Inhibitory effects of extracts from Eucalyptus gunnii on α-synuclein amyloid fibrils.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.

Effects of 1,5-anhydroglucitol on postprandial blood glucose and insulin levels and hydrogen excretion in rats and healthy humans.

The inhibition by 1,5-anhydro-d-glucitol (1,5-AG) was determined on disaccharidases of rats and humans. Then, the metabolism and fate of 1,5-AG was investigated in rats and humans. Although 1,5-AG inhibited about 50 % of sucrase activity in rat small intestine, the inhibition was less than half of d-sorbose. 1,5-AG strongly inhibited trehalase and lactase, whereas d-sorbose inhibited them very weakly. 1,5-AG noncompetitively inhibited sucrase. The inhibition of 1,5-AG on sucrase and maltase was similar between humans and rats. 1,5-AG in serum increased 30 min after oral administration of 1,5-AG (600 mg) in rats, and mostly 100 % of 1,5-AG was excreted into the urine 24 h after administration. 1,5-AG in serum showed a peak 30 min after ingestion of 1,5-AG (20 g) by healthy subjects, and decreased gradually over 180 min. About 60 % of 1,5-AG was excreted into the urine for 9 h following ingestion. Hydrogen was scarcely excreted in both rats and humans 24 h after administration of 1,5-AG. Furthermore, 1,5-AG significantly suppressed the blood glucose elevation, and hydrogen excretion was increased following the simultaneous ingestion of sucrose and 1,5-AG in healthy subjects. 1,5-AG also significantly suppressed the blood glucose elevation following the simultaneous ingestion of glucose and 1,5-AG; however, hydrogen excretion was negligible. The available energy of 1,5-AG, which is absorbed readily from the small intestine and excreted quickly into the urine, is 0 kJ/g (0 kcal/g). Furthermore, 1,5-AG might suppress the blood glucose elevation through the inhibition of sucrase, as well as intestinal glucose absorption.

Leg ulcer due to multiple arteriovenous malformations in the lower extremity of an elderly patient.

A 66-year-old woman with a history of deep vein thrombosis (DVT) presented with an irregularly shaped leg ulcer surrounded by pigmentation on the left lower limb. In addition, the circumference of her left thigh had gradually increased. The ulcer did not respond to topical treatment and enlarged, therefore, she visited our hospital. Arteriography of the left lower limb showed multiple arteriovenous malformations (AVMs), based on which we made a diagnosis of a leg ulcer due to multiple AVMs. Transcatheter arterial embolisation with a mixture of N-butyl-2-cyanoacrylate and lipiodol was performed six times in the period of about a year for treating the AVMs. The ulcer was managed with bed rest, surgical debridement, continuous pressure support with elastic wrap and topical treatment. After 15 months, the ulcer healed, leaving pigmentation and scarring. It is quite rare for AVMs to progress in the elderly. We speculate that the DVT had caused occult AVMs to become symptomatic following an increase in size.

Digestibility of new dietary fibre materials, resistant glucan and hydrogenated resistant glucan in rats and humans, and the physical effects in rats.

Resistant glucan (RG) and hydrogenated resistant glucan (HRG) are newly developed non-digestible carbohydrate materials that decrease lifestyle-related diseases. The bioavailability of RG and HRG was investigated by in vitro experiments using human and rat small intestinal enzymes and by in vivo experiments using rats in the present study. Oligosaccharides, which are minor components of RG and HRG, were hydrolysed slightly by small intestinal enzymes of humans and rats, and the hydrolysing activity was slightly higher in rats than in humans. The amount of glucose released from HRG was greater than that from RG. However, the high-molecular-weight carbohydrates of the main components were hardly hydrolysed. Furthermore, neither RG nor HRG inhibited disaccharidase activity. When rats were raised on a diet containing 5 % of RG, HRG, resistant maltodextrin or fructo-oligosaccharide (FOS) for 4 weeks, all rats developed loose stools and did not recover during the experiment, except for the FOS group. Body weight gain was normal in all groups and was not significantly different compared with the control group. Caecal tissue and content weights were significantly increased by feeding RG or HRG, although other organ and tissue weights were not significantly different among the groups. In conclusion, RG and HRG consist of small amounts of glucose and digestible and non-digestible oligosaccharides, and large amounts of glucose polymers, which were hardly hydrolysed by α-amylase and small intestinal enzymes. RG and HRG, which were developed newly as dietary fibre materials, had no harmful effects on the growth and development of rats.

Characteristic hydrolyzing of megalosaccharide by human salivary α-amylase and small intestinal enzymes, and its bioavailability in healthy subjects.

