Novel role of homogalacturonan region of pectin in disrupting the interaction between fibronectin and integrin ß1.

Pectin interacts with fibronectin (FN), a modular protein in the extracellular matrix. This interaction is significant as FN plays a pivotal role by binding to the receptor integrin α5ß1. However, the molecular mechanism underlying the pectin-FN interaction and its impact on integrin binding remains unknown. In this study, water-soluble pectins (WSPs) were extracted from three different pectin sources and subsequently characterized. These included Citrus WSP, which primarily comprises the homogalacturonan region, and Kaki and Yuzu WSPs, both of which are rich in rhamnogalacturonan regions. We investigated the molecular interactions between these WSPs and two FN fragments, Anastellin and RetroNectin, using surface plasmon resonance analysis. Citrus WSP exhibited a notable binding affinity to FN, with a dissociation constant (KD) of approximately 10-7 M. In contrast, Kaki and Yuzu WSPs displayed comparatively weaker or negligible binding affinities. The binding reactivity of Citrus WSP with FN was notably diminished following the enzymatic removal of its methyl-ester groups. Additionally, Citrus WSP disrupted the binding of integrin ß1 to RetroNectin without altering the affinity, despite its minimal direct binding to integrin itself. This study furthers our understanding of the intricate pectin-FN interaction and sheds light on their potential physiological relevance and impact on cellular responses.

Pectin Modulates Calcium Absorption in Polarized Caco-2 Cells via a Pathway Distinct from Vitamin D Stimulation.

Pectin, a type of soluble fiber, promotes morphological changes in the small intestinal villi. Although its physiological significance is unknown, we hypothesized that changes in villus morphology enhance the efficiency of nutrient absorption in the small intestine and investigated the effect of pectin derived from persimmon on calcium absorption using polarized Caco-2 cells. In polarized Caco-2 cells, pectin altered the mRNA expression levels of substances involved in calcium absorption and the regulation of intracellular calcium concentration and significantly reduced calcium absorption. Although this was comparable to the results of absorption and permeability associated with the addition of active vitamin D, the simultaneous action of pectin and active vitamin D did not show any additive effects. Furthermore, as active vitamin D significantly increases the activity of intestinal alkaline phosphatase (ALP), which is known to be involved in the regulation of intestinal absorption of calcium and lipids, we also investigated the effect of pectin on intestinal ALP activity. As a result, it was found that, unlike the effect of active vitamin D, pectin significantly reduced intestinal ALP activity. These results suggest that pectin stimulates polarized Caco-2 cells through a mechanism distinct from the regulation of calcium absorption by vitamin D, modulating total calcium absorption from the elongated villi through morphological changes in the small intestine by suppressing it at the cellular level.

The effect of edible bird's nests on the expression of MHC-II and costimulatory molecules of C57BL/6 mouse splenocytes.

The glutinous nest that builds by the saliva secretion of swiftlet is recognizable as an edible bird's nest (EBN). It enriched a medicinal value and was regarded as supplementary food that exerts various beneficial health effects, especially immune boosters. This study's objective was to determine the impact of EBN on the expression of MHC-II and costimulatory molecules (CD86 and CD80) related to the initiation of T-cell activation. Both rEBN and pEBN samples were prepared with simulated gastrointestinal digestion for enhancing the bioaccessibility of bioactive compounds. Our result showed that digested EBN samples slightly influence the upregulation of MHC-II, CD86, and CD80 in gene expression of LPS-stimulated Raw 264.7 cells. The concern of endotoxin contamination in EBN samples, which may cause a false-positive result, was measured by quantitative PCR. We found that the inflammatory genes (IL-1ß and TNF-α) were not induced by EBN treatments. Moreover, cell surface protein expression in splenocytes treated with EBN was assessed using flow cytometric analysis. Digested EBN samples demonstrated their capacity to promote the elevation of MHC-II, CD86, and CD80 cell surface protein expression. Finally, the digested-EBN-treated splenocytes only exhibited a specific response in the T-cells population. Thus, EBN is a source of the bioactive compound that has been proposed to exert a role in the stimulation of both MHC-II and costimulatory molecules for TCR/pMHC-II interaction leading to T-cell activation.

Histochemical Analysis of Heparan Sulfate 3-O-sulfotransferase Expression in Mouse Brain.

In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify the cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with non-radioactive nucleotides.

The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and ß-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and ß-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

Arabinogalactan in the side chain of pectin from persimmon is involved in the interaction with small intestinal epithelial cells.

