http://www.mdpi.com/section/biochemistry http://www.mdpi.com/1422-0067/11/3/1057/ This study aims to elucidate the effects of chrysin on human ER-negative breast cancer cell line, MDA-MB-231. The study demonstrated that treatment of MDA-MB-231 cells with 20 µM chysin for 48 h significantly inhibited the growth of MDA-MB-231 cells and induced cytoplasmic lipid accumulation in the cells, but that the observed of cell death was not caused by apoptosis. The expression of PPARalpha mRNA in chrysin-treated MDA-MB-231 cells was significantly increased, which was likely associated to the proliferation of the cells post chrysin treatment. http://www.mdpi.com/1422-0067/11/3/1057/ Thu, 11 Mar 2010 00:00:00 CET International Journal of Molecular Sciences 2010-03-11 11 3 Article 1057 1069 1422-0067 Potential Effects of Chrysin on MDA-MB-231 Cells 2010-03-11 doi: 10.3390/ijms11031057 Teh Ban Hong Anizah Rahumatullah Thaneswary Yogarajah Maimunah Ahmad Khoo Boon Yin http://www.mdpi.com/1422-0067/11/3/982/ The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener’s cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener’s cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK) isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer) from signalling (information transfer) within dissipative metabolic structures – intracellular energetic units (ICEU). Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via cyclic fluctuations of cytosolic ADP, Pi and Cr/PCr ensures metabolic stability necessary for normal function of cardiac cells. http://www.mdpi.com/1422-0067/11/3/982/ Mon, 08 Mar 2010 00:00:00 CET International Journal of Molecular Sciences 2010-03-08 11 3 Review 982 1019 1422-0067 Application of the Principles of Systems Biology and Wiener's Cybernetics for Analysis of Regulation of Energy Fluxes in Muscle Cells in Vivo 2010-03-08 doi: 10.3390/ijms11030982 Rita Guzun Valdur Saks http://www.mdpi.com/1422-0067/11/3/858/ The use of biocatalysts for the production of both consumer goods and building blocks for chemical synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technology and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chemicals, intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research. http://www.mdpi.com/1422-0067/11/3/858/ Tue, 02 Mar 2010 00:00:00 CET International Journal of Molecular Sciences 2010-03-02 11 3 Review 858 879 1422-0067 Miniaturization in Biocatalysis 2010-03-02 doi: 10.3390/ijms11030858 Pedro Fernandes http://www.mdpi.com/1422-0067/11/2/762/ Functionalization of porous solids plays an important role in many areas, including heterogeneous catalysis and enzyme immobilization. In this study, large-pore ordered mesoporous SBA-15 molecular sieves were synthesized with tetraethyl orthosilicate (TEOS) in the presence of the non-ionic triblock co-polymer Pluronic P123 under acidic conditions. These materials were grafted with 3 aminopropyltrimethoxysilane (ATS), 3-glycidoxypropyltrimethoxysilane (GTS) and with 3 aminopropyltrimethoxysilane and glutaraldehyde (GA-ATS) in order to provide covalent anchoring points for enzymes. The samples were characterized by nitrogen adsorption, powder X-ray diffraction, solid-state NMR spectroscopy, elemental analysis, diffuse reflectance fourier transform infrared spectroscopy and diffuse reflectance UV/Vis spectroscopy. The obtained grafted materials were then used for the immobilization of chloroperoxidase (CPO) and glucose oxidase (GOx) and the resulting biocatalysts were tested in the oxidation of indole. It is found that enzymes anchored to the mesoporous host by the organic moieties can be stored for weeks without losing their activity. Furthermore, the covalently linked enzymes are shown to be less prone to leaching than the physically adsorbed enzymes, as tested in a fixed-bed reactor under continuous operation conditions. http://www.mdpi.com/1422-0067/11/2/762/ Wed, 24 Feb 2010 00:00:00 CET International Journal of Molecular Sciences 2010-02-24 11 2 Article 762 778 1422-0067 Covalent Anchoring of Chloroperoxidase and Glucose Oxidase on the Mesoporous Molecular Sieve SBA-15 2010-02-24 doi: 10.3390/ijms11020762 Dirk Jung Carsten Streb Martin Hartmann http://www.mdpi.com/1422-0067/11/2/745/ RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2) protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA). The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site. http://www.mdpi.com/1422-0067/11/2/745/ Fri, 12 Feb 2010 00:00:00 CET International Journal of Molecular Sciences 2010-02-12 11 2 Article 745 753 1422-0067 Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies 2010-02-12 doi: 10.3390/ijms11020745 Vincenzo Salvatore Nicoletta Potenza Umberto Papa Valentina Nobile Aniello Russo http://www.mdpi.com/1422-0067/11/2/731/ 2-Bromopropane (2-BP), an alternative to ozone-depleting solvents, is used as a cleaning solvent. Here, we examined the cytotoxic effects of 2-bromopropane (2-BP) on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Mouse blastocysts were incubated in medium with or without 2-BP (2.5, 5 or 10 μM) for 24 h. Cell proliferation and growth were investigated with dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, and implantation and post-implantation development of embryos were assessed using in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 5 or 10 μM 2-BP displayed significantly increased apoptosis, and decreased inner cell mass (ICM) and trophectoderm (TE) cell number. Additionally, the implantation success rates of 2-BP-pretreated blastocysts were lower than those of untreated controls. In vitro treatment with 5 or 10 μM 2-BP was associated with increased resorption of postimplantation embryos, and decreased placental and fetal weights. Our results collectively indicate that in vitro exposure to 2-BP induces apoptosis, suppresses implantation rates after transfer to host mice, and retards early postimplantation development. http://www.mdpi.com/1422-0067/11/2/731/ Thu, 11 Feb 2010 00:00:00 CET International Journal of Molecular Sciences 2010-02-11 11 2 Article 731 744 1422-0067 Cytotoxic Effects of 2-Bromopropane on Embryonic Development in Mouse Blastocysts 2010-02-11 doi: 10.3390/ijms11020731 Wen-Hsiung Chan http://www.mdpi.com/1422-0067/11/2/719/ The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. There were reports to show that some microRNAs (miRNAs) may play a key role during embryo implantation in mouse. However, the miR-320 expression profiles in the rat uterus during peri-implantation are unknown. In the present study, we found that the expression level of miR-320 was lower on day 5 of gestation (g.d. 5) in rats than g.d.3 and g.d.4 and restored gradually from g.d.6. MiR-320 was specifically localized in glandular and luminal epithelia and decidua. The expression of miR-320 was not significantly different in the pseudopregnant uterus and decreased in the uteri of rats subjected to activation of delayed implantation. Artificial decidualization and treatment with progesterone increased the miR-320 expression. Thus, miR-320 was differentially expressed in the rat uterus during implantation. The expression level was affected by active blastocysts and decidualization during the window of implantation. Steroid hormones, progesterone stimulated miR-320 expression. http://www.mdpi.com/1422-0067/11/2/719/ Thu, 11 Feb 2010 00:00:00 CET International Journal of Molecular Sciences 2010-02-11 11 2 Article 719 730 1422-0067 Temporal and Spatial Regulation of miR-320 in the Uterus during Embryo Implantation in the Rat 2010-02-11 doi: 10.3390/ijms11020719 Hong-Fei Xia Xiao-Hua Jin Pei-Pei Song Yi Cui Chun-Mei Liu Xu Ma http://www.mdpi.com/1422-0067/11/2/521/ The nuclear factor kappa B is widely expressed in the distinct subpopulations of chorionic villi and deciduas of first-trimester pregnancies. We examined the cellular distribution and expression of nuclear factor kappa B in the human first-trimester chorionic villi and deciduas of women with early spontaneous miscarriage and viable pregnancy