Probes for INS

Reduced expression of GM1 and other major brain gangliosides GD1a, GD1b and GT1b have been reported in Parkinson's disease (PD) brain. Mechanisms underlying these changes are unclear but may be due to a deficit in the ganglioside biosynthetic process. The present study examined the extent to which deficits in gene expression of key biosynthetic enzymes involved in synthesis of GM1 and GD1b (B3galt4) and GD1a and GT1b (St3gal2) exist in neuromelanin-containing neurons in the PD substantia nigra (SN). In situ hybridization histochemistry was used to examine gene expression of B3GALT4 and ST3GAL2 in neuromelanin-containing neurons in the SN in 8 normal controls (61-92 yrs.) and 7 PD subjects (77-95 yrs). There was a significant decrease in both B3GALT4 and ST3GAL2 gene expression in residual neuromelanin-containing cells in the SN of PD patients compared to age-matched neurologically normal controls. These changes appeared to be cell-type specific as abundant B3GALT4 and ST3GAL2 gene expression was observed in non-neuromelanin containing neurons located outside of the SN in the PD brain. These data show that residual neuromelanin-containing neurons in the PD SN have decreased expression of the ganglioside biosynthetic genes B3GALT4 and ST3GAL2, consistent with previous reports of decreased levels of gangliosides GM1, GD1a, GD1b and GT1b in the PD SN. These changes may increase the vulnerability of these neurons to degeneration in response to a variety of potential stressors.

Abstract

Background and Aims

Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal sodium/hydrogen exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in ulcerative colitis (UC)-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors.

Methods

We assessed NHE8 expression in human CRCs by IHC and studied tumor burden in NHE8KO mice using an AOM/DSS colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NSG mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by PCR and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of β-catenin and c-Myc were also analyzed to evaluate Wnt/β-catenin activation.

Results

NHE8 was undetectable in human CRC tissues. Whereas only 9% of NHE8WT mice exhibited tumorigenesis in the AOM/DSS colon cancer model, almost ten times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared to HT29NHE8WT cells. Furthermore, NHE8 deficiency resulted in elevated Lgr5 expression in the colon, in HT29 derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/β-catenin activation.

Conclusions

NHE8 might be an intrinsic factor that regulates Wnt/β-catenin in the intestine.

The yolk sac and small intestine are 2 important organs responsible for the digestion and absorption of nutrients in chickens during the embryonic and posthatch periods, respectively. The peptide transporter PepT1 is expressed in both the yolk sac and small intestine and plays an important role in the transport of amino acids as short peptides. The objective of this study was to profile the spatial transcriptional patterns of PepT1 mRNA in the yolk sac and small intestine from embryonic and posthatch broilers. The distribution of PepT1 mRNA was investigated by in situ hybridization at embryonic (e) d 11, 13, 15, 17, 19 and day of hatch (doh) in the yolk sac and at e19, doh, and d 1, d 4, and d 7 posthatch in the small intestine. PepT1 mRNA was expressed in the endodermal cells of the yolk sac. PepT1 mRNA was barely detectable at e11, increased from e11 to e13, e15, and e17, and then gradually decreased from e19 to doh. In the small intestine, there was a rapid increase in expression of PepT1 mRNA in the enterocytes from e19 to doh, with expression relatively constant from d 1 to d 7. In addition, there was a differential increase in the heights of the villi in different parts of the small intestine from d 1 to 7, which may partially explain the temporal increase in PepT1 mRNA detected by qPCR. The villi in the duodenum showed the earliest increase in villus height and ultimately resulted in the highest villi at d 7. These results demonstrate that there are temporal changes in PepT1 mRNA expression in the yolk sac and the small intestine, which correspond with their expected role in nutrient uptake during the embryonic and posthatch periods.

