Heterologous expression and characterization of an Arabidopsis ß-l-arabinopyranosidase and α-d-galactosidases acting on ß-l-arabinopyranosyl residues.

The major plant sugar l-arabinose (l-Ara) has two different ring forms, l-arabinofuranose (l-Araf) and l-arabinopyranose (l-Arap). Although l-Ara mainly appears in the form of α-l-Araf residues in cell wall components, such as pectic α-1,3:1,5-arabinan, arabinoxylan, and arabinogalactan-proteins (AGPs), lesser amounts of it can also be found as ß-l-Arap residues of AGPs. Even though AGPs are known to be rapidly metabolized, the enzymes acting on the ß-l-Arap residues remain to be identified. In the present study, four enzymes, which we call ß-l-ARAPASE (APSE) and α-GALACTOSIDASE 1 (AGAL1), AGAL2, and AGAL3, are identified as those enzymes that are likely to be responsible for the hydrolysis of the ß-l-Arap residues in Arabidopsis thaliana. An Arabidopsis apse-1 mutant showed significant reduction in ß-l-arabinopyranosidase activity, and an apse-1 agal3-1 double-mutant exhibited even less activity. The apse-1 and the double-mutants both had more ß-l-Arap residues in the cell walls than wild-type plants. Recombinant APSE expressed in the yeast Pichia pastoris specifically hydrolyzed ß-l-Arap residues and released l-Ara from gum arabic and larch arabinogalactan. The recombinant AGAL3 also showed weak ß-l-arabinopyranosidase activity beside its strong α-galactosidase activity. It appears that the ß-l-Arap residues of AGPs are hydrolysed mainly by APSE and partially by AGALs in Arabidopsis.

Translational research into species differences of endocrine toxicity via steroidogenesis inhibition by SMP-028--for human safety in clinical study.

SMP-028 is a drug candidate developed for the treatment of asthma. In a 13-week repeated dose toxicity study of SMP-028 in rats and monkeys, differences of endocrine toxicological events between rats and monkeys were observed. In rats, these toxicological events mainly consisted of pathological changes in the adrenal, testis, ovary, and the other endocrine-related organs. On the other hand, in monkeys, no toxicological events were observed. The goal of this study is to try to understand the reason why only rats, but not monkeys, showed toxicological events following treatment with SMP-028 and to eventually predict the possible toxicological effect of this compound on human endocrine organs. Our results show that SMP-028 inhibits neutral cholesterol esterase more strongly than other steroidogenic enzymes in rats. Although SMP-028 also inhibits monkeys and human neutral cholesterol esterase, this inhibition is much weaker than that of rat neutral cholesterol esterase. These results indicate (1) that the difference in endocrine toxicological events between rats and monkeys is mainly due to inhibition of steroidogenesis by SMP-028 in rats, not in monkeys, and (2) that SMP-028 may not affect steroidogenesis in humans and therefore might cause no endocrine toxicological events in clinical studies.

Quantitative analysis of the skull in the Japanese wolf (Canis lupus hodophilax) using CT.

In this study using computed tomography (CT), the volumes of the internal cranial cavities, such as the braincase, frontal sinus and tympanic cavity, and the ratio of the volume of each cavity to the skull volume in Japanese wolves were quantified, and CT images of the frontal sinus were observed. The results were then compared with those of other wolf subspecies, including Akita, a dog breed, to clarify the characteristics of the internal cranial cavities in Japanese wolves. The present study revealed that the Japanese wolf had a relatively larger braincase volume and a relatively smaller frontal sinus volume than the wolf ssp. (a group of wild wolf subspecies except the Japanese wolf) and Akita. Moreover, the relative and absolute tympanic cavity volumes of the Japanese wolf and Akita were significantly smaller than those of the wolf ssp. In the CT image or macroscopic observations, the frontal sinuses of the wolf ssp. and Akita were relatively well developed to the caudal and dorsal directions, respectively, compared with that of the Japanese wolf, and the tympanic cavity of the wolf ssp. was more largely swelled ventrally and medially than that of other groups.

