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121.
High-yield isolation, expansion, and differentiation of murine bone marrow-derived mesenchymal stem cells using fibrin microbeads (FMB) 总被引:1,自引:0,他引:1
Rivkin R Ben-Ari A Kassis I Zangi L Gaberman E Levdansky L Marx G Gorodetsky R 《Cloning and stem cells》2007,9(2):157-175
Transplantation of adult mesenchymal stem cells (MSCs) could provide a basis for tissue regeneration. MSCs are typically isolated from bone marrow (BM) based on their preferential adherence to plastic, although with low efficiency in terms of yield and purity. Extensive expansion is needed to reach a significant number of MSCs for any application. Fibrin microbeads (FMB) were designed to attach mesenchymal cells and to provide a matrix for their expansion. The current study was aimed at isolating a high yield of purified BM-derived mouse MSCs based on their preferential adherence and proliferation on FMB in suspension cultures. MSCs could be downloaded to plastics or further expanded on FMB. The yield of MSCs obtained by the FMB isolation technique was about one order of magnitude higher than that achieved by plastic adherence, suggesting that these cells are more abundant than previously reported. FMB-isolated cells were classified as MSCs by their fibroblastic morphology, self-renewal ability, and expression profile of their surface antigens, as examined by flow cytometry and immunostaining. In cell culture, the isolated MSCs could be induced to differentiate into three different mesodermal lineages, as demonstrated by histochemical stains and by RT-PCR analyses of tissue-specific genes. MSCs were also able to differentiate into osteocytes while still cultured on FMB. Our results suggest that FMB might serve as an efficient platform for the isolation, expansion, and differentiation of mouse BM-derived MSCs to be subsequently implanted for tissue regeneration. 相似文献
122.
123.
Okun E Dikshtein Y Carmely A Saida H Frei G Sela BA Varshavsky L Ofir A Levy E Albeck M Sredni B 《The FEBS journal》2007,274(12):3159-3170
Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) is an organotellurium compound with pleiotropic functions that has been associated with antitumoral, immunomodulatory and antineurodegenerative activities. Tellurium compounds with a +4 oxidation state, such as AS101, react uniquely with thiols, forming disulfide molecules. In light of this, we tested whether AS101 can react with the amino acid homocysteine both in vitro and in vivo. AS101 conferred protection against homocysteine-induced apoptosis of HL-60 cells. The protective mechanism of AS101 against homocysteine toxicity was directly mediated by its chemical reactivity, whereby AS101 reacted with homocysteine to form homocystine, the less toxic disulfide form of homocysteine. Moreover, AS101 was shown here to reduce the levels of total homocysteine in an in vivo model of hyperhomocysteinemia. As a result, AS101 also prevented sperm cells from undergoing homocysteine-induced DNA fragmentation. Taken together, our results suggest that the organotellurium compound AS101 may be of clinical value in reducing total circulatory homocysteine levels. 相似文献
124.
Louw C Gordon A Johnston N Mollatt C Bradley G Whiteley CG 《Journal of enzyme inhibition and medicinal chemistry》2007,22(1):121-126
There is, at present, no definitive pre-mortem diagnostic tool for Alzheimer's disease, (AD) which relates to a poor understanding of its etiology. Brains of AD patients contain large amounts of the toxic plaque-forming beta-amyloid1-42 fragment in addition to elevated concentrations of the amino acid L-arginine. This work proposes that lowering levels of arginine in the astrocytes surrounding amyloid plaques may serve as a therapeutic tool in this neurodegenerative disorder. Arginine deiminase (ADI), from Pseudomonas aeruginosa, and peptidylarginine deiminase [PAD II], from bovine brain, are inhibited by amyloid peptides that contain arginine (amyloid1-42) and those that have no arginine (amyloid12-28/22-35). Enhanced activity of PAD II is noted with free L-arginine. 相似文献
125.
Finzi A Pacheco B Xiang SH Pancera M Herschhorn A Wang L Zeng X Desormeaux A Kwong PD Sodroski J 《Journal of virology》2012,86(17):8974-8986
Metastable conformations of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp120 to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gp41-mediated virus-cell membrane fusion and entry. Topological layers in the gp120 inner domain contribute to gp120-trimer association in the unliganded state and to CD4 binding. Here we describe similarities and differences between HIV-1 and SIVmac gp120. In both viruses, the gp120 N/C termini and the inner domain β-sandwich and layer 2 support the noncovalent association of gp120 with the envelope glycoprotein trimer. Layer 1 of the SIVmac gp120 inner domain contributes more to trimer association than the corresponding region of HIV-1 gp120. On the other hand, layer 1 plays an important role in stabilizing the CD4-bound conformation of HIV-1 but not SIVmac gp120 and thus contributes to HIV-1 binding to CD4. In SIVmac, CD4 binding is instead enhanced by tryptophan 375, which fills the Phe 43 cavity of gp120. Activation of SIVmac by soluble CD4 is dependent on tryptophan 375 and on layer 1 residues that determine a tight association of gp120 with the trimer. Distinct biological requirements for CD4 usage have resulted in lineage-specific differences in the HIV-1 and SIV gp120 structures that modulate trimer association and CD4 binding. 相似文献
126.
