Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4

Rhodococcus erythropolis strain PR4 has been isolated as an alkane-degrading bacterium. The strain harbours one linear plasmid, pREL1 (271 577 bp) and two circular plasmids, pREC1 (104 014 bp) and pREC2 (3637 bp), all with some sequence similarities to other Rhodococcus plasmids. For pREL1, pREC1 and pREC2, 298, 102 and 3 open reading frames, respectively, were predicted. Linear plasmid pREL1 has several regions homologous to plasmid pBD2 found in R. erythropolis BD2. Sequence analysis of pREL1 and pBD2 identified common metal-resistance genes on both, but pREL1 also encodes alkane-degradation genes not found on pBD2, with enzyme constituents some of which are quite different from those of other organisms. The alkane hydroxylase consisted of a cytochrome P450 monooxygenase, a 2Fe-2S ferredoxin, and a ferredoxin reductase. The ferredoxin reductase amino acid sequence resembles the AlkT (rubredoxin reductase) sequence. A zinc-containing alcohol dehydrogenase further oxydizes alkanols, alkane oxidation products catalysed by alkane hydroxylase. Of the circular plasmids, the pREC1 sequence is partially similar to the sequence of pREAT701, the virulence plasmid found in Rhodococcus equi. pREC1 has no pREAT701 virulence genes and encodes genes for beta-oxidation of fatty acids. Thus, joint actions of enzymes encoded by pREL1 and pREC1 may enable efficient mineralization of alkanes.     

Lung and buccal ventilation in the frog: uncoupling coupled oscillators

The frog, with two distinct ventilatory acts, provides a useful model to investigate the prospective interaction of two oscillators in generating the respiratory rhythm. Building on evidence supporting the existence of separate oscillators generating buccal and lung ventilation, we have attempted to uncouple the two rhythms in the isolated brain stem preparation. Opioid preferentially inhibits the lung rhythm, suggesting an uncoupling of the lung from the buccal oscillator. Reduction of the superfusate chloride concentration alters both the buccal and the lung rhythms. Joint application of opioid and reduced-chloride superfusate leads to an increase in the variability of the buccal burst-to-lung burst intervals. This increase in variability suggests that chloride-mediated mechanisms are involved in coupling the buccal oscillator to the lung oscillator. Given the results from these interventions, we propose a simple schematic model of the frog respiratory rhythm generator, outlining the coupling of the lung and buccal oscillators.     

Functional consequences of stenophylly for leaf productivity: comparison of the anatomy and physiology of a rheophyte, Farfugium japonicum var. luchuence, and a related non-rheophyte, F. japonicum (Asteraceae)

We investigated the anatomical and physiological characteristics of stenophyllous leaves of a rheophyte, Farfugium japonicum var. luchuence, and sun and shade leaves of a non-rheophyte, F. japonicum, comparing three different populations from coastal, forest floor, and riparian habitats. Light adaptation resulted in smaller leaves, and riparian adaptation resulted in narrower leaves (stenophylly). The light-saturated rate of photosynthesis (P _max) per unit leaf area corresponded to the light availability of the habitat. Irrespective of leaf size, the P _max per unit leaf mass was similar for sun and shade leaves. However, the P _max per mass of stenophyllous leaves was significantly lower than that of sun and shade leaves. This was because the number and size of mesophyll cells were greater than that required for intercellular CO₂ diffusion, which resulted in a larger leaf mass per unit leaf area. Higher cell density increases contact between mesophyll cells and enhances leaf toughness. Stenophyllous leaves of the rheophyte are frequently exposed to a strong water flow when the water level rises, suggesting a mechanical constraint caused by physical stress.     

Simple and sensitive determination of free and total morphine in human liver and kidney using gas chromatography-mass spectrometry

We developed a reliable, simple and sensitive method to determine free and total morphine in human liver and kidney, using gas chromatography-mass spectrometry (GC-MS). Free morphine or total morphine obtained by acid hydrolysis from 0.2g tissue sample was extracted using an Extrelut NT column with an internal standard, dihydrocodeine, followed by trimethylsilylation. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of morphine in both liver and kidney samples were linear in the concentration range from 0.005 to 5 microg/g. The lower limits of detection of morphine were 0.005 microg/g. This method proved successful when we determined free and total morphine in liver and kidney obtained from an autopsied man who was mis-ingested morphine compound in the hospital, which resulted in the cause of death being morphine intoxication.     