by confocal laser scanning microscope and immunohistochemistry. There is a greater nuclear translocation of nuclear factor kappa B is restricted to villous stromal cells, decidual stromal cells, glandular epithelial cells and vessel endothelial cells in early spontaneous miscarriage than in viable pregnancies. Collectively these observations suggest that over-activation of nuclear factor kappa B has a relationship with early spontaneous miscarriages. http://www.mdpi.com/1422-0067/11/2/521/ Wed, 03 Feb 2010 00:00:00 CET International Journal of Molecular Sciences 2010-02-03 11 2 Article 521 531 1422-0067 Nuclear Translocation of Nuclear Factor Kappa B in First Trimester Deciduas and Chorionic Villi in Early Spontaneous Miscarriage Women 2010-02-03 doi: 10.3390/ijms11020521 Li-qin Wang Xue-wen Yu Chun-fang Yan Xiang Wang http://www.mdpi.com/1422-0067/11/1/222/ The extracellular signal-regulated kinase (ERK) pathway is a member of the mitogen-activated protein kinase (MAPK) superfamily, which is an important, highly conserved family of enzymes associated with cell membrane receptors and regulative targets. In the central nervous system, there is almost no mature neuronal proliferation and differentiation, but the regulation of MAPK and its upstream and downstream molecular pathways is still widespread, with the ERK signaling pathway being one of the most actively studied signal transduction pathways. It is activated by a variety of cell growth factors and substances which promote mitotic activity, and transmits extracellular signals from the cell surface to the nucleus, which transmission plays an important role in the process of cell proliferation and differentiation. In recent years, accumulating evidence has shown that the ERK signaling pathway has an important link with the higher functions of learning and memory. http://www.mdpi.com/1422-0067/11/1/222/ Wed, 13 Jan 2010 00:00:00 CET International Journal of Molecular Sciences 2010-01-13 11 1 Review 222 232 1422-0067 ERK in Learning and Memory: A Review of Recent Research 2010-01-13 doi: 10.3390/ijms11010222 Sheng Peng Yan Zhang Jiannan Zhang Hua Wang Bingxu Ren http://www.mdpi.com/1422-0067/11/1/41/ Different concentrations of asafoetida extract were added to the medium of Pleurotus ferulic and the effects of the extract on growth of P. ferulic mycelium and fruiting bodies was observed. As the amount of asafoetida extract additive was increased, the growth of Pleurotus mycelium was faster, the time formation of buds was shorter and that yield of fruiting bodies was stimulated. However, overdosing of asafoetida extract hampered the growth of Pleurotus ferulic. The amino acid composition and volatile components in three kinds of pleurotus’ were contrasted, including wild pleurotus (WP), cultivated pleurotus with asafoetida extract (CPAE) and cultivated pleurotus without asafoetida extract (CP). CPAE with 2.3 g/100 g asafoetida extract addition had the highest content of total amino acids, as well as essential amino acids. WP had a higher content of total amino acids and essential amino acids than CP. In addition, CPAE with 2.3 g/100 g had the highest score of protein content of pleurotus fruiting bodies, while WP had a higher score than CP. In the score of essential amino acid components of pleurotus fruiting bodies, CP had the highest score, while CPAE was higher than WP. Asafoetida extract influenced the volatile components of Pleurotus ferulic greatly, making the volatile components of cultivated pleurotus more similar to those of wild pleurotus (WP). http://www.mdpi.com/1422-0067/11/1/41/ Tue, 29 Dec 2009 00:00:00 CET International Journal of Molecular Sciences 2009-12-29 11 1 Article 41 51 1422-0067 Effect of Asafoetida Extract on Growth and Quality of Pleurotus ferulic 2009-12-29 doi: 10.3390/ijms11010041 Zuoshan Feng Yujia Bai Fanglin Lu Wenshu Huang Xinmin Li Xiaosong Hu http://www.mdpi.com/1422-0067/10/12/5498/ Hyperthermophilic superoxide dismutases (SODs) are of particular interest due to their potential industrial importance and scientific merit in studying the molecular mechanisms of protein folding and stability. Compared to the mesophilic SODs, the hyperthermostable Fe-SODs (TcSOD and ApSOD) have an extended C-terminal helix, which forms an additional ion-pairing network. In this research, the role of the extended C-terminus in the structural stability of TcSOD was studied by investigating the properties of two deletion mutants. The results indicated that the ion-pairing network at the C-terminus had limited contributions to the stability of TcSOD against heat- and GdnHClinduced inactivation. The intactness of the C-terminal helix had dissimilar impact on the two stages of TcSOD unfolding induced by guanidinium chloride. The mutations slightly decreased the Gibbs free energy of the dissociation of the tetrameric enzymes, while greatly affected the stability of the molten globule-like intermediate. These results suggested that the additional ion-pairing network mainly enhanced the structural stability of TcSOD by stabilizing the monomers. http://www.mdpi.com/1422-0067/10/12/5498/ Wed, 23 Dec 2009 00:00:00 CET International Journal of Molecular Sciences 2009-12-23 10 12 Article 5498 5512 1422-0067 Contributions of the C-Terminal Helix to the Structural Stability of a Hyperthermophilic Fe-Superoxide Dismutase (TcSOD) 2009-12-23 doi: 10.3390/ijms10125498 Sha Wang Yong-Bin Yan Zhi-Yang Dong http://www.mdpi.com/1422-0067/10/12/5442/ Objective: To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-α (TNF-α) converting enzyme (TACE) on TACE activity and to develop an approach to interfere with inflammation processes. Methods: The cDNA encoding the fulllength ectodomain (T1300) and prodomain (T591) of TACE were amplified by RT-PCR. The expression plasmids (pET-28a (+)-T1300 and pET-28a (+)-T591) were constructed and transformed into E. coli BL21. After Ni2+-NTA resin affinity chromatography, the recombinant T591 protein was obtained and assayed. In order to detect its inhibiton of TACE activity, the mice in the LPS-induced endotoxemia model group were treated with the recombinant TACE prodomain protein prior to the injection of LPS. Murine peritoneal macrophages were isolated from mice abdominal cavity for FCM and the liver, kidney and lung were removed for traditionally histopathology sectioning. Results: The FCM results showed that the recombinant prodomain protein decreased the release of the sTNF-α, which mediated the accumulation of TNF-α on the surface of macrophage cells. HE staining proved that the recombinant protein can decrease the inflammatory response in internal organs of endotoxaemia mice. Conclusions: The recombinant prodomain of TACE has the ability to inhibit sTNF-α release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs. http://www.mdpi.com/1422-0067/10/12/5442/ Fri, 18 Dec 2009 00:00:00 CET International Journal of Molecular Sciences 2009-12-18 10 12 Article 5442 5454 1422-0067 The Study of the Inhibition of the Recombinant TACE Prodomain to Endotoxemia in Mice 2009-12-18 doi: 10.3390/ijms10125442 Xiaoou Li Yuan Yan Wei Huang Yuzhen Yang http://www.mdpi.com/1422-0067/10/10/4437/ Regulatory interaction networks are often studied on their dynamical side (existence of attractors, study of their stability). We focus here also on their robustness, that is their ability to offer the same spatiotemporal patterns and to resist to external perturbations such as losses of nodes or edges in the networks interactions architecture, changes in their environmental boundary conditions as well as changes in the update schedule (or updating mode) of the states of their elements (e.g., if these elements are genes, their synchronous coexpression mode versus their sequential expression). We define the generic notions of boundary, core, and critical vertex or edge of the underlying interaction graph of the regulatory network, whose disappearance causes dramatic changes in the number and nature of attractors (e.g., passage from a bistable behaviour