Identification of the p-coumaric acid biosynthetic gene cluster in Kutzneria albida: insights into the diazotization-dependent deamination pathway.

Recently, we identified the biosynthetic gene cluster of avenalumic acid (ava cluster) and revealed its entire biosynthetic pathway, resulting in the discovery of a diazotization-dependent deamination pathway. Genome database analysis revealed the presence of more than 100 ava cluster-related biosynthetic gene clusters (BGCs) in actinomycetes; however, their functions remained unclear. In this study, we focused on an ava cluster-related BGC in Kutzneria albida (cma cluster), and revealed that it is responsible for p-coumaric acid biosynthesis by heterologous expression of the cma cluster and in vitro enzyme assays using recombinant Cma proteins. The ATP-dependent diazotase CmaA6 catalyzed the diazotization of both 3-aminocoumaric acid and 3-aminoavenalumic acid using nitrous acid in vitro. In addition, the high efficiency of the CmaA6 reaction enabled us to perform a kinetic analysis of AvaA7, which confirmed that AvaA7 catalyzes the denitrification of 3-diazoavenalumic acid in avenalumic acid biosynthesis. This study deepened our understanding of the highly reducing type II polyketide synthase system as well as the diazotization-dependent deamination pathway for the production of avenalumic acid or p-coumaric acid.

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

Association between Lack of Social Support from Partner or Others and Postpartum Depression among Japanese Mothers: A Population-Based Cross-Sectional Study.

Lack of social support is a known risk factor for postpartum depression (PPD). However, the association between lack of social support from a partner or others and PPD remains unknown. We examined this association among Japanese mothers. We distributed an original questionnaire to mothers participating in a three- or four-month health check-up program over October to November 2012 in Aichi Prefecture, Japan. Of the 9707 eligible mothers, 6590 responded to the questionnaire (response rate: 68%). Social support from a partner or others was assessed based on whether the mother can consult with her partner or others (i.e., parents, relatives, and friends who are close by or far) on childcare. PPD was assessed with the Edinburgh Postnatal Depression Scale. The data were analyzed using multiple logistic regression analysis for four categories: no social support from either a partner/others, social support from a partner only, social support from others only, and social support from both, adjusted for possible covariates. Mothers who have no social support from either a partner/others, have social support from a partner only, and have social support from others only were 7.22 (95% confidence interval [CI], 1.76-29.6), 2.34 (95% CI, 1.37-3.98), and 3.13 (95% CI, 2.11-4.63) times more likely to show PPD, respectively, in comparison with mothers who have social support from both, after adjustment of possible covariates. Mothers with no social support from a partner, but have social support from others, showed significant risk for PPD, which may be invisible. Further prevention effort is needed to detect PPD cases, with a focus on mothers without support from their partner.

Probabilistic transition from unstable predator-prey interaction to stable coexistence of Dictyostelium discoideum and Escherichia coli.

Predator-prey interactions have been found at all levels within ecosystems. Despite their ecological ubiquity and importance, the process of transition to a stable coexistent state has been poorly verified experimentally. To investigate the stabilization process of predator-prey interactions, we previously constructed a reproducible experimental predator-prey system between Dictyostelium discoideum and Escherichia coli, and showed that the phenotypically changed E. coli contributed to stabilization of the system. In the present study, we focused on the transition to stable coexistence of both species after the phenotypic change in E. coli. Analysis of E. coli cells isolated from co-culture plates as single colony enabled us to readily identify the appearance of phenotypically changed E. coli that differed in colony morphology and growth rate. It was also demonstrated that two types of viscous colony, i.e., the dense-type and sparse-type, differing in spatial distribution of both species emerged probabilistically and all of the viscous colonies maintained stably were of the sparse-type. These results suggest that the phenotypically changed E. coli may produce two types of viscous colonies probabilistically. The difference in spatial distribution would affect localized interactions between both species and then cause probabilistic stabilization of predator-prey interactions.

Role of pre- and postsynaptic activity in thalamocortical axon branching.

