Functional characterization of Capsicum chinense vanillin aminotransferase: Detection of vanillylamine-forming activity from vanillin.

In capsaicin biosynthesis, vanillin aminotransferase (VAMT; EC 2.6.1.119) catalyzes the conversion of vanillin (V) to vanillylamine (VA). In vitro analysis of the recombinant VAMT enzyme has been reported; however, this enzyme catalyzed only the V-forming reaction and not the VA-forming reaction, which is inconsistent with the postulated pathway for capsaicin biosynthesis. In this study, we expressed, purified, and characterized functional recombinant VAMT of Capsicum chinense cv. Habanero from an Escherichia coli strain. The enzyme catalyzed reversible transamination between V and VA, and its VA-forming activity was high when γ-aminobutyric acid (GABA) was used as an amino donor. The enzyme exhibited maximum activity at pH 8.0 and 55 °C, and was stable up to 60 °C over a pH range from 4.5 to 8.0. The enzyme was stable in the presence of various chemicals and metal ions. The enzyme accepted several 4-8-carbon long primary amines and ω-amino acids with carbon chains longer than 4 as amino donors despite the narrow specificity of the amino acceptor. Based on its kinetic attributes and localization, VAMT appears to have evolved from GABA-aminotransferase to catalyze reversible transamination between V and VA, and is responsible for VA biosynthesis using GABA as an amino donor in the cytosol of capsicum fruit cells.

Effects of lipoprotein nanoparticles' composition and size on their internalization in plant and mammalian cells.

The internalization of engineered high-density lipoprotein nanoparticles (engineered lipoproteins [eLPs]) with different lipid and protein compositions, zeta potentials, and/or sizes were analyzed in representative plant and mammalian cells. The impact of the addition of a cell-penetrating peptide to eLPs on the internalization was very small in Bright Yellow (BY)-2 protoplasts compared with HeLa cells. When eLPs were prepared with one of the abundant lipids in BY-2 cells, digalactosyldiacylglycerol (DGDG) (eLP4), its internalization was dramatically increased only in HeLa cells. Such an increase in HeLa cells was also obtained for liposomes containing DGDG in a DGDG content-dependent manner. Increasing the size and zeta potential of eLPs improved their internalization in both HeLa cells and in BY-2 protoplasts but to quite varying degrees. Although eLPs tended to stay at the plasma membrane (PM) in BY-2 protoplasts with much less internalization, the PM-bound eLPs somehow promoted the internalization of coexisting nanobeads in cell culture media. These results provide fundamental insight into the future design of lipid nanoparticles for drug delivery in mammalian and plant cells.

Innovative Eco-Friendly Microwave-Assisted Rapid Biosynthesis of Ag/AgCl-NPs Coated with Algae Bloom Extract as Multi-Functional Biomaterials with Non-Toxic Effects on Normal Human Cells.

Harmful algal blooms impact human welfare and are a global concern. Sargassum spp., a type of algae or seaweed that can potentially bloom in certain regions of the sea around Thailand, exhibits a noteworthy electron capacity as the sole reducing and stabilizing agent, which suggests its potential for mediating nanoparticle composites. This study proposes an eco-friendly microwave-assisted biosynthesis (MAS) method to fabricate silver nanoparticles coated with Sargassum aqueous extract (Ag/AgCl-NPs-ME). Ag/AgCl-NPs-ME were successfully synthesized in 1 min using a 20 mM AgNO3 solution without additional hazardous chemicals. UV-visible spectroscopy confirmed their formation through a surface plasmon resonance band at 400-500 nm. XRD and FTIR analyses verified their crystalline nature and involvement of organic molecules. TEM and SEM characterization showed well-dispersed Ag/AgCl-NPs-ME with an average size of 36.43 nm. The EDS results confirmed the presence of metallic Ag+ and Cl- ions. Ag/AgCl-NPs-ME exhibited significant antioxidant activity against free radicals (DPPH, ABTS, and FRAP), suggesting their effectiveness. They also inhibited enzymes (tyrosinase and ACE) linked to diseases, indicating therapeutic potential. Importantly, the Ag/AgCl-NPs-ME displayed remarkable cytotoxicity against cancer cells (A375, A549, and Caco-2) while remaining non-toxic to normal cells. DNA ladder and TUNEL assays confirmed the activation of apoptosis mechanisms in cancer cells after a 48 h treatment. These findings highlight the versatile applications of Ag/AgCl-NPs-ME in food, cosmetics, pharmaceuticals, and nutraceuticals.

