Effect of platelet lysate on Schwann-like cell differentiation of equine bone marrow-derived mesenchymal stem cells.

Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 106/µl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time PCR analysis of the expression levels of neuronal markers showed that the ePL group tended to express higher levels of Nestin, Musashi1, and Pax3 than the FBS group. p75 was expressed at low levels in both groups. Immunostaining results showed localization of Nestin in both groups of differentiated cells, but the positive cell rate was significantly higher in the ePL group than in the FBS group. Overall, the ePL gro showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease. This knowledge could be applied translationaly in the treatment of amyotrophic lateral sclerosis in humans.Overall, the ePL group showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease and in the treatment of amyotrophic lateral sclerosis in humans.

Different pharmacoresistance of focal epileptic spasms, generalized epileptic spasms, and generalized epileptic spasms combined with focal seizures.

OBJECTIVE: Among standard treatments for infantile spasms, adrenocorticotropic hormone (ACTH) is reported as the best treatment, but ACTH is ineffective in one-half of the patients. To establish precision medicine, we examined pharmacoresistance of focal epileptic spasms (ES), generalized ES, and generalized ES combined with focal seizures, diagnosed based on the revised seizure classification of ILAE in 2017. METHODS: We conducted a retrospective nationwide study in Japan on the long-term seizure outcome of ES. Long-term seizure outcome was evaluated by seizure-free rate, seizure-free period, and Kaplan-Meier curve. Seizure-free was defined as seizure control for longer than 2 months. RESULTS: From the medical history of 501 patients, 325 patients had generalized ES only (GES group) at the start of the first treatment, 125 patients had generalized ES after focal seizure onset (FS-GES group), seven patients had focal ES after focal seizure onset (FS-FES group), and 24 patients had generalized ES combined with focal seizures after focal seizure onset (FS-GES + FS group). Seizure-free period of ES (generalized ES and focal ES) [mean (95% confidence interval)] was 2.7 (0.0-5.4) months in GES group, 1.1 (0.1-2.2) months in FS-GES group, 1.0 (0.2-1.9) months in FS-GES + FS group, and 0.1 (-0.2-0.5) months in FS-FES group. Seizure-free rate, seizure-free period, and Kaplan-Meier curve of generalized ES were almost the same in GES group and FS-GES group, with characteristics of superior response to ACTH. Mean seizure-free period of generalized ES combined with focal seizures was significantly shorter in FS-GES + FS group than in GES group. Mean seizure-free period of focal ES in FS-FES group was extremely short with exceedingly early relapse. SIGNIFICANCE: Pharmacoresistance was different in generalized ES, focal ES, and generalized ES combined with focal seizures. ES with focal features or with focal seizures may have focal lesions, thus consider surgical options earlier in the course.

Effect of a novel rice fermented extract on gastric ulcers in horses.

Gastric ulcers cause appetite loss, poor body condition, and colic in horses. This study investigated the protective effect of a rice fermented extract on the gastric mucosa in 17 healthy Thoroughbreds. For one month, horses in the rice fermented extract (nine horses) and control (eight horses) groups were orally administered a rice fermented extract (100%; 0.2 ml/kg, SID) and tap water (0.2 ml/kg), respectively. Gastric endoscopic images were obtained before and one month after rice fermented extract administration. The gastric ulcer score was lower after administration (median, 1; maximum, 2; minimum, 1) than before administration (median, 4; maximum, 4; minimum, 3) in the rice fermented extract group (P<0.05). In conclusion, the administration of a rice fermented extract for one month improves gastric mucosal lesions in Thoroughbreds with gastric ulcers.

Identification of Maple Anthocyanin and its Antiproliferative Activity against LLC, T47D and C3H10T1/2 Cells.

BACKGROUND: The genus Acer contains around 200 species, with more than 400 garden varieties. There is considerable diversity in these species and garden varieties, and each can be characterized by morphology and chemical composition. The red appearance of Acer leaves is due to anthocyanin compounds, including cyanidin glycosides, delphinidin glycosides, and galloylated anthocyanins. Few studies have investigated the diversity of anthocyanin compounds in garden varieties, and no studies have examined the pharmacological effects of these compounds. OBJECTIVE: The purpose of this study was to identify the anthocyanins of Acer palmatum cv. 'Chishio', a garden variety of A. palmatum and evaluate their antiproliferative and antioxidant activities. METHODS: A methanol extract of fresh leaves was partitioned with ethyl acetate. The extract was purified by column chromatography and compounds were subsequently identified by 1H and 13C NMR and ESI-HRMS. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt (MTS) colorimetric assay. The antioxidant assay was evaluated by scavenging activity using the stable radical DPPH. RESULTS: The anthocyanins cyanidin-3-O-(6''-O-α-rhamnopyranosyl- ß-glucopyranoside), cyanidin-3-O- ß- glucopyranoside, cyanidin-3-O-[2''-O-(galloyl)-6''-O-(rhamnosyl)-ß-glucoside], and cyanidin-3-O-[2''-O-(galloyl)- ß-glucopyranoside] were isolated from A. palmatum cv. 'Chishio'. All four anthocyanin compounds showed antiproliferative activity against LLC and T47D cells, and galloylated anthocyanin showed antiproliferative activity against C3H10T1/2 cells. All four anthocyanins inhibited the activity of DPPH radicals in a dosedependent manner. CONCLUSION: Maple anthocyanins could be a new cancer therapeutic agent or prophylactic medicine.

