[show abstract][hide abstract] ABSTRACT: The HIV-1 accessory protein Nef plays an active role in the pathogenesis of AIDS by its numerous cellular interactions that facilitate the release of virus particles. This 27 kDa protein is required for maintenance of the viral replication in HIV, and is also known to contribute to immune evasion, blocking of apoptosis in virus-infected cells and enhancement of virus infectivity. Nef has been shown to be secreted and is present on the surface of virus-infected cells. Recent studies from our laboratory have shown that the Nef protein is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (40-100 nm diam.) that do not contain virions. We have identified three amino-terminal domains of Nef as necessary for secretion: (i) the four arginine residues (17,19,21, 22) comprising the basic region; (ii) the phosphofurin acidic cluster sequence (PACS) composed of four glutamic acid residues (61-64); (iii) a previously unknown motif spanning amino acid residues 65-69 (VGFPV) which we named the secretion modification region (SMR). In this study, we have used population-based phylogeny data and sequence analysis to characterize the conservation of the Nef SMR domain that regulates vesicle secretion. We have performed in silico computational chemistry analysis involving molecular dynamic structure modeling of mutations in the SMR motif. Sequence analysis of Nef from HIV-1-infected patients, including slow progressors (SP), long term progressors (LTP) and long term non-progressors (LTNP) demonstrated 99 % conservation of the Nef SMR motif. Computational analysis including modeling of wild-type HIV-1 Nef and V66A Nef SMR mutant using structural homology and molecular dynamics of ligand-associated interactions indicated significant structural changes in the Nef mutant, thus supporting the importance of the SMR domain for mediating Nef vesicle secretion.
Journal of Molecular Modeling 05/2012; 18(10):4603-13. · 1.98 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes invasive properties. The transforming growth factor β (TGF-β) family member bone morphogenetic protein-3B (BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the regulatory mechanisms leading to its silencing have not been identified.
Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2 overexpression in H1299 cells increased wound healing response to TGFβ treatment.
Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells.
Molecular Cancer 04/2012; 11:27. · 5.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4⁺ T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef's SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties.
Journal of Virology 01/2012; 86(1):406-19. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: A series of CoMo/γ-Al2O3 catalysts was prepared with [Co/(Co + Mo)] ratios of 0.3, 0.4 and 0.5 while maintaining a total metal content of 19 wt%.
These catalysts were tested in a batch autoclave reactor after presulfiding with the objective of studying the influence of
Co/(Co + Mo) ratio on the hydrodesulfurization (HDS) pathways of benzothiophene (BT) and dibenzothiophene (DBT). The results
of this study have clearly demonstrated that the Co/Co + Mo ratio has a significant influence on the overall HDS of BT and
DBT as well on the direct desulfurization (DDS) pathway, but showed no influence on the hydrogenation pathway. A Co/Co + Mo
ratio of 0.4 was found to be optimum for both overall HDS as well as the HDS by DDS pathway.
[show abstract][hide abstract] ABSTRACT: The objective of our study was to assess patellofemoral measurements on MRI and to correlate the measurements with different grades of cartilage defect.
Axial and sagittal MR images of 100 patients with various pathologic knee conditions were analyzed. The patients were divided into two age groups: < 40 years and > or = 40 years. Patellar measurements of facet asymmetry, the patella-to-patellar tendon ratio, and the amount of patellotrochlear cartilage overlap were obtained in each subject. Similarly, trochlear measurements of the ventral trochlear prominence, trochlear depth, facet asymmetry, sulcus angle, and lateral inclination were obtained. Axial and sagittal MR images were reviewed to grade the severity of focal cartilage defects in the patellofemoral region on the basis of the depth of the lesion. Measurements in knees without a chondral defect were compared with knees with mild and severe chondral defects.
There was a statistically significant difference in the trochlear measurements of the ventral prominence (p = 0.012), trochlear depth (p = 0.001), sulcus angle (p = 0.208), and lateral inclination (p = 0.154) between normal knees and knees with severe cartilage defects in patients younger than 40 years. No significant difference was seen in the patellar measurements between normal knees and knees with severe cartilage defects.
There is an association between abnormal trochlear morphology and severe patellofemoral cartilage defects in patients younger than 40 years.
American Journal of Roentgenology 03/2010; 194(3):721-7. · 2.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.
AIDS research and human retroviruses 02/2010; 26(2):173-92. · 2.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epigenetic control of ribosomal RNA (rRNA) gene transcription by cell type-specific regulators, such as the osteogenic transcription factor Runx2, conveys cellular memory of growth and differentiation to progeny cells during mitosis. Here, we examined whether coregulatory proteins contribute to epigenetic functions that are mitotically transmitted by Runx2 in osteoblastic cells. We show that the transcriptional corepressor Transducin Like Enhancer-1 (TLE1) associates with rRNA genes during mitosis and interphase through interaction with Runx2. Mechanistically, depletion of TLE1 relieves Runx2-mediated repression of rRNA genes transcription and selectively increases histone modifications linked to active transcription. Biologically, loss of TLE-dependent rRNA gene repression coincides with increased global protein synthesis and enhanced cell proliferation. Our findings reinforce the epigenetic marking target genes by phenotypic transcription factors in mitosis and demonstrate a requirement for retention of coregulatory factors to sustain physiological control of gene expression during proliferation of lineage committed cells.
