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Abstract Objectives The growth and development of the epiglottis and preepiglottic space (PES) of the human larynx as it acquires the vocal tract were investigated. Methods Three newborns, one infant, four children (2, 7, 8, and 12 years old), and two adult normal larynges were investigated and compared using the whole organ serial section technique. Results The newborn PES occupied a small area just anterior to the epiglottis. It was composed of immature adipose tissue and areolar tissue. The epiglottis lay on a somewhat horizontal axis and is partially obscured behind the hyoid bone. The hyoid bone overlapped the thyroid cartilage, partially obscuring the superior thyroid notch. The newborn epiglottic cartilage was immature elastic cartilage, and the elastic fiber component was sparse. In the first 8 years of life, as the PES grew, the PES was located not only anterior to but also posterolateral and inferolateral to the epiglottic cartilage and thyroepiglottic ligament. Meanwhile, the epiglottic cartilage matured. Conclusions In order to develop the vocal tract for speech production, it is reported that the human larynx descends as the child grows in the first 9 years of life. This study showed that the PES, occupying a small area just anterior to the epiglottis, grew and existed astride the epiglottis as the larynx descended and the vocal tract developed. Consequently, its distribution allows the epiglottis to more effectively play the role of retroflection during swallowing in order to prevent aspiration. The human speech faculty likely develops in conjunction with swallowing physiology.

Abstract Objective Pretreatment systemic inflammation and nutrition‐based prognostic indices (SINBPI) have demonstrated significance. This study investigated the prognostic value of pretreatment SINBPI for patients with oropharyngeal cancer and identified unfavorable prognostic markers. Methods We retrospectively reviewed the data of 124 patients with oropharyngeal squamous cell carcinoma (OPSCC) who received definitive treatment between January 2010 and December 2018. The prognostic utility of the neutrophil‐to‐lymphocyte ratio, platelet‐to‐lymphocyte ratio (PLR), lymphocyte‐to‐monocyte ratio (LMR), prognostic nutritional index, and high‐sensitivity modified Glasgow prognostic score (HS‐mGPS) was assessed for disease‐free survival (DFS), disease‐specific survival (DSS), and overall survival (OS) using univariate and multivariate analyses. Results Multivariate analyses revealed that human papillomavirus (HPV) status and HS‐mGPS were significantly associated with DFS, DSS, and OS. Patients with a HS‐mGPS of 2 had a significantly higher rate of treatment‐related deaths than those with a HS‐mGPS of 0 or 1. The combination of the HS‐mGPS and PLR had more accurate predictive ability in DFS and OS compared with the HS‐mGPS alone, and the combination of the HS‐mGPS and LMR had more accurate predictive ability in DSS and OS. Conclusion Our results indicated that the HS‐mGPS was a useful prognostic marker for patients with OPSCC, and combined markers consisting of the HS‐mGPS and PLR or LMR may provide more accurate prognostic predictions. Level of Evidence: 3.

Abstract Objectives To analyze various aspects of complex tissue, there is increasing demand to study each sample at different length scales in biology. Correlative light and electron microscopy (CLEM) is the latest technique to correlate two different types of information on the exact same histological area of interest: histology (from light microscopy) and ultrastructure (from electron microscopy). The three‐dimensional fine structures of the maculae flavae (MFe) of the human vocal fold were investigated using CLEM. Methods Five normal human adult vocal folds as specimens embedded in paraffin, sectioned, and mounted on glass slides with/without a chemical digestion method (modified sodium hydroxide maceration method) were investigated. Observations using CLEM were performed. Results The fine structures of cells and extracellular matrices in the MFe and their peripheral regions were able to be observed on the exact same histological area of interest with the light microscope and field emission‐scanning electron microscope. Cobblestone‐like polygonal cells, vocal fold stellate cell‐like cells, and fibroblast‐like spindle cells were intermingled in the MFe of the human vocal fold. The extracellular matrices surrounding each three types of cell in the MFe differed, suggesting the cells were different in functional property. Conclusion CLEM is a useful technique to observe the three‐dimensional fine structures of the human vocal fold mucosa. The results of the present study are consistent with the hypothesis that the cells in the MFe of the human vocal fold have heterogeneity and each three types of cell have different properties.

