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101.
Huang C Das A Barker D Tholpady S Wang T Cui Q Ogle R Botchwey E 《Cell and tissue research》2012,347(3):553-566
Endogenous stem cell recruitment to the site of skeletal injury is key to enhanced osseous remodeling and neovascularization.
To this end, this study utilized a novel bone allograft coating of poly(lactic-co-glycolic acid) (PLAGA) to sustain the release
of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors, from calvarial allografts. Uncoated allografts,
vehicle-coated, low dose FTY720 in PLAGA (1:200 w:w) and high dose FTY720 in PLAGA (1:40) were implanted into critical size
calvarial bone defects. The ability of local FTY720 delivery to promote angiogenesis, maximize osteoinductivity and improve
allograft incorporation by recruitment of bone progenitor cells from surrounding soft tissues and microcirculation was evaluated.
FTY720 bioactivity after encapsulation and release was confirmed with sphingosine kinase 2 assays. HPLC-MS quantified about
50% loaded FTY720 release of the total encapsulated drug (4.5 μg) after 5 days. Following 2 weeks of defect healing, FTY720
delivery led to statistically significant increases in bone volumes compared to controls, with total bone volume increases
for uncoated, coated, low FTY720 and high FTY720 of 5.98, 3.38, 7.2 and 8.9 mm3, respectively. The rate and extent of enhanced bone growth persisted through week 4 but, by week 8, increases in bone formation
in FTY720 groups were no longer statistically significant. However, micro-computed tomography (microCT) of contrast enhanced
vascular ingrowth (MICROFIL?) and histological analysis showed enhanced integration as well as directed bone growth in both
high and low dose FTY720 groups compared to controls. 相似文献
102.
Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India
and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase
production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with
three reference strains, viz., Pseudomonas fluorescens (NCIM2099T), Pseudomonas aureofaciens (NCIM2026T), and Pseudomonas aeruginosa (MTCC2582T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with
three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged
to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing
strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving
(NH4)2SO4 precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity
at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg
(for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn2+, Cu2+, and Ni2+ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature. 相似文献
103.
104.
Zhang H Zhao Q Bhattacharya S Waheed AA Tong X Hong A Heck S Curreli F Goger M Cowburn D Freed EO Debnath AK 《Journal of molecular biology》2008,378(3):565-580
The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer α-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 Å. Using this structural information, we have utilized a structure-based rational design approach to stabilize the α-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced α-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein. 相似文献
105.
Bhattacharya S Zhang H Debnath AK Cowburn D 《The Journal of biological chemistry》2008,283(24):16274-16278
The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly. 相似文献
106.
During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development. 相似文献
107.
Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis
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Qing Yao Andrew I Jewett Yi‐Wei Chang Catherine M Oikonomou Morgan Beeby Cristina V Iancu Ariane Briegel Debnath Ghosal Grant J Jensen 《The EMBO journal》2017,36(11):1577-1589
FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram‐negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ‐like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ‐driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction. 相似文献
108.
Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, we have identified a link between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, we find that paxillin is redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy. 相似文献
109.
Autophagy (specifically macroautophagy) is an evolutionarily conserved catabolic process where the cytoplasmic contents of a cell are sequestered within double membrane vacuoles, called autophagosomes, and subsequently delivered to the lysosome for degradation. Autophagy can function as a survival mechanism in starving cells. At the same time, extensive autophagy is commonly observed in dying cells, leading to its classification as an alternative form of programmed cell death. The functional contribution of autophagy to cell death has been a subject of great controversy. However, several recent loss-of-function studies of autophagy (atg) genes have begun to address the roles of autophagy in both cell death and survival. Here, we review the emerging evidence in favor of and against autophagic cell death, discuss the possible roles that autophagic degradation might play in dying cells, and identify salient issues for future investigation. 相似文献
110.
Sathiyanarayanan Anusuya 《Archives Of Phytopathology And Plant Protection》2013,46(13):1611-1620
Germinating seeds tend to release a variety of proteins into their surrounding surfaces; some of which have an inhibitory action against plant pathogens. The aim of this study was to investigate and identify defence proteins present in the exudates from water-imbibed and chitosan-imbibed (0.1% w/v) seeds of chickpea (Cicer arietinum L). Chickpea seeds imbibed in chitosan released a higher amount of proteins in the exudate when compared to the seeds imbibed in water. The obtained exudates were analysed in regard to specific protein activities by enzymatic assays and SDS-PAGE analysis. Results showed that the exude obtained from chickpea seeds imbibed in chitosan solution exhibited a new isoform of chitinase, chitosanase and protease inhibitors. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus, Fusarium oxysporum f.sp. ciceri. Our results suggest that seed exudates protect seeds during their germination from soil pathogens. 相似文献