Preparation of O-Glycans from Mucins Using Hydrazine Treatment.

Mucin glycomic analysis is crucial owing to the participation of mucin O-glycans in several biological functions. Liquid chromatographic analysis of fluorescently labeled glycans is an effective tool for glycomic analysis. The first step of this analysis involves the release of O-glycans from mucins. As no enzyme is known to release all glycans, chemical methods are required for the process; therefore, hydrazine treatment is a commonly used chemical method. It enables the release of O-glycans from mucin while preserving the aldehyde group at the reducing end. This ensures that the reducing end can be modified using fluorescent reagents. However, it is also accompanied by the degradation of the glycans through a process called "peeling." Here, we describe a method for releasing glycans from mucins using hydrazine treatment with minimal "peeling."

MALDI-TOF MS/MS Analysis of Permethylated O-Glycan Alditols Released from Mucins.

Mucin glycans are associated with the function of mucin in maintaining mucosal homeostasis. Therefore, the glycomic analysis of mucins is crucial. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most suitable methods for the glycomic analysis of mucin O-glycans. In this chapter, we describe methods for analyzing permethylated O-glycan alditols released from mucins by MALDI-TOF MS and MALDI-TOF tandem mass spectrometry (MALDI-TOF MS/MS).

Improvement of spatial resolution in photoacoustic microscopy using transmissive adaptive optics with a low-frequency ultrasound transducer.

Maintaining a high spatial resolution in photoacoustic microscopy (PAM) of deep tissues is difficult due to large aberration in an objective lens with high numerical aperture and photoacoustic wave attenuation. To address the issue, we integrate transmission-type adaptive optics (AO) in high-resolution PAM with a low-frequency ultrasound transducer (UT), which increases the photoacoustic wave detection efficiency. AO improves lateral resolution and depth discrimination in PAM, even for low-frequency ultrasound waves by focusing a beam spot in deep tissues. Using the proposed PAM, we increased the lateral resolution and depth discrimination for blood vessels in mouse ears.

Comparison of Methods to Release Mucin-Type O-Glycans for Glycomic Analysis.

Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60 °C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, so-called "peeled" products. Ammonia treatment also yielded intact O-glycans but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yielded mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine and labeled with anthranilic acid had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans.

Transmissive liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy.

All aberrations produced inside a biospecimen can degrade the quality of a three-dimensional image in two-photon excitation laser scanning microscopy. Previously, we developed a transmissive liquid-crystal device to correct spherical aberrations that improved the image quality of a fixed-mouse-brain slice treated with an optical clearing reagent. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism. The motivation for this study is that asymmetric aberration can be induced by the shape of a biospecimen and/or by a complicated refractive-index distribution in a sample; this can considerably degrade optical performance even near the sample surface. The device's performance was evaluated by observing fluorescence beads. The device was inserted between the objective lens and microscope revolver and succeeded in improving the spatial resolution and fluorescence signal of a bead image that was originally degraded by asymmetric aberration. Finally, we implemented the device for observing a fixed whole mouse brain with a sloping surface shape and complicated internal refractive-index distribution. The correction with the device improved the spatial resolution and increased the fluorescence signal by ?2.4×. The device can provide a simple approach to acquiring higher-quality images of biospecimens.

Discrimination of rat Brunner's gland carbohydrate antigens by site-specific monoclonal antibodies.

