Analysis of the enzymes involved in PEN5 carbohydrate metabolism
Ontology highlight
ABSTRACT: Dr. Eric Vivier's lab would like to tackle the enzymes involved in PEN5 carbohydrate metabolism. We have generated these cell lines by repeated cell sorting and cell cloning of the parental HSB-2 T cell line and the parental JY B cell line. H+ are PEN5+ HSB-2 cells, H- are PEN5- HSB-2 cells; JY+ are PEN5+ HSB-2 cells, JY- are PEN5- HSB-2 cells. We have originally described a cell surface molecule called PEN5, as a sulfated lactosamine, that is selectively expressed on a mature subset of human NK cells (Vivier, J. Exp. Med. 1993). Later, we have shown that the PEN5 carbohydrate decorates PSGL-1, confering L-selectin binding properties (André et al. PNAS, 2000). We have recently performed a Glycan Array Screening via Core H (Glycomics 917). These studies indeed reveal a very nice binding of the anti-PEN5 mAb to a subset of sulfated lactosamines. We would like to tackle the enzymes involved in PEN5 carbohydrate metabolism. We thus requesting a Gene microarray analysis performed using RNA extracted from PEN5+ and PEN5- NK cells, as well from the H+, H-, JY+ and JY- stable cell lines. We have generated these cell lines by repeated cell sorting and cell cloning of the parental HSB-2 T cell line and the parental JY B cell line. H+ are PEN5+ HSB-2 cells, H- are PEN5- HSB-2 cells; JY+ are PEN5+ HSB-2 cells, JY- are PEN5- HSB-2 cells. RNA preparations from variants of the HSB-2 (human T leukemia) and JY (human B lymphoma) cell lines, which were express or not PEN5 were sent to Microarray Core (E). These stable variants are referred to as H+ (HSB-2 cells expressing PEN5), H- (HSB-2 cells NOT expressing PEN5), JY+ (JY cells expressing PEN5), and JY- (JY cells NOT expressing PEN5). Samples were prepared at 3 different time points. The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays. Data was sent to Dr. Eric Vivier for analysis.
Project description:Dr. Eric Vivier's lab would like to tackle the enzymes involved in PEN5 carbohydrate metabolism. We have generated these cell lines by repeated cell sorting and cell cloning of the parental HSB-2 T cell line and the parental JY B cell line. H+ are PEN5+ HSB-2 cells, H- are PEN5- HSB-2 cells; JY+ are PEN5+ HSB-2 cells, JY- are PEN5- HSB-2 cells.
Project description:Pluripotent cells can be derived from somatic cells by either overexpression of defined transcription factors (resulting in induced pluripotent stem cells (iPSCs)) or by nuclear transfer or cloning (resulting in NT-ESCs). To determine whether cloning further reprograms iPSCs, we used iPSCs as donor cells in nuclear transfer experiments. An iPSC clone derived from tail-tip fibroblasts using adenoviral vectors was used as donor cell in nuclear transfer experiments. RNA was isolated from both parental iPSC clone and derivative NT-ESCs lines and analyzed.
Project description:Numerous studies show dietary carbohydrates (C) affect the sensation of sweetness. However, protein (P) is one of the most critical macronutrients in the diet as well. It is still unclear how carbohydrates and proteins interact to influence sweet taste sensitivity. Here, we use the nutritional geometry framework (NGF) to tackle this problem in Drosophila melanogaster. Our results showed that the combination of high protein and low carbohydrates caused higher taste responses to sucrose stimuli in both sexes. Additionally, transcriptome analysis revealed that the gene expression of glycine, serine, and threonine pathway in the high-protein, low-carbohydrate diet was significantly upregulated, compared to a diet with low protein, and high carbohydrate. We confirmed that serine and threonine supplementation in the high-carbohydrate, low-protein diet enhanced the sucrose sensitivity of flies. Our results demonstrate that sucrose taste sensitivity is affected by the dietary balance of protein and carbohydrates possibly mediated by the change in serine, and threonine. The high protein, low carbohydrate diets enhanced sucrose taste sensitivity.
