Stability of amino acids, free and acyl-carnitine in stored dried blood spots.

BACKGROUND: Newborn screening of inborn errors of metabolism using tandem mass spectrometry has become a public health strategy in many developed countries. Retrospective analyses using stored dried blood specimens have been limited, mainly due to a lack of biochemical information on the long-term stability of acylcarnitines and amino acids in stored specimens. We studied the characteristic profiles of the stability of amino acid, free carnitine, and acyl carnitines in dried blood specimens stored in a refrigerator after newborn screening. METHODS: Dried blood specimens from 198 healthy newborns, which had been stored in a refrigerator at 5 °C after newborn screening, were prospectively subjected to tandem mass spectrometry analyses after 1, 3, 6 months, 1 and 2 years of storage. We also retrospectively re-analyzed the stored samples from 90 newborns, which had been analyzed and stored at 5 °C for 4 years. RESULTS: We found that proline (Pro) and tyrosine (Tyr) were stable for 2 years, and that alanine (Ala), arginine (Arg), and phenylalanine (Phe) decayed with linear regression. The C0 increased during the time-course of 2 years, whereas most acylcarnitines gradually decayed and some showed a linear correlation. The retrospective analysis of samples stored for 4 years revealed that Ala, Phe, Pro and Tyr were almost stable, leucine (Leu), valine (Val) decayed with linear regression, C0 increased, and C10, C12, C14, C14:1, C16, C18, C18:1 decreased, while maintaining a linear correlation. CONCLUSIONS: These data suggested that some metabolic parameters from refrigerator-stored dried blood specimens were applicable for the detection of inborn errors of metabolism.

Essential Oil of Citrus sudachi Suppresses T Cell Activation Both In Vitro and In Vivo.

The essential oil of Citrus sudachi (sudachi oil) is extracted from the peel of sudachi, a citrus plant. We investigated the effect of sudachi oil on immune function in both in vitro antigen (Ag) induced lymphocyte activation and in vivo Ag-specific immune response. In the in vitro study, the proliferative activity of splenocytes upon Ag-specific and non-specific stimulation was suppressed by treatment with sudachi oil in a dose-dependent manner. In addition, the expression level of Ag-presentation-related molecules and their Ag-presenting function on dendritic cells were suppressed by sudachi oil. To examine how sudachi oil regulates an Ag-specific immune response in vivo, mice were immunized with ovalbumin and the immune response of the mice was investigated. Ag-specific proliferation response of splenocytes from mice treated with sudachi essential oil was significantly suppressed. The results indicate that sudachi oil suppresses T cell and dendritic cell functions in vitro and Ag-specific T cell induction in vivo.

Generic selection criteria for safety and patient benefit [X]: Water-vapor permeability and peel force properties of brand-name and generic ketoprofen tapes.

Tape products containing ketoprofen have transdermal analgesic and anti-inflammatory effects. We compared the physicochemical properties (water-vapor permeability, peel force, peel force-time curve) between one brand-name product and eight generic products. Regarding the measurement of water-vapor permeability, the formulations using methacrylic acid n-butyl acrylate copolymer (MBA) adhesives showed higher water-vapor permeability than those using styrene isopropyl styrene block copolymer (SIS) adhesives. In the case of the formulation using SIS adhesive, the central part of the formulation had higher water-vapor permeability than both ends. In the 90-degree peel test using the methods of adhesion testing, significant differences were observed between the products, especially as the various application times (5 min, 30 min, 9 h and 24 h) increased. This may be because the longer the time of attachment to the adherend, the more the adhesive force with the adherend increased due to the "anchoring effect" of the adhesive. The measurement of the peel force-time curve showed different curves among the products, especially in the peel force curve of Teikoku after 24 h, which showed two characteristic peak curves. Furthermore, when the peel forces at 25°C and 40°C were compared, Mohrus and Toko showed significantly higher values at 40°C compared to 25°C. This study showed that there are many generic drugs with formulation characteristics different from those of brand-name drugs, and that there is a large difference among the products in terms of adhesion and detachment.