Proceedings of the National Academy of Sciences 02/2010; 107(9):4165-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Intrahepatic pancreatic pseudocyst extension is a rare but complex clinical entity requiring multimodality approach for management. There is no consensus regarding the optimal strategy for the treatment of intrahepatic pancreatic pseudocyst and the literature is limited to a few case reports. Most of the published cases were managed by surgical or percutaneous drainage.
We hereby report a case of intrahepatic pancreatic pseudocyst extension which failed to resolve by percutaneous drainage. Endoscopic transpapillary drainage was utilized which led to complete resolution of the intrahepatic pancreatic pseudocyst.
The excellent results obtained in our patient suggest that it should be considered as primary treatment and may obviate the need for more aggressive and potentially morbid procedures.
[show abstract][hide abstract] ABSTRACT: Nuclear microenvironments are architecturally organized subnuclear sites where the regulatory machinery for gene expression, replication, and repair resides. This compartmentalization is necessary to attain required stoichiometry for organization and assembly of regulatory complexes for combinatorial control. Combined and methodical application of molecular, cellular, biochemical, and in vivo genetic approaches is required to fully understand complexities of biological control. Here we provide methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.
Methods in molecular biology (Clifton, N.J.) 01/2010; 647:77-93.
[show abstract][hide abstract] ABSTRACT: The purposes of this study were to assess the degree of patellotrochlear chondral overlap (patellotrochlear index), correlate it with the Insall-Salvati and modified Insall-Salvati indexes, and determine the association between these measurements and patellofemoral chondral defects.
Sagittal 1.5-T and 3-T MR images of 100 consecutively registered patients with symptoms were analyzed, and the Insall-Salvati index, modified Insall-Salvati index, patellotrochlear index, and patellophyseal index (ratio of the height of patella above the physeal line to the length of the patellar articular cartilage) were calculated. The upper and lower limits of 2 SDs were used to define patella alta and baja, and the correlation coefficient curves were plotted to compare techniques. The indexes in normal knees were compared with those in knees with severe chondral defects.
The mean patellotrochlear index was 0.49 +/- 0.15 (SD) (range, 0-0.88). On the basis of calculation of 2 SDs, patella alta was determined to have a patellotrochlear index less than 0.18 and patella baja, an index greater than 0.80. Weak correlation was found between the measured patellotrochlear index and Insall-Salvati index (r = -0.224) and between the patellotrochlear index and modified Insall-Salvati index (r = -0.073). A strong correlation was found between the patellotrochlear index and patellophyseal index (r = -0.813). A statistically significant (p < 0.05) difference in the modified Insall-Salvati index and patellophyseal index was found between knees with normal and those with severe cartilage defects.
Our results indicate that the commonly used Insall-Salvati and modified Insall-Salvati indexes do not correlate with patellotrochlear articular cartilage congruence. We did find an association between the modified Insall-Salvati and patellophyseal indexes and the presence of severe chondral defects.
American Journal of Roentgenology 11/2009; 193(5):1361-6. · 2.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trichobezoars are usually without symptoms until they reach a large size. The "Rapunzel" syndrome is a trichobezoar with a long tail extending from the stomach to small bowel. We report the case of a 6-year-old girl with a history of trichotillomania and hair ingestion for three years, who presented with multiple jejunojejunal intussusceptions due to a trichobezoar with a long, 90-cm tail into the small bowel. To our knowledge, this is the first report of trichobezoars as a cause of jejunal intussusceptions, which should be suspected in the appropriate clinical circumstances.
Southern medical journal 05/2009; 102(4):416-8. · 0.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is growing awareness that the fidelity of gene expression necessitates coordination of transcription factor metabolism and organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture. The regulatory machinery that governs genetic and epigenetic control of gene expression is compartmentalized in nuclear microenvironments. Temporal and spatial parameters of regulatory complex organization and assembly are functionally linked to biological control and are compromised with the onset and progression of tumorigenesis. High throughput imaging of cells, tissues, and tumors, including live cell analysis, is expanding research's capabilities toward translating components of nuclear organization into novel strategies for cancer diagnosis and therapy.
Annals of the New York Academy of Sciences 03/2009; 1155:4-14. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epigenetic control is required to maintain competency for the activation and suppression of genes during cell division. The association between regulatory proteins and target gene loci during mitosis is a parameter of the epigenetic control that sustains the transcriptional regulatory machinery that perpetuates gene-expression signatures in progeny cells. The mitotic retention of phenotypic regulatory factors with cell cycle, cell fate, and tissue-specific genes supports the coordinated control that governs the proliferation and differentiation of cell fate and lineage commitment.
Biochemistry and Cell Biology 03/2009; 87(1):1-6. · 2.92 Impact Factor