The digestibility of Megalosaccharide® (newly developed carbohydrate comprising α-1,4-glucosaccharide) was investigated in vitro and in vivo. Isomaltosyl-megalosaccharide® (IMS) and nigerosyl-megalosaccharide® (NMS) contain 20% and 50% of the megalosaccharide fraction (degree of polymerization (DP) 10-35), respectively. IMS was hydrolyzed readily by α-amylase to oligosaccharides (DP ≤ 7), and a small amount of glucose was produced from oligosaccharides by small intestinal enzymes (SIEs). NMS was partially hydrolyzed by α-amylase to oligosaccharides, and a small amount of glucose produced by SIEs. When IMS and NMS were treated by SIEs after treatment with human saliva α-amylase for a few minutes, IMS and NMS were hydrolyzed readily to glucose. Plasma levels of glucose and insulin upon ingestion of 50 g of IMS or NMS were elevated the same as those for 50 g of glucose, and breath hydrogen was not excreted. These results suggest that IMS and NMS are digestible carbohydrates.

Fructooligosaccharide feeding during gestation to pregnant mice provided excessive folic acid decreases maternal and female fetal oxidative stress by increasing intestinal microbe-derived hydrogen gas.

Fructooligosaccharide (FOS) is fermented by intestinal microbes to generate intestinal microbe-derived hydrogen gas (IMDH). Oxidative stress increases during gestation, whereas hydrogen gas has antioxidant effects with therapeutic benefits. We have previously reported that the offspring from a pregnant, excessive folic acid mouse model (PEFAM) had abnormal glucose metabolism after growth. We hypothesized that IMDH by FOS feeding during gestation in PEFAM would suppress maternal and fetal oxidative stress. C57BL/6J mice on day 1 of gestation were divided into 3 groups and dissected at gestational day 18. The control (CONT) diet was AIN-93G containing folic acid 2 mg/kg diet; PEFAM was fed with an excessive folic acid (EFA) diet containing folic acid 40 mg/kg diet, and the EFA-FOS diet was replaced half of the sucrose in the EFA diet. Hydrogen gas concentrations in maternal livers and whole fetuses in EFA-FOS were significantly higher than those in CONT and EFA, respectively (P < .05). Maternal and fetal 8-hydroxy-2'-deoxyguanosine in EFA-FOS were not significantly different from those in the CONT group, whereas those in the EFA group were significantly increased compared with CONT and EFA-FOS (P < .05). In EFA-FOS, expression of protein and mRNA of superoxide dismutase and heme oxygenase 1 in mothers and superoxide dismutase in fetuses were not significantly different from those in CONT, whereas those in EFA were significantly increased (P < .05). The protein expression of Nrf2 in mothers and fetuses were not significantly different between EFA-FOS and CONT. Therefore, FOS feeding to PEFAM during gestation decreases maternal and fetal oxidative stress through IMDH.

Erythema multiforme induced by sorafenib for metastatic renal cell carcinoma.

OBJECTIVE: Sorafenib is one of the few standard agents for metastatic renal cell carcinoma. Although sorafenib-induced erythema multiforme is rarely reported, we evaluated the cases of erythema multiforme induced by sorafenib for metastatic renal cell carcinoma. METHODS: From November 2006 to November 2011, 36 eligible patients who had been treated with sorafenib were enrolled in this study. Patients received sorafenib 200 or 400 mg orally, twice daily, at 12 h intervals, on a continuous dosing schedule. All patients who experienced rash or erythema multiforme underwent a skin biopsy, and the histopathological diagnosis was confirmed. RESULTS: Twenty-eight patients (78%) experienced a skin reaction of any toxicity grade. Hand-foot skin reactions occurred in 17 (47%), erythema multiforme in 9 (25%), rash/desquamation in 6 (17%) and alopecia in 9 (25%). Skin biopsy was performed and histopathological diagnosis was confirmed for all nine patients (25%) who experienced erythema multiforme. All nine showed a positive reaction to sorafenib on a subsequent patch test. CONCLUSIONS: Sorafenib-induced erythema multiforme may not be rare in Japanese patients. Patients who once showed erythema multiforme after sorafenib treatment are never to be treated with sorafenib again. Patients treated with sorafenib should be monitored carefully, with a multidisciplinary approach. Consultation with a dermatologist is critical because some cases quickly become severe.

Treatment of intractable skin ulcers caused by vascular insufficiency with allogeneic cultured dermal substitute: a report of eight cases.

