The smell and odorous components of dried shiitake mushroom, <Emphasis Type="Italic">Lentinula edodes</Emphasis> I: relationship between sensory evaluations and amounts of odorous components

The smell of food is one of the most important factors in assessing its quality. Concerning the smell of dried shiitake mushrooms [Lentinula edodes (Berk.) Pegler], 1,2,3,5,6-pentathiepane, commonly known as lenthionine, has been reported as a key compound. However, other compounds have not been studied sufficiently in connection with smell. From the results of sensory intensity studies and sensory evaluations of dried shiitake mushrooms, a positive significant correlation at 1% risk was observed between sensory intensity and sulfur perception. This showed that the smell of dried shiitake mushrooms was characterized by a sulfurous smell. Also, comparing the sensory intensity with the amounts of volatile components showed positive significant correlations at 1% risk between sensory intensity and three compounds: 1,2,4-trithiolane, 1,2,4,6-tetrathiepane, and lenthionine. Furthermore, significant correlations at 5% risk were obtained between the amounts of these three compounds and sensory intensity by multiple regression analysis. This showed that the smell of dried shiitake mushrooms depended on these compounds. The partial regression coefficient of 1,2,4-trithiolane was larger than those of the others, and so it was proposed that 1,2,4-trithiolane could serve as an indicator to estimate the smell of dried shiitake mushroom.     

Expression of monocarboxylate transporter 1 (MCT1) in the dog intestine

In this study, the expression and distribution of monocarboxyolate transporter 1 (MCT1) along the intestines (duodenum, jejunum, ileum, cecum, colon and rectum) of dogs were investigated at both the mRNA and protein levels. The expression of MCT1 protein and its distribution were confirmed by Western blotting and immunohistochemical staining using the antibody for MCT1. We identified mRNA coding for MCT1 and a 43-kDa band of MCT1 protein in all regions from the duodenum to the rectum. Immunoreactive staining for MCT1 was also observed in epithelial cells throughout the intestines. MCT1 immunoreactivity was greater in the large intestine than in the small intestine. MCT1 protein was predominantly expressed on the basolateral membranes along intestinal epithelial cells, suggesting that MCT1 may play an important role in lactate efflux and transport of short-chain fatty acids (SCFAs) to the bloodstream across the basolateral membranes of the dog intestine.     

Toll-like receptor 2 and 9 are expressed and functional in gut-associated lymphoid tissues of presuckling newborn swine

To clarify the crucial role of Toll-like receptor (TLR) 2 and TLR9 in immature gut-associated lymphoid tissues (GALT), we focused on the expression of TLR2 and TLR9 and the immune responses induced by their ligands in the GALT of presuckling newborn swine. Quantitative real-time PCR revealed that TLR2 and TLR9 mRNA were expressed at detectable levels in all tested tissues (heart, thymus, lung, spleen, liver, kidney, skeletal muscle, duodenum, jejunum, ileum, ileal Peyer patches (Pps), and mesenteric lymph nodes (MLN)). In particular, in immature intestinal tissues and GALT, TLR2 and TLR9 mRNA were expressed at higher levels in ileal Pps and MLN than in the duodenum, jejunum, and ileum. We confirmed that the TLR2 and TLR9 proteins were also highly expressed and that their ligands were preferentially recognized by TLR2- or TLR9-expressing cells in the MLN and ileal Pps. Zymosan, CpG2006, and lactic acid bacteria could promote mitogenesis and production of multiple cytokines by the MLN and ileal Pps. In addition, double immunostaining for cytokeratin 18 and either TLR2 or TLR9 revealed that both TLR2 and TLR9 are strongly expressed in the columnar membranous (M) cells. Interestingly, while the apical membrane of the columnar M cells strongly expressed TLR2 protein and preferentially recognized zymosan, both "TLR2 expression on the apical membrane" and "TLR2-mediated zymosan binding" were negligible in neighboring enterocytes. These results indicate that TLR2 and TLR9 allow MLN and ileal Pps to respond to a variety of bacterial components immediately after birth, thereby providing newborns with a host defense system.     