Identification of tuliposides K-M in tulip bulbs via an enzyme reaction-based screening method using a tuliposide-converting enzyme.

Tuliposides (Pos) are major defense-related secondary metabolites in tulip, having 4-hydroxy-2-methylenebutanoyl and/or (3S)-3,4-dihydroxy-2-methylenebutanoyl groups at the C-1 and/or C-6 positions of d-glucose. The acyl group at the C-6 position is converted to antimicrobial lactones (tulipalins) by an endogenous Pos-converting enzyme. Based on this enzyme activity, we examined tulip bulb extracts and detected HPLC peaks that disappeared following the reaction by the Pos-converting enzyme. Spectroscopic analyses of the three purified compounds revealed that one of them was a glucose ester-type Pos, while the other two were identified as a glucoside ester-type Pos. These compounds were designated as PosK, L, and M. They were specific to bulbs, with the highest content in the outermost layer, but they were markedly less abundant than PosG, the minor bulb Pos we identified earlier. The study results suggest that tulip bulbs contain at least four minor Pos in addition to the major 6-PosA. Although PosK-M were present in almost all of the tested tulip cultivars, they were detected in only a few wild species, indicative of their potential utility as chemotaxonomic markers in tulip. Identification of PosK-M as 6-PosA derivatives unveils the biosynthetic diversity of Pos, the well-known group of secondary metabolites in tulip.

Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

High-transcriptional activation ability of bamboo SECONDARY WALL NAC transcription factors is derived from C-terminal domain.

The secondary cell wall, which is mainly composed of cellulose, hemicellulose, and lignin, constitutes woody tissues and gives physical strength and hydrophobic properties for resistance against environmental stresses. We cloned and functionally analyzed the homologous transcription factor (TF) genes of SECONDARY WALL NAC (SWN) proteins from Hachiku bamboo (Phyllostachys nigra; PnSWNs). An RT-PCR analysis showed that PnSWNs are expressed in young tissues in bamboo. Their transcriptional activation activities were higher than that of the Arabidopsis NAC SECONDARY WALL THICKENING PROMOTING FACTOR 3 (NST3) TF, which was equivalent to SWN TFs in monocot. PnSWNs preferred to activate the genes related to secondary cell wall formation but not the genes related to programmed cell death. When PnSWNs were expressed in Arabidopsis, they highly induced secondary cell wall formation, like previously-shown rice SWN1. Dissection analysis revealed that this high activity largely depends on C-terminal domain. These results demonstrate that the cloned bamboo SWNs function as regulators of secondary cell wall formation with strong activation ability derived from C-terminal domain, and could be served as new genetic tools for secondary cell wall manipulation.

First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-L-α-lysine (ε-PαL) and ε-oligo-L-ß-lysine (ε-OßL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OßL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OßL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.

BAHD acyltransferase induced by histone deacetylase inhibitor catalyzes 3-O-hydroxycinnamoylquinic acid formation in bamboo cells.

Suspension-cultured cells of a bamboo species (Bambusa multiplex; Bm) produce 3-O-feruloylquinic acid (3-FQA) and 3-O-p-coumaroylquinic acid (3-pCQA) by treatment with the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA). Acyltransferases catalyzing the formation of 5-O-hydroxycinnamoylquinic acid esters by transesterification from hydroxycinnamoyl-CoAs to the C-5 hydroxy group of quinic acid (hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, HQT) have been identified in the biosynthesis of chlorogenic acids and monolignols; however, an HQT that catalyzes the acylation of the C-3 hydroxy group of quinic acid has not been identified previously. In the present study, we purified a native HQT from SBHA-treated Bm cells. The purified enzyme preferentially accepted feruloyl-/p-coumaroyl-CoAs as acyl-donors and quinic acid as the acyl-acceptor, and the enzyme specifically formed 3-FQA and 3-pCQA but not 5-O-hydroxycinnamoylquinic acid esters or esters with shikimic acid. A cDNA (BmHQT1) encoding this HQT was isolated. Although BmHQT1 is a phylogenetically unique member of the BAHD acyltransferase superfamily that does not cluster with other HQTs, functional characterization of the recombinant enzyme verified that BmHQT1 catalyzes the regiospecific formation of 3-O-hydroxycinnamoylquinic acid esters. Transcript levels of BmHQT1 markedly increased in Bm cells cultured in the presence of SBHA. Moreover, elevated acetylation levels of histone H3 were observed in the coding region of BmHQT1 in the presence of SBHA, indicating that the induced accumulation of 3-FQA/3-pCQA by SBHA is caused by transcriptional activation of BmHQT1 by the action of SBHA as a histone deacetylase inhibitor. The results demonstrate the utility of HDAC inhibitors for discovery of cryptic secondary metabolites and unknown biosynthetic enzymes.