Although cancer tissue generally shows limited immune responses, some cancers abound with lymphocytes, which generally show favorable prognosis. These cancers, despite their rarity, are important in analyzing immune responses in cancer tissue. Transforming growth factor β1 (TFGβ1) is a multifunctional cytokine, generally having an immunosuppressive function. The present study analyzes the in situ TGFβ1 expression in 23 cases of lymphocyte-rich gastric carcinomas (Ly-rich GCs) using immunohistochemistry and in situ hybridization. Immunohistochemistry revealed that latency-associated peptide (LAP) of TGFβ1 was localized in mainly immune cells in all cases, which was more abundant than in control GCs. Expression of LAP by cancer cells was only focal. In situ hybridization also confirmed abundant TGFβ1 mRNA expression in the lymphoid stroma. Double immunofluorescent microscopy identified LAP+ cells as macrophages, dendritic cells, and part of T cells. Close cell-to-cell contact was observed between LAP+ dendritic-shaped cells and FoxP3+ regulatory T cells (Treg cells). Mature dendritic cells in Ly-rich GCs expressed LAP more frequently than those in the secondary lymphoid organs. Our data revealed abundant expression of TGFβ1 in immune cells with contact to Treg cells in lymphoid stroma, which is consistent with the notion that TGFβ1 is one of the immunosuppressive factors in cancer stroma.

In the brain, AMPA receptors (AMPARs)-mediated excitatory synaptic transmission is critically regulated by the receptor auxiliary subunits. Recent proteomic studies have identified that Ferric Chelate Reductase 1 Like protein (FRRS1L), whose mutations in human lead to epilepsy, choreoathetosis, and cognitive deficits, is present in native AMPAR complexes in the brain. Here we have characterized FRRS1L in both heterologous cells and in mouse neurons. We found that FRRS1L interacts with both GluA1 and GluA2 subunits of AMPARs, but does not form dimers/oligomers, in HEK cells. In mouse hippocampal neurons, recombinant FRRS1L at the neuronal surface partially co-localizes with GluA1 and primarily localizes at non-synaptic membranes. In addition, native FRRS1L in hippocampus is localized at dynein, but not kinesin5B, vesicles. Functionally, over-expression of FRRS1L in hippocampal neurons does not change glutamatergic synaptic transmission. In contrast, single-cell knockout (KO) of FRRS1L strongly reduces the expression levels of the GluA1 subunit at the neuronal surface, and significantly decreases AMPAR-mediated synaptic transmission in mouse hippocampal pyramidal neurons. Taken together, these data characterize FRRS1L in heterologous cells and neurons, and reveal an important role of FRRS1L in the regulation of excitatory synaptic strength.

The hormone fibroblast growth factor 23 (FGF23) is secreted from bone and is involved in phosphorus (P) metabolism. FGF23 mainly binds the FGF receptor, which interacts with αKlotho in the kidney or parathyroid and regulates Na-dependent phosphate co-transporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) expression, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity, and parathyroid hormone (PTH) secretion. In this study, we utilized hemi-nephrectomized rats fed a high-P diet (HP Nx), rats subjected to a partial nephrectomy (PN) and rats with doxorubicin-induced renal failure (DXR) as chronic kidney disease (CKD) animal models and analyzed the P metabolism and FGF23 expression in the kidneys in each CKD model. We cultured HK2 cells with a high level of P, 1,25(OH)2D3 or transforming growth factor-β1 (TGFβ1) to investigate the FGF23 expression mechanism. In both the HP Nx and PN rats, the blood FGF23 and PTH levels were increased. However, the 1,25(OH)2D3 level was increased in the HP Nx rats and decreased in the PN rats. In all three animal models, the mRNA expression of αKlotho, NaPi-IIa and NaPi-IIc was decreased, and the mRNA expression of TGFβ1, collagen1a1, osteopontin and FGF23 was elevated in the kidney. FGF23 protein and mRNA were expressed at high levels in the extended tubule epithelium, which was an osteopontin-positive region in the HP and PN rats. FGF23 and osteopontin mRNAs were expressed in HK2 cells incubated with TGFβ1; however, these levels were not altered in HK2 cells incubated with 1,25(OH)2D3 and high P levels in vitro. Altogether, FGF23 is expressed in the kidneys in CKD model rats. Following stimulation with TGFβ1, the injured renal tubular epithelial cells are strongly suspected to express both FGF23 and osteopontin. FGF23 produced in the kidney might contribute to P metabolism in subjects with CKD.