California poppy or golden poppy (Eschscholzia californica) is the iconic state flower of California, with native ranges from Northern California to Southwestern Mexico. It grows well as an ornamental plant in Mediterranean climates, but it might be invasive in many parts of the world. California poppy was also highly prized by Native Americans for its medicinal value, mainly due to its various specialized metabolites, especially benzylisoquinoline alkaloids (BIAs). As a member of the Ranunculales, the sister lineage of core eudicots it occupies an interesting phylogenetic position. California poppy has a short-lived life cycle but can be maintained as a perennial. It has a comparatively simple floral and vegetative morphology. Several genetic resources, including options for genetic manipulation and a draft genome sequence have been established already with many more to come. Efficient cell and tissue culture protocols are established to study secondary metabolite biosynthesis and its regulation. Here, we review the use of California poppy as a model organism for plant genetics, with particular emphasis on the evolution of development and BIA biosynthesis. In the future, California poppy may serve as a model organism to combine two formerly separated lines of research: the regulation of morphogenesis and the regulation of secondary metabolism. This can provide insights into how these two integral aspects of plant biology interact with each other.

Abstract 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.

Abstract Objectives Our previous investigations showed that tissue stem cells in the maculae flavae (a stem cell niche) form colonies in vivo like stem cells in vitro. However, the roles of colony‐forming cells in the maculae flavae in vivo have not yet been determined. This study investigated the metabolism of the colony‐forming tissue stem cells in the maculae flavae of the human adult vocal fold. Study design Histologic analysis of the human vocal folds. Methods Three normal human adult vocal folds were investigated under transmission electron microscopy and light microscopy including immunohistochemistry. Results Mitochondrial cristae of the colony‐forming cells in the maculae flavae were sparse. Hence, the microstructural features of the mitochondria suggested that their metabolic activity and oxidative phosphorylation were low. Colony‐forming cells strongly expressed glucose transporter‐1 and glycolytic enzymes (hexokinase II, glyceraldehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase A). The colony‐forming cells did not express phosphofructokinase‐1 but did express glucose‐6‐phosphate dehydrogenase indicating the cells relied more on the pentose phosphate pathway. Since the colony‐forming cells expressed lactate dehydrogenase A, cells seemed to rely more on anaerobic glycolysis in an anaerobic microenvironment. Conclusions The present study is consistent with the hypothesis that the colony‐forming tissue stem cells in the maculae flavae of the human adult vocal fold seemed to rely more on anaerobic glycolysis using the pentose phosphate pathway for energy supply in vivo. Microstructural features of the mitochondria and expressed glycolytic enzymes of the colony‐forming cells in the maculae flavae suggested that the oxidative phosphorylation activity was low. In an anaerobic microenvironment in vivo, there is likely a complex cross‐talk regarding the metabolism between the colony‐forming aggregated cells along the adhesion machinery and chemical signaling pathways, which reduces toxic oxygen species and is favorable to maintaining the stemness and undifferentiated states of the tissue stem cells. Level of Evidence NA.

