Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.     

Eyelid Opening with Trigeminal Proprioceptive Activation Regulates a Brainstem Arousal Mechanism

Eyelid opening stretches mechanoreceptors in the supratarsal Müller muscle to activate the proprioceptive fiber supplied by the trigeminal mesencephalic nucleus. This proprioception induces reflex contractions of the slow-twitch fibers in the levator palpebrae superioris and frontalis muscles to sustain eyelid and eyebrow positions against gravity. The cell bodies of the trigeminal proprioceptive neurons in the mesencephalon potentially make gap-junctional connections with the locus coeruleus neurons. The locus coeruleus is implicated in arousal and autonomic function. Due to the relationship between arousal, ventromedial prefrontal cortex, and skin conductance, we assessed whether upgaze with trigeminal proprioceptive evocation activates sympathetically innervated sweat glands and the ventromedial prefrontal cortex. Specifically, we examined whether 60° upgaze induces palmar sweating and hemodynamic changes in the prefrontal cortex in 16 subjects. Sweating was monitored using a thumb-mounted perspiration meter, and prefrontal cortex activity was measured with 45-channel, functional near-infrared spectroscopy (fNIRS) and 2-channel NIRS at Fp1 and Fp2. In 16 subjects, palmar sweating was induced by upgaze and decreased in response to downgaze. Upgaze activated the ventromedial prefrontal cortex with an accumulation of integrated concentration changes in deoxyhemoglobin, oxyhemoglobin, and total hemoglobin levels in 12 subjects. Upgaze phasically and degree-dependently increased deoxyhemoglobin level at Fp1 and Fp2, whereas downgaze phasically decreased it in 16 subjects. Unilateral anesthetization of mechanoreceptors in the supratarsal Müller muscle used to significantly reduce trigeminal proprioceptive evocation ipsilaterally impaired the increased deoxyhemoglobin level by 60° upgaze at Fp1 or Fp2 in 6 subjects. We concluded that upgaze with strong trigeminal proprioceptive evocation was sufficient to phasically activate sympathetically innervated sweat glands and appeared to induce rapid oxygen consumption in the ventromedial prefrontal cortex and to rapidly produce deoxyhemoglobin to regulate physiological arousal. Thus, eyelid opening with trigeminal proprioceptive evocation may activate the ventromedial prefrontal cortex via the mesencephalic trigeminal nucleus and locus coeruleus.     

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Ameliorative effects of mulberry (Morus alba L.) leaves on hyperlipidemia in rats fed a high-fat diet: induction of fatty acid oxidation, inhibition of lipogenesis, and suppression of oxidative stress

To determine the effects of mulberry (Morus alba L.) leaves on hyperlipidemia, we performed gene expression profiling of the liver. Rats were fed a high-fat diet and administered mulberry leaves for 7 weeks. Plasma triglyceride and non-esterified fatty acid levels were significantly lower in the rats treated with mulberry leaves as compared with the untreated rats. DNA microarray analysis revealed that mulberry leaves upregulated expression of the genes involved in α-, β- and ω-oxidation of fatty acids, mainly related to the peroxisome proliferator-activated receptor signaling pathway, and downregulated the genes involved in lipogenesis. Furthermore, treatment with mulberry leaves upregulated expression of the genes involved in the response to oxidative stress. These results indicate that consumption of fatty acids and inhibition of lipogenesis are responsible for the reduction in plasma lipids caused by mulberry administration. In addition, mulberry treatment maintains the body's oxidative state at a low level despite enhancing fatty acid oxidation.     

Determining important regulatory relations of amino acids from dynamic network analysis of plasma amino acids

The changes in the concentrations of plasma amino acids do not always follow the flow-based metabolic pathway network. We have previously shown that there is a control-based network structure among plasma amino acids besides the metabolic pathway map. Based on this network structure, in this study, we performed dynamic analysis using time-course data of the plasma samples of rats fed single essential amino acid deficient diet. Using S-system model (conceptual mathematical model represented by power-law formalism), we inferred the dynamic network structure which reproduces the actual time-courses within the error allowance of 13.17%. By performing sensitivity analysis, three of the most dominant relations in this network were selected; the control paths from leucine to valine, from methionine to threonine, and from leucine to isoleucine. This result is in good agreement with the biological knowledge regarding branched-chain amino acids, and suggests the biological importance of the effect from methionine to threonine.     

