Orally administered low-molecular weight agaro-oligosaccharides are absorbed into the plasma of healthy humans.

Agaro-oligosaccharides (AOSs) are known to have biological activities, such as anti-inflammatory, anti-tumor, and anti-obesity effects. Although existing evidence suggests the presence of AOSs in peripheral tissues after oral administration, whether AOSs permeate into the blood circulation remains unknown. Thus, we hypothesized that AOSs with low-molecular weight can permeate the human gastrointestinal tract. To test this hypothesis, the time course of absorption was examined by analyzing plasma samples before and 1, 2, and 4 h after ingestion. Analysis was performed using liquid chromatography/mass spectrometry after labeling with p-aminobenzoic ethyl ester. Our results showed that the plasma concentration of agarobiose (Abi) was higher than that of agarotetraose (Ate); however, agarohexaose was not detected. Additionally, plasma levels of Abi and Ate were proportional to the dose. These results suggest that permeation efficiency is dependent on the molecular weight and that the systemic absorption of Abi via the gastrointestinal tract is better than that of Ate. These findings will contribute to a better understanding of the bioactivity of orally administered AOSs in peripheral tissues.

Intestinal permeability of agaro-oligosaccharides: Transport across Caco-2 cell monolayers and pharmacokinetics in rats.

Agaro-oligosaccharides (AOSs), even-numbered oligosaccharides prepared from agar, are applied to various food, including supplements, drinks, and jellies because of their biological activities. This study aimed to evaluate the AOS permeation in the gastrointestinal tract in vivo and in vitro. Agarobiose (Abi), agarotetraose (Ate), and agarohexaose (Ahe) were detected in rat plasma after oral administration of AOSs. The detection level of agarobiose in the plasma was higher than that of agarohexaose, which was consistent with the permeation study using Caco-2 cell monolayers. Further, the adenosine triphosphate inhibitor (sodium azide) or endocytosis inhibitor (colchicine) did not inhibit AOS permeation through Caco-2 cell monolayers. Conversely, AOS permeation enhanced upon treatment with cytochalasin B, a tight junction disrupter, suggesting that AOSs might have passed mainly through the tight junctions between the intestinal epithelial cells. These results indicate that AOSs, especially agarobiose, can be absorbed as an intact form via the gastrointestinal tract across the intestinal epithelium through the paracellular pathway.

Methylnigakinone content determination and geographical origin discrimination for P. quassioides via fluorescence fingerprint and principal component analyses.

Picrasma quassioides is used as a bittersweet stomach medicine. Because it is a natural product obtained from various geographical regions, the production area is important when P. quassioides is used as a crude drug. Herein, we developed a method to determine the content of methylnigakinone, one of the major active ingredients in P. quassioides, and a protocol for discriminating the geographical origin of this natural product using a fluorescence fingerprint analysis and principal component analysis (PCA). Because methylnigakinone is fluorescent (excitation wavelength: 352 nm, emission wavelength: 458 nm), the content of this molecule can be determined in the concentration range of 0.1-1 µg/mL. The quantification results of methylnigakinone obtained using the developed method were similar to those obtained from an HPLC analysis. Furthermore, the PCA of the fluorescence fingerprint of P. quassioides produced a score plot with the three different geographical origins (Kyushu island (Japan), Shikoku island (Japan), and China) plotted in the regions. Thus, it was possible to discriminate the geographical origin of the P. quassioides samples. The developed method is simple, quick, and has a minimal environmental impact. Therefore, the developed method will be useful for confirming the origin of P. quassioides.

Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay.

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.

Simple and Rapid Evaluation of the Unique Manuka Factor in Manuka Honey Using Fluorescence Fingerprints and Principal Component Analysis.

The excellent antibacterial activity of manuka honey has been well-documented and is often evaluated according to the unique manuka factor (UMF) index. UMF is determined by an assay based on a bacterial culture, which is time-consuming and does not allow for quantitative analysis. This study developed a simple and rapid method for UMF evaluation using fluorescence fingerprints, principal component analysis (PCA), and partial least squares (PLS) regression. Manuka honey samples were diluted four times with water and fluorescence was observed at three wavelength combinations, namely 260-300 (excitation; ex) to 370 (emission; em) nm, 340 (ex) to 480 nm (em), and 440 (ex) to 520 nm (em), that are mainly attributed to lepteridine, leptosperin, 2-methoxybenzoic acid, and N-methyl phenazinium. Analyzing fluorescence fingerprints using PCA and PLS regression provided a reliable evaluation of the UMF in manuka honey and could be used to differentiate between manufacturers.

