The Cerebellar Cortex Receives Orofacial Proprioceptive Signals from the Supratrigeminal Nucleus via the Mossy Fiber Pathway in Rats.

Proprioceptive sensory information from muscle spindles is essential for the regulation of motor functions. However, little is known about the motor control regions in the cerebellar cortex that receive proprioceptive signals from muscle spindles distributed throughout the body, including the orofacial muscles. Therefore, in this study, we investigated the pattern of projections in the rat cerebellar cortex derived from the supratrigeminal nucleus (Su5), which conveys orofacial proprioceptive information from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 revealed that many bilateral axon terminals (rosettes) were distributed in the granular layer of the cerebellar cortex (including the simple lobule B, crus II and flocculus) in a various sized, multiple patchy pattern. We could also detect JCMS proprioceptive signals in these cerebellar cortical regions, revealing for the first time that they receive muscle proprioceptive inputs in rats. Retrograde tracer injections confirmed that the Su5 directly sends outputs to the cerebellar cortical areas. Furthermore, we injected an anterograde tracer into the external cuneate nucleus (ECu), which receives proprioceptive signals from the forelimb and neck muscle spindles, to distinguish between the Su5- and ECu-derived projections in the cerebellar cortex. The labeled terminals from the ECu were distributed predominantly in the vermis of the cerebellar cortex. Almost no overlap was seen in the terminal distributions of the Su5 and ECu projections. Our findings demonstrate that the rat cerebellar cortex receives orofacial proprioceptive input that is processed differently from the proprioceptive signals from the other regions of the body.

Cerebellar Nuclei Receiving Orofacial Proprioceptive Signals through the Mossy Fiber Pathway from the Supratrigeminal Nucleus in Rats.

Proprioception from muscle spindles is necessary for motor function executed by the cerebellum. In particular, cerebellar nuclear neurons that receive proprioceptive signals and send projections to the lower brainstem or spinal cord play key roles in motor control. However, little is known about which cerebellar nuclear regions receive orofacial proprioception. Here, we investigated projections to the cerebellar nuclei from the supratrigeminal nucleus (Su5), which conveys the orofacial proprioception arising from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 resulted in a large number of labeled axon terminals bilaterally in the dorsolateral hump (IntDL) of the cerebellar interposed nucleus (Int) and the dorsolateral protuberance (MedDL) of the cerebellar medial nucleus. In addition, a moderate number of axon terminals were ipsilaterally labeled in the vestibular group Y nucleus (group Y). We electrophysiologically detected JCMS proprioceptive signals in the IntDL and MedDL. Retrograde tracing analysis confirmed bilateral projections from the Su5 to the IntDL and MedDL. Furthermore, anterograde tracer injections into the external cuneate nucleus (ECu), which receives other proprioceptive input from forelimb/neck muscles, resulted in only a limited number of ipsilaterally labeled terminals, mainly in the dorsomedial crest of the Int and the group Y. Taken together, the Su5 and ECu axons almost separately terminated in the cerebellar nuclei (except for partial overlap in the group Y). These data suggest that orofacial proprioception is differently processed in the cerebellar circuits in comparison to other body-part proprioception, thus contributing to the executive function of orofacial motor control.

Enhanced Co-culture System Using Escherichia coli and Pichia pastoris (Komagataella phaffii) for Improved Microbial Production of Valuable Plant Alkaloids.

Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.

Transport engineering using tobacco transporter NtJAT1 enhances alkaloid production in Escherichia coli.

Transporters have been used in the production of plant metabolites in microorganisms. This study introduced a tobacco multidrug and toxic compound extrusion transporter, NtJAT1, into alkaloid-producing Escherichia coli cells. NtJAT1 expression enhanced alkaloid production secretion into the medium by 14 folds. Our findings further demonstrate the usefulness of the transport-engineering approach.

Overproduction of PGR5 enhances the electron sink downstream of photosystem I in a C4 plant, Flaveria bidentis.

C4 plants can fix CO2 efficiently using CO2 -concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C4 plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase-like complex (NDH) accumulated in the thylakoid membrane in leaves of C4 plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C4 plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C4 photosynthesis. In this study, PGR5 was overexpressed in a C4 dicot, Flaveria bidentis. In PGR5-overproducing (OP) lines, PGR5 levels were 2.3- to 3.0-fold greater compared with wild-type plants. PGR5-like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5-OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO2 assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.

Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production.

BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli-E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.

Disproportionality Analysis of Safety Signals for a Wide Variety of Opioid-Related Adverse Events in Elderly Patients Using the Japanese Adverse Drug Event Report (JADER) Database.

Opioids are widely used for the treatment of moderate/severe pain in cancer and noncancer patients. In this study, we searched for safety signals for a wide variety of opioid-related adverse events (AEs) in elderly patients by disproportionality analysis using the Japanese Adverse Drug Event Report (JADER) database. Data from the JADER database from April 2004 to May 2018 were obtained from the Pharmaceuticals and Medical Devices Agency website. Safety signal detection of opioid-related AEs in elderly patients was defined using the relative elderly reporting odds ratio (ROR). Among the analyzed AEs, opioid-induced neurotoxicity (OIN) was assessed based on the time to onset using the Weibull shape parameter. The following safety signals were detected in elderly patients: respiratory depression, somnolence, hallucinations, akathisia and OIN. Fentanyl, tramadol, oxycodone and morphine exhibited a large relative elderly ROR for OIN. The median time to onset of OIN of transdermal fentanyl, oral tramadol, oral oxycodone and oral morphine was 13.5, 6, 9, and 6 d, respectively. These opioids were classified as early failure types using the Weibull distribution. Our results showed that elderly patients who are administered opioids should be closely monitored for AEs, such as respiratory depression, OIN and akathisia.

Comparative analysis using the draft genome sequence of California poppy (Eschscholzia californica) for exploring the candidate genes involved in benzylisoquinoline alkaloid biosynthesis.

Genome characterization of California poppy (Eschscholzia californica cv. "Hitoezaki"), which produces pharmaceutically important benzylisoquinoline alkaloids (BIAs), was carried out using the draft genome sequence. The numbers of tRNA and rRNA genes were close to those of the other plant species tested, whereas the frequency of repetitive sequences was distinct from those species. Comparison of the predicted genes with those of Amborella trichopoda, Nelumbo nucifera, Solanum lycopersicum, and Arabidopsis thaliana, and analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway indicated that the enzyme genes involved in BIA biosynthesis were highly enriched in the California poppy genome. Further comparative analysis using the genome information of Papaver somniferum and Aquilegia coerulea, both BIA-producing plants, revealed that many genes encoding BIA biosynthetic enzymes, transcription factors, transporters, and candidate proteins, possibly related to BIA biosynthesis, were specifically distributed in these plant species.

Transgenerational lipid-reducing activity of benzylisoquinoline alkaloids in Caenorhabditis elegans.

Epigenetic mechanisms allow for transgenerational memory of an ancestor's environment and can affect the gene expression, physiology and phenotype of that ancestor's descendants, independent of DNA sequence alteration. Among many model organisms, Caenorhabditis elegans has been instrumental in studies of transgenerational inheritance, most of which have focused on the effects of external stressors of the parent worm on the life span and stress resistance of future generations. In this work, we used Nile red staining of accumulated lipids in C. elegans to investigate the transgenerational effect of two benzylisoquinoline alkaloids, namely, berberine and sanguinarine. Our results showed that a reduction in Nile red fluorescence can be propagated to subsequent worm generations. Using mutant worms, we found that the transgenerational effect requires the ASH-2 component of the histone H3K4me3 complex and the HRDE-1 worm Argonaute protein. Ash-2 is also required for transgenerational inheritance of the xenobiotic response in the worm. Our study offers new insights into transmissible drug effects across multiple generations and suggests the importance of such analyses in the drug development process.

