In vitro plant growth promoter assay of actinobacteria potential for tidal land farming

The use of tidal land for agricultural still faces constraints, mainly due to high iron content. Actinobacteria produce bioactive compound with many functions. The aim of this work was to assess the growth of actinobacteria at various iron concentrations and its capability as plant growth promoter. Four actinobacteria isolates (Cal31t, Dbi28t, Crc32t and Cal24h) were grown at various iron concentrations. The isolates were examined for their capability to produce IAA and fix N2 under in vitro assay. The growth of actinobacteria under stress conditions was examined by cultivating them in ISP2 medium at pH 4, 3% NaCl, 750 mg.L−1 AlCl3 and 8 concentrations of FeCl3, i.e. 0, 500, 1000, 2000, 4000, 8000, 16000, 32000 mg.L−1. Actinobacteria isolates were able to grow under iron stress condition up to 32.000 mg.L−1. Both Cal3t and Dbi28t produced higher cell biomass compared with the other two tested isolates. All isolates produced IAA when grown under iron stress condition up to 4000 mg.L−1 of FeCl3, were able to grow under N-free medium and capable to produce ammonia at various concentrations. Crc32t produced the highest number of ammonia (0,354 mg.L−1). Cal31t and Crc32t isolates have the potency as plant growth promoter in tidal land farming.

Imaging Promoter Assay of Adenylyl Cyclase A Gene in Dictyostelium discoideum during Fruiting Body Formation by Dual-Color Bioluminescence Microscopy

Cyclic adenosine monophosphate (cAMP), which is derived from adenosine triphosphate through adenylyl cyclase A (acaA), acts as an intracellular secondary messenger and an extracellular chemotactic substance in important biological processes. In the social amoebae Dictyostelium discoideum, cAMP mediates cell aggregation, development, and differentiation to spore and stalk cells during fruiting body formation. The acaA gene is transcribed under the control of three different alternative promoters. This study aimed to develop a promoter assay for acaA in D. discoideum using bioluminescence microscopy. Here, we inserted green- and red-emitting luciferase genes into downstream of promoter regions 1 and 3, respectively. Promoter activities were visualized by bioluminescence microscopy. We confirmed the differential expression of acaA under the control of promoters 1 and 3 at the different stages of D. discoideum development. We also demonstrated the application of dual-color bioluminescence imaging in the development of an imaging promoter assay.

PI3K/AKT/mTOR Signaling Pathway Regulates MMP9 Gene Activation via Transcription Factor NF-κB in Mammary Epithelial Cells of Dairy Cows

Background Matrix metalloproteinase 9 (MMP9) plays a pivotal role in mammary ductal morphogenesis, angiogenesis, and glandular tissue architecture remodeling. However, the molecular mechanism of MMP9 expression in mammary epithelial cells of dairy cows remains unclear. The current study aimed to explore the underlying mechanism of MMP9 expression. Results In this study, to determine whether the PI3K/AKT/mTOR/NF-κB signaling pathway participates in the regulation of MMP9 expression, we treated mammary epithelial cells with specific pharmacological inhibitors of PI3K (LY294002), mTOR (Rapamycin), or NF-κB (Celastrol), respectively. Western blotting results indicated that LY294002, Rapamycin, and Celastrol markedly decreased MMP9 expression and P65 nuclear translocation. Furthermore, we found that NF-κB (P65) overexpression resulted in elevated expression of MMP9 protein and activation of MMP9 promoter. In addition, we observed that Celastrol markedly decreases P65-overexpression-induced MMP9 promoter activity. Moreover, the results of the promoter assay indicated that the core regulation sequence for MMP9 promoter activation may be located -80bp to -420bp downstream from the transcription start site. Conclusions These observations indicated that the PI3K/AKT/mTOR signaling pathway is involved in MMP9 expression by regulating MMP9 promoter activity via NF-κB in the mammary epithelial cells of dairy cows.

Ethylene Induced by Sound Stimulation Enhances Anthocyanin Accumulation in Grape Berry Skin through Direct Upregulation of UDP-Glucose: Flavonoid 3-O-Glucosyltransferase

Global warming has resulted in the loss of anthocyanin accumulation in berry skin. Sound stimulation can be used as a potential method for enhancing fruit color development since many plants recognize sound vibration as an external stimulus and alter their physiological status in response to it. Sound stimulation (sine wave sound at 1000 Hz) enhanced anthocyanin accumulation in grape cultured cells and berry skins in field-grown grapevines at the early stage of ripening. The transcription of UFGT and ACO2, which encode the key enzymes in anthocyanin and ethylene biosynthesis, respectively, was upregulated in grape cultured cells exposed to sound stimulation. In contrast, the transcription of MybA1 and NCED1, which encode a transcription factor for UFGT and a key enzyme in abscisic acid biosynthesis, respectively, was not affected by the sound stimulation. A treatment with an ethylene biosynthesis inhibitor, aminoethoxyvinyl glycine hydrochloride, revered the enhancement of anthocyanin accumulation by sound stimulation. As the promoter assay using a GUS reporter gene demonstrated that UFGT promoter was directly activated by the ethylene-releasing compound ethephon, which enhanced anthocyanin accumulation in grape cultured cells, we conclude that sound stimulation enhanced anthocyanin accumulation through the direct upregulation of UFGT by ethylene biosynthesis. Our findings suggest that sound stimulation contributes to alleviating poor coloration in berry skin as a novel and innovative practical technique in viticulture.