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord.

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.

Hepatic Squamous Cell Carcinoma Diagnosed by Endoscopic Ultrasound-Guided Fine-Needle Aspiration.

Primary hepatic squamous cell carcinoma (SCC) is a rare malignancy with aggressive clinical features. This is the first case report of a primary hepatic SCC diagnosed by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), which is a reliable and safe procedure for the histopathological diagnosis of liver lesions, even if the percutaneous approach is difficult due to ascites or hypervascularity at the puncture site. A 52-year-old man presented to the emergency department of a tertiary referral hospital with right upper quadrant abdominal pain and abdominal distention. Given the laboratory data, a diagnosis of spontaneous bacterial peritonitis (SBP) was made. Concurrently, an abdominal computed tomography (CT) scan revealed an 8 cm hypodense mass with delayed peripheral enhancement in the left hepatic lobe and paraaortic and perihepatic lymphadenopathy. As persistent ascites precluded percutaneous liver biopsy, we performed EUS-FNA of the liver mass, and the obtained specimen showed SCC. As otorhinolaryngological consultation and whole-body investigations, including chest CT, upper and lower endoscopy, and positron emission tomography CT, were all unremarkable except for the liver lesion and lymph nodes, a diagnosis of primary hepatic SCC with systemic lymph node metastasis was made. After treatment of SBP with antibiotics, we initiated chemotherapy concurrent with radiation therapy, adapted to his liver function. Radiation and three cycles of chemotherapy were not effective as the disease progressed, as seen on the follow-up CT scan, and the patient died of hepatic failure on the 134th day after diagnosis. In conclusion, EUS-FNA was a reliable method for tissue sampling in liver malignancies, particularly in selected patients with contraindications for percutaneous biopsy.

Effects of nafamostat mesilate on 5-hydroxytryptamine release from isolated ileal tissues induced by anti-cancer drugs in rats.

Administration of cisplatin and methotrexate significantly increased 5-hydroxytryptamine (5-HT) release from intestinal tissues isolated at 72 h after administration in rats. Daily administration with nafamostat mesilate, a potent serine protease inhibitor, significantly inhibited the release of 5-HT induced by methotrexate, but not by cisplatin, in a dose-dependent manner. When applied to isolated ileal tissues in vitro, nafamostat mesilate also significantly inhibited the release of 5-HT induced by methotrexate, but not by cisplatin, in a concentration-dependent manner. These results suggest that serine proteases are involved in the mechanism of the methotrexate-induced release of 5-HT from the rat small intestine.

Predictors for bile duct stone recurrence after endoscopic extraction for naïve major duodenal papilla: A cohort study.

BACKGROUND: Predictors for bile duct stone recurrence after endoscopic stone extraction have not yet been clearly defined and a study investigating naïve major duodenal papilla is warranted because studies focusing only on naïve major duodenal papilla are rare. The aim of this study was to observe the long-term outcomes of endoscopic bile duct stone extraction for naïve major duodenal papilla and to assess the predictors for recurrence. METHODS: This was a retrospective cohort study that consisted of 384 patients with naïve papilla who underwent initial endoscopic bile duct stone extraction. Patients were followed up in outpatient department subsequent to complete stone clearance. Recurrence was defined as symptomatic repeated stone formation observed at least three months after the procedure. Stone recurrence, predictors of recurrence, and the recurrence rate, depending on each endoscopic treatment for major duodenal papilla, were examined. RESULTS: In this study, 34 patients (8.9%) developed stone recurrence. The median time to recurrence was 439 days. Periampullary diverticulum and multiple stones were strong predictors of bile duct stone recurrence (RR, 5.065; 95% CI, 2.435-10.539 and RR: 2.4401; 95% CI: 1.0946-5.4396, respectively). The above two factors were independent predictors of stone recurrence as per logistic regression analysis adjusted for confounders (Periampullary diverticulum: OR, 7.768; 95% CI, 3.27-18.471; multiple stones: OR, 4.144; 95% CI, 1.33-12.915). No recurrence was observed after endoscopic papillary large balloon dilatation (0/20), whereas recurrence was observed in 7 patients after endoscopic papillary balloon dilatation (7/45) and in 27 patients after endoscopic sphincterotomy (27/319). However, these differences were not statistically significant (p = 0.105). CONCLUSIONS: We determined that the presence of periampullary diverticulum and multiple stones are strong predictors for recurrence after endoscopic stone extraction. Moreover, endoscopic papillary large balloon dilatation tended to be correlated with non-recurrence of bile duct stone.