Abstract Objectives In our previous studies, the features of the mitochondria of tissue stem cells in the maculae flavae of the human vocal fold suggested that their metabolic activity and oxidative phosphorylation was low. This study investigated the metabolic activity, especially glycolysis of the tissue stem cells in the maculae flavae of the human adult vocal fold. Study Design Histologic analysis of the human vocal folds. Methods Three normal human adult vocal folds obtained from autopsy cases were investigated using immunohistochemistry. Results Among the three phenotypes of cells in the human adult maculae flavae, the vocal fold stellate cell‐like cells strongly expressed glucose transporter‐1. Three phenotypes of cells in the human adult maculae flavae expressed glycolytic enzymes (hexokinase II, glyceraldehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase A) indicating the tissue stem cells in the maculae flavae relied more on glycolysis. The cells did not express phosphofructokinase‐1 but did express glucose‐6‐phosphate dehydrogenase indicating the cells relied more on the pentose phosphate pathway. The cells expressed lactate dehydrogenase A indicating the maculae flavae of the human adult vocal fold was likely to be an anaerobic microenvironment. Conclusions The present study is consistent with the hypothesis that the tissue stem cells in the maculae flavae of the human vocal fold seem to rely more on anaerobic glycolysis, especially by the pentose phosphate pathway, for energy supply. The metabolism of the tissue stem cells in the maculae flavae of the human adult vocal fold is likely to prevent toxic reaction oxygen species and be favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cells system. Level of Evidence NA.

Abstract Objectives There is growing evidence that the cells in the maculae flavae (MFe) are tissue stem cells and the MFe are a stem cell niche of the human vocal fold mucosa. Heterogeneity and hierarchy of tissue stem cells in the MFe of newborn vocal fold were investigated in vivo. Study design Histologic analysis of the human vocal folds. Methods Five normal human newborn vocal folds were investigated under transmission electron microscopy and light microscopy. Results Cobblestone‐like polygonal cells, vocal fold stellate cell‐like cells, and fibroblast‐like spindle cells were intermingled in the newborn MFe in vivo, indicating that the cells in the MFe had heterogeneity. However, cobblestone‐like polygonal cells were predominant. Free ribosomes were well developed in the cytoplasm. The cells in some cases formed gap junctions with each other. The cells in some cases were attached to other cells and formed cell junctions with each other. These findings indicated cells in the newborn maculae flavae possessed features of mesenchymal cells (cells in mesenchyme). Colony‐forming‐unit‐like cell aggregate was observed, indicating the cells in the newborn MFe had stemness. The cobblestone‐like polygonal cells expressed SSEA‐3 (a human pluripotent stem cell marker), indicating they were at the top of a cellular hierarchy in the stem cell system. Conclusions The cells in the MFe of the human newborn vocal fold mucosa had heterogeneity and hierarchy in the stem cell system in vivo. At birth, newborn maculae flavae are ready to start the growth of the vocal fold mucosa as a vibrating tissue.

Abstract Objective Histopathology of the maxillary sinus mucosa with intractable odontogenic maxillary sinusitis (OMS) was investigated and the role endoscopic sinus surgery (ESS) plays in its pathophysiology was clarified. Study Design Histopathological analysis of the OMS mucosa. Methods Surgical specimens were obtained from 20 patients who underwent ESS for intractable OMS. For rigid endoscopic observation of the mucosae, a 70° rigid endoscope 4 mm in diameter with an attached high definition surgical camera was used. Histopathological analyses of the maxillary sinus mucosa were conducted by light and scanning electron microscopy. Results All the maxillary sinuses were filled, not with viscous, but with purulent secretions. The high‐definition camera showed that the maxillary sinus mucosa had gyrus‐like appearance. Light microscopic histopathological studies revealed that the surface of the maxillary sinus mucosa was convoluted. Light and scanning electron microscopic histopathological studies revealed that the ciliated cells of the epithelium had not decreased and their goblet cells were not hypertrophic, indicating that the damage of the ciliated columnar epithelium was not severe and they were not injured irreversibly. Conclusion The ciliated columnar epithelium with intractable OMS was not severely damaged and not irreversibly injured. Hence, the pathophysiology of intractable OMS is one of the reasons why ESS is highly indicated for maxillary sinusitis requiring surgery and the treatment results are exceptionally good when the ventilation and drainage of the maxillary sinus is successfully restored after surgery. Level of Evidence NA

Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

Transcription factors of the WRKY family play pivotal roles in plant defense responses, including the biosynthesis of specialized metabolites. Based on the previous findings of WRKY proteins regulating benzylisoquinoline alkaloid (BIA) biosynthesis, such as CjWRKY1—a regulator of berberine biosynthesis in Coptis japonica—and PsWRKY1—a regulator of morphine biosynthesis in Papaver somniferum—we performed genome-wide characterization of the WRKY gene family in Eschscholzia californica (California poppy), which produces various BIAs. Fifty WRKY genes were identified by homology search and classified into three groups based on phylogenetic, gene structure, and conserved motif analyses. RNA sequencing showed that several EcWRKY genes transiently responded to methyl jasmonate, a known alkaloid inducer, and the expression patterns of these EcWRKY genes were rather similar to those of BIA biosynthetic enzyme genes. Furthermore, tissue expression profiling suggested the involvement of a few subgroup IIc EcWRKYs in the regulation of BIA biosynthesis. Transactivation analysis using luciferase reporter genes harboring the promoters of biosynthetic enzyme genes indicated little activity of subgroup IIc EcWRKYs, suggesting that the transcriptional network of BIA biosynthesis constitutes multiple members. Finally, we investigated the coexpression patterns of EcWRKYs with some transporter genes and discussed the diversified functions of WRKY genes based on a previous finding that CjWRKY1 overexpression in California poppy cells enhanced BIA secretion into the medium.

Objectives There is growing evidence to suggest that the cells in the maculae flavae of the vocal fold mucosa are tissue stem cells of the human vocal fold. This study investigated the metabolic activity of the cells in the maculae flavae of the human vocal fold from the aspect of mitochondrial microstructure. Study Design Histologic analysis of the human vocal folds. Methods Five normal human adult vocal folds obtained from autopsy cases were investigated under transmission electron microscopy. Results Mitochondria were randomly distributed in the cytoplasm of the cells. The morphological features of the mitochondria consisted of a double‐membrane‐bounded body containing matrices and a system of cristae. In each mitochondria, the lamellar cristae were sparse. The intercristal space was occupied by a mitochondrial matrix which contained electron‐dense matrix granules, mitochondrial DNA, and ribonucleoprotein granules. Some mitochondria spread out over or fused to the surface of a lipid droplet in the cytoplasm. In addition, both the mitochondrial outer and inner membranes and the membranes of the lipid droplets had disappeared. Some close association between mitochondria and rough endoplasmic reticulum was present. Conclusions The features of the mitochondria of the cells in the maculae flavae of the human vocal fold suggested that their metabolic activity and oxidative phosphorylation were low and that they may have shifted to the utilization of lipids to some extent for their metabolic needs. Level of Evidence NA

This article presents small RNA sequencing data of Caenorhabditis elegans consist of P0 control worms (untreated), P0 worms treated with a plant alkaloid, sanguinarine, and its F3 offspring. The data were analyzed to identify microRNAs that were differentially expressed in both the sanguinarine-treated P0 and its descendants F3 worms. Targets of the identified miRNAs, gene function annotations and their functional clusters are shown. The data presented here will facilitate comparison with data from other researchers who are working on miRNAs profiling of xenobiotic-treated C. elegans. Keywords: Caenorhabditis elegans, Sanguinarine, MicroRNA, Benzylisoquinoline alkaloids, Transgenerational inheritance