Identification of a multi-component berberine 11-hydroxylase from Burkholderia sp. strain CJ1.

Berberine (BBR) is a protoberberine alkaloid extracted from plants such as Coptis japonica (Ranunculaceae). In a previous report, we demonstrated the existence of a 11-hydroxylation pathway employed by BBR-utilizing bacteria for metabolism of BBR. In the present study, we report the identification of the genes brhA, brhB, and brhC as encoding a multicomponent BBR 11-hydroxylase in Burkholderia sp. strain CJ1. BrhA is belonging to the Rieske non-heme iron oxygenase (RO) family, a class of enzymes known to catalyze the first step in bacterial aromatic-ring hydroxylation. We further demonstrate that BrhA activity requires BrhB (ferredoxin reductase) and BrhC (ferredoxin) as electron transport chain components. A BLAST search revealed that BrhA exhibits 38% and 33% sequence identity to dicamba O-demethylase (DdmC; AY786443) and chloroacetanilide herbicides N-dealkylase (CndA; KJ461679), respectively. To our knowledge, this work represents the first report of a bacterial oxygenase catalyzing the metabolism of a polycyclic aromatic-ring alkaloid.Abbreviations: BBR: berberine; D-BBR: demethyleneberberine; H-BBR: 11-hydroxyberberine; HD-BBR: 11-hydroxydemethyleneberberine; HDBA: 2-hydroxy-3,4-dimethoxybenzeneacetic acid; PAL: palmatine; H-PAL: 11-hydroxypalmatine; BRU: berberrubine; Fd: ferredoxin; FdR: ferredoxin reductase; ETC: electron transport chain.

Mining of the Uncharacterized Cytochrome P450 Genes Involved in Alkaloid Biosynthesis in California Poppy Using a Draft Genome Sequence.

Land plants produce specialized low molecular weight metabolites to adapt to various environmental stressors, such as UV radiation, pathogen infection, wounding and animal feeding damage. Due to the large variety of stresses, plants produce various chemicals, particularly plant species-specific alkaloids, through specialized biosynthetic pathways. In this study, using a draft genome sequence and querying known biosynthetic cytochrome P450 (P450) enzyme-encoding genes, we characterized the P450 genes involved in benzylisoquinoline alkaloid (BIA) biosynthesis in California poppy (Eschscholzia californica), as P450s are key enzymes involved in the diversification of specialized metabolism. Our in silico studies showed that all identified enzyme-encoding genes involved in BIA biosynthesis were found in the draft genome sequence of approximately 489 Mb, which covered approximately 97% of the whole genome (502 Mb). Further analyses showed that some P450 families involved in BIA biosynthesis, i.e. the CYP80, CYP82 and CYP719 families, were more enriched in the genome of E. californica than in the genome of Arabidopsis thaliana, a plant that does not produce BIAs. CYP82 family genes were highly abundant, so we measured the expression of CYP82 genes with respect to alkaloid accumulation in different plant tissues and two cell lines whose BIA production differs to estimate the functions of the genes. Further characterization revealed two highly homologous P450s (CYP82P2 and CYP82P3) that exhibited 10-hydroxylase activities with different substrate specificities. Here, we discuss the evolution of the P450 genes and the potential for further genome mining of the genes encoding the enzymes involved in BIA biosynthesis.

Roles of miR319 and TCP Transcription Factors in Leaf Development.

