Chiral inversion of RS-8359: a selective and reversible MAO-A inhibitor via oxido-reduction of keto-alcohol

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine is a selective and reversible MAO-A inhibitor. The (S)-enantiomer of RS-8359 has been demonstrated to be inverted to the (R)-enantiomer after oral administration to rats. In the current study, we investigated the chiral inversion mechanism and the properties of involved enzymes using rat liver subcellular fractions. The 7-hydroxy function of RS-8359 was oxidized at least by the two different enzymes. The cytosolic enzyme oxidized enantiospecifically the (S)-enantiomer with NADP as a cofactor. On the other hand, the microsomal enzyme catalyzed more preferentially the oxidation of the (S)-enantiomer than the (R)-enantiomer with NAD as a cofactor. With to product enantioselectivity of reduction of the 7-keto derivative, it was found that only the alcohol bearing (R)-configuration was formed by the cytosolic enzyme with NADPH and the microsomal enzyme with NADH at almost equal rate. The reduction rate was much larger than the oxidation rate of 7-hydroxy group. The results suggest that the chiral inversion might occur via an enantioselectivity of consecutive two opposing reactions, oxidation and reduction of keto-alcohol group. In this case, the direction of chiral inversion from the (S)-enantiomer to the (R)-enantiomer is governed by the enantiospecific reduction of intermediate 7-keto group to the alcohol with (R)-configuration. The enzyme responsible for the enantiospecific reduction of the 7-keto group was purified from rat liver cytosolic fractions and identified as 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) via database search of peptide mass data obtained by nano-LC/MS/MS.     

Chondromodulin-I and tenomodulin are differentially expressed in the avascular mesenchyme during mouse and chick development

Chondromodulin-I (ChM-I) and tenomodulin (TeM) are homologous angiogenesis inhibitors. We have analyzed the spatial relationships between capillary networks and the localization of these molecules during mouse and chick development. ChM-I and TeM proteins have been localized to the PECAM-1-negative avascular region: ChM-I is expressed in the avascular cartilage, whereas TeM is detectable in dense connective tissues, including tendons and ligaments. We have also examined the vasculature of chick embryos by injection with India ink and have performed in situ hybridization of the ChM-I and TeM genes. The onset of ChM-I expression is associated with chondrogenesis during mouse embryonic development. ChM-I expression is also detectable in precartilaginous or noncartilaginous avascular mesenchyme in chick embryos, including the somite, sclerotome, and heart. Hence, the expression domains of ChM-I and TeM during vertebrate development incorporate the typical avascular regions of the mesenchymal tissues. This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sport, Science, and Technology of Japan and by the Tanabe Medical Frontier Conference.     

Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori

The contribution of pyocyanin to the virulence of Pseudomonas aeruginosa against the silkworm Bombyx mori was studied. First, purified pyocyanin was injected into the hemocoel of B. mori. Acute toxicity was observed only when a high dose of pyocyanin was injected. The lethal dose 50% value of pyocyanin was found to be 9.52 microg per larva. Next, mutant strains of phzM and phzS, which encode putative phenazine-specific methytransferase and flavin-containing monooxygenase, respectively, were created, and their virulence was compared with that of the PAO1 parent strain. Although the ability to produce pyocyanin was completely lost in the phz-mutant strains, they maintained the same level of virulence as the PAO1 parent strain. In addition, the complementation of the corresponding gene in trans in the mutant strains did not have any effect on the virulence of those mutant strains. These results indicated that pyocyanin does not act as a virulence factor in B. mori after invasion, which was different from the results obtained in other Lepidopteran host models.     

Tenomodulin Expression in the Periodontal Ligament Enhances Cellular Adhesion

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.     

