Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates ...from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.
Introduction
Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how ...these aberrant myeloid cells and fibroblasts are produced remains unclear.
Aim and methods
In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination.
Results
Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms.
Conclusion
A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.
To explore useful natural compounds from indigenous medicinal plants, the cytotoxic properties from a methanolic extract of
Boehmeria sieboldiana
leaves against human cancer cell lines were isolated ...in the present study. After purification of the extract,
seco
-dehydroantofine B (
1
) together with two known phenanthroindolizine alkaloids,
seco
-dehydroantofine A (
2
) and septicine (
3
), were isolated. The structure of
seco
-dehydroantofine B was elucidated by performing comprehensive one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The cytotoxicity of these compounds against five human tumor cell lines was evaluated. Compound
3
exhibited anti-tumor activity at IC
50
values of 50.0, 66.9, 50.0, and 153.7 µM against MKN1, SAS, HL-60, and THP-1 cells, respectively.
Amomum Semen, the seed mass of
Amomum xanthioides,
has been imported from Southeast Asia and China and used for the treatment of gastric and intestinal disorders.
A. xanthioides
has been treated as a ...synonym of
A. villosum
var.
xanthioides
. Furthermore,
A. villosum
var.
villosum, A. villosum
var.
xanthioides
, or
A. longiligulare
have been described as the botanical origin of Amomi Fructus, which is a similar crude drug in Chinese Pharmacopoeia. Under these circumstances, the botanical origin of Amomum Semen was changed to
A. villosum
var.
xanthioides
,
A. villosum
var.
villosum,
or
A. longiligulare
in Supplement II to the 17th edition of the Japanese Pharmacopoeia. To develop an objective identification method for Amomum Semen and to confirm the phylogenetic relationship among
Amomum
taxa, the nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacer region and chloroplast DNA partial
mat
K-
trn
K and
trn
H-
psb
A intergenic spacer regions were determined in specimens collected from Southeast Asia and China, including those from the type localities of each taxon. Six taxa were divided into four groups.
A. xanthioides
from Myanmar belonging to group 1 was discriminated from
A. villosum
var.
xanthioides
from China of group 2.
A. villosum
and its varieties were divided into two groups: group 2 included those from China, and group 3 consisted of
A. villosum
from Laos.
A. longiligulare
from China and Laos and
A. uliginosum
from Laos belonged to group 3 and group 4, respectively. These findings illustrate the phylogenetic basis for the need for taxonomical reorganization among the
Amomum
species.
is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of
has shown accumulation ...of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of
, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of
with
identified 20,232 orthogroups, representing 30,633 unigenes of
, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.
Apricot and Peach Kernels are commercial crude drugs used in many formulas of traditional Japanese medicine, Kampo. Although their applications are quite different, it is difficult to distinguish ...them using conventional methods such as HPLC. The study aimed at near-infrared (NIR) metabolic profiling to discriminate Apricot and Peach Kernels (Armeniacae Semen and Persicae Semen) collected from Japanese markets. A fast, simple, non-destructive, and robust NIR measurement of kernel surface with no sample pre-treatment was achieved in situ. Principal component analysis and orthogonal partial least squares discriminant analysis (OPLS-DA) models showed discrimination between the two crude drugs with good fitting and prediction values. These results indicate that NIR metabolic profiling is useful for discriminating Apricot and Peach Kernels based on their chemical constituents using a simple and non-destructive procedure.
Epithelial–mesenchymal transition (EMT) has recently been associated with cancer invasion, metastasis, and resistance. In our previous study, we discovered nanaomycin K, a natural growth inhibitor ...for EMT-induced Madin Darby canine kidney (MDCK) cells, from the cultured broth of actinomycetes. However, the screening method was undeveloped, because the activity of nanaomycin K was discovered accidentally. In this study, we established a screening method by analyzing the characteristics of nanaomycin K in MDCK cells. Nanaomycin K showed the characteristic growth inhibitory activity on MDCK cells cultured under four conditions: medium containing dimethyl sulfoxide, SB431542, TGF-β, and a mixture of SB431542 and TGF-β. The activity was stronger in TGF-β-treated cells than in DMSO-treated cells. In the mixture of SB431542 and TGF-β-treated cells, the activity of nanaomycin K was suppressed. The anti-cancer agents, mitomycin C, cisplatin, and staurosporine, lacked the characteristics as that of nanaomycin K for these four treatment conditions. Since these four conditions distinguish between the effects of nanaomycin K and other anti-cancer agents in EMT-induced cells, the screening method was established. Among the 13,427 plant extracts tested,
Piper betle
leaf extract displayed growth inhibitory activity against EMT-induced cells. Through the purification of the extract via bio-guided fractionation, hydroxychavicol was isolated as an active compound. The cytotoxic activity of hydroxychavicol was stronger in EMT-induced MDCK cells than in control cells. However, its cytotoxic activity was suppressed in EMT-inhibited cells. Furthermore, hydroxychavicol exhibited same activity against SAS cells (human squamous cell carcinoma of the tongue). Thus, we have successfully established a screening method for growth inhibitors of EMT-induced cells and have discovered an inhibitor from plant-based sources.
Graphical abstract
A discriminant LC/MS quantitative analysis of ephedrine (EP) and pseudoephedrine (PEP) in Ephedrae herba was performed. Aerial parts of three
Ephedra
species were separated into internodes and ...extracted using Finger Masher with minimum loss. The contents of EP and PEP were measured by LC/MS/MS using the multiple reaction monitoring (MRM) method. Their contents in old-year branches were lower than those current-year branches and tended to be higher in the middle part than in the tip of each branch. The content ratio of EP and PEP was reversed in some branches depending on their extent of growth. In
E. sinica
, the contents were low at the first internode closest to the central main stem at each branch. The contents drastically increased from the second internode and were highest at the third internode. There was a strong correlation between the internode distance and alkaloid (EP + PEP) contents. The distribution of alkaloids in one internode was examined and the results showed that the part closest to the node had the lowest contents.
Graphic abstract
This study used two types of analyses and statistical calculations on powdered samples of Polygala root (PR) and Senega root (SR): (1) determination of saponin content by an independently developed ...quantitative analysis of tenuifolin content using a flow reactor, and (2) near-infrared spectroscopy (NIR) using crude drug powders as direct samples for metabolic profiling. Furthermore, a prediction model for tenuifolin content was developed and validated using multivariate analysis based on the results of (1) and (2). The goal of this study was to develop a rapid analytical method utilizing the saponin content and explore the possibility of quality control through a wide-area survey of crude drugs using NIR spectroscopy. Consequently, various parameters and appropriate wavelengths were examined in the regression analysis, and a model with a reasonable contribution rate and prediction accuracy was successfully developed. In this case, the wavenumber contributing to the model was consistent with that of tenuifolin, confirming that this model was based on saponin content. In this series of analyses, we have succeeded in developing a model that can quickly estimate saponin content without post-processing and have demonstrated a brief way to perform quality control of crude drugs in the clinical field and on the market.
Graphical abstract
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