Pectin in Diospyros kaki (persimmon) is a complex polysaccharide and is classified as a dietary fiber. Pectin is characterized by the presence of side chains of neutral sugars, such as galactose residues; however, the structure and properties of these sugars vary greatly depending on the plant species from which it is derived. Here, we report the structural features of pectin extracted from persimmon. The polysaccharide was low-methoxy pectin with a degree of methyl esterification <50% and ratio of side chain galactan to arabinan in the rhamnogalacturonan-I region of pectin of 3-20. To investigate the physiological function of pectin from persimmon, we performed a coculture assay using Caco-2 cells. As a result, it was shown that the proliferation of undifferentiated Caco-2 cells was promoted, and further, the importance of arabinogalactan among the pectin structures was shown.

Diospyros kaki extract protects myoblasts from oxidative stress-induced cytotoxicity via secretions derived from intestinal epithelium.

Under oxidative stress, reactive oxygen species (ROS) alter signal transduction and induce macromolecular damage in cells. Such oxidative damage can lead to sarcopenia, an age-related syndrome characterized by a progressive loss of mass and strength of skeletal muscles. Because food components do not directly come in contact with muscle cells, we focused on the effects of secretions produced by stimulated intestinal epithelial cells on oxidative stress in myoblast cells. An extract of Diospyros kaki was fractionated using different concentrations of ethanol. Each fraction showed different levels of antioxidant and phenolic compounds. The biological activity was evaluated using a Caco-2 cell coculture system. Secretions from Caco-2 cells exposed to 0.5 mg/mL D. kaki extract attenuated the oxidative stress-induced reduction of C2C12 cell viability, suggesting that the D. kaki extract could stimulate intestinal epithelial cells to produce secretions that reduce oxidative stress in myoblasts in vitro.

Senescence-accelerated mouse prone 8 mice exhibit specific morphological changes in the small intestine during senescence and after pectin supplemented diet.

Impairment of gastrointestinal function and reduction of nutrient absorption associated with aging contribute to increased risk of malnutrition in the elderly population, resulting in physical weakness and vulnerability to disease. The present study was performed to examine the relationships between aging-associated morphological changes of the small intestine and nutrient malabsorption using senescence-accelerated mouse prone 8 (SAMP8) mice. Comparison of the morphology of the small intestine of young (22-week-old) and senescent (43-week-old) SAMP8 mice showed no significant changes in villus length, while the mRNA expression levels of secretory cell marker genes were significantly reduced in senescent mice. In addition, crypts recovered from the small intestine of senescent mice showed a good capacity to form intestinal organoids ex vivo, suggesting that the regenerative capacity of intestinal stem cells (ISCs) was unaffected by accelerated senescence. These results indicated that changes induced by accelerated senescence in the small intestine of SAMP8 mice are different from changes reported previously in normal aging mouse models. Biochemical analyses of serum before and during senescence also indicated that senescent SAMP8 mice are not in a malabsorption state. Furthermore, a diet supplemented with persimmon pectin had a mild effect on the small intestine of senescent SAMP8 mice. Intestinal villus length was slightly increased in the medial part of the small intestine of pectin-fed mice. In contrast, intestinal crypt formation capacity was enhanced by the pectin diet. Organoid culture derived from the small intestine of mice fed pectin exhibited a greater number of lobes per organoid compared with those from mice fed a control diet, and Lyz1 and Olfm4 mRNA levels were significantly increased. In conclusion, accelerated senescence induced exclusive changes in the small intestine, which were not related to nutrient malabsorption. Therefore, the SAMP8 strain may not be a suitable model to evaluate the effects of aging on intestinal homeostasis and nutrient absorption impairment.

Dietary Fiber Pectin Ameliorates Experimental Colitis in a Neutral Sugar Side Chain-Dependent Manner.

Dietary fiber, with intake of soluble fibers in particular, has been reported to lower the risk for developing inflammatory bowel diseases (IBD). This is at least partly attributable to the fermentation of dietary fiber by the colonic microbiota to produce short chain fatty acids. Pectin, a widely consumed soluble fiber, is known to exert a protective effect in murine models of IBD, but the underlying mechanism remains elusive. Apart from having a prebiotic effect, it has been suggested that pectin direct influences host cells by modulating the inflammatory response in a manner dependent on its neutral sugar side chains. Here we examined the effect of the side chain content of pectin on the pathogenesis of experimental colitis in mice. Male C57BL/6 mice were fed a pectin-free diet, or a diet supplemented with characteristically high (5% orange pectin) or low (5% citrus pectin) side chain content for 10-14 days, and then administered 2,4,6-trinitrobenzene sulfonic acid or dextran sulfate sodium to induce colitis. We found that the clinical symptoms and tissue damage in the colon were ameliorated in mice that were pre-fed with orange pectin, but not in those pre-fed with citrus pectin. Although the population of CD4+Foxp+ regulatory T cells and CD4+RORγt+ inflammatory T cells in the colon were comparable between citrus and orange pectin-fed mice, colonic interleukin (IL)-1ß and IL-6 levels in orange pectin-fed mice were significantly decreased. The fecal concentration of propionic acid in orange pectin-fed mice was slightly but significantly higher than that in control and citrus pectin-fed mice but the cecal concentration of propionic acid after the induction of TNBS colitis was comparable between orange and citrus pectin-fed mice. Furthermore, the protective effect of orange pectin against colitis was observed even in mice treated with antibiotics. IL-6 production from RAW264.7 cells stimulated with the toll-like receptor agonist Pam3CSK4 or lipopolysaccharide was suppressed by pre-treatment with orange pectin in vitro. Taken together, these results suggest that the side chains of pectin not only augment prebiotic effects but also directly regulate IL-6 production from intestinal host cells in a microbiota-independent fashion to attenuate colitis.