Generation of Fusarium oxysporum-suppressive soil with non-soil carriers using a multiple-parallel-mineralization technique.

Disease-suppressive soils exist worldwide. However, the disease-suppression mechanism is unknown, and it's unclear how to produce such soils. The microbiota that develop in a multiple-parallel-mineralization system (MPM) can increase nutrient production efficiency and decrease root disease in hydroponic systems. Artificial media inoculated with MPM microorganisms can degrade organic matter to produce inorganic nutrients similarly to natural soil, but it's unknown whether they can also suppress pathogen growth. Here, we produced an artificial medium that inhibited root disease similarly to disease-suppressive soils. Microbial MPM culture solution was inoculated into non-soil carriers (rockwool, rice husk charcoal, and vermiculite) to test whether it could suppress growth of Fusarium oxysporum f. sp. lactucae J. C. Hubb. & Gerik. We inoculated F. oxysporum f. sp. conglutinans (Wollenweber) Snyder et Hansen strain Cong:11 and F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik into artificial media sown each with Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. var. capitata supplemented with MPM culture microbes. The MPM microorganisms suppressed F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik growth and prevented plant disease. Thus, MPM-inoculated non-soil carriers that can generate inorganic nutrients from organic matter may also suppress disease in the absence of natural soil. Our study shows novel creation of a disease-suppressive effect in non-soil media using the microbial community from MPM culture solution.

Occurrence of Z-2-oxo-4-methyl-3-pentene-1,5-dioic acid and its regioisomer 4-methylene-2-oxo-glutaric acid in tulip tissues.

Although Z-2-oxo-4-methyl-3-pentene-1,5-dioic acid (Z-OMPD) has been identified as a major dicarboxylic acid in tulip tissues, its biosynthetic pathway has not been elucidated. Herein, Z-OMPD was isolated from tulip leaves and chemically synthesized. Comparisons of these samples revealed that Z-OMPD exists as a tautomeric mixture at physiological pH. As a regioisomer of Z-OMPD, we enzymatically and chemically prepared 4-methylene-2-oxo-glutaric acid (4-MEOG) for the first time. Using these compounds as standards, the occurrence of Z-OMPD and 4-MEOG in various tissues of the tulip cultivar "Murasakizuisho" was evaluated directly and by 2,4-dinitrophenylhydrazone derivatization. Z-OMPD was found to be abundant in the aerial tissues, whereas 4-MEOG was almost absent from all tissues. Stability analyses of Z-OMPD and 4-MEOG revealed that no double bond isomerization occurred at physiological pH, suggesting that enzyme systems are responsible for Z-OMPD biosynthesis in tulip tissues.

Discovery of a Polyamino Acid Antibiotic Solely Comprising l-ß-Lysine by Potential Producer Prioritization-Guided Genome Mining.

While the genome mining approach has enabled the rational exploration of untapped bioactive natural products, in silico identifications of their biosynthetic genes are often unconnected to the actual production of the corresponding molecules in native strains due to the genetic dormancy. We report here the rational discovery of an unexplored cationic homo polyamino acid (CHPA) antibiotic by potential producer prioritization-guided genome mining. Mining the genome of γ-poly-d-diaminobutyric acid (poly-d-Dab)-producing Streptoalloteichus hindustanus NBRC 15115, which was selected based on the finding that the known CHPAs are universally co-produced in pairs, identified a putative CHPA synthetase, PblA, as a potential candidate being expressed actively. Bioinformatic and biochemical analyses of PblA provided the critical clue that its polymer product could be an unusual CHPA consisting of l-ß-lysine. Instrumental analyses of the metabolites from S. hindastanus indeed revealed the production of an unprecedented linear CHPA, ε-poly-l-ß-lysine, concomitantly with poly-d-Dab. The CHPA we discovered exerted excellent antimicrobial activity against a broad spectrum of microorganisms, including bacteria and fungi, and was revealed to show resistance against nonspecific proteolytic enzymes. This study marks the first report of the efficacy of the strain prioritization-guided genome mining strategy for the discovery of bioactive CHPAs.