to a unique periodic regime) or in the range of their basins of stability. The dynamic transition of states will be presented in the framework of threshold Boolean automata rules. A panorama of applications at different levels will be given: brain and plant morphogenesis, bulbar cardio-respiratory regulation, glycolytic/oxidative metabolic coupling, and eventually cell cycle and feather morphogenesis genetic control. http://www.mdpi.com/1422-0067/10/10/4437/ Mon, 19 Oct 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-10-19 10 10 Article 4437 4473 1422-0067 Robustness in Regulatory Interaction Networks. A Generic Approach with Applications at Different Levels: Physiologic, Metabolic and Genetic 2009-10-19 doi: 10.3390/ijms10104437 Jacques Demongeot Hedi Ben Amor Adrien Elena Pierre Gillois Mathilde Noual Sylvain Sené http://www.mdpi.com/1422-0067/10/10/4418/ In this study we investigated the effect of near-infrared fluorescent quantum dots (NIRF-QDs, QTracker) on the proliferation, adherence, invasion and chemotaxis of human tongue squamous cell carcinoma cell line Tca8113 in vitro. Cell proliferation and colony formation rate were determined by using a hemocytometer and culture plate. A transwell chamber assay was used to determine the cell invasion, adherence and chemotaxis. The results showed that there was no significant difference between the results of Tca8113 cells labeled with NIRF-QD800 and those of unlabeled Tca8113 cells, suggesting that the proliferation, invasion, adherence and chemotaxis of Tca8113 cells were not affected by NIRF-QD800. These results provide a basis for the further utilization of NIRF-QDs in non-invasive imaging and tracking of tumor cells in vivo. http://www.mdpi.com/1422-0067/10/10/4418/ Mon, 12 Oct 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-10-12 10 10 Article 4418 4427 1422-0067 Effect of Peptide-Conjugated Near-Infrared Fluorescent Quantum Dots (NIRF-QDs) on the Invasion and Metastasis of Human Tongue Squamous Cell Carcinoma Cell Line Tca8113 in Vitro 2009-10-12 doi: 10.3390/ijms10104418 Kai Yang Zhigang Li Yuan Cao Xiuli Yu Jie Mei http://www.mdpi.com/1422-0067/10/10/4330/ The ethanol extract of Teucrium manghuaense grown in Hainan province (China) was analysed by GC and GC/MS. Of the constituents 84–96% were identified on the basis of their GC retention times and their mass spectra in regard to authentic compounds. The results revealed that it contained 9-methyl-9-azabicyclo[4.2.1]nonane (7.43%), 2-methylpyrrolidine (19.42%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (10.84%), and squalene (28.55%), as major components, constituting 66.24% of the extract. The chemical characterization of the ethanol extract by GC/MS also allowed identification of 1-octen-3-one (3.41%), 2-pentyl-piperidine (2.25%), 1-(2-methyl-1-propenyl)-piperidine (4.63%), 2,2'-diethoxy-5,5'-bi-1-pyrroline (0.41%), (Z,Z,Z)-9,12,15-octadecatrieniic acid, 2,3-dihydroxypropyl ester (1.56%), vitamin E (2.95%) and stigmasterol (1.39%). Finally, the antioxidant and anti-tumor activities of the ethanol extract have been evaluated. Results show that the extract of Teucrium manghuaense leaf possesses strong DPPH-, hydroxyl radical scavenging activity and anti-tumor activity. http://www.mdpi.com/1422-0067/10/10/4330/ Fri, 09 Oct 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-10-09 10 10 Article 4330 4341 1422-0067 Extraction of Teucrium manghuaense and Evaluation of the Bioactivity of Its Extract 2009-10-09 doi: 10.3390/ijms10104330 Guihao Yin Huicai Zeng Mingjun He Mingyue Wang http://www.mdpi.com/1422-0067/10/10/4210/ Acrylamide is widely used worldwide in industry and it can also be produced by the cooking and processing of foods. It is harmful to human beings, and human brain CK (HBCK) has been proposed to be one of the important targets of acrylamide. In this research, we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inactivated at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The binding sites of acrylamide were located at the cleft between the N- and C-terminal domains of CK, and Glu232 was one of the key binding residues. The effects of acrylamide on CK were proposed to be isoenzyme- and species-specific, and the underlying molecular mechanisms were discussed. http://www.mdpi.com/1422-0067/10/10/4210/ Mon, 28 Sep 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-09-28 10 10 Article 4210 4222 1422-0067 Effects of Acrylamide on the Activity and Structure of Human Brain Creatine Kinase 2009-09-28 doi: 10.3390/ijms10104210 Qing Sheng He-Chang Zou Zhi-Rong Lü Fei Zou Yong-Doo Park Yong-Bin Yan Shan-Jing Yao http://www.mdpi.com/1422-0067/10/10/4198/ The structure and function of an enzyme can be altered by nanoparticles (NPs). The interaction between enzyme and NPs is governed by the key properties of NPs, such as structure, size, surface chemistry, charge and surface shape. Recent representative studies on the NP-enzyme interactions and the regulation of enzyme activity by NPs with different size, composition and surface modification are reviewed. http://www.mdpi.com/1422-0067/10/10/4198/ Mon, 28 Sep 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-09-28 10 10 Review 4198 4209 1422-0067 Regulation of Enzyme Activity through Interactions with Nanoparticles 2009-09-28 doi: 10.3390/ijms10104198 Zhaochun Wu Bin Zhang Bing Yan http://www.mdpi.com/1422-0067/10/9/4187/ Estrogen is believed to be pre-initiator in the risk of breast cancer. The BRCA1 is a tumor suppressor gene associated with breast and ovarian cancer risk. This report describes functional analysis of two BRCA1 missense mutations (Asp67Glu and Thr1051Ser) observed in the familial breast/ovarian cancer patients in Thailand. Levels of luciferase activity of the two mutations were relatively lower than in the wild-type BRCA1. It is indicated that mutants may fail to promote the estrogen receptor dependent functions.It is presumed that estrogen and insulin/IGF-1 regulate c-Myc and cyclin D1 during breast cancer cell proliferation. It is also likely to affect ubiquitination mechanism. Since three affected cancer families carry the Asp67Glu mutation, it is believed that this type of mutation could have some effect on breast/ovarian cancer progression. http://www.mdpi.com/1422-0067/10/9/4187/ Thu, 24 Sep 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-09-24 10 9 Article 4187 4197 1422-0067 Functional Analysis of Familial Asp67Glu and Thr1051Ser BRCA1 Mutations in Breast/Ovarian Carcinogenesis 2009-09-24 doi: 10.3390/ijms10094187 Malinee Pongsavee Pimpicha Patmasiriwat Grady F. Saunders http://www.mdpi.com/1422-0067/10/9/4116/ The objective of this study was to locate chromosomes for improving water and phosphorus-deficiency tolerance of wheat at the seedling stage. A set of Chinese Spring- Egyptian Red wheat substitution lines and their parent Chinese Spring (recipient) and Egyptian Red (donor) cultivars were measured to determine the chromosomal locations of genes controlling water use efficiency (WUE) and phosphorus use efficiency (PUE) under different water and phosphorus conditions. The results underlined that chromosomes 1A, 7A, 7B, and 3A showed higher leaf water use efficiency (WUEl = Pn/Tr; Pn = photosynthetic rate; Tr = transpiration rate) under W-P (Hoagland solution with1/2P), -W-P (Hoagland solution with 1/2P and 10% PEG). Chromosomes 7A, 3D, 2B, 3B, and 4B may carry genes for positive effects on individual plant water use efficiency (WUEp = biomass/TWC; TWC = total water consumption) under WP (Hoagland solution), W-P and -W-P treatment. Chromosomes 7A and 7D carry genes for PUE enhancement under WP, -WP (Hoagland solution with 10% PEG) and W-P treatment. Chromosome 7A possibly has genes for controlling WUE and PUE simultaneously, which indicates that WUE and PUE may share the same genetic background. Phenotypic and genetic analysis of the investigated traits showed that photosynthetic rate (Pn) and transpiration rate (Tr), Tr and WUEl showed significant positive and negative correlations