A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti

Transplantable neural progenitor populations derived from Rhesus monkey embryonic stem cells

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3% +/- 8.1%)populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65% - 80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor PI, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3'-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

Circulating immunoglobulin A- and immunoglobulin G-secreting hybridoma cells in peripheral blood preferably migrate to female genital tracts. The role of sex hormones

Antigen-specific circulating immunoglobulin-secreting cells (ISC) migrate to various secondary and tertiary lymphoid tissues. To understand the migration of the cells into the genital tract and its regulation by sex hormones, spleen-derived SG2 hybridoma cells secreting immunoglobulin G2b (IgG2b) and Peyer's patch-derived PA4 hybridoma cells secreting polymer IgA were labelled with (3) H-TdR, and intravenously injected into syngeneic mice of both sexes. Using flow cytometry, surface molecular markers of plasma cells, CD38 and CD138, and adhesion molecules, CD49d, CD162, and CD11a were found to be positive in SG2 and PA4 cells, but CD62L, alpha4beta7 and CD44 were not expressed on these cells. The relative distribution indexes (RDIs) of the cells in genital tract and other tissues were measured. The means of RDIs of SG2 and PA4 cells in female genital tissues were 6.5 and 4.5 times as many as the means in male genital tissues, respectively. The treatment of ovariectomized mice with beta-oestradiol significantly increased the RDIs of PA4 cells in cervix and vagina, but decreased the RDIs of SG2 cells in vagina, horn of uterus, uterus and rectum (P <0.05). Progesterone treatment increased the RDIs of PA4 cells in vagina and rectum (P <0.05). The treatment with testosterone significantly increased the RDIs of SG2 and PA4 cells in epididymis and accessory sex glands (P <0.05). These results demonstrate that the female genital tract is the preferable site for the migration of circulating hybridoma cells to the male genital tract, and sex hormones play an important role in regulation of the migration of circulating ISC to genital tracts.

Identification and characterization of a new class of human immunodeficiency virus type 1 recombinants comprised of two circulating recombinant forms, CRF07_BC and CRF08_BC, in China

We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences if 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new "second-generation" recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.

Expression, purification and characterization of recombinant Jerdonitin, a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains

Jerdonitin is a P-II class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. In this study, we established a high-level expression system in Pichia pastoris and developed a purification strategy for the recombinant Jerdonitin. This recombinant Jerdonitin degraded fibrinogen at a level of activity comparable with its wild type. The effects of recombinant Jerdonitin on inhibiting ADP-induced human platelet aggregation were in a dose-dependent manner with an IC50 of 248 nM. In addition, we reported here that Jerdonitin can significantly inhibit the growth of several cell lines, including human liver cancer cells (Bel7402), human leukemia cells (K562) and human gastric carcinoma cells (BGC823). This study offers recombinant Jerdonitin that will be valuable for further functional and structural studies of Jerdonitin. (C) 2009 Elsevier Ltd. All rights reserved.

Attenuated Salmonella Typhimurium lacking the pathogenicity island-2 type 3 secretion system grow to high bacterial numbers inside phagocytes in mice.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

Slowness and sparseness lead to place, head-direction, and spatial-view cells.

We present a model for the self-organized formation of place cells, head-direction cells, and spatial-view cells in the hippocampal formation based on unsupervised learning on quasi-natural visual stimuli. The model comprises a hierarchy of Slow Feature Analysis (SFA) nodes, which were recently shown to reproduce many properties of complex cells in the early visual system []. The system extracts a distributed grid-like representation of position and orientation, which is transcoded into a localized place-field, head-direction, or view representation, by sparse coding. The type of cells that develops depends solely on the relevant input statistics, i.e., the movement pattern of the simulated animal. The numerical simulations are complemented by a mathematical analysis that allows us to accurately predict the output of the top SFA layer.

The influence of 1D, meso- and crystal structures on charge transport and recombination in solid-state dye-sensitized solar cells

We have prepared single crystalline SnO2 and ZnO nanowires and polycrystalline TiO2 nanotubes (1D networks) as well as nanoparticle-based films (3D networks) from the same materials to be used as photoanodes for solid-state dye-sensitized solar cells. In general, superior photovoltaic performance can be achieved from devices based on 3-dimensional networks, mostly due to their higher short circuit currents. To further characterize the fabricated devices, the electronic properties of the different networks were measured via the transient photocurrent and photovoltage decay techniques. Nanowire-based devices exhibit extremely high, light independent electron transport rates while recombination dynamics remain unchanged. This indicates, contrary to expectations, a decoupling of transport and recombination dynamics. For typical nanoparticle-based photoanodes, the devices are usually considered electron-limited due to the poor electron transport through nanocrystalline titania networks. In the case of the nanowire-based devices, the system becomes limited by the organic hole transporter used. In the case of polycrystalline TiO2 nanotube-based devices, we observe lower transport rates and higher recombination dynamics than their nanoparticle-based counterparts, suggesting that in order to improve the electron transport properties of solid-state dye-sensitized solar cells, single crystalline structures should be used. These findings should aid future design of photoanodes based on nanowires or porous semiconductors with extended crystallinity to be used in dye-sensitized solar cells. © 2013 The Royal Society of Chemistry.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.