Generic selection criteria for safety and patient benefit [IX]: Evaluation of "feeling of use" of sodium hyaluronate eye drops using the Haptic Skill Logger (HapLog®) wearable sensor for evaluating haptic behaviors.

We compared the pharmaceutical properties, such as surface tension, drop volume, nozzle inner diameter, and force to push the drug product out of the container (squeeze force), of purified sodium hyaluronate eye drops preparations of one brand-name (Hyalein) and 11 generic drugs used for treatment of keratoconjunctiva epithelial disorders, and examined product selection based on the needs of the patient. The surface tension of Nissin (51.0 dyn/cm) and Nitten (52.3 dyn/cm) was significantly lower than that of Hyalein (62.8 dyn/cm), whereas Nitten PF (69.5 dyn/cm) was significantly higher than Hyalein. The drop volume of Tearbalance (42.4 mg), Nissin (43.7 mg), and Nitten (42.7 mg) was significantly lower than that of Hyalein (50.4 mg). We compared the squeeze force using a wearable touch sensor (Haptic Skill Logger: HapLog®) and digital force gauge (DF). The squeeze force of HapLog® showed values of about 1.7- to 3.5-fold higher than that of DF. Moreover, the squeeze force of Eyecare (34.0 N), Kyorin (35.4 N), and Nitten PF (44.3 N) by HapLog® was significantly higher than that of Hyalein (10.5 N). In contrast, the squeeze force of Kyorin (20.8 N) and Nitten PF (25.0 N) by DF was significantly higher than that of Hyalein (12.2 N). Two questionnaire surveys on the feeling of instillation of eye drops revealed a strong negative correlation between feeling of use and squeeze force.

Eucoleus garfiai (Gállego et Mas-Coma, 1975) (Nematoda: Capillariidae) infection in wild boars (Sus scrofa leucomystax) from the Amakusa Islands, Japan.

We examined lingual tissues of Japanese wild boars (Sus scrofa leucomystax) captured in the Amakusa Islands off the coast of Kumamoto Prefecture. One hundred and forty wild boars were caught in 11 different locations in Kamishima (n = 36) and Shimoshima (n = 104) in the Amakusa Islands, Japan between January 2016 and April 2018. Lingual tissues were subjected to histological examinations, where helminths and their eggs were observed in the epithelium of 51 samples (36.4%). No significant differences in prevalence were observed according to maturity, sex or capture location. Lingual tissues positive for helminth infection were randomly selected and intact male and female worms were collected for morphological measurements. Based on the host species, site of infection, and morphological details, we identified the parasite as Eucoleus garfiai (Gállego et Mas-Coma, 1975) Moravec, 1982 (syn. Capillaria garfiai). This is the first report from outside Europe of E. garfiai infection in wild boars. Phylogenetic analysis of the parasite using the 18S ribosomal RNA gene sequence confirmed that the parasite grouped with other Eucoleus species, providing additional nucleotide sequence for this genus. Since wild boar populations are widely distributed in Japan, continuing surveys on the epidemiology of the parasite and identifying possible intermediate host candidates are crucial for elucidating the transmission route of the parasite.

Generic selection criteria for safety and patient benefit [VIII]: Comparing the physicochemical and pharmaceutical properties of brand-name and generic diclofenac sodium tapes.

