pH regulation in spread cells and round cells

The aim of this work was to characterize the changes in pH regulation that lead to increased intracellular pH (pHi) in well-spread cells on tissue culture plastic relative to cells on a nonadhesive surface. Bicarbonate was not required for maintenance of a control steady state pHi or of the difference in pHi between round and spread cells. In the absence of bicarbonate, lowering the sodium content of the medium led to decreased pHi and elimination of the difference between round and spread cells. In the presence or absence of bicarbonate, adding ethylisopropyl amiloride lowered pHi and eliminated the difference between round and spread cells. Measurements of recovery from acute acidification in the absence of bicarbonate confirmed that Na+/H+ exchange was enhanced in spread cells. However, recovery from both acidification and alkalinization in the presence of bicarbonate showed that bicarbonate-dependent recovery in both directions, most likely due to sodium-dependent and -independent HCO3-/Cl- exchangers, was also stimulated in spread cells. We conclude that Na+/H+ exchange has a primary role in determining steady state pHi in 3T3 cells in serum and is responsible for the lower pHi in round cells. Bicarbonate-dependent pH regulatory mechanisms are also inhibited in round cells.     

Mechanotransduction in cells

Cell-matrix and cell-cell adhesions critically influence cell metabolism, protein synthesis, cell survival, cytoskeletal architecture and consequently cell mechanical properties such as migration, spreading and contraction. An important group of adhesive transmembrane receptors that mechanically link the ECM (extracellular matrix) with the internal cytoskeleton are integrins which are intimately connected with the FAs (focal adhesions) which consists of many proteins. The transient formation of FAs is greatly augmented either through externally applied tension to the cell or internally through myosin II-driven cell contractility. Exactly which protein(s) within FAs sense, transmit and respond to mechanical stress is currently debated and numerous candidates have been proposed.     

Regulatory T cells interfere with glutathione metabolism in dendritic cells and T cells

Naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) suppress proliferation of CD4(+)CD25(-) effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation.     

Estrogen-sensitive cells : Hormone receptors in tumors and cells in culture

A high affinity, specific estrogen-binding protein has been detected in the cytosol of pituitary tumor cells grown in culture. A similar estrogen-binding protein can be extracted from the nuclei by 0.5 M KCl; furthermore, the nuclei contain an estradiol-binding system that functions independently of the presence or absence of cytosol in the incubation medium and which cannot be solubilized by 0.5 M KCl. The cytosol ‘receptor’ does not directly facilitate the transfer of the hormone from the cytoplasm to the nucleus.     

Semaphorins in interactions between T cells and antigen-presenting cells

Although semaphorins were identified originally as guidance cues for developing neuronal axons, accumulating evidence indicates that several semaphorins are expressed also in the immune system. SEMA4D (CD100), which is expressed constitutively by T cells, enhances the activation of B cells and dendritic cells (DCs) through its cell-surface receptor, CD72. SEMA4A, which is expressed by DCs, is involved in the activation of T cells through interactions with TIM2. So, these semaphorins seem to function in the reciprocal stimulation of T cells and antigen-presenting cells (APCs). Emerging evidence indicates that additional semaphorins and related molecules are involved in T-cell-APC interactions also.     

体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells

Background: To evaluate the influence of hypoxia and molecular events in endothelial and embryonic stem cells.Materials and Methods: Human umbilical vein endothelial cells (HUVECs) and mouse embryoid body (EB) cells were subjected to hypoxic conditions for different time courses. DNA fragmentation assay, quantification of apoptotic cells by TUNEL assay measured by flowcytometry, and Western blot analysis for the molecular events of apoptosis were performed.Results: DNA fragmentation could be identified under hypoxic conditions in HUVECs and mouse EBs. The DNA fragmentation increased when the hypoxic interval was extended.In situ internucleosomal DNA fragmentation-TUNEL assay also found that the percentages of apoptotic cells increased gradually in HUVECs and mouse EBs when the hypoxic interval was extended. Furthermore, the levels of expression of p53 and Bax both increased in hypoxic conditions.Conclusions: Hypoxia increases both HUVEC and mouse EB apoptosis, which is associated with increase in p53/Bax expression.     

Hybridization of testis-derived stem cells with somatic cells and embryonic stem cells in mice

Somatic cell hybridization is widely used to study the control of gene regulation and the stability of differentiated states. In contrast, the application of this method to germ cells has been limited in part because of an inability to culture germ cells. In this study, we produced germ cell hybrids using germ-line stem (GS) cells and multipotent germ-line stem (mGS) cells. While GS cells are enriched for spermatogonial stem cell (SSC) activity, mGS cells are similar to embryonic stem (ES) cells and originally derived from GS cells. Hybrids were successfully obtained between GS cells and ES cells, between GS cells and mGS cells, and between mGS cells and thymocytes. All exhibited ES cell markers and a behavior similar to ES cells, formed teratomas, and differentiated into somatic cell tissues. However, none of the hybrid cells were able to reconstitute spermatogenesis after microinjection into seminiferous tubules. Analyses of the DNA methylation patterns of imprinted genes also showed that mGS cells do not possess a DNA demethylation ability, which was found in embryonic germ cells derived from primordial germ cells. However, mGS cells reactivated the X chromosome and induced Pou5f1 expression in female thymocytes in a manner similar to ES cells. These data show that mGS cells possess ES-like reprogramming potential, which predominates over-SSC activity.     

Tumorigenesis in cells derived from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells are an attractive source for potential cell-replacement therapy. However, transplantation of differentiated products harbors the risk of teratoma formation, presenting a serious health risk. Thus, we characterized Nanog-expressing (undifferentiated) cells remaining after induction of differentiation by cytological examination. To induce differentiation of iPS cells, we generated embryoid bodies (EBs) derived from iPS cells carrying a Nanog–green fluorescent protein (GFP) reporter and then injected GFP-positive and GFP-negative EBs into nude mice. GFP-positive EB transplantation resulted in the formation of immature teratoma grade 3, but no tumors were induced by GFP-negative EB. GFP-positive cells revealed significantly lower cytoplasmic area and higher nucleus/cytoplasm ratio than those of GFP-negative cells. Our results suggest that morphological analysis might be a useful method for distinguishing between tumorigenic and nontumorigenic iPS cells.