Analysis of Protein Digestion and Absorption Using a Ussing Chamber to Simulate the Environment in the Digestive Tract.

The Ussing chamber is a tool for analyzing drug absorption. We investigated whether the Ussing chamber can be used to analyze the process from digestion to absorption of protein in the gastrointestinal tract. Mixtures containing infant formula, whole cow's milk, processed soy milk, enteral nutrition, or human breast milk, were placed in the apical membrane side equipped with Caco-2 cells. After the addition of first pepsin then pancreatin, samples from the apical and basal membranes were collected. Infant formula showed the highest digestibility and absorption rate. This may be attributed to the presence of whey protein, which is rapidly digested and absorbed. The digestion and absorption of human breast milk showed different results in each donor, suggesting that digestion and absorption may vary among individuals. We concluded that the Ussing chamber can continuously analyze the process from digestion to absorption of proteins in the gastrointestinal tract.

Pyrylium based derivatization imaging mass spectrometer revealed the localization of L-DOPA.

Simultaneous imaging of l-dihydroxyphenylalanine (l-DOPA), dopamine (DA) and norepinephrine (NE) in the catecholamine metabolic pathway is particularly useful because l-DOPA is a neurophysiologically important metabolic intermediate. In this study, we found that 2,4,6-trimethylpyrillium tetrafluoroborate (TMPy) can selectively and efficiently react with target catecholamine molecules. Specifically, simultaneous visualization of DA and NE as metabolites of l-DOPA with high steric hinderance was achieved by derivatized-imaging mass spectrometry (IMS). Interestingly, l-DOPA showed strong localization in the brainstem, in contrast to the pattern of DA and NE, which co-localized with tyrosine hydroxylase (TH). In addition, to identify whether the detected molecules were endogenous or exogenous l-DOPA, mice were injected with l-DOPA deuterated in three positions (D3-l-DOPA), which was identifiable by a mass shift of 3Da. TMPy-labeled l-DOPA, DA and NE were detected at m/z 302.1, 258.1 and 274.1, while their D3 versions were detected at 305.0, 261.1 and 277.1 in mouse brain, respectively. l-DOPA and D3-l-DOPA were localized in the BS. DA and NE, and D3-DA and D3-NE, all of which are metabolites of L-DOPA and D3-l-DOPA, were localized in the striatum (STR) and locus coeruleus (LC). These findings suggest a mechanism in the brainstem that allows l-DOPA to accumulate without being metabolized to monoamines downstream of the metabolic pathway.

Optimization of the use of Py-Tag for next generation derivatization reagents in imaging mass spectrometry.

To demonstrate the accurate analysis of catecholamines and amino acid using derivatization reagents, we investigated the reaction conditions for 2,4,6-triethyl-3,5-dimethyl pyrylium trifluoromethanesulfonate (Py-Tag), derivatization of the targets dopamine (DA) and γ-aminobutyric acid (GABA) on tissue sections, and constructed an optimized reaction compartment. Ten different Py-Tag reaction conditions with the targets were considered. The optimal condition for the Py-Tag reaction with the targets was identified as a 70% methanol with 5% trimethylamine (v/v) solution at 60 °C under homogenous conditions. To reproduce this reaction on tissue sections, we constructed a reaction compartment to maintain humidity levels and facilitate the derivatization reaction. Moreover, visualization of DA and GABA was archived by derivatized-imaging mass spectrometry. Brain sections of unilateral 6-OHDA lesioned Parkinson's disease model rats showed Py-Tag DA (m/z 328.3) in the unilateral striatum and Py-Tag GABA (m/z 278.3) in the cerebral cortex, striatum, hippocampus and hypothalamus. Using the Parkinson's disease model rat brain, images with left-right differences were obtained for the localization of DA and GABA. These findings indicate that it is important to consider the reaction conditions that allow high reaction efficiency between DA or GABA and Py-Tag as well as high quality imaging of sections.