under WP, W-P, -WP and -W-P, W-P, -WP treatments, respectively. Dry mass (DM), WUEP, PUT (phosphorus uptake) all showed significant positive correlation under WP, W-P and -WP treatment. PUE and phosphorus uptake (PUT = P uptake per plant) showed significant negative correlation under the four treatments. The results might provide useful information for improving WUE and PUE in wheat genetics. http://www.mdpi.com/1422-0067/10/9/4116/ Fri, 18 Sep 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-09-18 10 9 Article 4116 4136 1422-0067 Chromosomal Location of Traits Associated with Wheat Seedling Water and Phosphorus Use Efficiency under Different Water and Phosphorus Stresses 2009-09-18 doi: 10.3390/ijms10094116 Hong-Xing Cao Zheng-Bin Zhang Cheng-Xu Sun Hong-Bo Shao Wei-Yi Song Ping Xu http://www.mdpi.com/1422-0067/10/9/3793/ Trehalose is a non-reducing disaccharide formed by two glucose molecules. It is widely distributed in Nature and has been isolated from certain species of bacteria, fungi, invertebrates and plants, which are capable of surviving in a dehydrated state for months or years and subsequently being revived after a few hours of being in contact with water. This disaccharide has many biotechnological applications, as its physicochemical properties allow it to be used to preserve foods, enzymes, vaccines, cells etc., in a dehydrated state at room temperature. One of the most striking findings a decade ago was the discovery of the genes involved in trehalose biosynthesis, present in a great number of organisms that do not accumulate trehalose to significant levels. In plants, this disaccharide has diverse functions and plays an essential role in various stages of development, for example in the formation of the embryo and in flowering. Trehalose also appears to be involved in the regulation of carbon metabolism and photosynthesis. Recently it has been discovered that this sugar plays an important role in plant-microorganism interactions. http://www.mdpi.com/1422-0067/10/9/3793/ Tue, 01 Sep 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-09-01 10 9 Review 3793 3810 1422-0067 Trehalose Metabolism: From Osmoprotection to Signaling 2009-09-01 doi: 10.3390/ijms10093793 Gabriel Iturriaga Ramón Suárez Barbara Nova-Franco http://www.mdpi.com/1422-0067/10/8/3655/ This Special Issue is a significant step in developing a new direction of metabolic research— Molecular System Bioenergetics, which itself is a part of Systems Biology. As a new paradigm of biological sciences, Systems Biology aims at understanding of biological functions by studies and description of new, system level properties, resulting from interactions between components of biological systems at any level of organization, from molecular to population. Metabolism is the way of life of cells by exchanging mass and energy with the surrounding medium, and understanding its mechanisms requires knowledge of the complex interactions between cellular systems and components. While studies of metabolism have a long history, new concepts of Systems Biology provide useful tools for metabolic research. According to Schrödinger, living cells need to be open systems with energy and mass exchange with the surrounding medium, with the aim of maintaining their high structural and functional organization and thus their internal entropy low, achieving this by means of increasing the entropy of the medium by catabolic reactions. Thus, Schrödinger wrote: “The essential thing in metabolism is that the organism succeeds in freeing itself from all entropy it cannot help producing while alive”. Thus, free energy conversion in the cells is an important, central part of metabolism, and understanding the complex mechanisms of its regulation is the aim of Molecular System Bioenergetics. In this Special Issue, several important problems in this field were analyzed. http://www.mdpi.com/1422-0067/10/8/3655/ Wed, 19 Aug 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-08-19 10 8 Editorial 3655 3657 1422-0067 Molecular System Bioenergetics—New Aspects of Metabolic Research 2009-08-19 doi: 10.3390/ijms10083655 Valdur Saks http://www.mdpi.com/1422-0067/10/8/3583/ The effect of biotic stress induced by viral infection (Potato virus Y, strain NTN and O) on NADP-malic enzyme (EC 1.1.1.40) in tobacco plants (Nicotiana tabacum L., cv. Petit Havana, SR1) was tested at the transcriptional, translational and activity level. The increase of enzyme activity in infected leaves was correlated with the increased amount of expressed protein and with mRNA of cytosolic NADP-ME isoform. Transcription of the chloroplastic enzyme was not influenced by viral infection. The increase of the enzyme activity was also detected in stems and roots of infected plants. The effect of viral infection induced by Potato virus Y, NTN strain, causing more severe symptoms, was compared with the effect induced by milder strain PVYO. The observed increase in NADP-malic enzyme activity in all parts of the studied plants was higher in the case of PVYNTN strain than in the case of strain PVYO. The relevance of NADP-malic enzyme in plants under stress conditions was discussed. http://www.mdpi.com/1422-0067/10/8/3583/ Thu, 13 Aug 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-08-13 10 8 Article 3583 3598 1422-0067 Effect of Potato Virus Y on the NADP-Malic Enzyme from Nicotiana tabacum L.: mRNA, Expressed Protein and Activity 2009-08-13 doi: 10.3390/ijms10083583 Veronika Doubnerová Karel Müller Noemi Čeřovská Helena Synková Petra Spoustová Helena Ryšlavá http://www.mdpi.com/1422-0067/10/8/3547/ Plants are particularly subject to environmental stress, as they cannot move from unfavourable surroundings. As a consequence they have to react in situ. In any case, plants have to sense the stress, then the signal has to be transduced to engage the appropriate response. Stress response is effected by regulating genes, by turning on molecular mechanisms to protect the whole organism and its components and/or to repair damage. Reactions vary depending on the type of stress and its intensity, but some are commonly turned on because some responses to different abiotic stresses are shared. In addition, there are multiple ways for plants to respond to environmental stress, depending on the species and life strategy, but also multiple ways within a species depending on plant variety or ecotype. It is regularly accepted that populations of a single species originating from diverse geographic origins and/or that have been subjected to different selective pressure, have evolved retaining the best alleles for completing their life cycle. Therefore, the study of natural variation in response to abiotic stress, can help unravel key genes and alleles for plants to cope with their unfavourable physical and chemical surroundings. This review is focusing on Arabidopsis thaliana which has been largely adopted by the global scientific community as a model organism. Also, tools and data that facilitate investigation of natural variation and abiotic stress encountered in the wild are set out. Characterization of accessions, QTLs detection and cloning of alleles responsible for variation are presented. http://www.mdpi.com/1422-0067/10/8/3547/ Thu, 13 Aug 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-08-13 10 8 Review 3547 3582 1422-0067 A Focus on Natural Variation for Abiotic Constraints Response in the Model Species Arabidopsis thaliana 2009-08-13 doi: 10.3390/ijms10083547 Valérie Lefebvre Seifollah Poormohammad Kiani Mylène Durand-Tardif http://www.mdpi.com/1422-0067/10/8/3371/ Chemical defences represent a main trait of the plant innate immune system. Besides regulating the relationship between plants and their ecosystems, phytochemicals are involved both in resistance against pathogens and in tolerance towards abiotic stresses, such as atmospheric pollution. Plant defence metabolites arise from the main secondary metabolic routes, the phenylpropanoid, the isoprenoid and the alkaloid pathways. In plants, antibiotic