Bisleuconothine A,an eburnane–aspidosperma bisindole alkaloid from Leuconotis griffithii

A new bisindole alkaloid, bisleuconothine A (1) consisting of an eburnane–aspidosperma type skeleton, was isolated from the bark of Leuconotis griffithii. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and X-ray analysis. Bisleuconothine A (1) showed cell growth inhibitory activity against various human cancer cell lines.     

The evolution of the regulatory mechanism of chloroplast division

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division, reminiscent of their free-living ancestor. However, chloroplasts can not divide by themselves, and the division is performed and controlled by proteins that are encoded by the host nucleus. The continuity of chloroplasts was originally established by synchronization of endosymbiotic cell division with host cell division, as seen in existent algae. In contrast, land plant cells contain multiple chloroplasts, the division of which is not synchronized, even in the same cell. Land plants have evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts (or other types of plastids) change along with their respective cellular function by changes in the division rate. We recently reported that PLASTID DIVISION (PDV) proteins, land-plant specific components of the chloroplast division apparatus, determined the rate of chloroplast division. The level of PDV protein is regulated by the cell differentiation program based on cytokinin, and the increase or decrease of the PDV level gives rise to an increase or decrease in the chloroplast division rate. Thus, the integration of PDV proteins into the chloroplast division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.Key words: chloroplast division, cell cycle, cell differentiation, cytokinin, endosymbiosis, evolution     

Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270) was obtained using sequence-based screening. From {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type {"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.Prolyl aminopeptidase (PAP) (EC 3.4.11.5), belonging to the S33 family, is an exopeptidase that catalyzes the hydrolysis of the N terminus prolyl residue of peptides or proteins. This family has catalytic Ser. To date, few applications of this enzyme for peptide synthesis have been reported. However, from the perspective of biotechnology, PAP might be a good tool for synthesizing proline-containing peptides by catalyzing aminolysis.Recently nutraceutical properties of peptides containing proline have received increasing attention. For example, prolyl hydroxyproline (Pro-Hyp) stimulates the growth of fibroblasts from mouse skin (11). Pro-Arg can protect against oxidative stress/damage and H₂O₂-induced human diploid fibroblast cell death (13). Furthermore, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro exhibit angiotensin I-converting enzyme-inhibiting activity (9). In addition to these dipeptides and tripeptides, a cyclic dipeptide (namely, diketopiperazine) containing proline shows several physiological functions. Cyclo[Pro-Pro] (cPP) exerts antibacterial activity against Micrococcus luteus and Pseudomonas aeruginosa (8). Caspase-3 activation by cyclo[Pro-Phe] in HT-29 cells has been described (3). However, its synthesis method has not been established. Enzymatic peptide synthesis presents a useful and desirable strategy because it can conduct specific reactions under milder conditions than those of chemical synthesis.Engineered endoserine proteases that have Cys substituted for catalytic Ser have also been applied for peptide synthesis since subtiligase was constructed by Abrahmsén et al. (1). Because of the weakened hydrolytic activity of the parent enzyme, it is considered that Ser/Cys-substituted protease can trap the substrate (acyl donor). Then, a nucleophilic reaction occurs between another substrate (acyl acceptor) and the trapped acyl donor (2). This is a so-called “aminolysis” reaction. Although aminolysis can conduct peptide synthesis in an aqueous solution, the problem of the necessity of using an N-protected amino acid as an acyl donor remains when using endoproteases.These problems would be solved using exoprotease as a catalyst, because N-terminal free amino groups of acyl donors are recognized by enzymes. It is rarely reported that exoprotease was applied for peptide synthesis, except in the report of Oshiro et al., in which Pro-Phe, Pro-Tyr, and Pro-Trp were synthesized (10). Recently our group reported that the Ser/Cys variant of exoprotease, aminolysin-S, has been constructed and has produced l-Phe-l-Phe ethyl ester and their derivatives from non-N-protected phenylalanine and phenylalanine ethyl ester as acyl donors in aqueous solution (12). However, aminolysin-S cannot produce proline-containing dipeptides.In this study, we describe a PAP from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 ({"type":"entrez-protein","attrs":{"text":"PAP14270","term_id":"1238921194","term_text":"PAP14270"}}PAP14270). Furthermore, synthesis of various proline peptides was attempted through catalysis by its Ser/Cys variant (scPAP14270) from proline ester and several amino acids and their esters in aqueous solution. A basic characterization to determine the effect of pH and the amount of substrate was conducted. Moreover, correlation was found between proline peptide synthesis and the log D value, which is the distribution coefficient between octanol and water, of acyl acceptors in aminolysis mediated by scPAP14270.