Extraction of urinary cell-free DNA by using triamine-modified silica particles for liquid biopsy.

The presence of approximately 200-bp cell-free DNA (cfDNA) in the urine has attracted attention as a biomarker for liquid biopsy. However, it is currently useful only for diagnoses of cancers in which a large amount of cfDNA is excreted in the urine. Therefore, the development of an efficient method for extracting cfDNA existing in small amounts in the urine is essential for diagnosing many other diseases. We examined the effect of particle size, small pore size (surface area), and surface modification of porous silica particles on the efficiency of DNA extraction. Our observations suggested that cfDNA could be captured by tertiary amine-modified particles and then removed from the particles by repeatedly washing with sodium bicarbonate (pH 11). Using this method with 30 mg of triamine-modified particles, we succeeded in extracting a few hundred nanograms of cfDNA from 15 mL urine. Furthermore, we could detect ~ 67 fg/mL caries DNA (211 bp) in 15 mL urine sample, suggesting that this method may be suitable for the extraction of genetic biomarkers for cfDNA-based liquid biopsy.

Trimethylammonium modification of a polymer-coated monolith column for rapid and simultaneous analysis of nanomedicines.

Drug-containing nanoparticles (nanomedicine) are ideal targeted-drug-delivery systems. However, methods for the simultaneous analysis of the drug within the nanoparticle and free drug in a short time are rather limited. In this study, we developed a polymer-modified monolithic column with cationic groups (trimethylammonium) for the simultaneous analysis of the drug within the nanoparticle and the free drug. The use of the acrylamide group was determined as the optimum connecting group, and the optimum concentration of the modifier was 6%. The prepared column retained the drug within the nanoparticle by anion exchange, and its elution time was controlled by the ionic concentration (tris(hydroxymethyl)aminomethane, Tris) of the mobile phase. The separation of two typical nanomedicines was studied on the prepared column. For DOXIL and Abraxane, the drugs within the nanoparticle were well separated from the free drugs, on the developed column. The developed polymer-coated monolithic column with trimethylammonium modification is expected to enable the rapid analysis of various nanomedicines.

A Simple and Easy Method of Monitoring Doxorubicin Release from a Liposomal Drug Formulation in the Serum Using Fluorescence Spectroscopy.

Formulation of a drug as liposomes facilitates its delivery to the disease target. Rightly, liposomes are gaining popularity in the medical field. In order for the drug to show efficacy, release of the encapsulated drug from the liposome at the target site is required. However, the release is affected by the permeability of the lipid bilayer of the liposome, and it is important to examine the effect of the surrounding environment on the permeability. In this study, we showed the usefulness of fluorescence analysis, especially fluorescence fingerprint, for a rapid and simple monitoring of release of an encapsulated anticancer drug (doxorubicin) from its liposomal formulation (DOXIL). Our result indicated that the release is accelerated by the existence of membrane permeable ions, such as tris(hydroxymethyl)aminomethane, and blood proteins like albumin. Hence, monitoring of doxorubicin release by fluorescence analysis is useful for the efficacy evaluation of DOXIL in a biomimetic environment.

Anti-inflammatory effects of new catechin derivatives in a hapten-induced mouse contact dermatitis model.

Contact dermatitis is a common skin disease, with various treatments available to dermatologists. According to general guidelines, the first line of treatment involves topical steroids; however, this treatment has application-site restrictions in order to avoid adverse cutaneous events. Accordingly, increased demand exists for the development of new treatments. In Japan, the recent use of catechin-containing health foods and their beneficial effects has attracted attention. Indeed, several studies have examined the anticancer, anti-obesity, anti-inflammatory, and antioxidant effects of catechins. In this study, we synthesized planar catechin (PC) from natural (+)-catechin, and further chemically modified it with the intent to clarify the anti-inflammatory and antioxidant effects of new catechin derivatives. Methylate-PC (methyl PC) and acetylate-PC (acetyl PC) were modified to increase lipid solubility. Their antioxidant effects were examined with electron spin resonance by evaluating the ability to remove hydroxyl radicals. In vitro, the antioxidant effects were in the order of PC > (+)-catechin > acetyl PC > methyl PC. In addition, we used a 1-fluoro-2,4-dinitrobenzene (DNFB)-induced allergic contact dermatitis model in BALB/c mice. Our results demonstrated that catechin derivatives inhibited ear swelling induced by DNFB, with acetyl PC demonstrating a greater inhibitory effect than PC and methyl PC. Moreover, acetyl PC downregulated the mRNA levels of inflammatory cytokines, including tumor necrosis factor-alpha, interleukin (IL)-1ß, and IL-4, as well as myeloperoxidase activity, in the ear tissue of DNFB-treated mice. Collectively, our novel findings suggest that catechin derivatives may be a promising new choice for the treatment of contact dermatitis.