Chronic leg ulcers have various causes and can be difficult to treat, although topical treatments, including basic fibroblast growth factor and PGE1, have been used. We applied an allogeneic cultured dermal substitute (CDS) to eight patients with intractable ulcers. The patients had various underlying diseases, including diabetes mellitus, systemic lupus erythematosus, antiphospholipid syndrome, necrobiosis lipoidica, stasis dermatitis, livedo vasculopathy, and rheumatoid arthritis. The CDS was prepared by seeding cultured human fibroblasts on a spongy matrix consisting of hyaluronic acid and atelocollagen. Good clinical results were achieved, as demonstrated by reepithelization, healthy granulation tissue formation, and a subsequent decrease in wound size. Daily dressing changes became unnecessary when the allogeneic CDS was used. Based on these results, we suggest that CDS may be useful for the treatment of intractable skin ulcers.

Metabolic Fate and Expectation of Health Benefits of [U-14C]-Sucrose Inhibited from Digestion Using Morus alba Leaf Extract.

Morus alba leaf extract (MLE), a strong inhibitor of sucrase, suppresses blood glucose elevation following sucrose ingestion. To investigate that sucrose inhibited from digestion using MLE is utilized through gut microbiota, [U-14C]-sucrose solutions with or without MLE were administered orally to conventional and antibiotic-treated rats, and the excretion of 14CO2 and H2 produced by gut microbiota was measured for 24 h. After an administration of [U-14C]-sucrose to conventional rats, the unit excreted 14CO2 peaked at 4 h, and the cumulative 14CO2 excreted over 24 h was approximately 60% of the radioactivity administered. No H2 was excreted. Following an administration of [U-14C]-sucrose and MLE in conventional rats, the unit excreted 14CO2 peaked later, at 8 h, and was significantly lower (p<0.05). The cumulative 14CO2 excreted over 24 h was equal in both groups, although there was a time lag of 2-3 h in rats given [U-14C]-sucrose and MLE. The amount of H2 excreted by these rats peaked 8 h after administration. Following the administration of [U-14C]-sucrose and MLE to antibiotic-treated rats, the unit excreted 14CO2 peaked lower, and the cumulative 14CO2 excretion over 24 h was approximately 40%. In this group, H2 was minimally excreted. H2 and 14CO2 produced by gut microbiota were excreted simultaneously. In conclusion, sucrose inhibited from digestion using MLE was fermented spontaneously by gut microbiota and was not excreted into feces. In addition, it confirmed that H2 excretion could be used directly to indicate the degree of fermentation of nondigestible carbohydrates.

Nestin expression in Bowen's disease and Bowen's carcinoma associated with human papillomavirus.

Human papillomaviruses (HPVs) have been detected in lesions of Bowen's disease (BD) and Bowen's carcinoma (BC); the invasive tumor retains the cytological characteristics of BD. Previous reports suggest that nestin-expressing hair follicle stem cells are undifferentiated and pluripotent, and nestin expression in some tumors indicates poor differentiation and high grade of malignancy. We identified HPV-DNA in BD (n=25) and BC (n=23) by in situ hybridization (ISH) analysis with INFORM(®) HPV III (Ventana Medical Systems. AZ, USA) and determined nestin expression by indirect immunohistochemical staining with anti-nestin polyclonal antibody (IBL, Gunma, Japan). We detected HPV-DNA in 68% of BD and in 87% of BC. In BD, 13 cases demonstrated the punctuate pattern, and four showed nestin expression. In BC, 19 cases showed the punctuate pattern and 16 showed nestin expression. HPV-DNA integrates into the host genome, and this is observed as the punctuate pattern on ISH. The nestin expression was statistically high in group of BC than BD (P<0.01). These results therefore suggest that HPV-DNA integrated in the genome of tumor cells of these diseases and contributed to malignant alteration. From the standpoint of tumorigenesis, BC might represent one type of poorly differentiated, high-grade squamous cell carcinoma.

Prognostic significance of the hair follicle stem cell marker nestin in patients with malignant melanoma.

Nestin is an intermediate filament protein, and serves as a hair follicle stem cell and neural stem cell marker. Recent studies have suggested that nestin expression is also important for tumorigenesis. Previous reports from our laboratory have revealed that nestin is a marker of HMB-45-negative melanoma cells in dermal invasive lesions of nodular malignant melanoma. The present study examines nestin expression in malignant melanoma and investigates the relationship between nestin expression and prognosis in patients. We immunohistochemically stained 78 formalin-fixed and paraffin-embedded malignant melanomas for nestin, HMB-45 and S100 reactivity. We found that nestin, HMB-45 and S100 protein were detected in 56.5%, 88.4% and 100% of malignant melanomas, respectively. The 5-year survival rate of stage I and II nestin-positive cases was significantly decreased compared to the nestin-negative cases (p < 0.05). In addition, the 5-year survival rate exceeded 80% in nestin-negative malignant melanomas at all stages of tumor development. We conclude that nestin expression may be a predictor of poor prognosis in patients with malignant melanoma.

