Analysis of Golgi pH in Chinese hamster ovary cells using ratiometric pH-sensitive fluorescent proteins

Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live-cell imaging revealed that the intracellular pH (pHi) of clones expressing the ratiometric pH-sensitive FPs converged to a specific pH range (cis-Golgi: 6.4–6.5; trans-Golgi: 5.9–6.0; and cytoplasm: 7.1–7.2). The pHi was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH-sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through pHi control.     

Structural properties of the seed bank and the two island diffusion

Journal of Mathematical Biology - We investigate various aspects of the (biallelic) Wright–Fisher diffusion with seed bank in conjunction with and contrast to the two-island model analysed...     

Measuring the adhesion limit of fibronectin for fibroblasts with a narrow-gap rotational rheometer

Bioprocess and Biosystems Engineering - We study the adhesion limit of 3T6 fibroblasts, cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37 °C...     

Novel identification of peripheral dopaminergic D2 receptor in male germ cells

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology.     

Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are mesoderm-derived cells that are considered a good source of somatic cells for treatment of many degenerative diseases. Previous studies have reported the differentiation of mesodermal MSCs into endodermal and ectodermal cell types beyond their embryonic lineages, including hepatocytes and neurons. However, the molecular pathways responsible for the direct or indirect cell type conversion and the functional ability of the differentiated cells remain unclear and need further research. In the present study, we demonstrated that valproic acid (VPA), which is a histone deacetylase inhibitor, induced an increase in the expression of endodermal genes including CXCR4, SOX17, FOXA1, FOXA2, GSC, c-MET, EOMES, and HNF-1β in human umbilical cord derived MSCs (hUCMSCs). In addition, we found that VPA is able to increase these endodermal genes in hUCMSCs by activating signal transduction of AKT and ERK. VPA pretreatment increased hepatic differentiation at the expense of adipogenic differentiation. The effects of VPA on modulating hUCMSCs fate were diminished by blocking AKT and ERK activation using specific signaling inhibitors. Together, our results suggest that VPA contributes to the lineage conversion of hUCMSCs to hepatic cell fate by upregulating the expression of endodermal genes through AKT and ERK activation.     

MicroRNA-27 promotes the differentiation of odontoblastic cell by targeting APC and activating Wnt/β-catenin signaling

MicroRNAs (miRNAs) play an essential role in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontoblastic cell differentaion is largely unknown. In the present study, we demonstrate that the expression of miR-27 was significantly increased during MDPC-23 odontoblastic cell differentiation. Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 odontoblastic cells and accelerates mineralization without cell proliferation. In addition, our results of target gene prediction revealed that the mRNA of adenomatous polyposis coli (APC) associated with Wnt/β-catenin signaling pathway has miR-27 binding site in the its 3′ UTR and is suppressed by miR-27. Subsequentially, the down-regulated APC by miR-27 triggered the activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Our data suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling. Therefore, miR-27 might be considered a critical candidate as an odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in the dental medicine.     

Requirement of the expression of 3-phosphoglycerate dehydrogenase for traversing S phase in murine T lymphocytes following immobilized anti-CD3 activation

Murine resting (G₀) T lymphocytes contained no detectable mRNA of 3-phosphoglycerate dehydrogenase (PHGDH) catalyzing the first step in the phosphorylated pathway of l-serine biosynthesis. Immobilized anti-CD3 activation of G₀ T cells expressed the PHGDH mRNA in G₁ with a maximum level in S phase. G₀ T cells activated with either immobilized anti-CD3 plus CsA or PBu₂, which failed to drive the activated T cells to enter S phase, did not express the PHGDH mRNA unless exogenous rIL-2 was added. Blocking of IL-2R signaling by adding anti-IL-2 and anti-IL-2Rα resulted in no expression of the PHGDH mRNA during immobilized anti-CD3 activation of G₀ T cells. Deprivation of l-serine from culture medium or addition of antisense PHGDH oligonucleotide significantly reduced [³H]TdR incorporation of activated T cells. These results indicate that the PHGDH gene expression, dictated by IL-2R signaling, is a crucial event for DNA synthesis during S phase of activated T cells.     

Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.Numerous studies have illustrated the relevant role of dissolved organic matter (DOM) present in soil solution and aquatic reservoirs (lakes, rivers, etc.) in the biological and chemical evolution of both natural and anthropogenic ecosystems (Stevenson, 1994; Tipping, 2002; Chen et al., 2004; Trevisan et al., 2011; Berbara and García, 2014; Canellas and Olivares, 2014; Mora et al., 2014a, 2014b). In many studies, DOM fractionation is made by using the methodology proposed by the International Humic Substances Society. Fractions obtained are operationally named humic acid (HA), fulvic acid, humin, and nonhumic fraction, which includes more hydrophilic compounds (polycarboxylic acids, aminoacids, sugars, etc.; Swift, 1996). Many studies have reported that HAs obtained from either organic materials (soils, soil sediments, composted wastes, etc.) or water reservoirs (rivers, lakes, etc.), extracted with alkaline water solutions, or isolated by resin fixation, reverse osmosis, or ultrafiltration (Alberts and Takács, 2004) affected the development of diverse plant species (for instance, cucumber [Cucumis sativus], tomato [Solanum lycopersicum], maize [Zea mays], wheat [Triticum aestivum], Arabidopsis [Arabidopsis thaliana], and rapeseed [Brassica Napus]) through common signaling pathways, which involved key phytoregulators, such as indole acetic acid (IAA)-nitric oxide (NO; Zandonadi et al., 2010; Canellas et al., 2011; Trevisan et al., 2011; Mora et al., 2012, 2014a), ethylene, and abscisic acid (ABA) in roots (Mora et al., 2012, 2014a) as well as cytokinins in shoots (Mora et al., 2010, 2014b). Recently, Mora et al., 2014a showed that the HA ability to enhance both shoot growth and ABA root concentration in cucumber was regulated by IAA and NO root signaling pathways. However, despite all of this information, the nature of a possible primary, common action on plant roots of HAs with diverse origin and structure remains elusive.Recently, Asli and Neumann (2010) described a new mechanism by which high concentrations of HAs extracted from diverse organic sources decreased shoot plant growth. This mechanism involved the reduction of root hydraulic conductivity (Lp_r) resulting from the fouling of root cell wall pores because of the accumulation and aggregation of HA molecules at root surface. Although the concentration of HAs used by Asli and Neumann (2010) (1 g L⁻¹) is much higher than that related to HA plant growth promotion ability (50–250 mg L⁻¹; Rose et al., 2014), the results do raise the hypothesis that the primary, still unknown event emerging from the interaction of humic substances with root surface cells might involve an unspecific, physical action on root permeability and water uptake. This event might trigger a chain of secondary events in the root that, in turn, would affect specific hormone signaling pathways, which may regulate shoot and root growth. This HA action on plant development would be positive (increasing) or negative (decreasing) depending on HAs concentration in the rhizosphere.To explore the suitability of this hypothesis, we have tested the potential role of Lp_r in the main mechanism by which HAs promote shoot growth in cucumber. To this end, we used a well-characterized and modeled sedimentary humic acid (SHA) at a concentration (100 mg of SHA organic carbon [C] L⁻¹) that was associated with plant shoot growth promotion in previous studies (Mora, 2009; Mora et al., 2014a, 2014b). We also investigated the functional relationships between these effects of SHA on Lp_r and shoot growth as well as in some shoot water-related parameters (leaf stomatal conductance [G_s] and ABA) and those caused by SHA on IAA-NO and ABA root signaling pathways. Finally, taking into account that root plasma membrane aquaporins (plasma membrane intrinsic proteins [PIPs]) are involved in the ABA regulation of Lp_r in other plant systems, we also studied the role of PIPs in SHA effects on plant shoot growth.The results obtained here show that SHA enhances shoot growth in cucumber through ABA-dependent increases in both Lp_r and root PIPs (CsPIPs) gene up-regulation.