Super-Moisturizing Materials from Morphological Deformation of Suprapolysaccharides.

The evaporative interface on polysaccharides has evolved to form hierarchical structures with moisture sensitivity to enable organisms to live in drying environments. Here, the discovery of the morphological instability of polysaccharides, especially the reversible self-assembly/disassembly between micron-fibers and microparticles in response to changes in aquatic environments, is reported. This is similar but different to the dynamic instability observed in cytoskeletal proteins, in terms of an accompanying the polymeric deformation. The formation of the polymeric fibers containing crystalline structures can be flexibly controlled by controlling the polymer concentration and salt concentration in aqueous mixtures. Moreover, the microparticles having crosslinking points in the interior acquire the ability to retain a larger number of water molecules in drying environments and behave as super-moisturizing materials.

Administration of mucuna beans (Mucuna pruriences (L.) DC. var. utilis) improves cognition and neuropathology of 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of extracellular amyloid-beta peptides (Aß) resulting in senile plaques and intracellular hyperphosphorylated tau protein resulting in neurofibrillary tangles (NFTs). Mucuna beans (Mucuna pruriences (L.) DC. var. utilis) are unique plants containing 3-9% L-3,4-dihydroxyphenylalanine (L-DOPA). Here we investigated the effect of the administration of Mucuna beans on AD prevention by feeding triple-transgenic mice (3 × Tg-AD mice) with a diet containing Mucuna beans for 13 months. The levels of Aß oligomers and detergent-insoluble phosphorylated tau decreased in the brain of mice fed with Mucuna beans (Mucuna group) compared to those of the Control group. Aß accumulation and phosphorylated tau accumulation in the brain in the Mucuna group were also reduced. In addition, administration of Mucuna beans improved cognitive function. These results suggest that administration of Mucuna beans may have a preventive effect on AD development in 3 × Tg-AD mice.

Modulatory Effect of Theaflavins on Apical Sodium-Dependent Bile Acid Transporter (ASBT) Activity.

Inhibiting apical sodium-dependent bile acid transporter (ASBT) has been identified as a potential strategy to reduce plasma cholesterol levels. Thus, in this study, we aimed to identify polyphenols that inhibited ASBT activity and to elucidate their mechanism. ASBT is responsible for most of the taurocholic acid (TC) uptake in Caco-2 cells. Of the 39 polyphenols examined, theaflavin (TF)-3-gallate (TF2A) and theaflavin-3'-gallate (TF2B) have been found to significantly reduce TC uptake in Caco-2 cells to 37.4 ± 2.8 and 33.8 ± 4.0%, respectively, of that in the untreated cells. The results from the TC uptake assay using N-acetylcysteine suggested that the inhibitory effect of TF2A and TF2B was attributed to the oxidization of their benzotropolone rings and their covalent bonding with ASBT's cysteine. TC uptake was reduced in the COS-7 cells expressing recombinant ASBT whose cysteine residues were mutated to alanine. Finally, the substrate concentration-dependent TC uptake assay showed that TFs competitively inhibited TC uptake.

Localization analysis of essential oils in perilla herb (Perilla frutescens var. crispa) using derivatized mass spectrometry imaging.

The localization of essential oils, including flavor components, in perilla herb (Perilla frutescens var. crispa) were visually determined using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging. The surface of a perilla leaf was peeled using a cyanoacrylate adhesion compound and contained oil glands that retained their morphology and chemical properties. We imaged the three essential oils perillaldehyde, ß-caryophyllene, and rosmarinic acid (RA). Perillaldehyde was derivatized using glycine to prevent evaporation and allow its detection and imaging while localized in oil glands. ß-caryophyllene also localized in the oil glands and not in the epidermis region. RA was detected throughout the leaf, including the oil glands. Quantitative data for the three essential oils were obtained by gas chromatography- or liquid chromatography-MS. The concentrations of perillaldehyde, ß-caryophyllene, and RA were 12.6 ± 0.62, 0.27 ± 0.02, and 0.16 ± 0.02 [mg/g] in the paste sample of perilla herb. Peeling using a cyanoacrylate adhesion compound, and derivatization of a target such as an aroma component have great potential for mass spectrometry imaging for multiple essential oils.

Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

Rosmarinic acid suppresses tau phosphorylation and cognitive decline by downregulating the JNK signaling pathway.

Rosmarinic acid (RA), a polyphenol found in Lamiaceae herbs, is a candidate of preventive ingredients against Alzheimer's disease (AD) as it potently suppresses the aggregation of amyloid ß (Aß); however, the effect of RA on tau phosphorylation and cognitive dysfunction remains unclear. The present study revealed that RA intake inhibited the pathological hallmarks of AD, including Aß and phosphorylated tau accumulation, and improved cognitive function in the 3 × Tg-AD mouse model. Additionally, RA intake suppressed hippocampal inflammation and led to the downregulation of the JNK signaling pathway that induces tau phosphorylation. Feeding with RA exerted an anti-inflammatory effect not only in the central nervous system but also in the periphery. Downregulation of the JNK signaling pathway in hippocampus may be a potential mechanism underlying the inhibition of progression of pathology and cognitive deficit by RA feeding.

Safety and efficacy of Melissa officinalis extract containing rosmarinic acid in the prevention of Alzheimer's disease progression.

We conducted a randomized placebo-controlled double-blind 24-week trial using Melissa officinalis (M. officinalis) extract richly containing rosmarinic acid (RA) on patients with mild dementia due to Alzheimer's disease (AD) with the aim to examine the safety and tolerability (primary endpoint) of RA (500 mg daily) and its clinical effects and disease-related biomarker changes (secondary endpoints). Patients (n = 23) diagnosed with mild dementia due to probable AD were randomized to either the placebo or M. officinalis extract group. No differences in vital signs or physical and neurologic examination results were detected between the M. officinalis and placebo groups. No serious adverse events occurred. There were no significant differences in cognitive measures; however, the mean Neuropsychiatric Inventory Questionnaire (NPI-Q) score improved by 0.5 points in the M. officinalis group and worsened by 0.7 points in the placebo group between the baseline and 24-week visit, indicating a significant difference (P = 0.012). No significant differences were apparent in disease-related biomarkers between the groups. M. officinalis extract containing 500 mg of RA taken daily was safe and well-tolerated by patients with mild dementia due to AD. Our results suggest that RA may help prevent the worsening of AD-related neuropsychiatric symptoms.Trial registration: The registration number for this clinical trial is UMIN000007734 (16/04/2012).

Effects of Kudoa septempunctata infections in a human intestinal epithelial model (Caco-2): a DNA microarray study.

Kudoa septempunctata, a myxosporean parasite infecting the trunk muscles of olive flounder (Paralichthys olivaceus), is reported to cause food poisoning in humans. The molecular mechanisms underlying the toxicity of K. septempunctata spores remain largely unknown. In the present study, we examine the molecular basis of such toxicity using DNA microarray analysis of K. septempunctata-inoculated human colon adenocarcinoma cells (Caco-2). We observed that the transepithelial resistance of the K. septempunctata-inoculated Caco-2 cell monolayers decreased markedly. DNA microarray analysis revealed that the mRNA expression profiles of control and inoculated cells clearly differed. Inflammatory and bacteria-related pathways, such as interleukin-8 (IL-8) production and MAPK/NF-kappa B pathway, were enriched. The concentrations of IL-8 and serotonin (5-HT) were higher in inoculated cells than in controls. K. septempunctata invasion damages the human intestinal epithelium, causing increased production of IL-8 and 5-HT, which likely results in the vomiting associated with K. septempunctata invasion.Abbreviations: AP-1: activator protein 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; ENS: enteric nervous system; FARMS: Factor Analysis for Robust Microarray Summarization; FDR: false discovery rate; GO: Gene Ontology; 5-HT: 5-hydroxytryptamine; IL-8: Interleukin-8; KEGG: Kyoto Encyclopedia of Genes and Genomes; K. septempunctata: Kudoa septempunctata; NF-kappa B: nuclear factor-kappa B; TJ: tight junction; TER: transepithelial electrical resistance.