Prebiotic effect of fructo-oligosaccharides on the inner ear of DBA/2 J mice with early-onset progressive hearing loss.

Nutrition and dietary habits contribute to the onset and progression of sensorineural hearing loss (SNHL). Fructo-oligosaccharides (FOS) are non-digestible oligosaccharides and are known as prebiotics, which enhance short-chain fatty acid (SCFA) production and antioxidant activity. Although a substantial number of studies have shown that FOS play a role in the prevention of lifestyle-related diseases as prebiotics, little is known about the effects on the inner ear. The purpose of this study is to investigate the effect of FOS on gene expression and spiral ganglion neuron (SGN) protection in the inner ear of DBA/2 J mice, which is a model for early-onset progressive hearing loss. DBA/2 J mice were fed either control diet or FOS diet contained 10% (w/w) of FOS for 8 weeks. Analysis of mice fed the FOS diet revealed a change in intestinal flora including an inversion of the ratio of Bacteroidetes and Firmicutes, which was followed by a significant increase in SCFAs in the cecum and a decrease in an oxidative stress marker in the serum. In the inner ear, gene expression of neurotrophin, brain-derived neurotrophic factor (BDNF), its receptor, tyrosine kinase receptor b (Trkb), and the SCFA receptor, free fatty acid receptor 3 (FFAR3), were increased by FOS. In addition, the survival rate of SGNs in the inner ear was maintained in FOS-fed mice. Altogether, these results suggest that a compositional variation of the intestinal flora due to a prebiotic effect may be involved in the progression of SNHL.

Metabolic fate of newly developed nondigestible oligosaccharide, maltobionic acid, in rats and humans.

Maltobionic acid (MA), formed by a gluconic acid and glucose linked by an α-1,4 bond, may have the properties of a nondigestible oligosaccharide. The objective of this study was to elucidate the bioavailability of MA in rats and humans by observing digestion of MA by small intestinal enzymes, the fermentation of MA by gut microbiota, and the effect of adaptation following prolonged ingestion of MA. MA digestion was assessed using brush border membrane vesicles (BBMV) from rat small intestine. A within-subject repeated measures design was used for ingestion experiments in 10 healthy female participants. After MA ingestion, postprandial plasma glucose and insulin levels, breath hydrogen excretion, and urinary MA were measured. The effect of adaptation following prolonged MA ingestion was investigated in rats. MA was minimally hydrolyzed by BBMV. Ingestion of 10 g of MA by healthy females did not elevate postprandial plasma glucose and insulin levels. Breath hydrogen and urinary MA were negligibly excreted over 8 hr following ingestion. Adaptation to prolonged MA ingestion produced no significant difference in exhaled hydrogen levels over 8 hr following administration compared with controls. MA is a new food material that is highly resistant to digestion and fermentation. It expresses the characteristics of a nondigestible oligosaccharide, including being low energy, improving the flavor of food and juice, and mineral solubilization.

Effects of cake made from whole soy powder on postprandial blood glucose and insulin levels in human subjects.

We investigated the glycemic index (GI) and the insulinemic index (II) of cake made from whole soy powder (SBC) and the suppressive effects of SBC on the postprandial blood glucose and insulin by other carbohydrate foods. Furthermore, breath hydrogen excretion was simultaneously investigated. Twenty subjects were given 114 g SBC, 144 g cooked paddy-rice, and 60 g SBC with 144 g cooked paddy-rice in random order using a within-subject, repeated-measures design. Blood and end-expiratory gas were collected at the indicated periods after ingestion. The GI and the II of SBC were 22+/-6 and 48+/-29, respectively. The elevation of blood glucose by cooked paddy-rice was significantly suppressed by the addition of 60 g SBC, although the insulin secretion did not decrease. Breath hydrogen excretion by the addition of SBC to 144 g cooked paddy-rice was not significantly increased in comparison with cooked paddy-rice alone. SBC was of low GI and low II, but the postprandial insulin secretion in response to cooked paddy-rice was not suppressed.

Dietary Fructooligosaccharide and Glucomannan Alter Gut Microbiota and Improve Bone Metabolism in Senescence-Accelerated Mouse.