Anthocyanins in perilla plants and dried leaves.

High-quality perilla leaves are purple on upper and lower surfaces and have a good aroma. The Japanese Pharmacopoeia specifies the content of essential oils in perilla leaves but not that of anthocyanins. Several reports have described the chemical species of anthocyanins in red perilla, but a complete analysis of anthocyanins in perilla has not been reported. In this study, the anthocyanins in the leaves of cultivated and wild species of perilla and those in commercially available perilla herbs were studied. Red perilla and most P. citriodora strains accumulate cyanidin derivatives that differ in the acyl group on the glucose moiety at the 3-O- and 5-O-positions of the anthocyanins. Several strains of P. citriodora contain cyanidin derivatives that are different from those in red perilla and most P. citriodora species. Green perilla and wild species other than P. citriodora do not contain foliar anthocyanins. The anthocyanins in commercially available perilla herbs and natural dyes made from red perilla were in agreement with those in fresh red perilla leaves and most P. citriodora samples. The amounts and types of anthocyanins were not associated with place of cultivation, although some changes occurred due to degradation during storage. These results provide clues regarding the biosynthesis of anthocyanins in perilla and the evolution of red perilla. The characteristics and stability of anthocyanins are discussed.

Molecular cloning and characterization of a Perilla frutescens cytochrome P450 enzyme that catalyzes the later steps of perillaldehyde biosynthesis.

Perilla produces the cyclohexanoid monoterpene perillaldehyde as a major constituent of an essential oil that is accumulated in its glandular trichomes. Perillaldehyde is a marker compound for quality control of soyo and has biological activities such as antibacterial, sedative, or vasodilatory effects. The predicted perillaldehyde formation involves the cyclization of geranyl diphosphate, hydroxylation, and oxidation, and cytochrome P450 plays a crucial role in perillaldehyde biosynthesis. In this study, a cytochrome P450-type enzyme with perillyl alcohol and perillaldehyde synthase activities was isolated by analyzing an expressed sequence tag library from several oil types of pure lines of perilla. A recombinant protein with a sequence that was highly specific for the type of perillaldehyde was expressed in Saccharomyces cerevisiae and evaluated by an in vitro enzymatic reaction. The recombinant protein catalyzed the hydroxylation and oxidation of limonene to perillyl alcohol and perillaldehyde. Cytochrome P450 limonene-7-hydroxylase cDNA from Perilla frutescens has been previously isolated. The cytochrome P450 isolated in this study shares 37% amino-acid identity with the previously isolated enzyme; however, it may have different characteristics.

Prevalence of oral health-related conditions that could trigger accidents for patients with moderate-to-severe dementia.

OBJECTIVE: This study was performed to determine the prevalence of oral health conditions unnoticed by doctors and ward staff that may increase risk of incidents and/or accidents in hospitalised patients with moderate-severe dementia. BACKGROUND DATA DISCUSSING THE PRESENT STATUS OF THE FIELD: Dementia patients may not recognise risks in the mouth, such as tooth mobility or ill-fitting dental prostheses and/or dentures. In addition to the risk of choking, injury by sharp edges of collapsed teeth or prosthodontics could pose risks. However, many previous publications were limited to case reports or series. MATERIALS AND METHODS: Ninety-two consecutive hospitalised dementia patients (M: 52, F: 40, median age: 82.5 years, range: 62-99 years, from 2011 to 2014), referred for dentistry for dysphagia rehabilitation, were enrolled in this study. Participants referred for dental treatment with dental problems detected by ward staff were excluded. All participants had a Global Clinical Dementia Rating Score >2. Their dental records were evaluated retrospectively for issues that may cause incidents and/or accidents. RESULTS: Problems in the mouth, for example tooth stumps, dental caries, and ill-fitting dentures, were detected in 51.1% of participants (47/92). Furthermore, 23.9% (22/92) showed risk factors that could lead to incidents and/or accidents, for example falling out of teeth and/or prosthodontics or injury by sharp edges of teeth and/or prosthodontics. CONCLUSIONS: Hospitalised moderate-severe dementia patients had a high prevalence of oral health conditions unnoticed by doctors and ward staff that may increase risk of incidents and/or accidents.

Synergistic effect of fragrant herbs in Japanese scent sachets.