With respect to diclofenac sodium-containing tape preparations of nonsteroidal antiphlogistic drug, we compared the pharmaceutical properties (pH, elongatedness, water-vapor permeability, adhesive force, and peeling-force) of 11 medicinal drugs (2 brand-name and 9 generic drugs) to obtain evidence for product selection in line with the needs of the patient. The elongatedness of the generic drugs Teikoku (1.39), Yutoku (1.40), and Nippon-zoki (1.43) were significantly higher than the brand-name drug Voltaren® (1.22). The adhesive force was measured using the probe tack test and the inclined ball tack test. The probe tack test results of Naboal® (6.8 N/cm2), Teikoku (6.1 N/cm2), Yutoku (5.9 N/cm2), Nippon-zoki (6.2 N/cm2), and Rakool (6.2 N/cm2) were higher than that of Voltaren (2.0 N/cm2). The inclined ball tack test results of Naboal (18.0), Teikoku (24.0), Yutoku (21.5), and Nippon-zoki (22.7) were also higher than that of Voltaren (7.2). Concerning peeling-force measurement, the 90° peeling-forces of Naboal (0.95 N), Teikoku (0.96 N), Yutoku (0.94 N), and Nippon-zoki (1.01 N) were higher than that of Voltaren (0.68 N). These results show that there were marked differences in the feeling of use of each product between the brand-name and generic drugs. The pharmacist indicates the basis for selection of a preparation according to the feeling of use desired by each patient. It has become possible to recommend products suitable for each patient, which will allow pharmacists to provide products according to the needs of each patient when a brand-name drug is changed to a generic one.

Generic selection criteria for safety and patient benefit [VII]: Comparing the physicochemical and pharmaceutical properties of brand-name and generic terbinafine hydrochloride cream.

We measured and compared the physicochemical properties (pH, yield value, and squeeze force) of a drug for dermatomycosis, terbinafine hydrochloride-containing cream (brand-name product), and 12 generic products to clarify the characteristics of each product. On pH measurement, the pH value of the brand-name product, Lamisil, was 4.8, and those of the generic products ranged from 4.3 to 5.5, showing no marked difference. Furthermore, the yield value of Lamisil, as an index of cream ductility, was 122.2 dyn/cm2, and those of the generic products ranged from 42.1 to 1,621.5 dyn/cm2. In particular, the value of a generic product, Taiyo (42.1 dyn/cm2), was significantly lower, whereas that of another one, Viras (1,621.0 dyn/cm2), was significantly higher. In addition, the squeeze force was measured by attaching a HapLog® to the thumb and second finger. The value of Lamisil was 12.9 N, and those of the generic products ranged from 8.0 to 15.4 N. The values of generic products, Mylan (8.6 N), Tebinaceil (9.0 N), and Kelger (8.0 N), were significantly lower, whereas that of another one, Viras (15.4 N), was significantly higher. These results showed that there were marked differences in the pharmaceutical properties between the generic and brand-name products. The above pharmaceutical characteristics of drugs facilitated the presentation of reasons for differences in the sense of use, which characterizes external preparations, suggesting that products appropriate for individual patients can be recommended.

Generic selection criteria for safety and patient benefit [VI]: Comparing the physicochemical and pharmaceutical properties of brand-name, generic, and OTC felbinac tapes.

We measured the pH, water-vapor permeability, adhesive force, peeling-force, elongation rate, support flexibility, and peeling time of medicinal and over-the-counter (OTC) tape preparations containing felbinac. When measuring the pH of each preparation, Felnabion (pH 4.5) was weakly acidic, and EMEC and Tokuon (pH 7.0) were neutral. When measuring the water-vapor permeability of each preparation, that of a generic product, EMEC (380 g/m2/24h), was twice as high as that of a brand-name product, Seltouch (189 g/m2/24 h). The adhesive force was measured using the ball tack test. The adhesive forces of OTC drugs, Salomethyl, Homepass, and Tokuhon (1.04 g), were higher than that of Seltouch (0.06 g). Concerning peeling-force measurement, the peeling-forces of a generic product, Falzy (4.15 N), and an OTC drug, Omuneed (4.89 N), were higher than that of Seltouch (0.91 N). The elongation rates of a generic product, Sumilu (319%), and OTC drugs, Nabolin (298%) and Homepass (299%), were higher than that of Seltouch (251%), but that of Tokuhon (72%) was lower. The support flexibilities of EMEC (150 degrees) and Tokuhon (131 degrees) were higher than that of Seltouch (96 degrees). In addition, the peeling time of Seltouch was 120 min or more, whereas those of EMEC and Nabolin were 1.4 and 0.2 min, respectively. These results suggest that the differences in pharmaceutical properties, such as the pH, water-vapor permeability, adhesive force, peeling-force, elongation rate, support flexibility, and peeling time, among the preparations markedly influence patients' subjective comfortableness. The results of this study facilitated individuals' comfortableness-matched drug selection.