The Effect of Polyphenols on Hypercholesterolemia through Inhibiting the Transport and Expression of Niemann-Pick C1-Like 1.

The Niemann-Pick C1-like 1 (NPC1L1) protein is a cholesterol transporter that is expressed in the small intestine. This report describes the discovery of NPC1L1, its transport properties, and the inhibitory effects of polyphenols on NPC1L1. NPC1L1 was identified in 2004 while searching for ezetimibe molecular targets. Excessive synthesis of cholesterol results in hyperlipidemia, which increases the amount of bile cholesterol excreted into the duodenum. The inhibition of NPC1L1 decreases blood cholesterol because food and bile cholesterol are also absorbed from NPC1L1 in the intestine. Some polyphenols, particularly luteolin, have been reported as NPC1L1-mediated anti-dyslipidemia constituents. Luteolin affects NPC1L1 through two mechanisms. Luteolin directly inhibits NPC1L1 by binding to it, which occurs in a short timeframe similar to that for ezetimibe. The other mechanism is the inhibition of NPC1L1 expression. Luteolin reduced the binding of Sterol-regulatory element-binding protein 2 (SREBP2) in the promoter region of the NPC1L1 gene and decreased mRNA levels of SREBP2 and hepatocyte nuclear factor 4α. These data suggest that luteolin decreases the expression of NPC1L1 through regulation of transcription factors. This review also explores the effect of other polyphenols on NPC1L1 and hypercholesterolemia.

Rosmarinic acid suppresses Alzheimer's disease development by reducing amyloid ß aggregation by increasing monoamine secretion.

A new mechanism is revealed by which a polyphenol, rosmarinic acid (RA), suppresses amyloid ß (Aß) accumulation in mice. Here we examined the brains of mice (Alzheimer's disease model) using DNA microarray analysis and revealed that the dopamine (DA)-signaling pathway was enhanced in the group fed RA versus controls. In the cerebral cortex, the levels of monoamines, such as norepinephrine, 3,4-dihydroxyphenylacetic acid, DA, and levodopa, increased after RA feeding. The expression of DA-degrading enzymes, such as monoamine oxidase B (Maob), was significantly downregulated in the substantia nigra and ventral tegmental area, both DA synthesis regions. Following in vitro studies showing that monoamines inhibited Aß aggregation, this in vivo study, in which RA intake increased concentration of monoamine by reducing Maob gene expression, builds on that knowledge by demonstrating that monoamines suppress Aß aggregation. In conclusion, RA-initiated monoamine increase in the brain may beneficially act against AD.

Intestinal Anti-Inflammatory Activity of Perillaldehyde.

Monoterpenoid perillaldehyde (PA) is the major component in Perilla frutescens leaf essential oil, but its function regarding anti-inflammatory effect is unclear. We explored the anti-inflammatory activity of PA in a dextran sulfate sodium (DSS)-induced colitis mouse model using relief of bodyweight loss (avg. 49.2% mitigation; P = 0.094) and colon damage (avg. 35.3% mitigation; P < 0.05) by administration of PA at a 100 mg/kg dosage. The PA administration resulted in suppression of DSS-induced expression of pro-inflammatory cytokine genes and matrix metalloproteinase-9 in the colon (e.g., avg. 60.6% mitigation for TNF-α mRNA levels; P < 0.05). These effects were confirmed in macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS). Application of PA induced cell suppression of LPS-induced expressions of genes and proteins of pro-inflammatory cytokines and induced activation of c-Jun N-terminal kinases (JNKs, p54 and p46; P < 0.05) but not nuclear factor-κB p65. The half maximal inhibitory concentration for decreased expression levels of TNF-α mRNA was 171.7 µM. We discuss the in vivo function of PA in amelioration of intestinal inflammation via JNK-mediated cytokine regulation.