Plants produce a large variety of low-molecular-weight and specialized secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used in the pharmaceutical industry. Although alkaloid chemistry has been intensively investigated, characterization of alkaloid biosynthesis, including biosynthetic enzyme genes and their regulation, especially the transcription factors involved, has been relatively delayed, since only a limited number of plant species produce these specific types of alkaloids in a tissue/cell-specific or developmental-specific manner. Recent advances in molecular biology technologies, such as RNA sequencing, co-expression analysis of transcripts and metabolites, and functional characterization of genes using recombinant technology and cutting-edge technology for metabolite identification, have enabled a more detailed characterization of alkaloid pathways. Thus, transcriptional regulation of alkaloid biosynthesis by transcription factors, such as basic helix–loop–helix (bHLH), APETALA2/ethylene-responsive factor (AP2/ERF), and WRKY, is well elucidated. In addition, jasmonate signaling, an important cue in alkaloid biosynthesis, and its cascade, interaction of transcription factors, and post-transcriptional regulation are also characterized and show cell/tissue-specific or developmental regulation. Furthermore, current sequencing technology provides more information on the genome structure of alkaloid-producing plants with large and complex genomes, for genome-wide characterization. Based on the latest information, we discuss the application of transcription factors in alkaloid engineering.

Abstract Plants produce many specific secondary metabolites as a response to environmental stress, especially biological stress. These compounds show strong biological activities and high stability against degradation by microbes and animals. Berberine, a benzylisoquinoline alkaloid, is found in many plant species and has strong antimicrobial activity, and is often included in traditional herbal medicines. We previously investigated how berberine is degraded in nature and we isolated two berberine-utilizing bacteria. In this study, we characterized the gene encoding the enzyme that degrades the 2,3-methylenedioxy ring of berberine; this ring is important for its activity and stability. Further characterization of several other berberine-utilizing bacteria and the genes encoding key demethylenation enzymes revealed that these enzymes are tetrahydrofolate dependent and similar to demethylation enzymes such as GcvT. Because the degradation of O-methyl groups or the methylenedioxy ring in phenolic compounds such as lignin, lignan and many other natural products, including berberine, is the key step for the catabolism of these compounds, our discovery reveals the common origin of the catabolism of these stable chemicals in bacteria.

Opiates—the gold standard for pain relief—are currently produced by extraction from opium poppies. Here the authors show that bacteria can serve as an efficient and flexible platform for the production of opiates by demonstrating the total synthesis of Thebaine and hydrocodone from stepwise fermentation in E. coli.

Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole alkaloids (MIAs), nicotine alkaloids, and benzylisoquinoline alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.

The effect of micro ridges on orientation of cultured cells has been studied in vitro. Several patterns of micro ridges have been fabricated on a transparent polydimethylsiloxane disk with the photo lithography technique. The ridges consist of several lines of rectangular column: the width of 0.003 mm, the interval of 0.007 mm. Variation has been made on the height of the ridge between 0.0003 mm and 0.0035 mm. C2C12 (mouse myoblast cell line originated with cross-striated muscle of C3H mouse) was cultured on the disk with the micro ridges for one week and was observed with an inverted phase contrast microscope. The experimental results show that cells adhere on the top of the ridge and align to the longitudinal direction of the micro ridges with the height between 0.0015 mm and 0.0025 mm.

Advanced Driver Assistance System (ADAS) and Automated Driving (AD) are the two major topics for the current and next generations of vehicles. To realize them in full-size vehicles equipped with a 12 V power supply, the need for electric power steering (EPS) is increasing. Currently, the steering system of full-size vehicles is equipped with hydraulic power steering due to electric power shortage. An auxiliary power supply system using a lithium-ion capacitor was developed in order to solve the concern. In addition, to mount the system in the vehicle with no cooling−heating equipment, development of expanding the operating temperature range of the lithium-ion capacitor was conducted. Several improvements have made the capacitor operate stably in high-temperature environments above 100 °C.

The response of cells to a flow has been studied in vitro. The response of cells was examined in two types of flow channels: a circumnutating flow in a donut-shaped open channel in a culture dish, and a one-way flow in a parallelepiped rhombus flow channel. Variation was made on the material of the parallelepiped channel to study on adhesion of cells to the plates: glass and polydimethylsiloxane. Behavior of cells on the plate was observed under a flow of a medium with an inverted phase-contrast-microscope. The shear stress on the plate is calculated with an estimated parabolic distribution of the velocity between the parallel plates. The adhesion of cells was evaluated with the cumulated shear, which is a product of the shear stress and the exposure time. The experimental results show that cells are responsive to the flow, which governs orientation, exfoliation, and differentiation. The response depends on the kinds of cells: endothelial cells orient along the stream line, although myocytes orient perpendicular to the stream line. The adhesion depends on the combination between scaffold and cell: myocytes are more adhesive to glass than cartilage cells, and fibroblasts are more adhesive to oxygenated polydimethylsiloxane than glass.