compounds can be both preformed (phytoanticipins) and inducible (phytoalexins), the former including saponins, cyanogenic glycosides and glucosinolates. Chronic exposure to tropospheric ozone (O3) stimulates the carbon fluxes from the primary to the secondary metabolic pathways to a great extent, inducing a shift of the available resources in favour of the synthesis of secondary products. In some cases, the plant defence responses against pathogens and environmental pollutants may overlap, leading to the unspecific synthesis of similar molecules, such as phenylpropanoids. Exposure to ozone can also modify the pattern of biogenic volatile organic compounds (BVOC), emitted from plant in response to herbivore feeding, thus altering the tritrophic interaction among plant, phytophagy and their natural enemies. Finally, the synthesis of ethylene and polyamines can be regulated by ozone at level of S-adenosylmethionine (SAM), the biosynthetic precursor of both classes of hormones, which can, therefore, mutually inhibit their own biosynthesis with consequence on plant phenotype. http://www.mdpi.com/1422-0067/10/8/3371/ Thu, 30 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-30 10 8 Review 3371 3399 1422-0067 Chemical Diversity and Defence Metabolism: How Plants Cope with Pathogens and Ozone Pollution 2009-07-30 doi: 10.3390/ijms10083371 Marcello Iriti Franco Faoro http://www.mdpi.com/1422-0067/10/8/3338/ The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP), as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and α-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects. http://www.mdpi.com/1422-0067/10/8/3338/ Wed, 29 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-29 10 8 Article 3338 3357 1422-0067 Inhibition of Citrinin-Induced Apoptotic Biochemical Signaling in Human Hepatoma G2 Cells by Resveratrol 2009-07-29 doi: 10.3390/ijms10083338 Chia-Chi Chen Wen-Hsiung Chan http://www.mdpi.com/1422-0067/10/8/3269/ Phytochelatins (PCs) play an important role in detoxification of heavy metals in plants. PCs are synthesized from glutathione by phytochelatin synthase (PCS), a dipeptidyltransferase. Sesbania rostrata is a tropical legume plant that can tolerate high concentrations of Cd and Zn. In this study, the S. rostrata PCS gene (SrPCS) and cDNAs were isolated and characterized. Southern blot and sequence analysis revealed that a single copy of the SrPCS gene occurs in the S. rostrata genome, and produces four different SrPCS mRNAs and proteins, SrPCS1-SrPCS4, by alternative splicing of the SrPCS pre-mRNA. The SrPCS1 and SrPCS3 proteins conferred Cd tolerance when expressed in yeast cells, whereas the SrPCS2 and SrPCS4 proteins, which lack the catalytic triad and the N-terminal domains, did not. These results suggested that SrPCS1 and SrPCS3 have potential applications in genetic engineering of plants for enhancing heavy metal tolerance and phytoremediation of contaminated soils. http://www.mdpi.com/1422-0067/10/8/3269/ Fri, 24 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-24 10 8 Article 3269 3282 1422-0067 Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms 2009-07-24 doi: 10.3390/ijms10083269 An-Ming Li Bing-Yun Yu Fu-Hua Chen Hui-Yan Gan Jian-Gang Yuan Rongliang Qiu Jun-Chao Huang Zhong-Yi Yang Zeng-Fu Xu http://www.mdpi.com/1422-0067/10/7/3255/ Genistein (4′, 5, 7-trihydroxyisoflavone) is an isoflavone compound obtained from plants that has potential applications in cancer therapy. However, the molecular mechanism of the action of genistein on cancer cell apoptosis is not well known. In this study, we investigated the effect of genistein on topoisomerase II-α (Topo IIα), an important protein involved in the processes of DNA replication and cell proliferation. The results revealed that inhibition of Topo IIα expression through the regulation of Specificity protein 1 and Specificity protein 3 may be one of the reasons for genistein’s induction of HeLa cell apoptosis. http://www.mdpi.com/1422-0067/10/7/3255/ Wed, 22 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-22 10 7 Article 3255 3268 1422-0067 Genistein Inhibition of Topoisomerase IIα Expression Participated by Sp1 and Sp3 in HeLa Cell 2009-07-22 doi: 10.3390/ijms10073255 Najing Zhou Yunli Yan Wenling Li Yanling Wang Lifen Zheng Shuo Han Yongxin Yan Yunzhi Li http://www.mdpi.com/1422-0067/10/7/3194/ Lysophosphatidic acid (LPA), a naturally occurring glycerophospholipid, can evoke various biological responses, including cell migration, proliferation and survival, via activation of G protein-coupled receptors (GPCRs). However, the role of LPA receptors and details of LPA signaling in migration are largely unexplored. In this study we detect the expression of LPA1 and LPA3 receptors in rat aortic smooth muscle cells (RASMCs). LPA stimulated RASMCs migration in a dose-dependent manner and induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell migration was significantly inhibited by specific LPA1/LPA3-receptor antagonist Dioctylglycerol pyrophosphate (8:0) (DGPP8.0) at higher concentration. Migration of cells toward LPA was partially, but significantly, reduced in the presence of SB-203580, a p38 MAPK inhibitor, but not PD98059, an ERK inhibitor. In addition, pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 MAPK,ERK phosphorylation and RASMCs migration. These data suggest that LPA-induced migration is mediated through the Gi-protein-coupled LPA1 receptor involving activation of a PTX-sensitive Gi / p38MAPK pathway. http://www.mdpi.com/1422-0067/10/7/3194/ Mon, 13 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-13 10 7 Article 3194 3208 1422-0067 Receptor-Mediated Vascular Smooth Muscle Migration Induced by LPA Involves p38 Mitogen-Activated Protein Kinase Pathway Activation 2009-07-13 doi: 10.3390/ijms10073194 Zhi-Bin Zhou Jian-Ping Niu Zhi-Jun Zhang http://www.mdpi.com/1422-0067/10/7/3168/ Land plants are anchored in one place for most of their life cycle and therefore must constantly adapt their growth and metabolism to abiotic stresses such as light intensity, temperature and the availability of water and essential minerals. Thus, plants’ subsistence depends on their ability to regulate rapidly gene expression in order to adapt their physiology to their environment. Recent studies indicate that post-transcriptional regulations of gene expression play an important role in how plants respond to abiotic stresses. We will review the different mechanisms of post-transcriptional regulation of nuclear genes expression including messenger RNA (mRNA) processing, stability, localization and protein translation, and discuss their relative importance for plant adaptation to abiotic stress. http://www.mdpi.com/1422-0067/10/7/3168/ Fri, 10 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-10 10 7 Review 3168 3185 1422-0067 Post-transcriptional Regulation of Gene Expression in Plants during Abiotic Stress 2009-07-10 doi: 10.3390/ijms10073168 Maïna Floris Hany Mahgoub Elodie Lanet Christophe Robaglia Benoît Menand http://www.mdpi.com/1422-0067/10/7/3149/ In Nature plants are constantly challenged by a variety of environmental stresses that could lead to disruptions in cellular homeostasis. Programmed cell death (PCD) is a fundamental cellular process that is often associated with defense responses to pathogens, during development and in response to abiotic stresses in fungi, animals and plants. Although there are many characteristics shared between different types of PCD events, it remains unknown whether a common mechanism drives various types of PCD in eukaryotes. One candidate regulator for such a mechanism is Bax Inhibitor-1 (BI-1), an evolutionary conserved, endoplasmic reticulum (ER)-resident protein that represents an ancient cell death regulator that potentially regulates PCD in all eukaryotes. Recent findings strongly suggested that BI-1 plays an important role in the conserved ER stress response pathway to modulate cell death induction in response to multiple types of cell death signals. As ER stress