Citrus pectin attenuates endotoxin shock via suppression of Toll-like receptor signaling in Peyer's patch myeloid cells.

Pectin, a water-soluble dietary fiber, has been found to improve survival in endotoxin shock. However, the underlying mechanism by which pectin exerts its protective effect against endotoxin shock remains unknown. Apart from its prebiotic effects, it has been suggested that pectin directly affects immune cells to regulate inflammatory responses. In this study, we investigated the direct effect of pectin in murine model of endotoxin shock. Citrus pectin solution was administered to male C57BL/6 mice for 10 days. Thereafter, hypothermia was induced in the mice with intraperitoneal injection of lipopolysaccharide (LPS). The pectin-treated mice showed attenuation of both the decrease in rectal temperature and increase in serum IL-6 level as compared to vehicle control mice. Simultaneously, the pectin-treated mice showed reduced levels of inflammatory cytokine mRNA in Peyer's patches and mesenteric lymph nodes, but not in the spleen. Peyer's patch cells from the pectin-treated mice were sorted and their levels of IL-6 production on LPS stimulation were measured. The results of ex vivo analysis indicated that IL-6 secretion from CD11c+ cells was suppressed by oral administration of pectin. Furthermore, IL-6 secretion from Toll-like receptor (TLR)-activated RAW264.7 cells was suppressed by pretreatment with pectin in vitro. This suppression was observed even with degraded pectin pretreatment but not with polygalacturonic acid, as the principal constituent of the pectin backbone. Taken together, these results suggest that pectin intake suppresses TLR-induced inflammatory cytokine expression in Peyer's patch myeloid cells, presumably through inhibition of TLR signaling by the pectin side chains.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages.

BACKGROUND: Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial-host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant. RESULTS: A putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages. CONCLUSIONS: Our results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

Characterization and comparison of recombinant honeybee chymotrypsin-like protease (HCLPase) expressed in Escherichia coli and insect cells.

We previously found a novel chymotrypsin-like protease in honeybee, designated as HCLPase. The recombinant enzyme expressed in insect cells was produced and compared to that in Escherichia coli. Both enzymes showed equivalent molecular size and specificity. However, HCLPase produced in insect cells showed higher specific activity. The C-terminal cleavage sites of HCLPase were phenylalanine, leucine, and tyrosine residues.

Effects of heat treatment on conformation and cell growth activity of alpha- lactalbumin and beta-lactoglobulin from market milk.

Heat processes, low temperature for long time (LTLT) pasteurization and ultra-heat treatment (UHT) sterilization, are essential for commercial market milk to improve the shelf life of raw milk and ensure microbial safety. We evaluated the effects of heat experience on the molecular properties of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) isolated from four types of market milk such as LTLT-A (66°C for 30 min), LTLT-B (65°C for 30 min), UHT-I (130°C for 2 s, indirect heating) and UHT-D (135°C for 2 s, direct heating) samples. We examined molecular conformations using circular dichroism spectrum measurement and cell growth activity using the WST-1 method for the proteins. α-LA isolated from each of these four types of market milk displayed no significant structural difference as compared to raw milk α-LA, while α-LA of UHT-I only inhibited cell growth of an intestinal epithelial cell line more potently than raw milk α-LA. In the case of ß-LG, only the UHT-I sample demonstrated a drastic change in structure, while it did not exhibit any cytotoxicity. We found that cell viability effects of α-LA and ß-LG are attributable to the type of UHT; indirect and direct. These findings indicate that the effect of heat treatment on whey proteins should carefully be investigated further.

White sesame seed water-soluble fraction enhances human neuroblast cell viability via an anti-apoptotic mechanism.

