High-Frequency Ultrasound Assessment of Antimicrobial Photodynamic Therapy In Vitro

Ultrasound imaging is proving to be an important tool for medical diagnosis of dermatological disease. Backscatter spectral profiles using high-frequency ultrasound (HFUS, 10–100 MHz) are sensitive to subtle changes in eukaryotic cellular morphology and mechanical properties that are indicative of early apoptosis, the main type of cell death induced following photodynamic therapy (PDT). We performed experiments to study whether HFUS could also be used to discern changes in bacteria following PDT treatment. Pellets of planktonic Staphylococcus aureus were treated with different PDT protocols and subsequently interrogated with HFUS. Changes in ultrasound backscatter response were found to correlate with antimicrobial effect. Despite their small size, distinct changes in bacterial morphology that are indicative of cell damage or death are detectable by altered backscatter spectra from bacterial ensembles using HFUS. This highlights the potential for HFUS in rapidly and non-invasively assessing the structural changes related to antimicrobial response.     

The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity.     

Directed differentiation of induced pluripotent stem cells towards T lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.     

Induction of high polyploidy in Phaseolus cell cultures by the protein kinase inhibitor,K-252a

Summary Somatic polyploidy of species-specific and tissue-specific degrees occurs in almost all plant species studied so far, but nearly nothing is known about the control mechanisms switching the mitotic cycle to an endoreduplication cycle. In order to search for a possible role of the cdc2 kinase, cell suspension cultures of the Runner bean, Phaseolus coccineus (Leguminosae) were treated with K-252a, an inhibitor of protein kinase activity. The treatment resulted in continuous cell cycles without mitosis, and hence induced polyploidy levels up to 2048C. It is, therefore, suggested that phosphorylation of a protein kinase, probably of the cell cycle-important p34^cdc2 type, is involved in the control of endoreduplication.     

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.     

植物磷脂酶D基因表达与衰老的关系

磷脂酶D (PLD)是一种重要的磷脂水解酶,在植物细胞中普遍存在。磷脂酶D能激活许多重要的细胞生理功能,包括调控细胞膜的重建、跨膜信号传导及细胞内调控、细胞骨架组装、防御反应以及种子萌发和植物的衰老等。对磷脂酶D的基本特性、磷脂酶D基因特异性表达模式及其活性抑制与植物衰老的关系进行了综述,并探讨和展望了今后植物磷脂酶D基因的研究方向。     

Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21 and Cyclin D2 in human colon adenoma cells

Epigenetic and posttranslational modifications of the expression of cell cycle-relevant genes or proteins like p21, e.g., by miRNAs are crucial mechanisms in the development or prevention of colon cancer. The present study investigated the influence of butyrate and trichostatin A (TSA) as histone deacetylase inhibitors on the expression of colon cancer-relevant miRNA (miR-135a, miR-135b, miR-24, miR-106b, miR-let-7a) in LT97 colon adenoma cells as a model of an early stage of colon carcinogenesis. The impact of distinct miRNAs (miR-106b, miR-135a) on butyrate-mediated regulation of p21 and Cyclin D2 gene and protein expression as well as the effect on LT97 cell proliferation (non-transfected, miR-106b and miR-135a mimic transfected) was analyzed. Butyrate and partial TSA reduced the expression of miR-135a, miR-135b, miR-24 and miR-let-7a (~0.5-fold, 24 h) and miR-24, miR-106b and miR-let-7a (~0.5–0.7-fold, 48 h) in LT97 cells. Levels of p21 mRNA and protein were significantly increased by butyrate and TSA (~threefold and 4.5-fold, respectively, 24 h) in non-transfected but not in miR-106b transfected LT97 cells. Levels of Cyclin D2 mRNA were significantly reduced by butyrate and TSA (~0.3-fold, 24 h) in non-transfected and miR-135a-transfected LT97 cells, whereas protein levels were predominantly not influenced. MiR-106b and miR-135a significantly reduced butyrate-/TSA-mediated inhibition of LT97 cell proliferation (72 h). These results indicate that butyrate is able to modify colon cancer-relevant miRNAs like miR-106b and miR-135a which are involved in the regulation of cell cycle-relevant genes like p21 and might influence inhibition of adenoma cell proliferation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0500-4) contains supplementary material, which is available to authorized users.     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.