Project description:Phytosulfokine (PSK) is a plant peptide hormone that contributes to plant signaling and induced various of effects, including growth, senescence, stress tolerant and defense responses. PSK is a pentapeptide with two tyrosine sulfated by tyrosylprotein sulfotransferase (TPST) enzyme. The sulfated PSK can bind to its cell surface receptors, but the transcriptional readouts remain largely unknown. Here, we treated tpst mutant that are unable to produce native sulfated PSK with synthetic active PSK to capture concentration-sensitive readouts of PSK signaling.
Project description:Phytosulfokine (PSK) is a plant peptide hormone that contributes to plant signaling and induced various of effects, including growth, senescence, stress tolerant and defense responses. PSK is a pentapeptide with two tyrosine sulfated by tyrosylprotein sulfotransferase (TPST) enzyme. The sulfated PSK can bind to its cell surface receptors, but the transcriptional readouts remain largely unknown. Here, we treated tpst mutant that are unable to produce native sulfated PSK with synthetic active PSK to capture time-series and tissue-specific readouts of PSK signaling.
Project description:The Jeryak is the hybrid offspring of yaks and Jersey cattle and exhibit improved milk and meat yields. Biomolecules carried within milk exosomes are important for cell growth, development, immune regulation, and various pathophysiological processes. Previous studies showed that miRNAs regulate mammary gland development, lactation, and milk quality. This study explored the relationship between milk exosomes miRNAs and lactation performance. A comparison of the milk content showed that yak milk was of a better quality compared to Jeryak milk (casein, fat, TS, SNF, lactose). Milk collected in December was superior to that collected in June for both yak and Jeryak, except for lactose concentrations. Exosomes were extracted by density gradient centrifugation and miRNA expression profiles in milk exosomes from three yaks and three Jeryaks collected in June and December were detected by small RNA sequencing. In all, 22, 120, 78, and 62 differentially expressed miRNAs (DEMs) were identified in Jun_ JY vs. Jun_ Y (P1: Jeryak in June vs. Yak in June), Jun_ JY vs. Dec_ JY (P2: Jeryak in June vs. Jeryak in December), Dec_ JY vs. Dec_ Y (P3: Jeryak in December vs. Yak in December), and Jun_ Y vs. Dec_ Y (P4: Yak in June vs. Yak in December) groups. These DEMs were enriched in functions and signaling pathways related to lactation performance. In conclusion, these findings are a reference tool to study the molecular basis of lactation performance.
Project description:In this study we have investigated the gene expression profiles of three different types of subclone all generated by single cell cloning of the same parental EBV positive Burkitt lymphoma cell line Awia-BL. These included EBV negative clones which have lost the virus episome, EBV positive clones with a conventional Latency I form of infection and EBV positive clones with an atypical Wp-restricted form of infection.
Project description:Syngeneic grafts of the D4M.3A.3 (parental) mouse melanoma cell line (derived from a Tyr::CreER;BrafCA;Ptenlox/lox mouse) in C56BL/6 mice model poorly immunogenic, low neoantigen human melanomas. The D3UV2 (UV2) cell line was derived by serial UVB irradiation and single cell cloning. The addition of UVB-induced putative neoantigens sensitizes UV2 syngeneic melanoma grafts to immune checkpoint inhibitors and triggers epitope skewing to tumor-lineage self-antigens, a phenomenon that can be successfully mimicked in parental melanomas through treatment combinations such as anti-PD-1 with ablative fractional photothermolysis and imiquimod. Our mouse models were used to characterize gene expression changes between neoantigen rich and neoantigen poor melanomas, and with immunotherapy.
Project description:The human genome contains at least 35 genes that encode Golgi sulfotransferases functioning in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Our insight into the sulfotransferases that serve glycoproteins and in particular GalNAc-type O-glycoproteins is limited, yet a number of important interactions with sulfated glycans by e.g. Selectins, Galectins, and Siglecs are thought to mainly rely on sulfated O-glycans. Moreover, sulfated mucins appear to be accumulated in respiratory diseases, arthritis and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, we have started to expand a cell-based glycan array in the human HEK293 cell line with sulfation capacities. Here, we stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knock-in of sulfotransferase genes, in combination with knockout of endogenous core2 and/or sialylation capacities, to enable production of mucin reporters with sulfated O-glycans. Expression of GAL3ST2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of GAL3ST4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan