A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101

p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides. To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited. In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized. The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76%), respectively. The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC. Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment. The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase. The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source. These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R. opacus SAO101.     

Decoding ABA and osmostress signalling in plants from an evolutionary point of view

The plant hormone abscisic acid (ABA) is fundamental for land plant adaptation to water-limited conditions. Osmostress, such as drought, induces ABA accumulation in angiosperms, triggering physiological responses such as stomata closure. The core components of angiosperm ABA signalling are soluble ABA receptors, group A protein phosphatase type 2C and SNF1-related protein kinase2 (SnRK2). ABA also has various functions in non-angiosperms, however, suggesting that its role in adaptation to land may not have been angiosperm-specific. Indeed, among land plants, the core ABA signalling components are evolutionarily conserved, implying their presence in a common ancestor. Results of ongoing functional genomics studies of ABA signalling components in bryophytes and algae have expanded our understanding of the evolutionary role of ABA signalling, with genome sequencing uncovering the ABA core module even in algae. In this review, we describe recent discoveries involving the ABA core module in non-angiosperms, tracing the footprints of how ABA evolved as a phytohormone. We also cover the latest findings on Raf-like kinases as upstream regulators of the core ABA module component SnRK2. Finally, we discuss the origin of ABA signalling from an evolutionary perspective.     

Attachment structures in Rhizotrochus (Scleractinia): macro‐ to microscopic traits and their evolutionary significance

Marine sessile benthic organisms living on hard substrates have evolved a variety of attachment strategies. Rhizotrochus (Scleractinia, Flabellidae) is a representative azooxanthellate solitary scleractinian coral with a wide geographical distribution and unique attachment structures; it firmly attaches to hard substrates using numerous tube‐like rootlets, which are extended from a corallum wall, whereas most sessile corals are attached by stereome‐reinforced structures at their corallite bases. Detailed morphological and constructional traits of the rootlets themselves, along with their evolutionary significance, have not yet been fully resolved. Growth and developmental processes of spines in Truncatoflabellum and rootlets in Rhizotrochus suggest that these structures are homologous, as they both develop from the growth edges of walls and are formed by transformation of wall structures and their skeletal microstructures possess similar characteristics, such as patterns of rapid accretion and thickening deposits. Taking molecular phylogeny and fossil records of flabellids into consideration, Rhizotrochus evolved from a common free‐living ancestor and invaded hard‐substrate habitats by exploiting rootlets of spines origin, which were adaptive for soft‐substrate environments.     

Synthesis and Juvenile Hormone Activity of Some Terpenoid Phenyl Ethers

Thirty eight new compounds with juvenile hormone (JH) activity were synthesized and evaluated biologically on Bombyx mori L. Among them, the activities of 7,8-epoxy-4,8-dimethyl-1 -(p-ethylphenoxy)-3-undecene and 7,8-epoxy-4,8-dimethyl-l-(p-methylphenoxy)-3-undencene were proved to be more than two thousand times as high as that of the C₁₈-Cecropia JH (mixed isomers). Structure-activity relationship of these compounds was discussed.     

FCGR3A-158 polymorphism influences the biological response to infliximab in Crohn’s disease through affecting the ADCC activity

An association between FCGR3A-158 V/F polymorphism and biological responses to infliximab has been reported in Crohn’s disease (CD) in Western countries. However, little is known about the mechanism by which gene polymorphism affects the responses to infliximab. The aims of this study were to confirm the association in Japanese CD patients and to reveal the effect of gene polymorphism on biological responses to infliximab. Japanese CD patients were examined retrospectively at weeks 8 and 30. Clinical and biological responses were assessed by the Crohn’s disease activity index and C-reactive protein levels, respectively. The infliximab-binding affinity of natural killer (NK) cells from FCGR3A-158 V/V, V/F and F/F donors was examined. Infliximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activities were also determined using transmembrane TNF-α-expressing Jurkat T cells as target cells and peripheral blood mononuclear cells (PBMCs) from V/V, V/F and F/F donors as effector cells. Biological responses at week 8 were statistically higher in V/V patients, whereas no significant differences were observed in either clinical responses at weeks 8 and 30 or biological responses at week 30 among the three genotypes. NK cells and PBMCs from V/V patients also showed higher infliximab-binding affinity and infliximab-mediated ADCC activity, respectively. Our results suggest that FCGR3A-158 polymorphism is a predicting factor of biological responses to infliximab in the early phases. FCGR3A-158 polymorphism was also found to affect the infliximab-binding affinity of NK cells and infliximab-mediated ADCC activity in vitro, suggesting that an effect on ADCC activity influences biological responses to infliximab in CD patients.     

A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.     

Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with multiple eyespots

We have isolated a new Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with from one up to more than four eyespots cell^?1. It was designated mes (multiple eyespots)‐10 A wild‐type cell has a single eyespot, located under the chloroplast envelope, at a certain position near the cell's equator where the chloroplast envelope is in contact with the cell membrane. The eyespot(s) in mes‐10, however, are located at various positions on its chloroplast. The mes‐10 cells displayed negative phototaxis to 480–500 nm light. This behavior differed from that of a similar mutant, ptx4, which has been shown to have multiple eyespots and display no phototaxis (Pazour et al., J. Cell Biol. 1995; 131 : 427–40). Mes‐10 may retain a functional photoreceptor and a photosignal transduction system independently of its multiple eyespots. This mutant should be useful for studying how C. reinhardtii responds to light signals, as well as how eyespots are formed in the cell.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.