Bonding of Lignin and Coniferyl Alcohol by a Redox Shuttle of Low-Molecular-Weight Lignols in Enzymatic Oxidative Dehydrogenative Polymerization.

Lignin is an aromatic polymer that constitutes plant cell walls. The polymerization of lignin proceeds by radical coupling, and this process requires radicalization of the phenolic end of lignin by enzymes. However, due to the steric hindrance between enzymes, lignin, and polysaccharides, the direct oxidation of the phenolic end of lignin by the enzyme would be difficult, and the details of the growth of lignin are still unknown. In this study, enzymatic dehydrogenative polymerization experiments were conducted using coniferyl alcohol (CA) and the deuterium-labeled lignin model compound (D-LM) under a noncontact condition in which horseradish peroxidase cannot directly oxidize D-LM due to separation by a dialysis membrane. Analysis of deuterium-labeled degraded compounds obtained by a combination of methylation and thioacidolysis revealed the formation of the bond between the phenolic end of D-LM and CA, suggesting that membrane-permeable, low-molecular-weight lignols functioned as a redox shuttle mediator.

Semaporin3A inhibitor ameliorates renal fibrosis through the regulation of JNK signaling.

Renal fibrosis is the common pathological pathway in progressive renal diseases. In the present study, we analyzed the roles of semaphorin 3 A (SEMA3A) on renal fibrosis and the effect of SEMA3A inhibitor (SEMA3A-I) using a unilateral ureteral obstruction (UUO) mouse model. Expression of SEMA3A in the proximal tubulus and neuropilin-1, a recepor of SEMA3A, in fibloblast and tubular cells were increased in UUO kidneys. The expression of myofibroblast marker tenascin-C and fibronection as well as renal fibrosis were increased in UUO kidneys, all of which were ameliorated by SEMA3A-I. In addition, the JNK signaling pathway, known as the target of SEMA3A signaling, was activated in proximal tubular cells and fibroblast cells after UUO surgery, and SEMA3A-I significantly attenuated the activation. In vitro, treatments with SEMA3A as well as transforming growth factor-ß1 (TGF-ß1) in human proximal tubular cells lost epithelial cell characteristics, and SEMA3A-I significantly ameliorated this transformation. The JNK inhibitor SP600125 partially reversed SEMA3A and TGF-ß1-induced cell transformation, indicating that JNK signaling is involved in SEMA3A-induced renal fibrosis. In addition, treatment with SEMA3A in fibroblast cells activated expression of tenascin-C, collagen type I, and fibronection, indicating that SEMA3A may accelerate renal fibrosis through the activation of fibroblast cells. Analysis of human data revealed the positive correlation between urinary SEMA3A and urinary N-acetyl-ß-d-glucosaminidase, indicating the association between SEMA3A and tubular injury. In conclusion, SEMA3A signaling is involved in renal fibrosis through the JNK signaling pathway and SEMA3A-I might be a therapeutic option for protecting from renal fibrosis.NEW & NOTEWORTHY Renal fibrosis is the common pathological pathway in the progression of renal diseases. This study, using a unilateral ureteral obstruction (UUO) mouse model, indicated increased semaphorin3A (SEMA3A) signaling in renal tubular cells as well as fibroblast cells under UUO surgery, and SEMA3A inhibitor ameliorated UUO-induced renal fibrosis through the regulation of JNK signaling. The study proposes the potential therapeutic option of SEMA3A inhibitor to treat renal fibrosis.

Localised laccase activity modulates distribution of lignin polymers in gymnosperm compression wood.

The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression-wood-specific S2 L cell wall layer with lignins highly enriched with p-hydroxyphenyl (H)-type units derived from H-type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)-type units from G-type monolignol with a trace amount of H-type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H-type and G-type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa). We performed a series of chemical-probe-aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases. Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H-type and G-type monolignols were precisely localised to distinct cell wall layers in which H-type and G-type lignin units were preferentially produced during the development of compression wood tracheids. We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood.

Pembrolizumab-induced hypothyroidism caused reversible increased serum creatinine levels: a case report.