Mucus produced and secreted by gastrointestinal mucosa contains various types of mucins equipped with unique sugar chains considered to play critical roles in protecting mucous membranes; therefore, the identification and verification of mucin sugar chains is important for understanding the underlying mechanisms. In our previous work, we generated three monoclonal antibodies (mAbs), RGM22, RGM26, and RGM42, which recognize sugar chains in rat gastric mucin. Here, we immunohistochemically analyzed the rat gastrointestinal mucosa and found that the antigens recognized by RGM22 and RGM42 were expressed in the rat antrum and Brunner's glands, whereas that recognized by RGM26 was detected in the antrum, but rarely in Brunner's glands. We found that these antibodies reacted with porcine gastric mucin-derived oligosaccharides bearing a common structure: GalNAcα1-3(Fucα1-2)Galß1-4GlcNAcß1-6GalNAc-ol. Moreover, epitope analysis revealed that RGM42 and RGM22 recognized α-linked GalNAc and GalNAcα1-3Gal, respectively, on the GalNAcα1-3(Fucα1-2)Gal structure, whereas RGM26 was specific for GalNAcα1-3(Fucα1-2)Gal. These results indicate that rat Brunner's glands express specific antigens bearing GalNAcα1-3Gal that are recognized by RGM22 and RGM42. Thus, RGM22, RGM26, and RGM42 with their unique antigen specificities could be useful tools for investigation of oligosaccharide diversity among mucins.

Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions.

Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.

Analysis of a segmented q-plate tunable retarder for the generation of first-order vector beams.

In this work we study a prototype q-plate segmented tunable liquid crystal retarder device. It shows a large modulation range (5π rad for a wavelength of 633 nm and near 2π for 1550 nm) and a large clear aperture of one inch diameter. We analyze the operation of the q-plate in terms of Jones matrices and provide different matrix decompositions useful for its analysis, including the polarization transformations, the effect of the tunable phase shift, and the effect of quantization levels (the device is segmented in 12 angular sectors). We also show a very simple and robust optical system capable of generating all polarization states on the first-order Poincaré sphere. An optical polarization rotator and a linear retarder are used in a geometry that allows the generation of all states in the zero-order Poincaré sphere simply by tuning two retardance parameters. We then use this system with the q-plate device to directly map an input arbitrary state of polarization to a corresponding first-order vectorial beam. This optical system would be more practical for high speed and programmable generation of vector beams than other systems reported so far. Experimental results are presented.

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy.

Two-photon excitation laser scanning microscopy has enabled the visualization of deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations that degrade spatial resolution and the fluorescence signal, especially during observation at deeper regions. Recently, we developed transmissive liquid-crystal devices for correcting spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. In this study, the device was inserted in front of the objective lens and supplied with the appropriate voltage according to the observation depth. First, we evaluated the device by observing fluorescent beads in single- and two-photon excitation laser scanning microscopes. Using a 25× water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, the device recovered the spatial resolution and the fluorescence signal degraded within a depth of 0.6 mm. Finally, we implemented the device for observation of a mouse brain slice in a two-photon excitation laser scanning microscope. An optical clearing reagent with a refractive index of 1.42 rendered the fixed mouse brain transparent. The device improved the spatial resolution and the yellow fluorescent protein signal within a depth of 0-0.54 mm.

Two-photon excitation STED microscopy by utilizing transmissive liquid crystal devices.

Transmissive liquid crystal devices (tLCDs) enable the modification of optical properties, such as phase, polarization, and laser light intensity, over a wide wavelength region at a high conversion efficiency. By utilizing tLCDs, we developed a new two-photon excitation stimulated emission depletion microscopy technique based on a conventional two-photon microscope. Spatial resolution was improved by compensating for phase shifts distributed in the optical path. Using this technique, we observed the fine structures of microtubule networks in fixed biological cells.

In-line FINCH super resolution digital holographic fluorescence microscopy using a high efficiency transmission liquid crystal GRIN lens.

We report a new optical arrangement that creates high-efficiency, high-quality Fresnel incoherent correlation holography (FINCH) holograms using polarization sensitive transmission liquid crystal gradient index (TLCGRIN) diffractive lenses. In contrast, current universal practice in the field employs a reflective spatial light modulator (SLM) to separate sample and reference beams. Polarization sensitive TLCGRIN lenses enable a straight optical path, have >90% transmission efficiency, are not pixilated, and are free of many limitations of reflective SLM devices. For each sample point, two spherical beams created by a glass lens in combination with a polarization sensitive TLCGRIN lens interfere and create a hologram and resultant super resolution image.

