Automated high-performance liquid chromatographic method for the determination of antipyrine and its metabolites in urine : Some preliminary results obtained from smokers and non-smokers

A simple, accurate and fully automated high-performance liquid-chromatographic method was developed for the simultaneous determination of antipyrine (AP), 3-hydroxymethylantipyrine (3HMA), 4-hydroxyantipyrine (4OHA) and norantipyrine (NORA) in urine. This method requires no extraction step and only one chromatographic run with the use of a reversed-phase system. The coefficient of variation (%) (n = 8 each) was: 4.14 for AP, 2.31 for 3HMA, 3.48 for 4OHA, and 2.71 for NORA. The method was applied to studies on AP metabolism in three smokers and three non-smokers who received an oral 10 mg/kg dose of AP. These preliminary results suggest that smokers appear to excrete more 4OHA and NORA in the urine than non-smokers.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

Tumour necrosis factor and the lysosomal enzymes of macrophages or macrophage-like cell line

Summary The relationship between tumour necrosis factor (TNF) and macrophages or macrophage-like cell line, especially the lysosomal enzymes was investigated. The serum lysosomal enzymes and LDH activities were increased in proportion to the TNF production even in different strains of mice. Lysosomal enzymes and TNF activity were released into the supernatant of the culture medium of macrophage-enriched peritoneal exudate cells (PEC) or spleen cells derived from Propionibacterium acnes-primed mice after addition of lipopolysaccharide (LPS). After passage through a Sephadex G-10 column, TNF activity could not be detected in the supernatant of these spleen cells after addition of LPS. Also TNF activity could not be detected in the supernatant following destruction of PEC. These results suggest that TNF producibility is strongly related to the degree of activation of macrophages, especially the lysosomal enzymes. The murine macrophage-like cell line, J 774, also released TNF activity and lysosomal enzymes after addition of LPS.     

Phylogeny and molecular epidemiology of Sporothrix schenckii in Japan

Mitochondrial DNA(mtDNA) diversity was investigated in 257 clinical isolants of Sporothrix schenckii obtained from 4 districts in Japan. S. schenckii was classified into 10 types based on Hae III restriction profiles. Phylogeny of types constructed by the method of Fitch and Margoliash [1] on the estimated sequence divergence within mtDNA using the methods of Nei and Li [2], showed that S. schenckii are grouped into 2 clusters, one group consisting of types 1, 2 and 3, and the other group consisting of the other seven types. In addition, types 1, 2, and 5 were correlated with their geographic origin, whereas type 4 was present throughout Japan.     

Kin recognition affects plant communication and defence

The ability of many animals to recognize kin has allowed them to evolve diverse cooperative behaviours; such ability is less well studied for plants. Many plants, including Artemisia tridentata, have been found to respond to volatile cues emitted by experimentally wounded neighbours to increase levels of resistance to herbivory. We report that this communication was more effective among A. tridentata plants that were more closely related based on microsatellite markers. Plants in the field that received cues from experimentally clipped close relatives experienced less leaf herbivory over the growing season than those that received cues from clipped neighbours that were more distantly related. These results indicate that plants can respond differently to cues from kin, making it less likely that emitters will aid strangers and making it more likely that receivers will respond to cues from relatives. More effective defence adds to a growing list of favourable consequences of kin recognition for plants.     

Niche Separation of Methanotrophic Archaea (ANME-1 and -2) in Methane-Seep Sediments of the Eastern Japan Sea Offshore Joetsu

In this study, we investigated the diversity and spatial distribution of anaerobic methanotrophic archaea (ANMEs) in sediments of a gas hydrate field off Joetsu in the Japan Sea. Distribution of ANMEs in sediments was identified by targeting the gene for methyl coenzyme M reductase alpha subunit (mcrA), a phylogenetically conserved gene that occurs uniquely in methanotrophic and methanogenic archaea, in addition to 16S rRNA genes. Quantitative PCR analyses of mcrA genes in 14 piston core samples suggested that members of ANME-1 group would dominate AOM communities in sulfate-depleted sediments, even below the sulfate-methane interface, while ANME-2 archaea would prefer to populate in shallower sediments containing comparatively higher sulfate concentrations. These results suggest that, although the potential electron acceptors in sulfate-depleted habitats remain elusive, the niche separation of ANME-1 and -2 may be controlled by in situ concentration of sulfate and the availability in sediments.     

Evidence for expression of 11β-hydroxysteroid dehydrogenase type3 (HSD11B3/HSD11B1L) in neonatal pig testis

11β-hydroxysteroid dehydrogenase (HSD11B) catalyzes the interconversion between active and inactive glucocorticoid, and is known to exist as two distinct isozymes: HSD11B1 and HSD11B2. A third HSD11B isozyme, HSD11B1L (SCDR10b), has recently been identified. Human HSD11B1L, which was characterized as a unidirectional NADP⁺-dependent cortisol dehydrogenase, appears to be specifically expressed in the brain. We previously reported that HSD11B1 and abundant HSD11B2 isozymes are expressed in neonatal pig testis and the Km for cortisol of NADP⁺-dependent dehydrogenase activity of testicular microsomes obviously differs from the same activity catalyzed by HSD11B1 from pig liver microsomes. Therefore, we hypothesized that the neonatal pig testis also expresses the third type of HSD11B isozyme, and we herein examined further evidence regarding the expression of HSD11B1L. (1) The inhibitory effects of gossypol and glycyrrhetinic acid on pig testicular microsomal NADP⁺-dependent cortisol dehydrogenase activity was clearly different from that of pig liver microsomes. (2) A highly conserved human HSD11B1L sequence was observed by RT-PCR in a pig testicular cDNA library. (3) mRNA, which contains the amplified sequence, was evaluated by real-time PCR and was most strongly expressed in pig brain, and at almost the same levels in the kidney as in the testis, but at lower levels in the liver. Based on these results, neonatal pig testis appears to express glycyrrhetinic acid-resistant HSD11B1L as a third HSD11B isozyme, and it may play a physiologically important role in cooperation with the abundantly expressed HSD11B2 isozyme in order to prevent Leydig cell apoptosis or GC-mediated suppression of testosterone production induced by high concentrations of activated GC in neonatal pig testis.     

EXTL2, a Member of the EXT Family of Tumor Suppressors,Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz ¹H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a “quality control system” for proteoglycans.