signaling pathways has been suggested to play important roles not only in the control of ER homeostasis but also in other biological processes such as the response to pathogens and abiotic stress in plants, BI-1 might function to control the convergence point that modulates the level of the “pro-survival and pro-death” signals under multiple stress conditions. http://www.mdpi.com/1422-0067/10/7/3149/ Fri, 10 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-10 10 7 Review 3149 3167 1422-0067 Bax Inhibitor-1, a Conserved Cell Death Suppressor, Is a Key Molecular Switch Downstream from a Variety of Biotic and Abiotic Stress Signals in Plants 2009-07-10 doi: 10.3390/ijms10073149 Naohide Watanabe Eric Lam http://www.mdpi.com/1422-0067/10/7/3076/ Stress responses are of particular importance to microorganisms, because their habitats are subjected to continual changes in temperature, osmotic pressure, and nutrients availability. Stressors (and stress factors), may be of chemical, physical, or biological nature. While stress to microorganisms is frequently caused by the surrounding environment, the growth of microbial cells on its own may also result in induction of some kinds of stress such as starvation and acidity. During production of fresh-cut produce, cumulative mild processing steps are employed, to control the growth of microorganisms. Pathogens on plant surfaces are already stressed and stress may be increased during the multiple mild processing steps, potentially leading to very hardy bacteria geared towards enhanced survival. Cross-protection can occur because the overlapping stress responses enable bacteria exposed to one stress to become resistant to another stress. A number of stresses have been shown to induce cross protection, including heat, cold, acid and osmotic stress. Among other factors, adaptation to heat stress appears to provide bacterial cells with more pronounced cross protection against several other stresses. Understanding how pathogens sense and respond to mild stresses is essential in order to design safe and effective minimal processing regimes. http://www.mdpi.com/1422-0067/10/7/3076/ Wed, 08 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-08 10 7 Review 3076 3105 1422-0067 Bacterial Stressors in Minimally Processed Food 2009-07-08 doi: 10.3390/ijms10073076 Vittorio Capozzi Daniela Fiocco Maria Luisa Amodio Anna Gallone Giuseppe Spano http://www.mdpi.com/1422-0067/10/7/3033/ Gastric cancer is the second most common cause of cancer deaths worldwide. The underlying molecular mechanisms of its carcinogenesis are relatively poorly characterized. Hedgehog (Hh) signaling, which is critical for development of various organs including the gastrointestinal tract, has been associated with gastric cancer. The present study was undertaken to reveal the underlying mechanism by which Hh signaling controls gastric cancer cell proliferation. Treatment of gastric cancer cells with cyclopamine, a specific inhibitor of Hh signaling pathway, reduced proliferation and induced apoptosis of gastric cancer cells. Cyclopamine treatment induced cytochrome c release from mitochondria and cleavage of caspase 9. Moreover, Bcl-2 expression was significantly reduced by cyclopamine treatment. These results suggest that Hh signaling regulates the survival of gastric cancer cells by regulating the expression of Bcl-2. http://www.mdpi.com/1422-0067/10/7/3033/ Mon, 06 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-06 10 7 Article 3033 3043 1422-0067 Hedgehog Signaling Regulates the Survival of Gastric Cancer Cells by Regulating the Expression of Bcl-2 2009-07-06 doi: 10.3390/ijms10073033 Myoung-Eun Han Young-Suk Lee Sun-Yong Baek Bong-Seon Kim Jae-Bong Kim Sae-Ock Oh http://www.mdpi.com/1422-0067/10/7/3012/ The secretome of a tobacco cell suspension culture was investigated by a combined proteomic and metabolomic approach. Protein analysis from 2-DE gels led to identification of 32 out of 60 spots from culture medium. Identified proteins were mainly involved in stress defence and cell regeneration processes. Among them three putative new isoforms, e.g. for chitinase, peroxidase and β-1,4-xylosidase were identified, not yet present in available protein databases for the genus Nicotiana. GC-MS analysis of time course experiments revealed significant changes for metabolites involved in energy transport, signalling and cell development. Among them, the most significant increase was found for putrescine in the medium of cultures entering the exponential phase. Results showed strong abundance of stress associated proteins and metabolites in the absence of elicitors or additional stress treatments. http://www.mdpi.com/1422-0067/10/7/3012/ Mon, 06 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-06 10 7 Article 3012 3032 1422-0067 Protein and Metabolite Analysis Reveals Permanent Induction of Stress Defense and Cell Regeneration Processes in a Tobacco Cell Suspension Culture 2009-07-06 doi: 10.3390/ijms10073012 Rico Lippmann Stephanie Kaspar Twan Rutten Michael Melzer Jochen Kumlehn Andrea Matros Hans-Peter Mock http://www.mdpi.com/1422-0067/10/7/2999/ Tb (Tbilisi), the reference Brucellaphage strain, was classified as a member of the Podoviridae family with icosahedral capsids (57 ± 2 nm diameter) and short tails (32 ± 3 nm long). Brucellaphage DNA was double stranded and unmethylated; its molecular size was 34.5 kilobase pairs. Some sequences were found through RAPD analysis, TA cloning technology, and structural proteins were observed by using SDS-PAGE. Thus, the results have laid the foundation for the wider use of Brucellaphage’s basic mechanisms and practical applications. http://www.mdpi.com/1422-0067/10/7/2999/ Fri, 03 Jul 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-07-03 10 7 Article 2999 3011 1422-0067 Molecular Characterization of Tb, a New Approach for an Ancient Brucellaphage 2009-07-03 doi: 10.3390/ijms10072999 Cai-Zhong Zhu Hong-Yan Xiong Jing Han Bu-Yun Cui Dong-Ri Piao Ya-Fei Li Hai Jiang Qian Ren Xiang-Yu Ma Ya-Ming Chai Xia Huang Hong-Yan Zhao Lan-Yu Li http://www.mdpi.com/1422-0067/10/7/2896/ We investigated the presence of carbonic anhydrase in root and hypocotyl of etiolated soybean using enzymatic, histochemical, immunohistochemical and in situ hybridization approaches. In parallel, we used in situ hybridization and immunolocaliza-tion to determine the expression pattern and localization of phosphoenolpyruvate carboxylase. Their co-localization in the root tip as well as in the central cylinder, suggests that a large fraction of the CO2 may be re-introduced into C4 compounds. GmPK3 expression, coding for a cytoplasmic isoform of pyruvate kinase, was detected in all different root cell types, suggesting that both phosphoenolpyruvate-utilizing enzymes are involved in phosphoenolpyruvate metabolism in etiolated soybean roots; a case indicative of the necessary flexibility plant metabolism has to adopt in order to compensate various physiological conditions. http://www.mdpi.com/1422-0067/10/7/2896/ Mon, 29 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-29 10 7 Article 2896 2910 1422-0067 Co-localization of Carbonic Anhydrase and Phosphoenol-pyruvate Carboxylase and Localization of Pyruvate Kinase in Roots and Hypocotyls of Etiolated Glycine max Seedlings 2009-06-29 doi: 10.3390/ijms10072896 Maria Dimou Anca Paunescu Georgios Aivalakis Emmanouil Flemetakis Panagiotis Katinakis http://www.mdpi.com/1422-0067/10/6/2849/ Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated that like many other mesophilic enzymes, T-PPase could be fully inactivated at a low SDS concentration of 2 mM. Using an enzyme activity assay, SDS was shown to act as a mixed type reversible inhibitor, suggesting T-PPase contained specific SDS binding sites. At high SDS concentrations, T-PPase was denatured via a two-state process without the accumulation of any intermediate, as revealed by far-UV CD and intrinsic fluorescence. A comparison of the inactivation and unfolding data suggested that