Flow injection determination of hydrogen peroxide using catalytic effect of cobalt(II) ion on a dye formation reaction.

A novel flow injection photometric method was developed for the determination of hydrogen peroxide in rainwater. This method is based on a cobalt(II)-catalyzed oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) as a modified Trinder's reagent to produce intensely colored dye (λ(max)=530nm) in the presence of hydrogen peroxide at pH 8.4. In this method, 1,2-dihydroxy-3,5-benzenedisulfonic acid (Tiron) acted as an activator for the cobalt(II)-catalyzed reaction and effectively increased the peak height for hydrogen peroxide. The linear calibration graphs were obtained in the hydrogen peroxide concentration range 5×10(-8) to 2.2×10(-6)mol dm(-3) at a sampling rate of 20h(-1). The relative standard deviations for ten determinations of 2.2×10(-6) and 2×10(-7)mol dm(-3) hydrogen peroxide were 1.1% and 3.7%, respectively. The proposed method was successfully applied to the determination of hydrogen peroxide in rainwater samples and the analytical results agreed fairly well with the results obtained by different two reference methods; peroxidase method and hydrogen peroxide electrode method.

Rapid and specific alterations of goblet cell mucin in rat airway and small intestine associated with resistance against Nippostrongylus brasiliensis reinfection.

The intestinal parasitic nematode Nippostrongylus brasiliensis is expelled rapidly from the rat in reinfection challenge compared with that of the primary infection owing to the host defense mechanisms raised against the pre-intestinal- and intestinal-stage larvae. We examined the relationship between the mucin alterations in airway and jejunal mucosae and the worm expulsion after third-stage larva reinfection. When rats had been inoculated with fourth-stage larvae and immunized with only the intestinal-stage worms for more than 8 days, the challenge larvae were expelled during the intestinal stage along with a rapid increase of the specific sialomucin in jejunal mucosa, without any effect on the bronchial mucus. When rats had been infected with third-stage larvae and immunized with only the pre-intestinal stage larvae by killing with antihelminthic, the challenge larvae were rejected during the pre-intestinal stage along with marked goblet cell hyperplasia and Muc5AC mucin hyperproduction on the bronchial mucosa, but not as a result of jejunal mucin alteration. Taking these finding together, immunization with pre-intestinal- and intestinal-stage worms independently increases the airway and intestinal goblet cell mucins, respectively, and in both cases, the mucin alterations may contribute to rapid worm expulsion upon reinfection.

The monoclonal antibody HCM31 specifically recognises the Sd(a) tetrasaccharide in goblet cell mucin.

Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis (N.b). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide-alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b-infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß- (Sd(a) blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sd(a) antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sd(a) tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sd(a) antigen.

Lateral resolution enhancement of laser scanning microscopy by a higher-order radially polarized mode beam.

We demonstrate that the lateral resolution of confocal laser scanning microscopy is dramatically improved by a higher-order radially polarized (HRP) beam with six concentric rings. This beam was generated simply by inserting liquid crystal devices in front of an objective lens. An HRP beam visualized aggregated 0.17 µm beads individually and is also applicable to biological imaging. This method can extend the capability of conventional laser scanning microscopes without modification of the system, with the exception of the addition of the liquid crystal devices in the optical path.

Protective effect of geranylgeranylacetone against loxoprofen sodium-induced small intestinal lesions in rats.