Enrichment of liposomal nanomedicines using monolithic solid phase extraction discs following preactivation with bivalent metal ion solutions.

Silicate is an excellent adsorbent because of its large surface area and amenability to surface modification. In this study, the representative liposome nanomedicines DOXIL® and AmBisome® were enriched using a silica monolith disc (diameter 4.2 mm, length 1.5 mm) with bimodal pores. Although the nanoparticles passed through the disc without retention when water was used as the preactivation solution, they were strongly retained by the disc when a 1 M bivalent metal (such as Mg2+, Ca2+, and Ni2+) solution was used. Notably, strong affinity was observed to DOXIL, a pegylated liposomal nanoparticle, by the disc composed of 5 µm and 10 nm through- and meso pores, respectively, and nearly 100% of DOXIL was recovered from a 40× diluted solution. Overall, the results demonstrate that monolithic discs are effective for the enrichment of liposomal nanomedicines.

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin.

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.

Novel assay of antibacterial components in manuka honey using lucigenin-chemiluminescence-HPLC.

Five components (hydrogen peroxide, methylglyoxal, dihydroxyacetone, fructose and glucose) of New Zealand manuka honey (Leptospermum scoparium) were analyzed using lucigenin chemiluminescence high-performance liquid chromatography (lucigenin-CL-HPLC). We focused on active oxygen species produced from the components in order to easily detect these five components contained in manuka honey. H2O2 and O2- generated from these components were identified by lucigenin-CL and electron spin resonance (ESR), and the bactericidal effect of ROS was confirmed using E. coli. The previously reported assays for Manuka honey components have low specificities and require complicated preprocessing methods. As our results, the detection and identification of these components were possible within 30 min in lucigenin-CL-HPLC system, without any special treatment. It is considered that lucigenin-CL-HPLC is useful for the quality control and the analysis of various honey.

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

Detection of telomerase activity using microchip electrophoresis.

Telomerase participates in malignant transformation or immortalization of cells and thus has attracted attention as an anticancer drug target and diagnostic tumor marker. The telomeric repeat amplification protocol (TRAP) and improved TRAP methods (TRAP-fluorescence, TRAP-hybridization, etc.) are widely used forms of this telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplification of telomeric repeats by polymerase chain reaction (PCR), making these TRAP methods time consuming and technically demanding. In this study we developed a novel telomerase assay using microchip electrophoresis for rapid and highly sensitive detection of telomerase activity in cancer cells. The mixed gel of 0.8% hydroxypropyl methylcellulose (HPMC) and 0.3% polyethylene oxide (PEO) with SYBR Gold (fluorescent reagent) was used for microchip electrophoresis. As a result, the product amplified by a telomerase-positive cell could be measured in one cell per assay and detected with high reproducibility (CV=0.67%) in the short time of 100s.

Fermented soybean powder with rice mold in the absence of salt stimulates the cellular immune system and suppresses the humoral immune response in mice.

The immunomodulatory effect of fermented non-salty soybean powder (NSBP) was investigated in C3H/HeN mice. The number of splenic CD11b(+), CD49b(+), and interferon (IFN)-γ(+)CD4(+) cells increased significantly, while that of interleukin (IL)-4(+)CD4(+) and CD19(+) cells decreased significantly in cultures containing NSBP. Similarly, in the spleen and Peyer's patches of mice fed a diet containing NSBP, the number of IL-12(+)CD11b(+), CD49b(+), and IFN-γ(+)CD4(+) cells increased noticeably, whereas the number of splenic IL-4(+)CD4(+) and CD19b(+) cells was lower compared to mice fed an NSBP-free diet. Superoxide production by peritoneal macrophages was significantly higher in mice fed an NSBP-containing diet. Both intestinal total IgA and serum total IgG levels declined in mice fed the NSBP-containing diet. Microarray analysis of mRNAs extracted from Peyer's patch cells of mice fed the NSBP-containing diet indicated an increase in the expression of several genes related to cellular immune responses, while the expression of genes related to immunoglobulin production decreased. These results indicate that NSBP stimulates the cellular immune response, but suppresses the acquired humoral immune response in C3H/HeN mice.

Anti-allergic properties of a matured fruit extract of the date palm tree (Phoenix dactylifera L.) in mite-sensitized mice.