Partial cDNA sequence of a relaxin‐like factor (RLF) receptor,LGR8 and possible existence of the RLF ligand‐receptor system in goat testes

Relaxin‐like factor (RLF), also known as insulin‐like factor 3 (INSL3), is produced by testicular Leydig cells, but its specific receptor LGR8 (leucine‐rich repeat family of G‐protein‐coupled receptor 8) has not been identified in goats. This study aimed to identify complementary DNA (cDNA) sequences of goat LGR8, and characterize the expression of both RLF and LGR8 in goat testes by RT‐PCR and immunohistochemistry. Testes were collected from immature (3‐month‐old) and mature (24‐month‐old) Saanen goats, and partial cDNA sequences of the goat homologue of human LGR8 were identified. The sequence encoded a reduced peptide sequence of 167 amino acids, which corresponded to transmembrane regions 2 through 5, followed by the beginning of intracellular loop 3 of human LGR8. Expression of both LGR8 and RLF genes was drastically increased in mature testes compared with immature ones. Although RLF protein was restricted to Leydig cells, LGR8 protein was detected in both Leydig cells and seminiferous epithelial cells (possibly germ cells and Sertoli cells). These results reveal a possible existence of the RLF‐LGR8 ligand‐receptor system within the goat testis, suggesting that RLF may play a role in testicular function through LGR8 on Leydig cells and seminiferous epithelial cells in an autocrine and/or paracrine manner.     

Properties of acetolactate synthase from sulfonylurea-resistant Scirpus juncoides Roxb. var. ohwianus T. Koyama

Properties of acetolactate synthase (EC 4.1.3.18; ALS) from sulfonylurea-resistant (SUR) Scirpus juncoides Roxb. var. ohwianus T. Koyama were studied biochemically and physiologically in comparison with those from sulfonylurea-susceptible weed (SUS). GR₅₀ values for growth inhibition and I₅₀ values for ALS inhibition by imazosulfuron were determined for both SUR and SUS. Imazosulfuron controlled the SUS above 80% at the dosage more than 10 g a.i./ha but did not control the SUR at the even great dosage of 1000 g a.i./ha. The rates required for 50% growth inhibition of the SUR relative to the SUS (R/S ratio) were 271-fold. The I₅₀ value for inhibition of ALS from the SUS was 15 nM, compared to I₅₀ of >3000 nM for inhibition of ALS from the SUR. These results suggest that a resistance may due to an altered ALS that is insensitive to imazosulfuron. The K_m (pyruvate) value of ALS from the SUR was similar to the K_m for ALS from the SUS, suggesting that a mutation resulting in resistance does not change the affinity of the enzyme for pyruvate. The specific activity of the SUR ALS was similar to that of the SUS ALS, which indicates that resistance is not an over-expression of the enzyme. ALS activity from both biotypes was inhibited by isoleucine, valine, and leucine in this order. However, the SUR ALS was less sensitive to inhibition by valine than the SUS ALS.     

Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa.     

Mycobacterium ulcerans infection in an Indian flap-shelled turtle (Lissemys punctata punctata)

We report an atypical mycobacterial infection in an Indian flap-shelled turtle, Lissemys punctata punctata, that died in an aquarium in Japan. At necropsy, the turtle showed multiple white nodules on the capsular surface and parenchyma of various organs such as the liver, spleen, intestine, and lung. Histologically, granulomatous inflammation surrounding a central zone of necrosis was observed. Sections stained by the Ziehl-Neelsen method revealed numerous acid-fast bacilli in the cytoplasm of macrophages and in the central area of necrosis. The organisms were identified as a mycobacterial species by PCR and nucleotide sequence analysis and revealed 98-100% homology to M. ulcerans. This is, to our knowledge, the first report of mycobacteriosis due to M. ulcerans in a turtle.     

Comparison of the glucosinolate-myrosinase systems among daikon (Raphanus sativus, Japanese white radish) varieties

Myrosinase is a cytosolic plant enzyme present in daikon ( Raphanus sativus, Japanese white radish) roots that hydrolyzes 4-methylthio-3-butenyl glucosinolate (MTBGLS) into the natural pungent agent 4-methylthio-3-butenyl isothiocyanate (MTBITC), which possesses antimicrobial, antimutagenic, and anticarcinogenic properties. The concentration of MTBGLS, myrosinase activity, and production of MTBITC in seven daikon varieties (one conventional and six heirlooms) were determined to rank the activity of the glucosinolate-myrosinase system and identify critical factors influencing the production of MTBITC. The six heirloom varieties produced 2.0-11.5 times higher levels of MTBITC as compared to the conventional variety, Aokubi, which is consumed by the present Japanese population. The myrosinase was located exclusively in the outer epidermal layer in Aokubi, and MTBGLS was widely distributed throughout the root tissue. Although the skin is a potentially rich source of myrosinase in Aokubi, the skin is usually peeled off in the current practice of preparing daikon for cooking. New practices are therefore proposed for the preparation of daikon tubers that eliminate the peeling of the skin to avoid removing the enzyme needed to convert MTBGLS to the health-beneficial MTBITC. It is also concluded that the consumption of heirloom daikon varieties in addition to changes in food preparation will optimize the health benefits of daikon.     

Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.