Generic selection criteria for safety and patient benefit [V]: Comparing the pharmaceutical properties and patient usability of original and generic nasal spray containing ketotifen fumarate.

The pH, osmotic pressure (cryoscopy), viscosity, squeeze force, spray angle, and spraying frequency of nasal spray containing ketotifen fumarate (1 brand-name product and 8 generic products) were measured. Based on the results of pH measurement, all products were weakly acidic (4.0 to 5.1). For all products, the osmotic pressure ratio to physiological saline was approximately 1. The viscosity of various products ranged from approximately 1.0 to 1.5 mPa·s. The spray angle of drug solution differed among the products: minimum, 46 degrees (Sawai and Fusachol); and maximum, 68.7 degrees (Sekiton). In particular, TOA, Sawai, Fusachol, and TYK showed significantly smaller angles compared to Zaditen (brand-name product). Container properties varied among the products: minimum squeeze force, 19.0 N (Sekiton); and maximum squeeze force, 43.1 N (Sawai). Based on these results, although all the above products are identical in dosage form and active ingredient, the differences in pharmaceutical properties, such as container operations and drug-solution spraying/attachment, may markedly influence patients' subjective opinions.

Cloning and characterization of hypoxia-inducible factor-1 subunits from Ascaris suum - a parasitic nematode highly adapted to changes of oxygen conditions during its life cycle.

The parasitic nematode Ascaris suum successfully adapts to a significant decrease in oxygen availability during its life cycle by altering its metabolic system dramatically. However, little is known about the regulatory mechanisms of adaptation to hypoxic environments in A. suum. In multicellular organisms, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1ß subunits, is a master regulator of genes involved in adaptation to hypoxia. In the present study, cDNAs encoding HIF-1α and HIF-1ß were cloned from A. suum and characterized. The full-length A. suum hif-1α and hif-1ß cDNAs contain open reading frames encoding proteins with 832 and 436 amino acids, respectively. In the deduced amino acid sequences of A. suum HIF-1α and HIF-1ß, functional domains essential for DNA-binding, dimerization, and oxygen-dependent prolyl hydroxylation were conserved. The interaction between A. suum HIF-1α and HIF-1ß was confirmed by the yeast two-hybrid assay. Both A. suum hif-1α and hif-1ß mRNAs were expressed at all stages examined (fertilized eggs, third-stage larvae, lung-stage larvae, young adult worms, and adult muscle tissue), and most abundantly in the aerobic free-living third-stage larvae, followed by a gradual decrease after infection of the host. hif-1 mRNA transcription was not sensitive to the oxygen environment in either third-stage larvae or adult worms (muscle tissue), and was regulated in a stage-specific manner. High expression of hif-1 mRNAs in third-stage larvae suggests its contribution to pre-adaptation to a hypoxic environment after infection of their host. Sequence analysis of 5'-upstream regions of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) genes, which show stage-specific expression and play an important role in oxygen adaptation during the life cycle, revealed that all subunits except for the adult-type flavoprotein subunit (Fp) possess putative hypoxia-responsive elements (HREs), suggesting that they are hif-1 target genes.

The γ-secretase inhibitor DAPT increases the levels of gangliosides at neuritic terminals of differentiating PC12 cells.