The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH) transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA) and by a chromatin immunoprecipitation (ChIP) assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

Shikonin derivatives are specialized lipophilic metabolites, secreted in abundant amounts from the root epidermal cells of Lithospermum erythrorhizon. Because they have anti-microbial activities, these compounds, which are derivatives of red naphthoquinone, are thought to serve as a chemical barrier for plant roots. The mechanism by which they are secreted from cells is, however, largely unknown. The shikonin production system in L. erythrorhizon is an excellent model for studying the mechanism by which lipophilic compounds are secreted from plant cells, because of the abundant amounts of these compounds produced by L. erythrorhizon, the 0 to 100% inducibility of their production, the light-specific inhibition of production, and the visibility of these products as red pigments. To date, many factors regulating shikonin biosynthesis have been identified, but no mechanism that regulates shikonin secretion without inhibiting biosynthesis has been detected. This study showed that inhibitors of membrane traffic strongly inhibit shikonin secretion without inhibiting shikonin production, suggesting that the secretion of shikonin derivatives into the apoplast utilizes pathways common to the ADP-ribosylation factor/GTPase-activating protein (ARF/GAP) system and actin filament polymerization, at least in part. These findings provide clues about the machinery involved in secreting lipid-soluble metabolites from cells.

An effect of an electric field on proliferation and on differentiation of cultured muscle cells has been studied in vitro. C2C12 (the mouse myoblast cell line originated with the cross-striated muscle of C3H mouse) was exposed to electric stimuli. In the first experiment, the adhered cells were exposed to the electric field between two electrodes made of platinum wire of 0.2 mm diameter dipped in the medium at 37 degrees Celsius for 72 hours. The electric pulses at a period of one second with a pulse width of 0.1 second were generated with a function generator. Variation was made on the pulse amplitude < 12 V. The number of adhered cells was counted after exposure to electric stimulation. In the second experiment, the cells were cultivated for 96 hours without electric stimulation in an incubator, after electric stimulation of 0.1 V for 72 hours. After incubation, the movement of myotubes was observed with electric stimulation at a period of one second with a pulse width of one millisecond of 30 V. The experimental results show that cells adhere and proliferate under electric pulses lower than 8 V, and that differentiation accelerates with the electric pulses of 0.1 V.

California poppy or golden poppy (Eschscholzia californica) is the iconic state flower of California, with native ranges from Northern California to Southwestern Mexico. It grows well as an ornamental plant in Mediterranean climates, but it might be invasive in many parts of the world. California poppy was also highly prized by Native Americans for its medicinal value, mainly due to its various specialized metabolites, especially benzylisoquinoline alkaloids (BIAs). As a member of the Ranunculales, the sister lineage of core eudicots it occupies an interesting phylogenetic position. California poppy has a short-lived life cycle but can be maintained as a perennial. It has a comparatively simple floral and vegetative morphology. Several genetic resources, including options for genetic manipulation and a draft genome sequence have been established already with many more to come. Efficient cell and tissue culture protocols are established to study secondary metabolite biosynthesis and its regulation. Here, we review the use of California poppy as a model organism for plant genetics, with particular emphasis on the evolution of development and BIA biosynthesis. In the future, California poppy may serve as a model organism to combine two formerly separated lines of research: the regulation of morphogenesis and the regulation of secondary metabolism. This can provide insights into how these two integral aspects of plant biology interact with each other. [ABSTRACT FROM AUTHOR]