A method for estimating radioactive cesium concentrations in cattle blood using urine samples.

In the region contaminated by the Fukushima nuclear accident, radioactive contamination of live cattle should be checked before slaughter. In this study, we establish a precise method for estimating radioactive cesium concentrations in cattle blood using urine samples. Blood and urine samples were collected from a total of 71 cattle on two farms in the 'difficult-to-return zone'. Urine 137 Cs, specific gravity, electrical conductivity, pH, sodium, potassium, calcium, and creatinine were measured and various estimation methods for blood 137 Cs were tested. The average error rate of the estimation was 54.2% without correction. Correcting for urine creatinine, specific gravity, electrical conductivity, or potassium improved the precision of the estimation. Correcting for specific gravity using the following formula gave the most precise estimate (average error rate = 16.9%): [blood 137 Cs] = [urinary 137 Cs]/([specific gravity] - 1)/329. Urine samples are faster to measure than blood samples because urine can be obtained in larger quantities and has a higher 137 Cs concentration than blood. These advantages of urine and the estimation precision demonstrated in our study, indicate that estimation of blood 137 Cs using urine samples is a practical means of monitoring radioactive contamination in live cattle.

International collaborative survey on epidemiology of hepatitis E virus in 11 countries.

We conducted seroepidemiological studies on antibody prevalence to hepatitis E virus (HEV) in 5,233 sera from 11 countries to ascertain the present state of HEV infection on a global basis. The prevalence of anti-HEV IgG increased with age in these tested countries, but the rate of antibody positivity was over 20% in the 16-30 year-old group in most of the participating countries, except for Japan, the USA, and Spain. Of patients with acute hepatitis of unknown etiology from Nepal, 56% (14/25) were positive for the IgM class of anti-HEV antibody. In addition, HEV RNAs in the serum from 3 Nepali patients who had the IgM antibody were detected by nested PCR and all of the HEV genes isolated belonged to genotype 1. Our results indicate that HEV is spreading worldwide, not only in developing countries, but also in more industrialized countries than previously thought.

Successful Conservative Treatment of a Cholecystoduodenal Fistula Caused by a Cytomegalovirus-associated Duodenal Ulcer.

An 87-year-old woman on oral prednisolone was diagnosed with a cholecystoduodenal fistula (CDF) caused by a cytomegalovirus-associated duodenal ulcer (DU) and was managed conservatively. A CDF caused by a DU is extremely rare. Although surgical repair is recommended for the treatment of a CDF caused by cholecystolithiasis, appropriate treatment for CDF caused by a DU remains controversial. This case report of a CDF caused by a DU suggests that conservative treatment is feasible in the absence of DU-associated complications, such as an untreatable hemorrhage or obstruction; this finding is compatible with previously reported cases that were conservatively treated.

Epidermoid Cyst in an Intrapancreatic Accessory Spleen: Case Report and Literature Review of the Preoperative Imaging Findings.