Activation of Cryptic Secondary Metabolite Biosynthesis in Bamboo Suspension Cells by a Histone Deacetylase Inhibitor.

Plants have evolved a diverse array of secondary metabolite biosynthetic pathways. Undifferentiated plant cells, however, tend to biosynthesize secondary metabolites to a lesser extent and sometimes not at all. This phenomenon in cultured cells is associated with the transcriptional suppression of biosynthetic genes due to epigenetic alterations, such as low histone acetylation levels and/or high DNA methylation levels. Here, using cultured cells of bamboo (Bambusa multiplex; Bm) as a model system, we investigated the effect of histone deacetylase (HDAC) inhibitors on the activation of cryptic secondary metabolite biosynthesis. The Bm suspension cells cultured in the presence of an HDAC inhibitor, suberoyl bis-hydroxamic acid (SBHA), exhibited strong biosynthesis of some compounds that are inherently present at very low levels in Bm cells. Two major compounds induced by SBHA were isolated and were identified as 3-O-p-coumaroylquinic acid (1) and 3-O-feruloylquinic acid (2). Their productivities depended on the type of basal culture medium, initial cell density, and culture period, as well as the SBHA concentration. The biosynthesis of these two compounds was also induced by another HDAC inhibitor, trichostatin A. These results demonstrate the usefulness of HDAC inhibitors to activate cryptic secondary metabolite biosynthesis in cultured plant cells.

Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol ß-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase.

Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol ß-primeveroside (4-VPP) and 4-vinylguaiacol ß-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.

[A Case in Which Multiple Post-Operatively Recurring Hepatocellular Carcinomas Showed a Positive Response to Sorafenib-Hepatic Arterial Infusion Chemotherapy (HAIC)Sequential Therapy].

A male patient in his 70s underwent a right lobectomy because of a hepatocellular carcinoma(HCC)located in the right lobe(S6)of his liver. Eleven months after surgery, contrast-enhanced CT showed multiple masses in the residual liver, which were diagnosed as HCC recurrence. He was then treated with hepatic arterial infusion chemotherapy(HAIC). Ten months after the recurrence, the liver tumors progressed. Therefore, treatment was switched to sorafenib(400 mg/day orally)and HAIC(low-dose FP: 5-FU 250 mg plus CDDP 5 mg 5 days/week 4 weeks)sequential therapy. The patient received 2 cycles of sorafenib-HAIC sequential therapy for 11 months, and his liver tumors shrunk considerably. Unfortunately, 24 months after the recurrence of HCC, he died of respiratory failure. The cause of his death was officially determined to be primary lung cancer. An autopsy revealed that most tissues were necrotic, and only a small number of viable tumor cells were present in the liver tumors. This suggests that sorafenib-HAIC sequential therapy was significantly effective in targeting the multiple HCCs in this case.

Intraductal Papillary Mucinous Neoplasm with Pancreatogastric Fistula.

We herein report a rare case of intraductal papillary mucinous neoplasm with a pancreatogastric fistula in an elderly Japanese man admitted to our hospital. The pancreatogastric fistula was confirmed using endoscopic retrograde pancreatography via a cannulated guidewire placed in the stomach. Six months after admission, the patient was diagnosed with intraductal papillary mucinous carcinoma. A pancreatogastric fistula is generally a rare complication of intraductal papillary mucinous neoplasm. It was caused by mechanical penetration in this case. Interestingly, we also observed endoscopic and histochemical mucosal changes in the fistula.

A rare case of Helicobacter pylori-uninfected intramucosal poorly differentiated adenocarcinoma that occurred in the gastric fornix.