BACKGROUND: The advent of immune checkpoint inhibitors (ICIs) has significantly improved the prognosis of patients with advanced malignancies. On the other hand, these drugs might cause immune-related adverse events (irAEs) including endocrinopathies and nephropathies. Thyroid dysfunction is one of the most common irAEs. For ICIs-induced nephropathies, most cases are due to tubulointerstitial nephritis, which might require steroid treatment. Here, we report a patient with non-small cell lung cancer treated with ICI who developed increased serum creatinine (s-Cr) levels due to ICIs-induced hypothyroidism. CASE PRESENTATION: A 57-year-old Asian man with refractory non-small cell lung cancer under ICIs therapy (pembrolizumab, an anti-programmed cell death-1 monoclonal antibody) developed increased s-Cr levels 5 months after the pembrolizumab initiation. His laboratory data, renal biopsy, and Gallium-67 scintigraphy findings denied pembrolizumab-induced tubulointerstitial nephritis. His renal function was correlated with thyroid function. Despite the increase of s-Cr levels, serum cystatin C levels were normal, which could be explained by the hypothyroidism. Levothyroxine treatment improved renal function as well as thyroid function. Then pembrolizumab was resumed, and both his thyroid and renal function remained normal level. Ultimately, we concluded that the increased s-Cr levels were caused by pembrolizumab-induced hypothyroidism. CONCLUSION: All clinicians involved in ICI treatment need to recognize the possible increase in s-Cr levels caused by ICIs-induced hypothyroidism, and we propose monitoring serum cystatin C levels to differentiate ICIs-induced hypothyroidism from tubulointerstitial nephritis before invasive renal biopsies or steroid treatment, which are recommended by the prescribing information for pembrolizumab, are performed.

Sodium Glucose Co-Transporter 2 Inhibitor Ameliorates Autophagic Flux Impairment on Renal Proximal Tubular Cells in Obesity Mice.

Obesity is supposed to cause renal injury via autophagy deficiency. Recently, sodium glucose co-transporter 2 inhibitors (SGLT2i) were reported to protect renal injury. However, the mechanisms of SGLT2i for renal protection are unclear. Here, we investigated the effect of SGLT2i for autophagy in renal proximal tubular cells (PTCs) on obesity mice. We fed C57BL/6J mice with a normal diet (ND) or high-fat and -sugar diet (HFSD) for nine weeks, then administered SGLT2i, empagliflozin, or control compound for one week. Each group contained N = 5. The urinary N-acetyl-beta-d-glucosaminidase level in the HFSD group significantly increased compared to ND group. The tubular damage was suppressed in the SGLT2i-HFSD group. In electron microscopic analysis, multi lamellar bodies that increased in autophagy deficiency were increased in PTCs in the HFSD group but significantly suppressed in the SGLT2i group. The autophagosomes of damaged mitochondria in PTCs in the HFSD group frequently appeared in the SGLT2i group. p62 accumulations in PTCs were significantly increased in HFSD group but significantly suppressed by SGLT2i. In addition, the mammalian target of rapamycin was activated in the HFSD group but significantly suppressed in SGLT2i group. These data suggest that SGLT2i has renal protective effects against obesity via improving autophagy flux impairment in PTCs on a HFSD.

Semaphorin3A-Inhibitor Ameliorates Doxorubicin-Induced Podocyte Injury.

Podocyte injury is an independent risk factor for the progression of renal diseases. Semaphorin3A (SEMA3A), expressed in podocytes and tubular cells in the mammalian adult kidneys, has been reported to regulate diverse biological functions and be associated with renal diseases. Here, we investigated pathological roles of SEMA3A signaling on podocyte injury using a doxorubicin (Dox)-induced mouse model and examined the therapeutic effect of SEMA3A-inhibitor (SEMA3A-I). We demonstrated that Dox caused massive albuminuria and podocyte apoptosis as well as an increase of SEMA3A expression in podocytes, all of which were ameliorated with SEMA3A-I treatment. In addition, c-Jun N-terminal kinase (JNK), known as a downstream of SEMA3A signaling, was activated in Dox-injected mouse podocytes while SEMA3A-I treatment partially blocked the activation. In vitro, SEMA3A-I protected against Dox-induced podocyte apoptosis and recombinant SEMA3A caused podocyte apoptosis with activation of JNK signaling. JNK inhibitor, SP600125, attenuated SEMA3A-induced podocyte apoptosis, indicating that the JNK pathway would be involved in SEMA3A-induced podocyte apoptosis. Furthermore, the analysis of human data revealed a positive correlation between levels of urinary SEMA3A and protein, suggesting that SEMA3A is associated with podocyte injury. In conclusion, SEMA3A has essential roles in podocyte injury and it would be the therapeutic target for protecting from podocyte injury.

Localization of (+)-Catechin in Picea abies Phloem: Responses to Wounding and Fungal Inoculation.