Sophisticated regulation of gene expression, including microRNAs (miRNAs) and their target genes, is required for leaf differentiation, growth, and senescence. The impact of miR319 and its target TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) genes on leaf development has been extensively investigated, but the redundancies of these gene families often interfere with the evaluation of their function and regulation in the developmental context. Here, we present the genetic evidence of the involvement of the MIR319 and TCP gene families in Arabidopsis (Arabidopsis thaliana) leaf development. Single mutations in MIR319A and MIR319B genes moderately inhibited the formation of leaf serrations, whereas double mutations increased the extent of this inhibition and resulted in the formation of smooth leaves. Mutations in MIR319 and gain-of-function mutations in the TCP4 gene conferred resistance against miR319 and impaired the cotyledon boundary and leaf serration formation. These mutations functionally associated with CUP-SHAPED COTYLEDON genes, which regulate the cotyledon boundary and leaf serration formation. In contrast, loss-of-function mutations in miR319-targeted and nontargeted TCP genes cooperatively induced the formation of serrated leaves in addition to changes in the levels of their downstream gene transcript. Taken together, these findings demonstrate that the MIR319 and TCP gene families underlie robust and multilayer control of leaf development. This study also provides a framework toward future researches on redundant miRNAs and transcription factors in Arabidopsis and crop plants.

Novel Compound Induces Erythropoietin Secretion through Liver Effects in Chronic Kidney Disease Patients and Healthy Volunteers.

BACKGROUND: TP0463518 is a novel hypoxia-inducible factor prolyl hydroxylase inhibitor developed to aid in the treatment of anemia associated with chronic kidney disease (CKD) and is expected to increase erythropoietin (EPO) derived from liver. Two phase I studies were conducted in healthy volunteers (HV) and CKD patients undergoing hemodialysis (i.e., HD patients) or those not undergoing dialysis (i.e., ND patients). METHODS: Pharmacokinetics, pharmacodynamics, and safety profiles of TP0463518 were assessed. Forty HV received single oral doses of TP0463518 at 3, 6, 11, 20, and 36 mg or placebo. Twenty ND patients received single doses of TP0463518 at 1, 6, and 11 mg and 9 HD patients received TP0463518 at 1 and 11 mg doses. To identify the source organ of EPO, glycosylation patterns were determined using percentage migrated isoform (PMI) values. RESULTS: Declining renal function slowed elimination of TP0463518 and increased the mean AUC0-∞. ∆Emax of serum EPO in 11-mg groups of HV, ND patients, and HD patients were 24.37 ± 11.37, 201.57 ± 130.34, and 1,324.76 ± 1,189.24 mIU/mL respectively. A strong correlation was -observed between logarithm conversions of ∆Emax and AUC0-∞ with correlation coefficients of 0.945. PMI values of blood after TP0463518 administration were elevated to similar or higher levels in comparison with those of umbilical cord blood, which mainly contains liver-derived EPO. CONCLUSIONS: TP0463518 induced dose-dependent EPO production, mainly derived from the liver in HV and CKD patients. These results suggest that TP0463518 is a new strategy for treating anemia in CKD, which can be used regardless of renal functions.

Comparison of rhythmic masticatory muscle activity during non-rapid eye movement sleep in guinea pigs and humans.

Rhythmic masticatory muscle activity can be a normal variant of oromotor activity, which can be exaggerated in patients with sleep bruxism. However, few studies have tested the possibility in naturally sleeping animals to study the neurophysiological mechanisms of rhythmic masticatory muscle activity. This study aimed to investigate the similarity of cortical, cardiac and electromyographic manifestations of rhythmic masticatory muscle activity occurring during non-rapid eye movement sleep between guinea pigs and human subjects. Polysomnographic recordings were made in 30 freely moving guinea pigs and in eight healthy human subjects. Burst cycle length, duration and activity of rhythmic masticatory muscle activity were compared with those for chewing. The time between R-waves in the electrocardiogram (RR interval) and electroencephalogram power spectrum were calculated to assess time-course changes in cardiac and cortical activities in relation to rhythmic masticatory muscle activity. In animals, in comparison with chewing, rhythmic masticatory muscle activity had a lower burst activity, longer burst duration and longer cycle length (P < 0.05), and greater variabilities were observed (P < 0.05). Rhythmic masticatory muscle activity occurring during non-rapid eye movement sleep [median (interquartile range): 5.2 (2.6-8.9) times per h] was preceded by a transient decrease in RR intervals, and was accompanied by a transient decrease in delta elelctroencephalogram power. In humans, masseter bursts of rhythmic masticatory muscle activity were characterized by a lower activity, longer duration and longer cycle length than those of chewing (P < 0.05). Rhythmic masticatory muscle activity during non-rapid eye movement sleep [1.4 (1.18-2.11) times per h] was preceded by a transient decrease in RR intervals and an increase in cortical activity. Rhythmic masticatory muscle activity in animals had common physiological components representing transient arousal-related rhythmic jaw motor activation in comparison to human subjects.

