The I/LWEQ Domain in RapGAP3 Required for Posterior Localization in Migrating Cells

Cell migration requires a defined cell polarity which is formed by diverse cytoskeletal components differentially localized to the poles of cells to extracellular signals. Rap-GAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of migrating cells. Here, we examined localization of truncated RapGAP3 proteins and found that the I/LWEQ domain in the central region of RapGAP3 was sufficient for posterior localization in migrating cells, as opposed to leading-edge localization of full-length Rap-GAP3. All truncated proteins accumulated at the leading edge of migrating cells exhibited clear translocation to the cell cortex in response to stimulation, whereas proteins localized to the posterior in migrating cells displayed no translocation to the cortex. The I/LWEQ domain appears to passively accumulate at the posterior region in migrating cells due to exclusion from the extended front region in response to chemoattractant stimulation rather than actively being localized to the back of cells. Our results suggest that posterior localization of the I/LWEQ domain of RapGAP3 is likely related to F-actin, which has probably different properties compared to newly formed F-actin at the leading edge of migrating cells, at the lateral and posterior regions of the cell.     

Pretreatment with recombinant human interleukin 2 (rhIL-2) Up-regulates PCNA-positive cells after partial hepatectomy in rat liver

We prepared recombinant human interleukin-2 (rhIL-2) and studied its pretreated influence on liver regeneration and the blood profile in partially (67%) hepatectomized (PH) male Sprague-Dawley rats. Rats were injected in the tail vein with rhIL-2 three times per day for 3 consecutive days and 67% underwent a partial hepatectomy (PH). Five days after the PH, liver tissue and blood samples were analyzed for liver regeneration and hematological changes. The weight of the liver in the rhIL-2 pretreated groups increased in a dose-dependent manner; with the highest treatment (24 × 10⁴ IU/kg), the maximum liver weight of 88.6% was exhibited. The control group showed a gradual increase to 76.3% of the original liver weight. A histological analysis of the liver showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells in rhIL-2 pretreated rat livers. The rate of hepatocyte proliferation also increased significantly in primary cultured rat liver cells following rhIL-2 treatment. These results suggest that pretreatment with rhIL-2 may play adjuvant roles in liver regeneration after PH.     

Drosophila melanogaster S2 cells are more suitable for the production of recombinant COX-1 than Trichoplusia ni BTI TN-5B1-4 cells

Recombinant human cyclooxygenase-1 (COX-1) was expressed from stably transfected Trichoplusia ni BTI TN-5B1-4 (TN-5B1-4) and Drosophila melanogaster S2 cells. Two kinds of recombinant COX-1 with molecular weights (MWs) of 68 and 74 kDa were expressed in the intracellular fractions of stably transfected TN-5B1-4/ COX-1 and S2/COX-1 cells, due to glycosylation. The recombinant COX-1 secreted to medium fractions has a MW of 72 kDa. Recombinant COX-1 in the intracellular fractions was purified to homogeneity using a one-step Ni-NTA affinity fractionation method. Recombinant COX-1 purified from TN-5B1-4/COX-1 and S2/COX-1 cells contained 11,389 and 33,850 Unit/mg of specific peroxidase activity, respectively. The maximum productions of intracellular recombinant COX-1 were 1.7 and 5.6 μg/10⁷ cells in the T-flask cultures of TN-5B1-4/COX-1 and S2/COX-1 cells, respectively. Taken together, our findings indicate that S2 cells can be more suitable system to produce recombinant COX-1, compared to TN-5B1-4 cells.     

Ninjurin1 Enhances the Basal Motility and Transendothelial Migration of Immune Cells by Inducing Protrusive Membrane Dynamics

Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.     

Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts

Human mesenchymal stromal cells (MSCs) offer great hope for the treatment of tissue degenerative and immune diseases, but their phenotypic similarity to dermal fibroblasts may hinder robust cell identification and isolation from diverse tissue harvests. To identify genetic elements that can reliably discriminate MSCs from fibroblasts, we performed comparative gene and microRNA expression profiling analyses with genome-wide oligonucleotide microarrays. When taken globally, both gene and microRNA expression profiles of MSCs were highly similar to those of fibroblasts, accounting well for their extensive phenotypic and functional overlaps. Scattered expression differences were pooled to yield an MSC-specific molecular signature, consisting of 64 genes and 21 microRNAs whose expressions were at least 10-fold and two-fold higher, respectively, in MSCs compared with fibroblasts. Genes either encoding transmembrane proteins or associated with tumors were relatively abundant in this signature. These data should provide the molecular basis not only for the discovery of novel diagnostic markers discriminating MSCs from fibroblasts, but also for further studies on MSC-specific signaling mechanisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sohyun Bae and Jung Hoon Ahn contributed equally to this work. This research was supported in part by a grant (SC-2140) from Stem Cell Research Center of the 21st Century Frontier Research Program, and in part by Korea Science and Engineering Foundation grant (M10641000037) funded by the Ministry of Education, Science and Technology, Republic of Korea.     

Protistan community patterns within the brine and halocline of deep hypersaline anoxic basins in the eastern Mediterranean Sea

Environmental factors restrict the distribution of microbial eukaryotes but the exact boundaries for eukaryotic life are not known. Here, we examine protistan communities at the extremes of salinity and osmotic pressure, and report rich assemblages inhabiting Bannock and Discovery, two deep-sea superhaline anoxic basins in the Mediterranean. Using a rRNA-based approach, we detected 1,538 protistan rRNA gene sequences from water samples with total salinity ranging from 39 to 280 g/Kg, and obtained evidence that this DNA was endogenous to the extreme habitat sampled. Statistical analyses indicate that the discovered phylotypes represent only a fraction of species actually inhabiting both the brine and the brine-seawater interface, with as much as 82% of the actual richness missed by our survey. Jaccard indices (e.g., for a comparison of community membership) suggest that the brine/interface protistan communities are unique to Bannock and Discovery basins, and share little (0.8–2.8%) in species composition with overlying waters with typical marine salinity and oxygen tension. The protistan communities from the basins’ brine and brine/seawater interface appear to be particularly enriched with dinoflagellates, ciliates and other alveolates, as well as fungi, and are conspicuously poor in stramenopiles. The uniqueness and diversity of brine and brine-interface protistan communities make them promising targets for protistan discovery.     

Prevention of in Vitro thermal aggregation and inactivation of foreign proteins by the hyperthermophilic group II chaperonin α-subunit from Aeropyrum pernix K1

In this study, we report that the recombinant α subunit chaperonin protein (ApCpnA) from Aeropyrum pernix K1 can efficiently prevent the thermal aggregation and inactivation of foreign model proteins, such as citrate synthase (CS) from= porcine heart, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (four 37.5 kDa subunits), and malate dehydrogenase (MDH) from Thermus flavus (two 67 kDa subunits)K=In the presence of ApCpnA and ATP, the thermal aggregation of CS and ADH were prevented by 90 and 65%, respectively, at each 43 and 50°C. Also, the activities of CS, ADH, and MDH under the thermal inactivation conditions were stably maintained at higher than 80% by addition of ApCpnA and ATP, while the activities of those enzymes in the absence of ApCpnA and ATP were dramatically inactivated and decreased below 20% within 30 min. Based on these results, we propose that the α subunit chaperonin from the hyperthermophilic archaeon, A. pernix K1 can be utilized to enhance the durability and cost effectiveness of high-temperature biocatalysts.