Nonsteroidal anti-inflammatory drugs induce small intestinal ulcers but the preventive measures against it remain unknown. So we evaluated the effect of geranylgeranylacetone (GGA), a mucosal protectant, on both the mucus content and loxoprofen sodium-induced lesions in the rat small intestine. Normal male Wistar rats were given GGA (200 or 400mg/kg p.o.) and euthanized 3h later for measurement of mucin content and immunoreactivity. Other Wistar rats were given loxoprofen sodium (30mg/kg s.c.) and euthanized 24h later. GGA (30-400mg/kg p.o.) was administered twice: 30min before and 6h after loxoprofen sodium. The total mucin content of the small intestinal mucosa increased, especially the ratio of sialomucin, which increased approximately 20% more than the control level after a single dose of GGA. Loxoprofen sodium provoked linear ulcers along the mesenteric margin of the distal jejunum, accompanied by an increase in enterobacterial translocation. Treatment of the animals with GGA dose-dependently prevented the development of intestinal lesions, and bacterial translocation following loxoprofen sodium was also significantly decreased. GGA protects the small intestine against loxoprofen sodium-induced lesions, probably by inhibiting enterobacterial invasion of the mucosa as a result of the increase in the mucosal barrier.

Nippostrongylus brasiliensis: increase of sialomucins reacting with anti-mucin monoclonal antibody HCM31 in rat small intestinal mucosa with primary infection and reinfection.

Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.

A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin.

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

Quantitative determination of gland mucous cells-type mucin using a monoclonal antibody, HIK1083: its pathophysiological changes in human gastric juice.

BACKGROUND: Pathological alteration in gastric mucosa is caused by Helicobacter pylori infection and is detectable by histological analysis. In particular, the alteration of gland mucous cells (GMCs)-type mucin, which plays a protective role against H. pylori infection, is critical in the pathogenesis of H. pylori-related gastritis. We established an assay for GMCs-type mucin and quantitatively assessed the pathophysiological changes in its content in human gastric juice samples. METHODS: The assay method for GMCs-type mucin was based on ELISA using a monoclonal antibody (HIK1083), and was used it to measure GMCs-type mucin in gastric juice obtained from patients with or without H. pylori infection. RESULTS: All the basic characteristics of the current method were satisfactory to quantify the GMCs-type mucin content in gastric juice. The GMCs-type mucin content, but not total mucin content, was significantly higher in patients with H. pylori infection (n=17; 437+/-476 U, mean+/-SD) than in those without H. pylori infection (n=55; 168+/-322 U, p<0.05). CONCLUSIONS: The current method is suitable for the quantitative analysis of GMCs-type mucin in gastric juice. The change in GMCs-type mucin content in gastric juice may be possibly implicated in the pathophysiology of the gastric mucosa and in the patient's gastric mucosal lesions.

Nocturnal enuresis and overactive bladder in children: an epidemiological study.

AIMS: To investigate the prevalence and characteristics of nocturnal enuresis (NE) and to examine the prevalence of overactive bladder (OAB) symptoms in primary schoolchildren. METHODS: After conducting an anonymous questionnaire survey about voiding habits and bowel habits in primary schoolchildren, a total of 6917 schoolchildren belonging to 11 primary schools were randomly enrolled in the survey. According to the International Continence Society, we defined NE as any involuntary loss of urine during sleep, occurring more frequently than once per month. Children with NE were subdivided into two groups, monosymptomatic NE (MNE) and enuretic syndrome (ES). To evaluate the characteristic differences of MNE and ES, we assessed the relationships between NE and voiding habits, and episodes of cystitis and constipation. Overactive bladder was defined as increased daytime frequency and/or urge urinary incontinence, and its prevalence was investigated. RESULTS: The response rate to the questionnaire was 76.4%. The prevalence of NE was 5.9% and was inversely related to increasing age. Monosymptomatic NE comprised 59.4% of NE cases. The annual spontaneous resolution rate of MNE was higher than that of ES. Increased daytime frequency, a history of cystitis and infrequent bowel habits were not related to MNE, but were significantly related to ES. The prevalence of OAB was 17.8%. Children with a history of cystitis had a significantly higher rate of OAB than children without it. CONCLUSIONS: Overall, NE and OAB were detected in 5.9% and 17.8% of primary schoolchildren, respectively. The link between NE and OAB symptoms, urinary tract infections and constipation deserves more attention.