Eggshell membrane powder ameliorates intestinal inflammation by facilitating the restitution of epithelial injury and alleviating microbial dysbiosis.

Gut microbiota is an essential factor in the shaping of intestinal immune system development and driving inflammation in inflammatory bowel disease (IBD). We report the effects and microbe-host interactions underlying an intervention using fine powder of eggshell membrane (ESM) against IBD. ESM attenuated lipopolysaccharide-induced inflammatory cytokine production and promoted the Caco-2 cell proliferation by up-regulating growth factors in vitro. In a murine model of dextran sodium sulphate-induced colitis, ESM significantly suppressed the disease activity index and colon shortening. These effects were associated with significant ameliorations of gene expressions of inflammatory mediators, intestinal epithelial cell proliferation, restitution-related factors and antimicrobial peptides. Multifaceted integrated omics analyses revealed improved levels of energy metabolism-related genes, proteins and metabolites. Concomitantly, cecal metagenomic information established an essential role of ESM in improving dysbiosis characterized by increasing the diversity of bacteria and decreasing absolute numbers of pathogenic bacteria such as Enterobacteriaceae and E. coli, as well as in the regulation of the expansion of Th17 cells by suppressing the overgrowth of segmented filamentous bacteria. Such modulations have functional effects on the host; i.e., repairing the epithelium, regulating energy requirements and eventually alleviating mucosal inflammation. These findings are first insights into ESM's modulation of microbiota and IBD suppression, providing new perspectives on the prevention/treatment of IBD.

Drying-Induced Self-Similar Assembly of Megamolecular Polysaccharides through Nano and Submicron Layering.

We propose a self-similar assembly to generate planar orientation of megamolecular polysaccharides on the nanometer scale and submicron scale. Evaporating the aqueous liquid crystalline (LC) solution on a planar air-LC interface induces polymer layering by self-assembly and rational action of macroscopic capillary forces between the layers. To clarify the mechanisms of nanometer- and submicron-scale layering, the polymer films are investigated by electron microscopy.

Thermal Disparity between Fingers after Cold-water Immersion of Hands: A Useful Indicator of Disturbed Peripheral Circulation in Raynaud Phenomenon Patients.

OBJECTIVE: To devise an effective method to assess the peripheral circulation using an infrared thermographic analysis. METHODS: Sequential measurements of the skin temperature before and after cold-water immersion of the hands were analyzed by a thermographic examination in healthy controls and patients diagnosed to have Raynaud phenomenon (RP). The skin temperatures of the dorsum of all fingernail folds and the metacarpophalangeal (MCP) joints were measured at baseline. Then the hands were immersed in 10°C water for 10 s, and the skin temperatures were measured at 0, 3, 5, 10, 15, 20 and 30 min after immersion. The mean temperature, recovery rate and disparity (coefficient of variation) of the nail fold temperatures were calculated. The distal-dorsal difference (DDD) was calculated by subtracting the mean MCP temperature from the mean nail fold temperature. Receiver operating characteristic (ROC) curves were generated to compare these parameters in terms of their capability to differentiate patients with RP. RESULTS: Thirty-one RP patients and 25 controls were included in the study. The baseline nail fold temperature was significantly lower in RP patients than in the controls. The RP patients had a lower recovery rate, lower DDD and higher disparity than the controls. The disparity and DDD were negatively correlated (r=-0.63, p<0.01), whereas the recovery rate and DDD were positively correlated (r=0.91, p<0.01). The ROC curve analysis revealed that the disparity in nail fold temperature effectively differentiated RP patients from controls (area under the curve: recovery rate 0.72; disparity 0.88; DDD 0.79). CONCLUSION: The temperature disparity between fingers is a useful thermographic parameter for evaluating disturbed peripheral circulation in patients with Raynaud phenomenon.

