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981.
There is growing evidence supporting the need for a broad scale investigation of the proteins and protein modifications in the organic matrix of bone and the use of these measures to predict fragility fractures. However, limitations in sample availability and high heterogeneity of bone tissue cause unique experimental and/or diagnostic problems. We addressed these by an innovative combination of laser capture microscopy with our newly developed liquid chromatography separation methods, followed by gel electrophoresis and mass spectrometry analysis. Our strategy allows in-depth analysis of very limited amounts of bone material, and thus, can be important to medical sciences, biology, forensic, anthropology, and archaeology. The developed strategy permitted unprecedented biochemical analyses of bone-matrix proteins, including collagen modifications, using nearly nanoscale amounts of exceptionally homogenous bone tissue. Dissection of fully mineralized bone-tissue at such degree of homogeneity has not been achieved before. Application of our strategy established that: (1) collagen in older interstitial bone contains higher levels of an advanced glycation end product pentosidine then younger osteonal tissue, an observation contrary to the published data; (2) the levels of two enzymatic crosslinks (pyridinoline and deoxypiridinoline) were higher in osteonal than interstitial tissue and agreed with data reported by others; (3) younger osteonal bone has higher amount of osteopontin and osteocalcin then older interstitial bone and this has not been shown before. Taken together, these data show that the level of fluorescent crosslinks in collagen and the amount of two major noncollagenous bone matrix proteins differ at the level of osteonal and interstitial tissue. We propose that this may have important implications for bone remodeling processes and bone microdamage formation. 相似文献
982.
Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA) 总被引:2,自引:0,他引:2
Sander JD Dahlborg EJ Goodwin MJ Cade L Zhang F Cifuentes D Curtin SJ Blackburn JS Thibodeau-Beganny S Qi Y Pierick CJ Hoffman E Maeder ML Khayter C Reyon D Dobbs D Langenau DM Stupar RM Giraldez AJ Voytas DF Peterson RT Yeh JR Joung JK 《Nature methods》2011,8(1):67-69
Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations. 相似文献
983.
Anjana Bhatia Saroj Arora Bikram Singh Gurveen Kaur Avinash Nagpal 《Phytochemistry Reviews》2011,10(3):309-323
Plants used in traditional medicine have stood up to the test of time and contributed many novel compounds for preventive
and curative medicine to modern science. India is sitting on a gold mine of well recorded and traditionally well practiced
knowledge of herbal medicine. Specially, plants growing at high altitude in Himalayan pastures are time-honored sources of
health and general well being of local inhabitants. As of today, Himalayan plants are a major contributor to the herbal pharmaceutical
industry both of India and other countries. Plants growing at higher altitudes are subjected to an assault of diverse testing
situations including higher doses of mutagenic UV-radiation, physiological drought, desiccation and strong winds. Plants interact
with stressful environments by physiological adaptation and altering the biochemical profile of plant tissues and producing
a spectrum of secondary metabolites. Secondary metabolites are of special interest to scientists because of their unique pharmacophores
and medicinal properties. Secondary metabolites like polyphenols, terpenes and alkaloids have been reported to possess antimutagenic
and anticancer properties in many studies. The fundamental aspiration of the current review is to divulge the antimutagenic/anticancer
potential of five alpine plants used as food or medicine by the populations living at high altitudes. 相似文献
984.
985.
Joel R. Meyerson Tommi A. White Donald Bliss Amy Moran Alberto Bartesaghi Mario J. Borgnia M. Jason V. de la Cruz David Schauder Lisa M. Hartnell Rachna Nandwani Moez Dawood Brianna Kim Jun Hong Kim John Sununu Lisa Yang Siddhant Bhatia Carolyn Subramaniam Darrell E. Hurt Laurent Gaudreault Sriram Subramaniam 《Journal of visualized experiments : JoVE》2011,(58)
Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Å resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.Download video file.(47M, mov) 相似文献
986.
Deepak B Thimiri Govinda Raj Bart Ghesquière Arun Kumar Tharkeshwar Katrijn Coen Rita Derua Dieter Vanderschaeghe Evelien Rysman Murali Bagadi Pieter Baatsen Bart De Strooper Etienne Waelkens Gustaaf Borghs Nico Callewaert Johan Swinnen Kris Gevaert Wim Annaert 《Molecular systems biology》2011,7(1)
We manufactured a novel type of lipid‐coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high‐resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N‐glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two‐thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications. 相似文献
987.
Andre Loboda Michael V Nebozhyn James W Watters Carolyne A Buser Peter Martin Shaw Pearl S Huang Laura Van’t Veer Rob AEM Tollenaar David B Jackson Deepak Agrawal Hongyue Dai Timothy J Yeatman 《BMC medical genomics》2011,4(1):1-10
Background
Copy number alterations (CNA) play a key role in cancer development and progression. Since more than one CNA can be detected in most tumors, frequently co-occurring genetic CNA may point to cooperating cancer related genes. Existing methods for co-occurrence evaluation so far have not considered the overall heterogeneity of CNA per tumor, resulting in a preferential detection of frequent changes with limited specificity for each association due to the high genetic instability of many samples.Method
We hypothesize that in cancer some linkage-independent CNA may display a non-random co-occurrence, and that these CNA could be of pathogenetic relevance for the respective cancer. We also hypothesize that the statistical relevance of co-occurring CNA may depend on the sample specific CNA complexity. We verify our hypotheses with a simulation based algorithm CDCOCA (complexity dependence of co-occurring chromosomal aberrations).Results
Application of CDCOCA to example data sets identified co-occurring CNA from low complex background which otherwise went unnoticed. Identification of cancer associated genes in these co-occurring changes can provide insights of cooperative genes involved in oncogenesis.Conclusions
We have developed a method to detect associations of regional copy number abnormalities in cancer data. Along with finding statistically relevant CNA co-occurrences, our algorithm points towards a generally low specificity for co-occurrence of regional imbalances in CNA rich samples, which may have negative impact on pathway modeling approaches relying on frequent CNA events. 相似文献988.
Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH2(40)) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH2(40) bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH2(40) blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine. 相似文献
989.
990.