The sedative activity of eight aromatic natural medicines that are traditionally used in Japanese scent sachets was examined using an open field test with mice. Galangal (Kaempferia galanga), patchouli (Pogostemon cablin), sandalwood (Santalum album), spikenard (Nardostachys chinensis), cinnamon (Cinnamomum cassia), clove (Syzygium aromaticum), star anise (Illicium verum), and borneol (Dryobalanops aromatica) distilled oils were used. These natural medicines have various pharmacological effects. For example, galangal has insecticidal activity and clove extracts possess strong total antioxidant activity. Aromatherapy, a well-known complementary medicine system that uses inhalation, has recently attracted much attention. The sedative activity of inhaled aromatic compounds or essential oils has been examined by measuring the spontaneous motor activity of mice in an open field test. The galangal, patchouli, sandalwood, spikenard, and borneol oils showed significant sedative effects. The effect was stronger for a mixture of the five oils than for any of the single oils. This suggests that the oil mixture may have synergistic activity. Sedative activity was not observed when inactive oils (cinnamon, clove, and star anise) were added to the mixture of the five active oils.

PCDH19-related female-limited epilepsy: further details regarding early clinical features and therapeutic efficacy.

Abnormalities in the protocadherin 19 (PCDH19) gene cause early-onset epilepsy exclusively in females. We aimed to explore the genetic and clinical characteristics of PCDH19-related epilepsy by focusing on its early features and treatment efficacy. PCDH19 was analyzed in 159 Japanese female patients with early-onset epilepsy via direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis. We identified 17 patients with PCDH19 abnormalities: point mutations were observed in 14 patients and whole PCDH19 deletions were detected in 3 patients. One affected sister of a proband with a mild phenotype was also analyzed. The frequency of PCDH19 deletion among all probands identified in Japan was 12.5% (3/24, including 7 probands reported previously by us). Clinical features included early onset (mean age at onset, 8.6 months), recurrent clusters of brief seizures (17/18), fever sensitivity (18/18), tonic seizures (13/18, probably including focal tonic seizures), tonic-clonic seizures (8/18), focal seizures often with subsequent generalization (17/18), intellectual disabilities (15/18), and autistic traits (13/18). Three patients exhibited delay in motor milestones before seizure onset. In 16 patients, seizures appeared in clusters from the onset of the disease. Among 6 patients for whom detailed information at onset was available, 2 onset patterns were identified: a biphasic course of short seizure clusters (each within days) in 2 patients and a prolonged course of clusters (from weeks to a month) in 4 patients. In both cases, initial seizures started during fever and transiently disappeared with the decline of fever; however, afebrile clusters recurred. In the former patients, motor development was delayed before onset, and seizures appeared in strong clusters from the onset of the disease. In the latter patients, initial development was normal and initial seizures were mild, but were followed by strong clusters lasting several weeks, even without fever. Treatment using phenytoin, potassium bromide, and clobazam showed high efficacy. Although focal seizures were the main feature in PCDH19-epilepsy, the efficacy of carbamazepine was poor. This study highlighted the significance of PCDH19 deletion, a unique pattern of initial seizure clusters, and the efficacy of antiepileptic drugs. Our data will facilitate early diagnosis and development of a treatment strategy for better clinical management of patients with PCDH19-related epilepsy.

[Clinical aspects of epilepsy in children with periventricular leukomalacia].

UNLABELLED: We investigated the frequency and characteristics of epilepsy in 63 children (39 males and 24 females) with cerebral palsy caused by periventricular leukomalacia, who were born preterm at <34 weeks' gestation and followed for more than five years (duration: 5-18 years, mean: 9.6 years). While seven (11%) of the 63 patients had febrile convulsions (FC), 11 (17%) were associated with symptomatic localization-related epilepsy (SLRE) and 8 (13%) with West syndrome (WS). The gestational ages of the WS group were significantly (p<0.05) longer than in FC group. The DQ of the SLRE and WS groups were significantly (p<0.01) lower than in the N-S group. The frequency of spastic quadriplegia was 19%, 29%, 36%, 50% in the N-S, FC, SLRE, WS groups, respectively. Among the 11 SLER patients, 5 had one seizure type, while 3 had two and 3 had three seizure types. The seizure patterns included complex partial seizures (CPS) in 8, secondarily generalized partial epileptic seizures in 8, and simple partial seizures in 4. One patients in the WS group developed CPS, and another patient developed epilepsy undetermined after infancy. Regarding the main localizing symptoms of SLRE, oculogyric seizures were observed in 7 patients and hemi-facial seizures were observed in 8 patients. In all WS patients, the location of the epileptiform discharges was in the parieto-occipital area, while 8 of 11 patients with SLES had it in the central area. IN CONCLUSION: 30% of all patients with PVL were associated with epilepsy. WS developed in 13% during early infancy and SLRE developed in 17% after infancy. The most common epileptic seizure in the patients with PVL was complex partial seizure.

Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka (Oryzias latipes).

Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YP1 and YP2 on gel filtration were 270 and 380 kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YP1 and YP2 were similar to those of Vg1 and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YP1 formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YP1 is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17beta (E2). The measurable range of both CLIAs was from 0.975 to 1000 ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (

Carp (Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay.

Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp.

Purification and identification of a second form of vitellogenin from ascites of medaka (Oryzias latipes) treated with estrogen.

Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1.