To understand the positional and temporal defense mechanisms of coniferous tree bark at the tissue and cellular levels, the phloem topochemistry and structural properties were examined after artificially induced bark defense reactions. Wounding and fungal inoculation with Endoconidiophora polonica of spruce bark were carried out, and phloem tissues were frequently collected to follow the temporal and spatial progress of chemical and structural responses. The changes in (+)-catechin, (-)-epicatechin, stilbene glucoside, and resin acid distribution, and accumulation patterns within the phloem, were mapped using time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS), alongside detailed structural (LM, TEM, SEM) and quantitative chemical microanalyses of the tissues. Our results show that axial phloem parenchyma cells of Norway spruce contain (+)-catechins, the amount of which locally increases in response to fungal inoculation. The preformed, constitutive distribution and accumulation patterns of (+)-catechins closely follow those of stilbene glucosides. Phloem phenolics are not translocated but form a layered defense barrier with oleoresin compounds in response to pathogen attack. Our results suggest that axial phloem parenchyma cells are the primary location for (+)-catechin storage and synthesis in Norway spruce phloem. Chemical mapping of bark defensive metabolites by cryo-ToF-SIMS, in addition to structural and chemical microanalyses of the defense reactions, can provide novel information on the local amplitudes and localizations of chemical and structural defense mechanisms and pathogen-host interactions of trees.

A Patient with Type 3 Autoimmune Polyglandular Syndrome who Developed Systemic Lupus Erythematosus 8 years after the Diagnosis of Autoimmune Hepatitis.

Eight years prior to her present admission, a 61-year-old Japanese woman was diagnosed with autoimmune hepatitis, slowly progressive insulin-dependent diabetes mellitus, and chronic thyroiditis; she had been treated with oral prednisolone (PSL). After she suddenly discontinued PSL, she newly developed systemic lupus erythematosus. A combination therapy of oral PSL and intravenous cyclophosphamide resulted in remission. She was finally diagnosed with autoimmune polyglandular syndrome (APS) type 3 (3A ,3B, 3D), complicated with four different autoimmune diseases. Since patients with type 3 APS may present many manifestations over a long period of time, they should be carefully monitored.

In Planta Localization of Stilbenes within Picea abies Phloem.

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.

Lignin-rich enzyme lignin (LREL), a cellulase-treated lignin-carbohydrate derived from plants, activates myeloid dendritic cells via Toll-like receptor 4 (TLR4).

Lignin-carbohydrates, one of the major cell wall components, are believed to be the structures that form chemical linkage between lignin and cell wall polysaccharides. Due to the molecular complexity of lignin-containing substances, their isolation and the assignment of their biological activities have so far remained a difficult task. Here, we extracted two lignin-containing carbohydrates, lignin-rich enzyme lignin (LREL) and pure enzyme lignin (PEL), from barley husk and demonstrated that they act as immune stimulators of dendritic cells (DCs), which are particularly important in linking innate and adaptive immunity. Thioacidolysis, acid hydrolysis, and mild alkali hydrolysis of both LREL and PEL revealed that their immunostimulatory activities depended on the lignin structure and/or content, neutral sugar content (especially the characteristic distribution of galactose and mannose), and presence of an ester bond. Furthermore, we showed that the immunostimulatory potency of the lignin-carbohydrate depended on its molecular weight and degree of polymerization. We also demonstrated that the LREL-induced activation of DCs was mediated via TLR4. Thus, LREL-induced increases in the expression levels of several cell surface marker proteins, production of inflammatory cytokines IL-12p40 and TNF-α, and activation and nuclear translocation of transcription factors, as was observed in the WT DCs, were completely abrogated in DCs derived from the TLR4(-/-) mice but not in DCs derived from the TLR2(-/-), TLR7(-/-), and TLR9(-/-) mice. We further demonstrated that LRELs isolated from other plant tissues also activated DCs. These immunostimulatory activities of lignin-carbohydrates, extracted from edible plant tissues, could have potential relevance in anti-infectious immunity and vaccine adjuvants.

The accumulation pattern of ferruginol in the heartwood-forming Cryptomeria japonica xylem as determined by time-of-flight secondary ion mass spectrometry and quantity analysis.