Gut microbiota improved using prebiotics may delay the onset of senescence-related health problems. We hypothesized that prolonged intake of prebiotics delays senile osteoporosis. Forty-five male senescence-accelerated mouse prone 6 (SAMP6) aged four weeks were raised on 5% fructooligosaccharide (FOS), 5% glucomannan (GM), or a control diet for 31 weeks. Gut microbiota were identified using culture-dependent analytical methods. Mineral content in femoral bone was analyzed using atomic absorption spectrophotometry. Bone metabolism and inflammatory markers were measured using enzyme-linked immunosorbent assay. The numbers of Lactobacillus and Bacteroides in cecal contents were significantly higher in the FOS than in the control group ( p < 0.05); the number of Clostridium was significantly higher in the GM than in the control group ( p < 0.05). Calcium content was significantly higher in the femoral bones of the FOS group (30.5 ± 0.8 mg) than in the control group (27.5 ± 1.5 mg) ( p < 0.05). There was no difference between the GM (29.1 ± 2.0 mg) and control groups. During senescence, urinary deoxypyridinoline and serum high-sensitivity C-reactive protein levels significantly decreased in the FOS (1.2 ± 0.2 nmol/3 d and 80 ± 6.1 ng/100 mL) and GM groups (1.2 ± 0.2 nmol/3 d and 80 ± 6.1 ng/100 mL) compared with the control group (1.8 ± 0.5 nmol/3 d and 93 ± 7.4 ng/100 mL) ( p < 0.05). Thus, dietary FOS and GM modified gut microbiota and reduced bone resorption by reducing systemic inflammation in SAMP6.

Metabolism and bioavailability of newly developed dietary fiber materials, resistant glucan and hydrogenated resistant glucan, in rats and humans.

BACKGROUND: Resistant glucan (RG) and hydrogenated resistant glucan (HRG) are new dietary fiber materials developed to decrease the risk of metabolic syndrome and lifestyle-related diseases. We investigated the metabolism and bioavailability of RG and HRG using rats and humans. METHODS: Purified RG and HRG were used as test substances. After 25 Wistar male rats (270 g) were fed with an experimental diet (AIN93M diet with the cellulose replaced by ß-corn starch) ad libitum for 1 week, they were used for the experiment involving blood collection and circulating air collection. Ten participants (5 males, 22.5 y, BMI 20.4 kg/m(2); 5 females, 25.8 y, BMI 20.9 kg/m(2)) voluntarily participated in this study. The study was carried out using a within-subject, repeated measures design. Effects of RG and HRG on the response for blood glucose and insulin and hydrogen excretion were compared with those of glucose and a typical nondigestible and fermentable fructooligosaccharide (FOS) in rats and humans. Available energy was evaluated using the fermentability based on breath hydrogen excretion. RESULTS: When purified RG or HRG (400 mg) was administered orally to rats, blood glucose and insulin increased slightly, but less than when glucose was administration (P < 0.05). Hydrogen started to be excreted 120 min after administration of RG with negligibly small peak at 180 min, thereafter excreted scarcely until 1440 min. Hydrogen excretion after HRG administration showed a larger peak than RG at 180 min, but was markedly less than FOS. RG and HRG were excreted in feces, but not urine. When purified RG or HRG (30 g) were ingested by healthy humans, blood glucose and insulin levels increased scarcely. Breath hydrogen excretion increased slightly, but remarkably less than FOS. Ingestion of purified RG or HRG (5 g) to evaluate available energy, increased scarcely glucose and insulin levels and breath hydrogen excretion. Available energy was evaluated as 0 kcal/g for purified RG and 1 kcal/g for HRG. CONCLUSION: The bioavailability was very low in both humans and rats, because oligosaccharide of minor component in purified RG and HRG was metabolized via intestinal microbes but major components with higher molecular weight were metabolized scarcely. Moreover, the ingestion of 30 g of RG or HRG did not induce apparent acute side effects in healthy adults. RG and HRG might potentially be used as new dietary fiber materials with low energy.

Non-pigmented fixed drug eruption induced by eprazinone hydrochloride.

A 68-year-old woman developed an upper respiratory tract infection in November 2002 and was treated with eprazinone hydrochloride, serrapeptase, carbocysteine and clarithromycin. Three days after the start of treatment, the patient noted erythema on her axilla, buttock and inguinal regions. The erythema subsided in 7 days although slight pigmentation remained. However, 7 days later the pigmentation completely disappeared. Oral eprazinone hydrochloride was given as a challenge, and 1 day later the erythema re-appeared in the same areas as on initial presentation (axilla, buttock, and inguinal regions). A fixed erythema without lasting pigmentation is attributed to eprazinone hydrochloride. Therefore, the patient was diagnosed as having a nonpigmented fixed drug eruption associated with eprazinone hydrochloride.