Signaling Pathway of Histamine H1 Receptor-Mediated Histamine H1 Receptor Gene Upregulation Induced by Histamine in U-373 MG Cells

Histamine H1 receptor (H1R) is one of the targets of histamine in the nervous system and the peripheral tissues. Protein kinase Cδ (PKCδ) signaling is involved in histamine-induced upregulation of H1R gene expression in HeLa cells. Histamine also upregulates H1R gene expression in U-373 MG cells. However, the molecular signaling of this upregulation is still unclear. Here, we investigated the molecular mechanism of histamine-induced H1R gene upregulation in U-373 MG cells. Histamine-induced H1R gene upregulation was inhibited by H1R antagonist d-chlorpheniramine, but not by ranitidine, ciproxifan, or JNJ77777120, and H2R, H3R, or H4R antagonists, respectively. Ro-31-8220 and Go6976 also suppressed this upregulation, however, the PKCδ selective inhibitor rottlerin and the PKCβ selective inhibitor Ly333531 did not. Time-course studies showed distinct kinetics of H1R gene upregulation in U-373 MG cells from that in HeLa cells. A promoter assay revealed that the promoter region responsible for H1R gene upregulation in U-373 MG cells was different from that of HeLa cells. These data suggest that the H1R-activated H1R gene expression signaling pathway in U-373 MG cells is different from that in HeLa cells, possibly by using different promoters. The involvement of PKCα also suggests that compounds that target PKCδ could work as peripheral type H1R-selective inhibitors without a sedative effect.

Snail Upregulates Transcription of FN, LEF, COX2, and COL1A1 in Hepatocellular Carcinoma: A General Model Established for Snail to Transactivate Mesenchymal Genes

SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

868 PRELP-facilitated enhancement of MHC class I surface expression in B16F10 melanoma cells

BackgroundPRELP (proline arginine-rich end leucine-rich repeat protein; also Prolargin), a small leucine-rich proteoglycan, functions as a molecule anchoring basement membranes to connective tissues via the interaction with collagens and heparin. PRELP facilitates the binding of cells to glycosaminoglycans as an important regulator of cell adhesion and thus displays pathophysiological features. Melanoma is an immunogenic tumor, whose relationship with immune cells resident in the microenvironment significantly influences cancer cell proliferation, progression and metastasis. Evasion from immune surveillance is a hallmark of melanoma progression. While our laboratory reported that the proteoglycan biglycan (BGN) was enhancing MHC class I in tumor cells,1 the role of PRELP in tumor immunology has not been studied.MethodsThe murine metastatic melanoma cell line B16F10, characterized by a reduced expression of MHC class I surface antigens was chosen for this study. B16F10 cells were transiently transfected with PRELP as well as co-transfected with BGN. Expression of antigen processing machinery (APM) components and PRELP was determined by qPCR and MHC class I surface expression by flow cytometry. Promoter activity of APM components was analysed by luciferase reporter assays. XTT assays were used to determine cell proliferation. The association of PRELP and MHC class I was studied by bioinformatics in a mixed melanoma dataset of 83 samples.2ResultsOver-expression of PRELP in B16F10 cells enhanced the expression of MHC class I surface antigens, which was due to a PRELP-mediated transcriptional upregulation of components of the MHC class I APM components TAP1, TAP2 and TAPBP as determined by qPCR and promoter assay in PRELP transfectants versus mock controls. Furthermore, MHC class I surface expression was even more pronounced upon BGN co-transfection with PRELP. PRELP overexpression was able to inhibit the proliferation of the B16F10 cells. Bioinformatics analyses demonstrated a positive correlation of PRELP with HLA-A , -B and -C alleles in human melanoma.ConclusionsOur findings demonstrated that overexpression of PRELP correlates with higher MHC class I expression and inhibits cell proliferation. For the first time, co-transfections of the two proteoglycans PRELP and BGN had a synergistic effect on upregulating MHC class I expression. Therefore, PRELP can serve as a novel therapeutic strategy that deserves further investigation.AcknowledgementsThe project is supported by Wilhelm-Sander-Stiftung (No: 2019.076.1) and by a Roux grant (FKZ: PK37) of the Medical Faculty of the Martin-Luther-University Halle-Wittenberg.ReferencesSubbarayan K, Leisz S, Wickenhauser C, Bethmann D, Massa C, Steven A, Seliger B. Biglycan-mediated upregulation of MHC class I expression in HER-2/neu-transformed cells. Oncoimmunology 2018;7:e1373233.Xu L, Shen SS, Hoshida Y, Subramanian A, Ross K, Brunet JP, Wagner SN, Ramaswamy S, Mesirov JP, Hynes RO. Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases. Molecular Cancer Research 2008;6:760-769.