Axonal branching is thought to be regulated not only by genetically defined programs but also by neural activity in the developing nervous system. Here we investigated the role of pre- and postsynaptic activity in axon branching in the thalamocortical (TC) projection using organotypic coculture preparations of the thalamus and cortex. Individual TC axons were labeled with enhanced yellow fluorescent protein by transfection into thalamic neurons. To manipulate firing activity, a vector encoding an inward rectifying potassium channel (Kir2.1) was introduced into either thalamic or cortical cells. Firing activity was monitored with multielectrode dishes during culturing. We found that axon branching was markedly suppressed in Kir2.1-overexpressing thalamic cells, in which neural activity was silenced. Similar suppression of TC axon branching was also found when cortical cell activity was reduced by expressing Kir2.1. These results indicate that both pre- and postsynaptic activity is required for TC axon branching during development.

Phenotypic plasticity of Escherichia coli at initial stage of symbiosis with Dictyostelium discoideum.

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E. coli cells with increased GFP concentration appeared, and when the formation of mucoidal colonies housing the coexisting two species began, most E. coli cells were from the new population. Further experiments suggest that the physiological change is induced by interaction with D. discoideum cells and is reversible, although the processes of the changes in both directions seem to proceed gradually. The observed phenotypic plasticity, together with natural selection under a co-cultivation environment, may be important for leading to the evolution of a new symbiotic system.

Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching.

Target and activity-dependent mechanisms of axonal branching were studied in the thalamocortical (TC) projection using organotypic cocultures of the thalamus and cortex. TC axons were labeled with enhanced yellow fluorescent protein (EYFP) by a single-cell electroporation method and observed over time by confocal microscopy. Changes in the firing activity of cocultures grown on multielectrode dishes were also monitored over time. EYFP-labeled TC axons exhibited more branch formation in and around layer 4 of the cortical explant during the second week in vitro, when spontaneous firing activity increased in both thalamic and cortical cells. Time-lapse imaging further demonstrated that branching patterns were generated dynamically by addition and elimination with a bias toward branch accumulation in the target layer. To examine the relationship between neural activity and TC branch formation, the dynamics of axonal branching was analyzed under various pharmacological treatments. Chronic blockade of firing or synaptic activity reduced the remodeling process, in particular, branch addition in the target layer. However, extension of branches was not affected by this treatment. Together, these findings suggest that neural activity can modify the molecular mechanisms that regulate lamina-specific TC axon branching.

Molecular mechanisms of thalamocortical axon targeting.

The thalamocortical (TC) projection in the mammalian brain is a well characterized system in terms of laminar specificity of neocortical circuits. To understand the mechanisms that underlie lamina-specific TC axon targeting, we studied the role of extracellular and cell surface molecules that are expressed in the upper layers of the developing cortex in in vitro culture techniques. The results demonstrated that multiple upper layer molecules co-operated to produce stop behaviour of TC axons in the target layer. Activity dependency of TC axon branching was also investigated in organotypic co-cultures of the thalamus and cortex. TC axon branches were formed dynamically by addition and elimination during the second week in vitro, when spontaneous firing increased in thalamic and cortical cells. Pharmacological blockade of firing or synaptic activity reduced the remodelling process, in particular branch addition, in the target layer. Together, these findings suggest that TC axon targeting mechanisms involve the regulation with multiple lamina-specific molecules and modification of the molecular mechanisms via neural activity.

Trends in new drug interactions for pharmaceutical products in Japan.

PURPOSE: The aim of the present study was to elucidate the trends in drug interactions for pharmaceutical products in Japan by examining safety profile updates. METHODS: All 12 422 prescription drugs currently on the Japanese market were included in the study. Revisions to their product information (or package insert: PI) were investigated from January 2000 to December 2003. The publication 'Drug Safety Update,' which is a summary of the revisions made to the precautions in PIs and is issued by The Society of Japanese Pharmacopoeia and The Federation of Pharmaceutical Manufacturers' Associations of Japan under the supervision of the Ministry of Health, Labour and Welfare (MHLW), was used as a data source. The revised drug interactions were categorized according to measures, mechanisms, and evidence from published references. RESULTS AND CONCLUSIONS: The results revealed 426 new interactions, including 75 contraindicative combinations, during the survey period. About 45% and 27% of the new interactions involved metabolic and pharmacological processes, respectively, with metabolic interactions involving cytochrome P450 3A4 being the dominant reason for the revision of PIs. Only 37% of the new interactions cited scientific journals and/or books, and 58% of these references cited were published more than 5 years prior to the date of revision. In conclusion, metabolic interactions were the major reasons for the update of safety information after 2000. Published references should be provided in order to assist with clinical management and avoid the undesirable effects of new drug interactions.