Mutations in presenilins are the major cause of early onset familial Alzheimer disease. It has recently been argued that clinical presenilin mutations work as loss-of-function but not toxic gain-of-function. To investigate whether presenilins are involved in the regulation of the distribution of neuronal membrane lipids, we treated neuronally differentiated PC12 cells with DAPT, an inhibitor of presenilin-dependent γ-secretase, and performed lipid analyses of neuritic terminals, which is an initial site of Aß deposition in brains, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). With DAPT treatment, levels of sphingomyelin, phosphatidylcholine, and cholesterol remained unchanged. However, DAPT treatment increased the ganglioside levels in PC12 neuritic terminals. Together with a previous finding that accumulation of gangliosides at neuritic terminals facilitates Aß assembly and deposition, the present data suggest that the loss-of-function of presenilins, i.e., a decrease in γ-secretase activity, has an impact on neuronal membrane architecture in a way that eventually exacerbates Alzheimer pathology.

A pilot, randomized, placebo-controlled, double-blind phase 0/biomarker study on effect of artepillin C-rich extract of Brazilian propolis in frequent colorectal adenoma polyp patients.

OBJECTIVE: Brazilian propolis, a folk medicine, is used worldwide as an alternative medicine to prevent colon cancer. The objective of the study was to test in a small pilot biomarker study in a high-risk group the safety and efficacy of propolis for colon cancer prevention, which has not been evaluated in humans. METHODS: Subjects with adenoma polyps recently removed from the colon were randomly assigned to a propolis group of 15 and a placebo group of 16. In a double-blind study, the propolis group received capsules containing 165 µmol artepillin C and 150 µmol other polyphenols per day for 3 months. Prior to and at the end of the experiments, their blood was analyzed using biochemical tests, and specimens from the normal-appearing sigmoid colon mucosa were biopsied endoscopically to examine the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and mRNA expressions of proliferating cell nuclear antigen, cyclin D1, and Bax. RESULTS: Propolis extract significantly increased the mRNA level of cyclin D1 in the sigmoid colon mucosa, and the other biomarkers remained unchanged. Blood biochemical tests showed significantly higher activity of creatine phosphokinase (CPK), 143 ± 52 units/ml in the propolis group and 104 ± 38 units/ml in the placebo group (p = 0.026), at the end of the study. The increase in CPK activity in the propolis group was due to the increase of the myocardial band form of CPK. On the other hand, laxative treatment prior to endoscopic biopsy significantly increased 8-OHdG levels. CONCLUSIONS: The results from our pilot study did not provide evidence that Brazilian propolis was effective in preventing changes occurring during early stages of colon cancer. In contrast, propolis may have detrimental side effects on muscle tissue, including myocardial cells.

Soft magnetic material based localized magnetic stimulation to cultured neuronal cells and modulation of network activities.

Magnetic stimulation is able to modulate the neuronal network activity using the non-invasive magnetically induced current. However, it is unknown how stimulation modulates the neuronal network activity. Therefore, we considered that precise stimulation and evaluation of the modulation of network activities in the vicinity of stimulated sites is required. Here, to establish precisely magnetic stimulation, we developed a Mu-metal that has high magnetic permeability soft magnetic material based localized magnetic stimulation (LMS) system with micro-fabricated dual cell-culture chambers. And, combining this device with a microelectrode array (MEA) permitted the evaluation of the stimulus effects at the stimulated and non-stimulated sites. Here, the dual cell-culture chambers were arranged in a concentric circle manner. Between the inner and outer chambers, 4, 8 and 12 connecting microfluid channels were fabricated using polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in outer and inner chambers. Through the micro-conduits, functional synaptic connections were formed. Mu-metal was aligned along the outer circle, which allowed us of focal magnetic stimulation to the cells in the outer chamber. Applying low frequency magnetic field to the Mu-metal, induced currents were generated and the electrical activity of the cells in the outer chamber was modified depending on the stimulation intensity. Following the modified activity in the outer circles, the cells in the inner chamber also showed slightly depressed activity patterns. These results suggested that our system would be promising for highly regulated neural stimulation.