We report a rare case of undifferentiated-type intramucosal gastric cancer that occurred in the fornix of the stomach without Helicobacter pylori infection, which consisted mainly of poorly differentiated adenocarcinoma. A 49-year-old man visited our hospital for a follow-up endoscopic examination of a small depressed lesion of the gastric fornix detected by surveillance esophagogastroduodenoscopy. On magnifying endoscopy with blue laser imaging, the depressed lesion (approximately 10 mm in diameter) was regarded as undifferentiated-type early gastric cancer that proved to be a poorly differentiated adenocarcinoma by histological examination of biopsied specimens. The cancerous lesion was successfully treated with endoscopic submucosal dissection and microscopically showed an intramucosal cancer that invaded the whole mucosal layer with predominant growth of a poorly differentiated adenocarcinoma component. The patient status was verified as Helicobacter pylori-naïve according to the strict diagnostic criteria, thereby confirming this case as an undifferentiated-type Helicobacter pylori-uninfected gastric cancer. Helicobacter pylori-uninfected intramucosal poorly differentiated adenocarcinoma occurring in the gastric fornix has not been previously reported.

Bioproduction of glucose conjugates of 4-hydroxybenzoic and vanillic acids using bamboo cells transformed to express bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase.

Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14 g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.

Identification of tuliposide G, a novel glucoside ester-type tuliposide, and its distribution in tulip.

Tuliposides (Pos) are major defensive secondary metabolites in tulip (genus Tulipa), having 4-hydroxy-2-methylenebutanoyl and/or (3S)-3,4-dihydroxy-2-methylenebutanoyl groups at the C-1 and/or C-6 positions of d-glucose. The acyl group at the C-6 position is converted to antimicrobial lactones, tulipalins, by tuliposide-converting enzymes (TCEs). In the course of a survey of tulip tissue extracts to identify novel Pos, we found a minute high-performance liquid chromatography peak that disappeared following the action of a TCE, and whose retention time differed from those of known Pos. Spectroscopic analyses of the purified compound, as well as its enzymatic degradation products, revealed its structure as 5″-O-(6-O-(4'-hydroxy-2'-methylenebutanoyl))-ß-d-glucopyranosyl-(2″R)-2″-hydroxymethyl-4″-butyrolactone, which is a novel glucoside ester-type Pos. We gave this compound the trivial name 'tuliposide G' (PosG). PosG accumulated in bulbs, at markedly lower levels than 6-PosA (the major Pos in bulbs), but was not found in any other tissues. Quantification of PosG in bulbs of 52 types of tulip, including 30 cultivars (Tulipa gesneriana) and 22 wild Tulipa spp., resulted in the detection of PosG in 28 cultivars, while PosG was present only in three wild species belonging to the subgenus Tulipa, the same subgenus to which tulip cultivars belong, suggesting the potential usefulness of PosG as a chemotaxonomic marker in tulip.

Isolation and identification of tuliposides D and F from tulip cultivars.

6-Tuliposides A (6-PosA) and B (6-PosB) are major defensive secondary metabolites in tulip cultivars (Tulipa gesneriana), having an acyl group at the C-6 position of d-glucose. Although some wild tulip species produce 1,6-diacyl-glucose type of Pos (PosD and PosF), as well as 6-PosA/B, they have not yet been isolated from tulip cultivars. Here, aiming at verifying the presence of PosD and PosF in tulip cultivars, tissue extracts of 25 cultivars were analyzed by high-performance liquid chromatography (HPLC). Although no HPLC peaks for PosD nor PosF were detected in most cultivars, we found two cultivars giving a minute HPLC peak for PosD and the other two cultivars giving that for PosF. PosD and PosF were then purified from petals of cultivar 'Orca' and from pistils of cultivar 'Murasakizuisho', respectively, and their identities were verified by spectroscopic analyses. This is the first report that substantiates the presence of 1,6-diacyl-glucose type of Pos in tulip cultivars.

Granulocyte Colony-stimulating Factor- and Interleukin-6-producing Large-cell Carcinoma of the Lung with Sarcomatoid Changes Suggestive of Epithelial-mesenchymal Transition: An Autopsy Case Report.

A rare case of lung cancer with the simultaneous production of granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) is reported. A 79-year-old man was admitted to our hospital due to cachectic symptoms and an increased inflammatory response. Laboratory tests and imaging studies suggested metastatic lung cancer with high serum levels of G-CSF and IL-6. He died of progressive disease, and an autopsy showed that the lung tumor had positive protein expression of both cytokines and a solid growth of large-cell carcinoma with sarcomatoid changes, possibly resulting from the epithelial-mesenchymal transition mediated by IL-6 and leading to widespread metastases.