Postharvest heat and conditioning treatments activate different molecular responses and reduce chilling injuries in grapefruit 总被引:4,自引:0,他引:4
Sapitnitskaya M Maul P McCollum GT Guy CL Weiss B Samach A Porat R 《Journal of experimental botany》2006,57(12):2943-2953
A combination of hot water (a rinse at 62 degrees C for 20 s) and conditioning (pre-storage at 16 degrees C for 7 d) treatments synergistically reduced chilling injury development in grapefruit (Citrus paradisi, cv. "Star Ruby") during cold storage at 2 degrees C, suggesting that the treatments may activate different chilling tolerance responses. To study the molecular mechanisms involved, chilling- and conditioning-responsive genes were isolated by polymerase chain reaction (PCR) cDNA subtraction, cDNA libraries were constructed from hot water- and conditioning-treated fruit, and cDNA sequencing was used to identify putative stress-responsive and chilling tolerance genes. PCR cDNA subtraction revealed the identification of 17 chilling-responsive and heat- and conditioning-induced genes, and the expression patterns of 11 additional stress-related genes, antioxidant defensive genes, and genes encoding enzymes involved in membrane lipid modifications were characterized. It was found that hot water and conditioning treatments had little effect on gene expression by themselves, but rather had a priming effect, and enabled the fruit to activate their defence responses after subsequent exposure to chilling. RNA gel blot hybridizations revealed that the expression patterns of eight genes, including HSP19-I, HSP19-II, dehydrin, universal stress protein (USP), EIN2, 1,3;4-beta-D-glucanase, and superoxide dismutase (SOD), were specifically regulated by the heat treatment, and four genes, including fatty acid desaturase2 (FAD2) and lipid transfer protein (LTP), were specifically regulated by the conditioning treatment. Furthermore, four more genes were identified, including a translation initiation factor (SUI1), a chaperonin, and alcohol dehydrogenase (ADH), that were commonly regulated by both heat and conditioning treatments. According to these data, it is suggested that pre-storage heat and conditioning treatments may enhance fruit chilling tolerance by activating different molecular mechanisms. The hot water treatment activates mainly the expression of various stress-related genes, whereas the conditioning treatment activates mainly the expression of lipid membrane modification enzymes. 相似文献
127.
Orly Dym Shira Albeck Yoav Peleg Alon Schwarz Yigal Burstein 《Journal of molecular biology》2009,393(4):937-717
We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs. 相似文献
128.
The high genomic G+C group of Actinobacteria possesses a variety of physiological and metabolic properties, and exhibits diverse lifestyles and ecological distribution. In recent years, Actinobacteria have been found to frequently dominate samples obtained from freshwater samples. Furthermore, phylogenetic analyses have shown that 16S rRNA genes from uncultured actinobacterial freshwater samples cluster in four distinct lineages. While these lineages are abundant, little is known about them and currently no pure‐culture representatives or genomic fragments of them are available. In a screen of a genomic library from the moderately eutrophic freshwater Lake Kinneret, five fosmid clones containing actinobacterial genomic fragments were found. Three ~40 kb genomic fragments were chosen for sequencing. Fosmids K003 and K005 showed high similarity and were affiliated with the acIV actinobacterial freshwater lineage. Fosmid K004 was affiliated with the highly abundant acI lineage. A comparative genomic analysis revealed high synteny between the two freshwater clones K003 and K005 but a lower synteny between these two and the K004 fosmid. Fosmids K003 and K005 share an identical arrangement of arginine biosynthesis gene while K004 showed a slightly different arrangement by lacking the argF gene. Fosmid Ant4E12, an Antarctic actinobacterial clone, showed a higher synteny with K003/5 than K004 and a similar arginine operon, but in a different genomic context. The Clusters of Orthologous Groups categories assignment of the three fosmids yielded genes that were mostly involved in amino acid and nucleotide metabolism, as well as transport and ribosomal RNA translation, structure and biogenesis. These genomic fragments represent the first sequences to be published from these lineages, providing a cornerstone for future work on this environmentally dominant group. 相似文献
129.
The glyoxylate bypass allows Escherichia coli to grow on carbon sources with only two carbons by bypassing the loss of carbons as CO2 in the tricarboxylic acid cycle. The flux toward this bypass is regulated by the phosphorylation of the enzyme isocitrate dehydrogenase (IDH) by a bifunctional kinase–phosphatase called IDHKP. In this system, IDH activity has been found to be remarkably robust with respect to wide variations in the total IDH protein concentration. Here, we examine possible mechanisms to explain this robustness. Explanations in which IDHKP works simultaneously as a first-order kinase and as a zero-order phosphatase with a single IDH binding site are found to be inconsistent with robustness. Instead, we suggest a robust mechanism where both substrates bind the bifunctional enzyme to form a ternary complex. 相似文献
130.
Herschhorn A Marasco WA Hizi A 《Journal of immunology (Baltimore, Md. : 1950)》2010,185(12):7623-7632
HIV selectively downregulates HLA-A and -B from the surfaces of infected cells to avoid detection by the immune system. In contrast, the HLA-C molecules are highly resistant to this downregulation. High expression level of HLA-C on the cell surface, which correlates with a single nucleotide polymorphism, is also associated with lower viral loads and slower progression to AIDS. These findings strongly suggest that HIV-1-derived peptides are efficiently presented by HLA-C and trigger the elimination of infected cells. Accordingly, the ability to detect these HLA-C-peptide complexes may be used for therapeutic targeting of HIV-1-infected cells and for measuring effective presentation of vaccine candidates after immunization with HIV-1-related proteins or genes. However, low level of HLA-C expression on the cell surface has impeded the development of such complex-recognizing reagents. In this study, we describe the development of a high-affinity human Ab that specifically interacts, at low pM concentrations, with a conserved viral T cell epitope derived from HIV-1 Nef protein and presented by HLA-C. The human Ab selectively detects this complex on different cells and does not interact with a control complex that differed only in the presented peptide. Engineering lentiviruses to display this Ab endowed them with the same specificity as the Ab, whereas coexpressing the Ab and Fas ligand enables the lentiviruses to kill specifically Nef-presenting cells. Abs and pseudoviruses with such specificity are likely to be highly valuable as building blocks for specific targeting and killing of HIV-1-infected cells. 相似文献