The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02

Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the Delta dotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of Delta dotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.     

Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells

We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.     

Differential protein expressions induced by adenovirus-mediated p16 gene transfer into Balb/c nude mouse

To evaluate the safety of adenovirus-mediated gene transfer, we investigated differential protein expression after transducing adenoviral vector containing the p16(INK4a) tumor suppressor gene (Ad5CMV-p16) into Balb/c nude mice. We found that adenovirus-mediated p16(INK4a) gene transfer inhibited experimental lung metastasis, and that the intratumoral injection of Ad5CMV-p16 resulted in regression of A549 cell xenografted tumors in Balb/c nude mice. We investigated changes in protein expression after intratumoral injection of Ad5CMV-p16 or Ad5CMV (10(10) plaque-forming units) into A549 cell xenografted Balb/c nude mice by two-dimensional gel electrophoresis /matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Compared with the control (serum-free medium treated tumor cells) Ad5CMV-p16 gene transfer changed the expression of 29 proteins including heterogeneous nuclear ribonucleoprotein, protein phosphatase 2, 14-3-3 zeta protein, alpha-tubulin, and glutathione-S-transferase P1. Moreover, both Ad5CMV-p16 and Ad5CMV up-regulated the expression of glutathione-S-transferase P1. In addition, Ad5CMV-p16 gene transfer did not seem to increase the expression of tumorigenicity-related protein in Balb/c nude mice. Further studies will be needed to investigate the effect of Ad5CMV-p16 on normal human cells and tissues for safety evaluation. These results suggest that the p16 gene seems to have an important role in apoptosis as well as in cell cycle arrest in non-small cell lung cancer.     

Clinically Relevant Characterization of Lung Adenocarcinoma Subtypes Based on Cellular Pathways: An International Validation Study

Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient''s prognosis. Additional results suggest the ability to use a similar approach to determine a patient''s sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.     

Detecting protein dissimilarities in multiple alignments using Bayesian variable selection

MOTIVATION: We present an application of Bayesian variable selection to the novel detection of sequence elements that confer negative design to protein structure and function. As an illustration, we analyze the different dimer interfaces between the CXCL8 chemokine family with the CCL4 and CCL2 chemokine families to discover the changes that disfavor CXCL8 of quaternary structure. RESULTS: In comparison with known experimental results, our method identifies evolutionarily conserved sequence changes in the CC families that inhibit CXCL8 quaternary structure. Therefore, we find positive selection of negative design elements. Furthermore, our approach predicts that a two-residue deletion conserved in the CCL4 chemokine family disfavors CXCL8 dimerization. AVAILABILITY: The Matlab code for the Bayesian variable selection is freely available at http://stat.tamu.edu/~mvannucci/webpages/codes.html     

The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans

The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.     

Identification of bronchioalveolar stem cells in normal lung and lung cancer

Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). Identified at the bronchioalveolar duct junction, BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties. Furthermore, BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo. These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma. Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma, this work points to BASCs as the putative cells of origin for this subtype of lung cancer.     

Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development

Although V(D)J recombination is thought to be regulated by changes in the accessibility of chromatin to the recombinase machinery, the mechanisms responsible for establishing "open" chromatin are poorly understood. We performed a detailed study of the acetylation status of histones associated with 11 V(H) gene segments, their flanking regions, and various intergenic elements during B-cell development and ontogeny, when V(D)J recombination is highly regulated. Histone H4 shows higher and more-regulated acetylation than does histone H3 in the V(H) locus. In adult pro-B cells, V(H) gene segments are acetylated prior to V(D)J rearrangement, with higher acetylation associated with J(H)-distal V(H) gene segments. While large regions of the V(H) locus have similar patterns of histone acetylation, acetylation is narrowly confined to the gene segments, their flanking promoters, and recombinase signal sequence elements. Thus, histone acetylation in the V(H) locus is both locally and globally regulated. Increased histone acetylation accompanies preferential recombination of J(H)-proximal V(H) gene segments in early B-cell ontogeny, and decreased histone acetylation accompanies inhibition of V-DJ recombination in a transgenic model of immunoglobulin heavy-chain allelic exclusion. Thus, changes in histone acetylation appear to be important for both promotion and inhibition of V-DJ rearrangement during B-cell ontogeny and development.     

Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells

Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit⁺ Lin^? HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit⁺ Lin^? HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.