Defatted sesame seed flour is recovered as a byproduct after oil extraction and is usually considered a waste product. Previously, we showed that water-soluble fractions purified from defatted white and gold sesame seed flour exhibited good antioxidant activity in vitro. We also identified ferulic acid and vanillic acid as the bioactive antioxidants in both white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively). In this study, we hypothesized that WS-wsf and GS-wsf may have neuroprotective effects due to their antioxidant potential. Treatment with WS-wsf for 24 hours enhanced human neuroblastoma SH-SY5Y cell viability and proliferation, while GS-wsf, ferulic acid, and vanillic acid did not show similar effects. In addition, WS-wsf (1-3 mg/mL) significantly and dose-dependently protected SH-SY5Y cells against camptothecin-induced apoptosis, suggesting the involvement of an anti-apoptosis mechanism in the neuroprotective effects of WS-wsf. In fact, treatment with WS-wsf significantly decreased the mRNA expression levels of pro-apoptotic Bax and p53 genes. WS-wsf also enhanced Bcl-2 protein level and Akt phosphorylation. Taken together, this study showed that WS-wsf has interesting neuroprotective potential via an anti-apoptotic mechanism, which is independent from its antioxidant capacity.

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress.

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H2O2) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H2O2, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3 (‡).

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.

Pectin from Prunus domestica L. induces proliferation of IEC-6 cells through the alteration of cell-surface heparan sulfate on differentiated Caco-2 cells in co-culture.

Dietary fiber intake provides various physiological and metabolic effects for human health. Pectin, a water-soluble dietary fiber, induces morphological changes of the small intestine in vivo. However, the molecular mechanisms underlying pectin-derived morphological alterations have not been elucidated. Previously, we found that pectin purified from Prunus domestica L. altered the sulfated structure of cell-surface heparan sulfate (HS) on differentiated Caco-2 cells via fibronectin and α5ß1 integrin. In this study, we investigated the biological significance of the effect of pectin on HS in differentiated Caco-2 cells. An in vitro intestinal epithelium model was constructed by co-culture of differentiated Caco-2 cells and rat IEC-6 cells, which were used as models of intestinal epithelium and intestinal crypt cells, respectively. We found that pectin-treated differentiated Caco-2 cells promoted growth of IEC-6 cells. Real-time RT-PCR analysis and western blotting showed that relative mRNA and protein expression levels of Wnt3a were upregulated by pectin treatment in differentiated Caco-2 cells. Analysis by surface plasmon resonance spectroscopy demonstrated that pectin-induced structural alteration of HS markedly decreased the interaction with Wnt3a. However, depression in the secretion of Wnt3a from Caco-2 cells by anti-Wnt3a antibody did not affect the proliferation of IEC-6 cells in co-culture system. These observations indicated that pectin altered the sulfated structure of cell-surface HS to promote secretion of Wnt3a from differentiated Caco-2 cells and Wnt3a indirectly stimulated the proliferation of IEC-6 cells.

Water-soluble extracts from defatted sesame seed flour show antioxidant activity in vitro.

Defatted white and gold sesame seed flour, recovered as a byproduct after sesame oil extraction, was extracted with 70% ethanol to obtain polar-soluble crude extracts. The in vitro antioxidant activity of the extract was evaluated by DPPH free radical scavenging activity and oxygen radical absorbing capacity (ORAC). The polar-soluble crude extracts of both sesame seed types exhibited good antioxidant capacity, especially by the ORAC method with 34,720 and 21,700 µmol Trolox equivalent/100g of white and gold sesame seed extract, respectively. HPLC, butanol extraction, and UPLC-MS analyses showed that different compounds contributed to the antioxidant activity of the polar-soluble crude extracts. Sesaminol glycosides were identified in the butanol-soluble fractions; whereas, purified water-soluble fraction contained ferulic and vanillic acids. This study shows that hydrophilic antioxidants in the purified water-soluble fraction contributed to the antioxidant activity of white and gold sesame seed polar-soluble crude extracts.

Histochemical analysis of heparan sulfate 3-O-sulfotransferase expression in mouse brain.

In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with nonradioactive nucleotides.

Development of a photoreactive probe-based system for detecting heparin.

We previously identified a peptide heparin-associated peptide Y (HappY) that binds specifically to heparin. In this article, we report a novel heparin detection system using chemically modified HappY as a probe. The photoreactive HappY probe was serially diluted and dispensed into a 96-well plate coated with biotinylated heparin. After ultraviolet irradiation, the HappY probe crosslinked to the heparin on the plate was detected with fluorescein isothiocyanate-conjugated streptavidin. Furthermore, the photoreactive HappY probe was used to stain cutaneous tissue sections obtained from dermatitis-affected or mastocytoma-affected cats and dogs. The photoreactive HappY probe stained limited resident mast cells in the connective tissue of skin compared with the anti-heparan sulfate monoclonal antibody 10E4, suggesting that the probe can be used to distinguish the structure of heparin in tissues. The interactions between glycosaminoglycans and proteins in vivo tend to be weak. Therefore, our method for enhancing such weak interactions may be a promising tool for intermolecular interaction studies in glycobiology research.