Chemokines in tumor development and progression

Chemokines were originally identified as mediators of the inflammatory process and regulators of leukocyte trafficking. Subsequent studies revealed their essential roles in leukocyte physiology and pathology. Moreover, chemokines have profound effects on other types of cells associated with the inflammatory response, such as endothelial cells and fibroblasts. Thus, chemokines are crucial for cancer-related inflammation, which can promote tumor development and progression. Increasing evidence points to the vital effects of several chemokines on the proliferative and invasive properties of tumor cells. The wide range of activities of chemokines in tumorigenesis highlights their roles in tumor development and progression.     

Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.     

Photosensitivity of Kinase Activation by Blue Light Involves the Lifetime of a Cysteinyl-Flavin Adduct Intermediate,S390, in the Photoreaction Cycle of the LOV2 Domain in Phototropin,a Plant Blue Light Receptor

Phototropin (phot) is a light-regulated protein kinase that mediates a variety of photoresponses in plants, such as phototropism, chloroplast positioning, and stomata opening. Arabidopsis has two homologues, phot1 and phot2, that share physiological functions depending on light intensity. A phot molecule has two photoreceptive light oxygen voltage-sensing domains, LOV1 and LOV2, and a Ser/Thr kinase domain. The LOV domains undergo a photocycle upon blue light (BL) stimulation, including transient adduct formation between the chromophore and a conserved cysteine (S390 intermediate) that leads to activation of the kinase. To uncover the mechanism underlying the photoactivation of the kinase, we have introduced a kinase assay system composed of a phot1 LOV2-linker-kinase polypeptide as a light-regulated kinase and its N-terminal polypeptide as an artificial substrate (Okajima, K., Matsuoka, D., and Tokutomi, S. (2011) LOV2-linker-kinase phosphorylates LOV1-containing N-terminal polypeptide substrate via photoreaction of LOV2 in Arabidopsis phototropin1. FEBS Lett. 585, 3391–3395). In the present study, we extended the assay system to phot2 and compared the photochemistry and kinase activation by BL between phot1 and phot2 to gain insight into the molecular basis for the different photosensitivities of phot1 and phot2. Photosensitivity of kinase activation by BL and the lifetime of S390 of phot1 were 10 times higher and longer, respectively, than those of phot2. This correlation was confirmed by an amino acid substitution experiment with phot1 to shorten the lifetime of S390. The present results demonstrated that the photosensitivity of kinase activation in phot involves the lifetime of S390 in LOV2, suggesting that the lifetime is one of the key factors for the different photosensitivities observed for phot1 and phot2.     

The rare terrestrial orchid Nervilia nipponica consistently associates with a single group of novel mycobionts

Nervilia nipponica is a tuberous terrestrial orchid that has a highly restricted distribution within common secondary evergreen forest communities in central and western Japan. Such a limited occurrence could be attributable to a requirement for a specific mycorrhizal fungus. As part of a broader examination of this hypothesis, we sought to elucidate the mycorrhizal associations of N. nipponica. Seventy-five samples of mycorrhizae from forty individuals were collected at ten populations throughout the orchid’s range in Japan. The identity of mycorrhizal fungi was investigated by sequencing two genetic markers (nrDNA ITS and nrDNA 28S LSU) and their relationships were assessed via phylogenetic analyses. The most frequently encountered mycorrhizal fungi consisted of four closely related Agaricomycetes that infected an average of 78.7 % of individuals per population. All four formed a discrete, monophyletic clade with low sequence homology to other fungi registered in GenBank, indicating that they belong to a novel, unnamed family. Two additional fungal groups, belonging to Ceratobasidiaceae and “Group B” Sebacinales, were found in 22.0 and 21.5 % of individuals per population, respectively. The orchid probably uses these two groups opportunistically, because they were found at lower densities and always in combination with the unidentified Agaricomycete. These findings suggest that a group of novel Agaricomycete fungi constitutes the dominant mycobiont of N. nipponica.     

Turbidity and Hardness of a Heat-induced Gel of Hen Egg Ovalbumin

The turbidity and hardness of a heat-induced gel prepared from ovalbumin were examined at various pHs and ionic strengths. Depending on the conditions of the medium, a transparent solution, transparent gel, turbid gel, or turbid suspension was obtained by heating. The hardness was a maximum with the conditions that gave a transparent or slightly turbid gel. The gel and coagulums were solubilized by 1% SDS, but not by 6 m urea or 50 mm mercaptoethanol. The solution obtained by SDS treatment contained polymers shorter than octamers.