A case of cutaneous Paecilomyces formosus infection in an extremely premature infant.

BACKGROUND: Many types of weak pathogenic microorganisms often cause opportunistic infections in extremely preterm infants. Paecilomyces formosus is one such opportunistic fungus that can lead to a serious infection. Here, we report the clinical course of P. formosus infection in an extremely preterm infant. CASE PRESENTATION: An extremely preterm male infant was born at 23 weeks of gestation. Six days after birth, he developed yellowish-brown nodules on the skin of the back extending to the buttocks. P. formosus was identified by culture of samples from the cutaneous lesions. We treated the infection with intravenous micafungin and lanoconazole ointment application. The skin lesions improved dramatically and healed without scar tissue formation. CONCLUSION: Neonatologists should consider opportunistic P. formosus infections. This is the first report to describe that micafungin is effective for P. formosus cutaneous infection in extremely premature infants.

Cholesterol-Lowering Effect of Calcium Alginate in Rats.

We examined whether calcium alginate (Ca-Alg) reduces blood cholesterol levels in rats fed a high-cholesterol diet. First, we examined taurocholate adsorption in vitro by various types of sodium alginate (Na-Alg). High molecular-weight, guluronic acid-rich Na-Alg showed the greatest adsorption of taurocholate, and therefore the corresponding Ca-Alg was chosen for the in vivo study. Rats were fed a high-cholesterol diet or a Ca-Alg-containing diet for 2 weeks. Body weight and diet intake were measured, and the general condition of the animals was monitored during this period. After 14 d, the plasma concentration of cholesterol, portal plasma concentration of bile acid, and bile acid in feces were measured. The plasma concentration of cholesterol was significantly reduced in rats fed a 2% Ca-Alg-containing diet. Furthermore, the portal concentration of bile acid was significantly lowered in the 2% Ca-Alg group. A tendency for a Ca-Alg concentration-dependent increase in fecal excretion of bile acid was also seen, although it was not statistically significant. While several changes in biochemical parameters and histopathological findings were observed, all the values remained within the physiological range. These results indicate that Ca-Alg is effective in reducing plasma cholesterol. A possible mechanism would be enhanced fecal excretion of bile acid due to reduced intestinal reabsorption, which in turn might stimulate bile acid synthesis from cholesterol in the liver, leading to a decrease in plasma cholesterol.

In Vitro Inactivation of Kudoa septempunctata Spores Infecting the Muscle of Olive Flounder Paralichthys olivaceus.

Kudoa septempunctata, a myxosporean parasite infecting the trunk muscles of olive flounder (Paralichthys olivaceus), has been recently reported to be the causative agent of a type of food poisoning in humans. Patients exhibited acute diarrhea and vomiting after ingestion of the raw flesh of infected flounder. A recent increase in the number of food-poisoning cases has prompted us to develop a control strategy of this parasite. In this study, we evaluated the efficacy of several temperature and chemical treatments for inactivating K. septempunctata spores in vitro using the vital staining assay with the fluorescent dyes Hoechst 33342 and propidium iodide (PI). Screening tests of treatment methods against K. septempunctata suggested that 25% ethanol for 5 min, 80°C for 10 s, limonene at 10 µL/mL for 5 min, and salinities at 0‰ and 160‰ for 5 min were effective for killing spores. To verify toxicity loss in K. septempunctata spores after the treatments, tight junction barrier integrity assays with Caco-2 cells were conducted. The results of the Caco-2 assays corresponded well with those of the Hoechst 33342-PI staining assay. Further studies are required to determine a practical treatment procedure for inactivating spores considering the treatment application in the production process of cultured olive flounder.