The expression of programed death ligand-1 could be related with unfavorable prognosis in salivary duct carcinoma.

BACKGROUND: Salivary duct carcinoma (SDC) is a rare tumor occurring in the salivary gland. SDC is a highly aggressive tumor and its prognosis is extremely poor. Effective treatments in advanced SDC have not yet been established. Recently, immune checkpoint inhibitors have paved the way for the treatment of various malignancies. We examined the expressions of programed death ligand (PD-L) 1/PD-L2 and programed death (PD-1), and the correlation of clinicopathological findings. METHODS: We examined 18 cases of SDC and conducted immunohistochemical staining using formalin-fixed paraffin-embedded full-face sections. RESULTS: The expression of PD-L1 and PD-L2 in tumor cells was observed in nine cases (50%) and 14 cases (78%), respectively. Cases with a high expression of PD-L1 and PD-L2 were found in four (22%) and seven cases (39%), respectively. The cases with a high expression of PD-L1 showed significantly shorter overall survival compared to those with low PD-L1 expression and null expression. We also examined the expression of PD-L1/PD-L2 and PD-1 of tumor-infiltrating mononuclear cells (TIMC) in stroma. The expressions of PD-L1 in tumor cells and stroma had a significant correlation. Association between the expressions of PD-L1 in tumor cells and those of PD-1 in stroma was significant. However, PD-L2 expression in the tumor had no significant correlation with expression in TIMCs. PD-L1, PD-L2 and PD-1 expressions in stroma were not associated with patient prognosis. CONCLUSIONS: High PD-L1 expression in SDC was strongly associated with unfavorable prognosis, indicating that PD-1/PD-L1 inhibitors could be effective in SDC.

Unraveling Additional O-Methylation Steps in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica).

California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.

Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus.

The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.

In vivo system for analyzing the function of the PsbP protein using Chlamydomonas reinhardtii.

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein-protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-∆15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII-LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII-LHCII supercomplex from green algae.

Laboratory-scale production of (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids, using a bacterial-based method.

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.

Increased intramyocellular lipid/impaired insulin sensitivity is associated with altered lipid metabolic genes in muscle of high responders to a high-fat diet.

The accumulation of intramyocellular lipid (IMCL) is recognized as an important determinant of insulin resistance, and is increased by a high-fat diet (HFD). However, the effects of HFD on IMCL and insulin sensitivity are highly variable. The aim of this study was to identify the genes in muscle that are related to this inter-individual variation. Fifty healthy men were recruited for this study. Before and after HFD for 3 days, IMCL levels in the tibialis anterior were measured by (1)H magnetic resonance spectroscopy, and peripheral insulin sensitivity was evaluated by glucose infusion rate (GIR) during the euglycemic-hyperinsulinemic clamp. Subjects who showed a large increase in IMCL and a large decrease in GIR by HFD were classified as high responders (HRs), and subjects who showed a small increase in IMCL and a small decrease in GIR were classified as low responders (LRs). In five subjects from each group, the gene expression profile of the vastus lateralis muscle was analyzed by DNA microarray analysis. Before HFD, gene expression profiles related to lipid metabolism were comparable between the two groups. Gene Set Enrichment Analysis demonstrated that five gene sets related to lipid metabolism were upregulated by HFD in the HR group but not in the LR group. Changes in gene expression patterns were confirmed by qRT-PCR using more samples (LR, n = 9; HR, n = 11). These results suggest that IMCL accumulation/impaired insulin sensitivity after HFD is closely associated with changes in the expression of genes related to lipid metabolism in muscle.