Physiological and environmental parameters associated with mass spectrometry-based salivary metabolomic profiles

Mass spectrometry (MS)-based metabolomic methods enable simultaneous profiling of hundreds of salivary metabolites, and may be useful to diagnose a wide range of diseases using saliva. However, few studies have evaluated the effects of physiological or environmental factors on salivary metabolomic profiles. Therefore, we used capillary electrophoresis-MS to analyze saliva metabolite profiles in 155 subjects with reasonable oral hygiene, and examined the effects of physiological and environmental factors on the metabolite profiles. Overall, 257 metabolites were identified and quantified. The global profiles and individual metabolites were evaluated by principle component analysis and univariate tests, respectively. Collection method, collection time, sex, body mass index, and smoking affected the global metabolite profiles. However, age also might contribute to the bias in sex and collection time. The profiles were relatively unaffected by other parameters, such as alcohol consumption and smoking, tooth brushing, or the use of medications or nutritional supplements. Temporomandibular joint disorders had relatively greater effects on salivary metabolites than other dental abnormalities (e.g., stomatitis, tooth alignment, and dental caries). These findings provide further insight into the diversity and stability of salivary metabolomic profiles, as well as the generalizability of disease-specific biomarkers.     

Psychiatric disorder‐related abnormal behavior and habenulointerpeduncular pathway defects in Wnt1‐cre and Wnt1‐GAL4 double transgenic mice

The neural crest is a unique structure in vertebrates. Wnt1‐cre and Wnt1‐GAL4 double transgenic (dTg) mice have been used in a variety of studies concerning neural crest cell lineages in which the Cre/loxP or GAL4/UAS system was applied. Here, we show psychiatric disorder‐related behavioral abnormalities and histologic alterations in a neural crest‐derived brain region in dTg mice. The dTg mice exhibited increased locomotor activity, decreased social interaction, and impaired short‐term spatial memory and nesting behavior. The choline acetyltransferase‐ and vesicular glutamate transporter 2‐immunoreactive habenulointerpeduncular fiber tracts that project from the medial habenular nucleus of the epithalamus to the interpeduncular nucleus of the midbrain tegmentum appeared irregular in the dTg mice. Both the medial habenula nucleus and the interpeduncular nucleus were confirmed to be derived from the neural crest. The findings of this study suggest that neural crest‐derived cells have pathogenic roles in the development of psychiatric disorders and that the dTg mouse could be a useful animal model for studying the pathophysiology of mental illness such as autism and schizophrenia. Scientists that use the dTg mice as a cre‐transgenic deleter line should be cautious in its possible toxicity, especially if behavioral analyses are to be performed.     

Evolution of the diverse array of phosphagen systems present in annelids

Annelids as a group express a variety of phosphagen kinases including creatine kinase (CK), glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and a unique arginine kinase (AK) restricted to annelids. In prior work, we have determined and compared the intron/exon organization of the annelid genes for cytoplasmic GK, LK, AK, and mitochondrial TK and LK (MiTK and MiLK, respectively), and found that these annelid genes, irrespective of cytoplasmic or mitochondrial, have the same 8-intron/9-exon organization strikingly similar to mitochondrial CK (MiCK) genes. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids. To gain a greater understanding of the evolutionary processes leading to the diversity of annelid phosphagen kinases, we determined for the first time the intron/exon organization of a cytoplasmic CK gene from a polychaete as well as that of another polychaete MiCK gene. These gene structures, coupled with a phylogenetic analyses of annelid enzymes and assessment of the fidelity of substrate specificity of some these phosphagen kinases, provide insight into the pattern of radiation of the annelid enzymes. Annelid phosphagen kinases appeared to have diverged in the following order (earliest first): (1) cytoplasmic AK, LK and TK, (2) GK, and (3) mitochondrial MiLK and MiTK. Interestingly, phylogenetic analyses showed that the above phosphagen kinases appear to be basal to all CK isoforms (mitochondrial, cytoplasmic and flagellar CKs). This somewhat paradoxical placement of CKs most likely reflects a higher rate of evolution and radiation of the annelid-specific LK, TK and GK genes than the CK isoform genes.     

Chondromodulin I is a bone remodeling factor

Chondromodulin I (ChM-I) was supposed from its limited expression in cartilage and its functions in cultured chondrocytes as a major regulator in cartilage development. Here, we generated mice deficient in ChM-I by targeted disruption of the ChM-I gene. No overt abnormality was detected in endochondral bone formation during embryogenesis and cartilage development during growth stages of ChM-I(-/-) mice. However, a significant increase in bone mineral density with lowered bone resorption with respect to formation was unexpectedly found in adult ChM-I(-/-) mice. Thus, the present study established that ChM-I is a bone remodeling factor.     

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua

Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 +/- 0.45 microm and 2.84 +/- 0.14 microm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 +/- 0.21 microm x 2.00 +/- 0.11 microm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.