the inhibition might be caused by the specific binding of the SDS molecules to the enzyme, while the unfolding might be caused by the cooperative non-specific binding of SDS to T-PPase. The possible molecular mechanisms underlying the mixed type inhibition by SDS was proposed to be caused by the local conformational changes or altered charge distributions. http://www.mdpi.com/1422-0067/10/6/2849/ Tue, 23 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-23 10 6 Article 2849 2859 1422-0067 Inactivation and Unfolding of the Hyperthermophilic Inorganic Pyrophosphatase from Thermus thermophilus by Sodium Dodecyl Sulfate 2009-06-23 doi: 10.3390/ijms10062849 Hang Mu Sheng-Mei Zhou Yong Xia Hechang Zou Fanguo Meng Yong-Bin Yan http://www.mdpi.com/1422-0067/10/6/2838/ We review the studies on the folding mechanism of the β-hairpin tryptophan zipper 2 (trpzip2) and present some additional computational results to refine the picture of folding heterogeneity and pathways. We show that trpzip2 can have a two-state or a multi-state folding pattern, depending on whether it folds within the native basin or through local state basins on the high-dimensional free energy surface; Trpzip2 can fold along different pathways according to the packing order of tryptophan pairs. We also point out some important problems related to the folding mechanism of trpzip2 that still need clarification, e.g., a wide distribution of the computed conformations for the transition state ensemble. http://www.mdpi.com/1422-0067/10/6/2838/ Mon, 22 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-22 10 6 Review 2838 2848 1422-0067 Folding Mechanism of Beta-Hairpin Trpzip2: Heterogeneity, Transition State and Folding Pathways 2009-06-22 doi: 10.3390/ijms10062838 Yi Xiao Changjun Chen Yi He http://www.mdpi.com/1422-0067/10/6/2763/ A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided. http://www.mdpi.com/1422-0067/10/6/2763/ Thu, 18 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-18 10 6 Review 2763 2788 1422-0067 Yeast Two-Hybrid, a Powerful Tool for Systems Biology 2009-06-18 doi: 10.3390/ijms10062763 Anna Brückner Cécile Polge Nicolas Lentze Daniel Auerbach Uwe Schlattner http://www.mdpi.com/1422-0067/10/6/2733/ The wild wheat Aegilops tauschii Coss. has extensive natural variation available for breeding of common wheat. Drought stress tolerance is closely related to abscisic acid (ABA) sensitivity. In this study, 17 synthetic hexaploid wheat lines, produced by crossing the tetraploid wheat cultivar Langdon with 17 accessions of Ae. tauschii, were used for comparative analysis of natural variation in drought tolerance and ABA sensitivity. Ae. tauschii showed wide natural variation, with weak association between the traits. Drought-sensitive accessions of Ae. tauschii exhibited significantly less ABA sensitivity. D-genome variations observed at the diploid genome level were not necessarily reflected in synthetic wheats. However, synthetic wheats derived from the parental Ae. tauschii accessions with high drought tolerance were significantly more tolerant to drought stress than those from drought-sensitive accessions. Moreover, synthetic wheats with high drought tolerance showed significantly higher ABA sensitivity than drought-sensitive synthetic lines. In the hexaploid genetic background, therefore, weak association of ABA sensitivity with drought tolerance wasobserved. To study differences in gene expression patterns between stress-tolerant and -sensitive lines, levels of two Cor/Lea and three transcription factor gene transcripts were compared. The more tolerant accession of Ae. tauschii tended to accumulate more abundant transcripts of the examined genes than the sensitive accession under stress conditions. The expression patterns in the synthetic wheats seemed to be additive for parental lines exposed to drought and ABA treatments. However, the transcript levels of transcription factor genes in the synthetic wheats did not necessarily correspond to the postulated levels based on expression in parental lines. Allopolyploidization altered the expression levels of the stress-responsive genes in synthetic wheats. http://www.mdpi.com/1422-0067/10/6/2733/ Thu, 18 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-18 10 6 Article 2733 2751 1422-0067 Variation in Dehydration Tolerance, ABA Sensitivity and Related Gene Expression Patterns in D-Genome Progenitor and Synthetic Hexaploid Wheat Lines 2009-06-18 doi: 10.3390/ijms10062733 Yumeto Kurahashi Akihiro Terashima Shigeo Takumi http://www.mdpi.com/1422-0067/10/6/2501/ Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3121 (or its enantiomorph P3221) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 Å and γ = 120°. The crystals were obtained at room temperature and diffracted to 2.0 Å resolution. http://www.mdpi.com/1422-0067/10/6/2501/ Wed, 03 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-03 10 6 Article 2501 2509 1422-0067 Purification, Crystallization and Preliminary X-ray Crystallographic Studies of RAIDD Death-Domain (DD) 2009-06-03 doi: 10.3390/ijms10062501 Tae-ho Jang Hyun Ho Park http://www.mdpi.com/1422-0067/10/6/2487/ Single nucleotide polymorphisms (SNPs) are important markers which can be used in association studies searching for susceptible genes of complex diseases. High-throughput methods are needed for SNP genotyping in a large number of samples. In this study, we applied polyacrylamide gel-based microarray combined with dual-color hybridization for association study of four BDNF polymorphisms with autism. All the SNPs in both patients and controls could be analyzed quickly and correctly. Among four SNPs, only C270T polymorphism showed significant differences in the frequency of the allele (χ2 = 7.809, p = 0.005) and genotype (χ2 = 7.800, p = 0.020). In the haplotype association analysis, there was significant difference in global haplotype distribution between the groups (χ2 = 28.19，p = 3.44e-005). We suggest that BDNF has a possible role in the pathogenesis of autism. The study also show that the polyacrylamide gel-based microarray combined with dual-color hybridization is a rapid, simple and high-throughput method for SNPs genotyping, and can be used for association study of susceptible gene with disorders in large samples. http://www.mdpi.com/1422-0067/10/6/2487/ Tue, 02 Jun 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-06-02 10 6 Article 2487 2500 1422-0067 Association Study between BDNF Gene Polymorphisms and Autism by Three-Dimensional Gel-Based Microarray 2009-06-02 doi: 10.3390/ijms10062487 Lu Cheng Qinyu Ge Pengfeng Xiao Beili Sun Xiaoyan Ke Yunfei Bai Zuhong Lu http://www.mdpi.com/1422-0067/10/6/2476/ The voltage-dependent anion channel (VDAC) is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana. http://www.mdpi.com/1422-0067/10/6/2476/ Tue, 26 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-26 10 6 Article 2476 2486 1422-0067 Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2) Is Involved in ABA-Mediated Early Seedling Development 2009-05-26 doi: 10.3390/ijms10062476 Jinping Yan Han He Shibo Tong Wanrong Zhang Jianmei Wang Xufeng Li Yi Yang http://www.mdpi.com/1422-0067/10/5/2431/ The present study found that ricin toxicity did not only manifest itself as inhibition of protein synthesis, but also induced apoptosis of immune cells and played an extremely significant role in intestinal injury. In this report, we describe a novel method to estimate binding events occurring on intestinal brush border membranes (BBM) based on SPR technology in an attempt to mimic the real intestinal surface capable of interacting physically and/or actively with certain biological molecules. Combined with HPCE-ESI-MS indentification, we obtained 28 kinds of proteins in BBM that interacted with ricin. http://www.mdpi.com/1422-0067/10/5/2431/ Fri, 22 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-22 10 5 Article 2431 2439 1422-0067 Analysis of Intestinal Injuries Induced by Ricin in Vitro Using SPR Technology and MS Identification 