A formation mechanism for 8-hydroxy-2'-deoxyguanosine mediated by peroxidized 2'-deoxythymidine.

The oxidative formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA is closely associated with the induction of degenerative diseases, including cancer. However, the oxidant species participating in the formation of 8-OHdG has yet to be fully clarified. On the basis that peroxyl radicals are a strong candidate for this species, we employed 2,2'-azobis(2-amidinopropane) (AAPH) as a peroxyl radical generator. Exposure of calf thymus DNA to AAPH formed 8-OHdG, but the exposure of 2'-deoxyguanosine (dG) alone did not. From the exposure of various combinations of nucleotides, 8-OHdG was formed only in the presence of dG and thymidine (dT). A mix of dG with an oxidation product of dT, 5-(hydroperoxymethyl)-2'-deoxyuridine, produced 8-OHdG, but the amount formed was small. In contrast, 8-OHdG was produced abundantly by the addition of dG to peroxidized dT with AAPH. Thus, the formation of 8-OHdG was mediated by the peroxidized dT. Instead of artificial AAPH, endogenous peroxyl radicals are known to be lipid peroxides, which are probably the oxidant species for 8-OHdG formation mediated by thymidine in vivo.

Yellow tea is more potent than other types of tea in suppressing liver toxicity induced by carbon tetrachloride in rats.

The present study compared the effects of six Chinese teas categorized by their production process: green, white, yellow, oolong, black and pu-erh teas, on carbon tetrachloride (CCl4)-induced liver injury. Wistar rats were given ad libitum the Chinese teas prepared according to the home-style methods for 1 week, and then intraperitoneally injected with CCl4 (1 mg/kg body weight) or olive oil as a vehicle. The yellow tea significantly ameliorated the increase in the activity of the alanine- and aspartate-aminotransferases in plasma. Thus, the drinking of yellow tea may contribute to protection against liver injury.

Effects of combined administration of quercetin, rutin, and extract of white radish sprout rich in kaempferol glycosides on the metabolism in rats.

Quercetin, rutin, the extract of white radish sprout rich in kaempferol glycosides, and their combination were intragastrically administered to Wistar rats to investigate the interactive metabolism of these flavonoids. The combined administration of these flavonoids changed the concentrations of the metabolites in plasma as compared with the concentrations after the administration of a single compound.

Cloning and sequencing of the aldehyde oxidase gene from Methylobacillus sp. KY4400.

The aldehyde oxidase genes (aods) from Methylobacillus sp. KY4400 were cloned, and sequenced. The sequences for small (aodS, 489 bp), medium (aodM, 993 bp), and large (aodL, 2,328 bp) subunit genes were determined. At least one additional ORF was indispensable for the expression of enzyme activity. The structural genes contained two [2Fe-2S] centers, an FAD binding site, and a molybdenum cofactor binding site.

Production of aldehyde oxidases by microorganisms and their enzymatic properties.

In order to establish an efficient process to decompose environmentally toxic aldehydes, dioxygen-dependent aldehyde oxidase (ALOD) from microorganisms was first sought, and some bacteria and actinomycetes were found to produce the enzyme in their cells. Methylobacillus sp., Pseudomonas sp. and Streptomyces moderates were selected as the representative ALOD-producing strains and their enzymes were partially purified and characterized. The three ALODs could oxidize a wide range of aldehydes including formaldehyde, aliphatic aldehydes, and aromatic aldehydes, though their preferences differ depending on their producing strains. The other enzymatic properties were also determined with regard to their producing strains. Methylobacillus sp. ALOD had the most acidic optimum pH for its activity and stability and Pseudomonas sp. ALOD had the highest stability against heat treatment. Three native ALODs had molecular weights ranging from 140 to 148 kDa and were composed of three subunits of different sizes: large (85 to 88 kDa), medium-sized (37 to 39 kDa) and small (18 to 23 kDa).