An epidermoid cyst arising within an intrapancreatic accessory spleen (ECIAS) is rare, and also difficult to correctly diagnose before surgery. It is mostly misdiagnosed as a cystic tumor, such as a mucinous cystic neoplasm or as a solid tumor with cystic degeneration, such as a neuro endocrine tumor. We herein report a case of ECIAS and also perform a literature review of 35 reports of ECIAS. Although the preoperative diagnosis of ECIAS using conventional imaging is relatively difficult to make, careful preoperative examinations of the features on computed tomography and magnetic resonance imaging could lead to a correct preoperative diagnosis of ECIAS which might thereby reduce the number of unnecessary resections.

Diagnostic utility of endoscopic ultrasound-guided fine-needle aspiration biopsy for glomus tumor of the stomach.

A 52-year-old man was referred for further investigation of a gastric submucosal tumor on the greater curvature of the antrum. Endoscopic ultrasonography demonstrated a hypoechoic solid mass, which was primarily connected to the muscular layer of the stomach. We performed endoscopic ultrasound-guided fine-needle aspiration biopsy. The pathological examination showed proliferation of oval-shaped cells with nest formation, which stained strongly positive for muscle actin, and negative for c-kit, CD34, CD56, desmin, S-100, chromogranin, and neuron-specific enolase. Therefore, we performed laparoscopy and endoscopy cooperative surgery based on the preoperative diagnosis of glomus tumor of the stomach. The final histological diagnosis confirmed the preoperative diagnosis. Although preoperative diagnosis of glomus tumor of the stomach is difficult with conventional images and endoscopic biopsy, endoscopic ultrasound-guided fine-needle aspiration biopsy is an essential tool to gain histological evidence of glomus tumor of the stomach for early diagnosis.

Efficacy of repeated cytology of pancreatic juice obtained by endoscopic nasopancreatic drainage tube for early diagnosis of pancreatic cancer: a case series including a case of carcinoma in situ.

Repeated cytology of pancreatic juice obtained by endoscopic nasopancreatic drainage (ENPD) tube has been highlighted as an early diagnostic method for small pancreatic cancer, including carcinoma in situ. We report two cases of early-stage pancreatic cancer diagnosed using repeated cytology; both cases underwent curative resection. No significant masses were found on conventional imaging in either case, with only pancreatic duct strictures being observed. ENPD tubes were placed to collect pancreatic juice in both cases. In case 1, two of five pancreatic juice samples showed adenocarcinoma. Therefore, distal pancreatectomy was performed, and a PanIN3 grade neoplasm (carcinoma in situ) was identified at the branch duct near the distal stricture. In case 2, two of seven pancreatic juice samples (collected during the second tube placement) showed adenocarcinoma. Therefore, distal pancreatectomy was performed, and a PanIN3 neoplasm was identified primarily in the pancreatic duct at a narrow section with fibrosis. Partial microinvasion (<1 mm) was observed at the branch duct. Repeated cytology of pancreatic juice obtained by ENPD tube is effective for early diagnosis of pancreatic cancer, especially in cases without mass formation. However, some issues, including the appropriate number of samples, should be addressed in large prospective studies.

Distribution of radioactive cesium and stable cesium in cattle kept on a highly contaminated area of Fukushima nuclear accident.

Radioactivity inspection of slaughtered cattle is generally conducted using a portion of the neck muscle; however, there is limited information about the distribution of radioactive cesium in cattle. In this study, therefore, we measured not only radioactive cesium but also stable cesium in various tissues of 19 cattle that had been kept in the area highly contaminated by the Fukushima nuclear accident. Skeletal muscles showed approximately 1.5-3.0 times higher concentration of radioactive cesium than internal organs. Radioactive cesium concentration in the tenderloin and top round was about 1.2 times as high as that in the neck muscle. The kidney showed the highest concentration of radioactive cesium among internal organs, whereas the liver was lowest. Radioactive cesium concentration in the blood was about 8% of that in the neck muscle. Characteristics of stable cesium distribution were almost the same as those of radioactive cesium. Correlation coefficient between radioactive cesium and stable cesium in tissues of individual cattle was 0.981 ± 0.012. When a suspicious level near 100 Bq/kg is detected in the neck of slaughtered cattle, re-inspection should be conducted using a different region of muscle, for example top round, to prevent marketing of beef that violates the Food Sanitation Act.