2009-05-22 doi: 10.3390/ijms10052431 Linna Liu Hongwei Gao Jiping Li Ying Dong Ning Liu Jiayu Wan Wensen Liu Yucheng Sun Ming Xu http://www.mdpi.com/1422-0067/10/5/2412/ The OB-fold is a diverse structure superfamily based on a β-barrel motif that is often supplemented with additional non-conserved secondary structures. Previous deletion mutagenesis and NMR hydrogen exchange studies of three OB-fold proteins showed that the structural stabilities of sites within the conserved β-barrels were larger than sites in non-conserved segments. In this work we examined a database of 80 representative domain structures currently classified as OB-folds, to establish the basis of this effect. Residue-specific values were obtained for the number of Cα-Cα distance contacts, sequence hydrophobicities, crystallographic B-factors, and theoretical B-factors calculated from a Gaussian Network Model. All four parameters point to a larger average flexibility for the non-conserved structures compared to the conserved β-barrels. The theoretical B-factors and contact densities show the highest sensitivity.Our results suggest a model of protein structure evolution in which novel structural features develop at the periphery of conserved motifs. Core residues are more resistant to structural changes during evolution since their substitution would disrupt a larger number of interactions. Similar factors are likely to account for the differences in stability to unfolding between conserved and non-conserved structures. http://www.mdpi.com/1422-0067/10/5/2412/ Fri, 22 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-22 10 5 Article 2412 2430 1422-0067 Relative Stabilities of Conserved and Non-Conserved Structures in the OB-Fold Superfamily 2009-05-22 doi: 10.3390/ijms10052412 Kaitlyn M. Guardino Sarah R. Sheftic Robert E. Slattery Andrei T. Alexandrescu http://www.mdpi.com/1422-0067/10/5/2348/ Aβ1-40 coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aβ (a hallmark of Alzheimer’s disease). We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aβ aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid. http://www.mdpi.com/1422-0067/10/5/2348/ Wed, 20 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-20 10 5 Article 2348 2366 1422-0067 Microscopic Investigation of Reversible Nanoscale Surface Size Dependent Protein Conjugation 2009-05-20 doi: 10.3390/ijms10052348 Kazushige Yokoyama Hyunah Cho Sean P. Cullen Matthew Kowalik Nicole M. Briglio Harold J. Hoops Zhouying Zhao Michael A. Carpenter http://www.mdpi.com/1422-0067/10/5/2336/ The goal of present work was to investigate the use of bioerodible polymeric nanoparticles as carriers of retinoic acid (RA), which is known to induce differentiation of several cell lines into neurons. A novel method, named “Colloidal-Coating”, has been developed for the preparation of nanoparticles based on a copolymer of maleic anhydride and butyl vinyl ether (VAM41) loaded with RA. Nanoparticles with an average diameter size of 70 nm and good morphology were prepared. The activity of the encapsulated RA was evaluated on SK-N-SH human neuroblastoma cells, which are known to undergo inhibition of proliferation and neuronal differentiation upon treatment with RA. The activity of RA was not affected by the encapsulation and purification processes. http://www.mdpi.com/1422-0067/10/5/2336/ Tue, 19 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-19 10 5 Article 2336 2347 1422-0067 A Novel Method for the Preparation of Retinoic Acid-Loaded Nanoparticles 2009-05-19 doi: 10.3390/ijms10052336 Cesare Errico Matteo Gazzarri Federica Chiellini http://www.mdpi.com/1422-0067/10/5/2252/ Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED), which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell’s ability to do work and control the intracellular Ca2+ homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS), mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD). However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect) is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis. http://www.mdpi.com/1422-0067/10/5/2252/ Tue, 19 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-19 10 5 Review 2252 2303 1422-0067 Mitochondria and Energetic Depression in Cell Pathophysiology 2009-05-19 doi: 10.3390/ijms10052252 Enn Seppet Marju Gruno Ants Peetsalu Zemfira Gizatullina Huu Phuc Nguyen Stefan Vielhaber Manfred H.P. Wussling Sonata Trumbeckaite Odeta Arandarcikaite Doreen Jerzembeck Maria Sonnabend Katharina Jegorov Stephan Zierz Frank Striggow Frank N. Gellerich http://www.mdpi.com/1422-0067/10/5/2158/ Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single- and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and β-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single- or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single- and multi-amiRNA expression vectors was comparable. http://www.mdpi.com/1422-0067/10/5/2158/ Thu, 14 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-14 10 5 Communication 2158 2168 1422-0067 Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells 2009-05-14 doi: 10.3390/ijms10052158 Tao Hu Qiong Fu Ping Chen Li Ma Onsam Sin Deyin Guo http://www.mdpi.com/1422-0067/10/5/2054/ We recently described Rolly Protein (ROLP), a small protein synthesized by substrate-adherent cells in a broad range of tissues. In a first set of experiments performed taking advantage of bone forming tibial cartilage as an experimental model we showed that ROLP transcription is associated to cells in an active proliferation state, whereas its downregulation is observed when cell proliferation decreases. Taking advantage of siRNA technology we also documented the expression modulation of some apoptosis-related genes in ROLP-silenced cells. In this work we search for the possible molecular interactors of ROLP by using both the antibody array approach as well as the co-immunoprecipitation approach. Results suggest the occurrence of an interaction of ROLP with Erythrocyte membrane Protein Band 4.1/3(Epb4.1/3), an oncosuppressor downregulated in tumor development and in metastatic tissues; in addition we report experimental results that keep in line also with a potential interaction of ROLP with other PDZ-containing proteins. We also present experimental evidences supporting a role played by ROLP in cell adhesion thus supporting the existence of a biologically relevant link between ROLP and Epb4.1/3. We here suggest that ROLP might exert its biological role cooperating with Epb4.1/3, a protein that is involved in biological pathways that are often inhibited in tumor metastasis. Given the role of Epb4.1/3 in contrasting cancerogenesis we think that its cooperation with ROLP might be relevant in cancer studies and deserves further investigation. http://www.mdpi.com/1422-0067/10/5/2054/ Tue, 12 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-12 10 5 Article 2054 2065 1422-0067 Rolly Protein (ROLP)-Epb4.1/3: A Potential Protein-Protein Interaction Relevant for the Maintenance of Cell Adhesion 2009-05-12 doi: 10.3390/ijms10052054 Manuele Castelnuovo Massimiliano Monticone Sara Massone Irene Vassallo Federico Tortelli Ranieri Cancedda Aldo Pagano http://www.mdpi.com/1422-0067/10/5/2066/ The folding of protein molecules in the GroEL inner cavity under the co-chaperonin GroES lid is widely accepted as a crucial event of GroEL-assisted protein folding. This review is focused on the data showing that GroEL-assisted protein folding may proceed out of the complex with the chaperonin. The models of GroEL-assisted protein folding assuming ligand-controlled dissociation of nonnative proteins from the GroEL surface and their folding in the bulk solution are also discussed. http://www.mdpi.com/1422-0067/10/5/2066/ Tue, 12 May 2009 00:00:00 CEST International Journal of Molecular Sciences 2009-05-12 10 5 Review 2066 2083 1422-0067 GroEL-Assisted Protein Folding: Does It Occur Within the Chaperonin Inner Cavity? 2009-05-12 doi: 10.3390/ijms10052066 Victor V. Marchenkov Gennady V. Semisotnov