The effects of oral ingestion of a hot water extract of matured fruit of the date palm tree (Phoenix dactylifera L.) on allergic responses were investigated in mite-sensitized mice. Sneezing and nose rubbing events in mice given a date extract-added diet were significantly lower than in those given an extract-free (control) diet. The serum total and mite antigen-specific immunoglobulin (Ig) E levels, and the number of spleen interleukin-4(+)CD4(+), IgE(+)B220(+) and FcεRIα(+)CD117(+) cells was significantly lower in mice given the date extract-added diet than in those given the control diet. Chlorogenic acid, pelargonin and ferulic acid significantly reduced the number of IgE(+)B220(+) cells, while chlorogenic acid and pelargonin significantly decreased the number of FcεRIα(+)CD117(+) cells in mouse spleen cell cultures. These results suggest that some polyphenols in the date may reduce mite-induced allergic symptoms in mice via a decrease in the number of IgE-producing plasma cells and high-affinity IgE receptor-expressing mast cells.

[Effect of the active adult learning/patient oriented clerkship on affective reaction of students ∼ from the results of student survey].

We have previously reported the efficacy of the Patient Oriented Clerkship (POC) in the clinical clerkship in Showa University Hospitals, by a trial with old four-year pharmacy program students. In the unique clerkship, each student has a patient in charge, and follows his/her clinical conditions throughout the rotation. The aim of the POC is that having the students learn spontaneously (Active Learning) and actively (Adult Learning) promoted by student's commitment and responsibility by communicating with patients and health professionals in a team. As the POC requires students both Active Learning and Adult Learning, we define the POC as Active Adult Learning (AAL). Having a patient in charge for each student gives them many opportunities to participate in the medical team and foster their problem solving skills. Our previous study eventually showed positive results of the POC in the one-month short clerkship in the four-year program. On the other hand, the effect of the unique hospital clerkship in the new six-year program is not known. We conducted a student survey to clarify the learning effect in the new six-year education system which was revised and 2.5 month clinical clerkship was scheduled according to the model core clerkship curriculum. This report is the first report to show a challenge of the AAL/POC clerkship in the new six-year pharmacy education program.

A matured fruit extract of date palm tree (Phoenix dactylifera L.) stimulates the cellular immune system in mice.

The immunomodulatory effects of a hot water extract from matured fruit of the date palm tree (Phoenix dactylifera L.) were investigated in comparison to those of prune and fig fruit in mice. The number of spleen IFN-γ(+)CD4(+), IFN-γ(+)CD49b(+) and IL-12(+)CD11b(+) cells was highest in mice given the date extract-added diet. Polyphenols identified in the date extract, such as chlorogenic acid, caffeic acid, pelargonin and ferulic acid, stimulated IFN-γ mRNA expression significantly in mouse Peyer's patch cell cultures. Chlorogenic acid and caffeic acid also increased the number of IFN-γ(+)CD4(+) cells significantly, while some polyphenols increased the number of IFN-γ(+)CD49b(+) and IL-12(+)CD11b(+) cells significantly. On the other hand, a 70% ethanol-insoluble date extract treated with trypsin increased the number of IFN-γ(+)CD49b(+) and IL-12(+)CD11b(+) cells significantly. These results indicate that some polyphenols and polysaccharides present in date fruit stimulate the cellular immune system in mice.

Suppressor of Hairless (Su(H)) is required for foregut development in the sea urchin embryo.

In sea urchin embryos, Notch signaling is required to segregate non-skeletogenic mesoderm from early endomesoderm, and is involved in endoderm development. To further investigate the role of Notch signaling in the endoderm cell lineage, we cloned a cDNA for the Hemicentrotus pulcherrimus ortholog of Suppressor of Hairless (HpSu(H)), which is a major mediator of the Notch signaling pathway, examined the expression during development and performed a functional analysis. HpSu(H) mRNA was ubiquitously expressed up to the unhatched blastula stage, and expression was exclusively detected in the vegetal plate region from the hatched blastula stage and then in the archenteron at the gastrula stage. Perturbation of HpSu(H) by injection of the dominant negative form of HpSu(H) (dn-HpSu(H)) mRNA into fertilized eggs led to the disappearance of secondary mesenchyme cells at the tip of the archenteron in the gastrula and pigment cells in the pluteus larva, confirming that Notch signaling is required for non-skeletogenic me soderm specification. In addition, injection of relatively high amounts of dn-HpSu(H) mRNA caused a defect or atrophy of the foregut in the archenteron at the pluteus stage. This result strongly suggests that Notch signaling is involved in foregut development during sea urchin development.

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis.

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.