Xanthofulvin, a novel semaphorin inhibitor produced by a strain of Penicillium.

A new semaphorin inhibitor xanthofulvin was isolated from the cultured broth of a fungus Penicillium sp. SPF-3059 along with a known compound vinaxanthone by solvent extraction and bioassay-guided fractionation. The tautomeric structure of xanthofulvin was determined by spectroscopic analyses. The two compounds exhibited significant semaphorin inhibitory activity with IC50 values of 0.09 and 0.1 microg/ml, respectively, in semaphorin3A-induced growth cone collapse assay using cultured chick dorsal root ganglia neurons.

Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes.

BACKGROUND: Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype. RESULTS: Based on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains. CONCLUSIONS: Gene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.

Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition.

BACKGROUND: Rats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the "rewiring" of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment. RESULTS: Treatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles). CONCLUSIONS: This study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

Gastrointestinal graft-versus-host disease in recipients of autologous hematopoietic stem cells: incidence, risk factors, and outcome.

Graft-versus-host disease (GVHD) is seen in skin, intestinal mucosa, and liver after autologous stem cell transplantation. We reviewed 681 consecutive patients to estimate the probability of gastrointestinal (GI) GVHD, response to treatment, risk factors for development, and effect on survival. GI GVHD was defined by persistent symptoms, mucosal abnormalities at endoscopy, and histology showing apoptotic crypt cells with or without lymphoid infiltrates. The proportion of patients with GI GVHD was 90/681 (13%). Nausea and vomiting occurred in 90% and diarrhea in 40%. The mean time to developing symptoms was day +15, that to histologically proven diagnosis was day +42, and that to starting prednisone treatment was day +45 after stem cell infusion. Treatment with a short course of prednisone effected durable responses in 79% of patients, and an additional 18% responded to a second course of prednisone. A multivariable logistic regression model demonstrated that the combined factor of a diagnosis of breast cancer or hematologic malignancy and female sex was statistically significantly associated with the probability of GI GVHD (P = .003). Survival in patients with GI GVHD was not statistically different than that in those without GVHD. We conclude that women with breast cancer or hematologic malignancy are more likely to develop GI GVHD after autologous transplantation, and that treatment with prednisone was effective.

In vitro and in vivo characterization of a novel semaphorin 3A inhibitor, SM-216289 or xanthofulvin.

SM-216289 (xanthofulvin) isolated from the fermentation broth of a fungal strain, Penicillium sp. SPF-3059, was identified as a strong semaphorin 3A (Sema3A) inhibitor. Sema3A-induced growth cone collapse of dorsal root ganglion neurons in vitro was completely abolished in the presence of SM-216289 at levels less than 2 mum (IC50 = 0.16 mum). When dorsal root ganglion explants were co-cultured with Sema3A-producing COS7 cells in a collagen gel matrix, SM-216289 enabled neurites to grow toward the COS7 cells. SM-216289 diminished the binding of Sema3A to its receptor neuropilin-1 in vitro, suggesting a direct interference of receptor-ligand association. Moreover, our data suggest that SM-216289 interacted with Sema3A directly and blocked the binding of Sema3A to its receptor. We examined the efficacy of SM-216289 in vivo using a rat olfactory nerve axotomy model, in which strong Sema3A induction has been reported around regenerating axons. The regeneration of olfactory nerves was significantly accelerated by a local administration of SM-216289 in the lesion site, suggesting the involvement of Sema3A in neural regeneration as an inhibitory factor. SM-216289 is an excellent molecular probe to investigate the function of Sema3A, in vitro and in vivo, and may be useful for the treatment of traumatic neural injuries.