两种毒株的NSP4,通过激光扫描共聚焦显微镜初步观察了它对细胞内钙离子浓度的影响.结果表明:两种来源的NSP4均可使细胞内钙离子浓度明显升高,在48h时大致升高3.1～3.4倍,96h时升高5.6～5.8倍,但两种毒株之间的差别并不明显.研究证实,人轮状病毒NSP4与以往报道的动物轮状病毒NSP4一样,可以引起细胞内钙离子增高,即可能与病毒的致病性有关.但重症腹泻毒株SZ1 NSP4第131～146位氨基酸位点出现的变异并未提高其毒力.轮状病毒的毒力改变可能与其它因素有关.     

Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer

To target tumors overexpressing low-density lipoprotein receptors (LDLr), a pyropheophorbide cholesterol oleate conjugate was synthesized and successfully reconstituted into the low-density lipoprotein (LDL) lipid core. Laser scanning confocal microscopy studies demonstrated that this photosensitizer-reconstituted LDL can be internalized via LDLr by human hepatoblastoma G(2) (HepG(2)) tumor cells.     

Molecular phylogeny of 48 species of damselfishes (Perciformes: Pomacentridae) using 12S mtDNA sequences

Phylogenetic relastionships within the family pomacentridae teleostei: Perciformes) were inferred by analyzing a portion of the 12S mitochondrial ribosomal DNA gene. Thirty-four pomacentrid species were sequenced for this study and the resulting data were combined with previously published pomacentrid sequence data to form a combined matrix of 53 pomacentrids representing 48 different species in 18 genera. Four outgroup species were also drawn from published data; these taxa were taken from the other three putative families of the suborder Labroidei, as well as a single representative of the family Moronidae. The data set contained 1053 data columns after alignment according to ribosomal secondary structure and the removal of all ambiguously aligned positions. The resulting strict consensus tree topology generally agreed with the previous molecular hypothesis, and recovers a monophyletic Pomacentridae and subfamily Amphiprioninae. The two other subfamilies included, Chrominae and Pomacentrinae, were found to be polyphyletic. A monophyletic group consisting of the Amphiprioninae, Pomacentrus, Acanthochromis, Amblyglyphidodon, Neoglyphidodon, Chrysiptera, Neopomacentrus, and Teixeirichthys was found. This group was recovered as the sister group to a clade consisting of a paraphyletic Chromis and a monophyletic Dascyllus. A sister-group relationship between the genus Pomacentrus and the subfamily Amphiprioninae was observed.     

Protection against diet-induced obesity and obesity- related insulin resistance in Group 1B PLA2-deficient mice

Group 1B phospholipase A2 (PLA2) is an abundant lipolytic enzyme that is well characterized biochemically and structurally. Because of its high level of expression in the pancreas, it has been presumed that PLA2 plays a role in the digestion of dietary lipids, but in vivo data have been lacking to support this theory. Our initial study on mice lacking PLA2 demonstrated no abnormalities in dietary lipid absorption in mice consuming a chow diet. However, the effects of PLA2 deficiency on animals consuming a high-fat diet have not been studied. To investigate this, PLA2(+/+) and PLA2(-/-) mice were fed a western diet for 16 wk. The results showed that PLA2(-/-) mice were resistant to high-fat diet-induced obesity. This observed weight difference was due to decreased adiposity present in the PLA2(-/-) mice. Compared with PLA2(+/+) mice, the PLA2(-/-) mice had 60% lower plasma insulin and 72% lower plasma leptin levels after high-fat diet feeding. The PLA2(-/-) mice also did not exhibit impaired glucose tolerance associated with the development of obesity-related insulin resistance as observed in the PLA2(+/+) mice. To investigate the mechanism by which PLA(2)(-/-) mice exhibit decreased weight gain while on a high-fat diet, fat absorption studies were performed. The PLA(2)(-/-) mice displayed 50 and 35% decreased plasma [(3)H]triglyceride concentrations 4 and 6 h, respectively, after feeding on a lipid-rich meal containing [(3)H]triolein. The PLA(2)(-/-) mice also displayed increased lipid content in the stool, thus indicating decreased fat absorption in these animals. These results suggest a novel role for PLA(2) in the protection against diet-induced obesity and obesity-related insulin resistance, thereby offering a new target for treatment of obesity and diabetes.     

A fast empirical approach to binding free energy calculations based on protein interface information

Three useful variables from the interfaces of 20 protein-protein complexes were investigated. These variables are the side-chain accessible number (N(b)), the number of hydrophilic pairs (N(pair)) and buried a polar solvent accessible surface areas (DeltaDeltaASA(apol)). An empirical model based on the three variables was developed to describe the free energy of protein associations. As the results show, the side-chain accessible numbers characterize the loss of side-chain conformational entropy of protein interactions and the effective empirical function presented here has great capability for estimating the binding free energy. It was found that the variables of interface information capture most of the significant features of protein-protein association. Also, we applied the model based on the variables as a rescoring function to docking simulations and found that it has the potential to distinguish the 'true' binding mode. It is clear that the simple and empirical scale developed here is an attractive target function for calculating binding free energy for various biological processes to rational protein design.     

Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life

MDC9 (ADAM9/meltrin gamma) is a widely expressed and catalytically active metalloprotease-disintegrin protein that has been implicated in the ectodomain cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and as an alpha secretase for the amyloid precursor protein. In this study, we evaluated the expression of MDC9 during development and generated mice lacking MDC9 (mdc9(-/-) mice) to learn more about the function of this protein during development and in adults. During mouse development, MDC9 mRNA is ubiquitously expressed, with particularly high expression levels in the developing mesenchyme, heart and brain. Despite the ubiquitous expression of MDC9, mdc9(-/-) mice appear to develop normally, are viable and fertile, and do not have any major pathological phenotypes compared to wild-type mice. Constitutive and stimulated ectodomain shedding of HB-EGF is comparable in embryonic fibroblasts isolated from mdc9(-/-) and wild-type mice, arguing against an essential role of MDC9 in HB-EGF shedding in these cells. Furthermore, there were no differences in the production of the APP alpha and gamma secretase cleavage product (p3) and of beta- and gamma-secretase cleavage product (A beta) in cultured hippocampal neurons from mdc9(-/-) or wild-type mice, arguing against an essential major role of MDC9 as an alpha-secretase in mice. Further studies, including functional challenges and an evaluation of potential compensation by, or redundancy with, other members of the ADAM family or perhaps even with other molecules will be necessary to uncover physiologically relevant functions for MDC9 in mice.     

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.