BACKGROUND AND AIMS: Heartwood formation is a unique phenomenon of tree species. Although the accumulation of heartwood substances is a well-known feature of the process, the accumulation mechanism remains unclear. The aim of this study was to determine the accumulation process of ferruginol, a predominant heartwood substance of Cryptomeria japonica, in heartwood-forming xylem. METHODS: The radial accumulation pattern of ferruginol was examined from sapwood and through the intermediate wood to the heartwood by direct mapping using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The data were compared with quantitative results obtained from a novel method of gas chromatography analysis using laser microdissection sampling and with water distribution obtained from cryo-scanning electron microscopy. KEY RESULTS: Ferruginol initially accumulated in the middle of the intermediate wood, in the earlywood near the annual ring boundary. It accumulated throughout the entire earlywood in the inner intermediate wood, and in both the earlywood and the latewood in the heartwood. The process of ferruginol accumulation continued for more than eight annual rings. Ferruginol concentration peaked at the border between the intermediate wood and heartwood, while the concentration was less in the latewood compared with the earlywood in each annual ring. Ferruginol tended to accumulate around the ray parenchyma cells. In addition, at the border between the intermediate wood and heartwood, the accumulation was higher in areas without water than in areas with water. CONCLUSIONS: TOF-SIMS clearly revealed ferruginol distribution at the cellular level. Ferruginol accumulation begins in the middle of intermediate wood, initially in the earlywood near the annual ring boundary, then throughout the entire earlywood, and finally across to the whole annual ring in the heartwood. The heterogeneous timing of ferruginol accumulation could be related to the distribution of ray parenchyma cells and/or water in the heartwood-forming xylem.

Compositional changes of human hair melanin resulting from bleach treatment investigated by nanoscale secondary ion mass spectrometry.

BACKGROUND/PURPOSE: It is important to understand the influence of bleach treatment on human hair because it is one of the most important chemical treatments in hair cosmetic processes. A comparison of the elemental composition of melanin between virgin hair and bleached hair would provide important information about the structural changes of melanin. To investigate the elemental composition of melanin granules in virgin black hair and bleached hair, these hair cross-sections are analyzed by using a nanoscale secondary ion mass spectrometry (NanoSIMS). METHODS: The virgin black hair and bleached hair samples were embedded in resin and smooth hair cross-sections were obtained using an ultramicrotome. NanoSIMS measurements were performed using a Cs(+) primary ion beam to detect negative secondary ions. RESULTS: More intensive (16) O(-) ions were detected from the melanin granules of bleached hair than from those of virgin black hair in NanoSIMS (16) O(-) ion image. In addition, it was indicated that (16) O(-) ion intensity and (16) O(-) /(12) C(14) N(-) ion intensity ratio of melanin granules in bleached hair were higher than those in virgin black hair. CONCLUSION: Nanoscale secondary ion mass spectrometry analysis of the cross-sections of virgin black hair and bleached hair indicated that the oxygen content in melanin granules was increased by bleach treatment.

A case of membranous nephropathy complicated by Cronkhite-Canada syndrome successfully treated with mizoribine.

Cronkhite-Canada syndrome (CCS) is a non-hereditary disorder characterized by non-neoplastic hamartomatous gastrointestinal polyposis, hair loss, nail atrophy, hyperpigmentation, and diarrhea. While the relationship between CCS and nephritis remains unclear, seven cases of nephritis complicated by CCS have been reported to date, all of which were membranous nephropathy (MN). A 57-year-old man presented with taste disturbance, hair loss, nail plate atrophy, skin pigmentation, and frequent diarrhea. Endoscopic findings showed multiple polyposis of the stomach and large intestine. Given the above, he was diagnosed with CCS. The symptoms gradually improved with prednisolone treatment, although urinary protein and hypoproteinemia appeared during the tapering of prednisolone. He was diagnosed with MN using a renal biopsy, and immunofluorescence microscopy with IgG subclass staining showed predominantly diffuse granular capillary wall staining of IgG4. The cause of secondary MN was not found, including malignant tumors. Nephrotic-range proteinuria persisted despite treatment with prednisolone and cyclosporine. Additional treatment with mizoribine resulted in incomplete remission type 1 of nephrotic syndrome, suggesting that mizoribine may be a treatment option for patients with CCS with steroid-resistant MN. Considering a high prevalence of hypoproteinemia due to chronic diarrhea and protein-losing enteropathy in patients with CCS, proteinuria might be overlooked; thus, follow-up urinalysis would be recommended in patients with CCS.

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry.

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.

Preventive effect of culture supernatant of epithelial-like peritoneal mesothelial cells on peritoneal fibrosis.

Peritoneal fibrosis (PF) is a primary reason for discontinuing peritoneal dialysis, which involves characteristic changes of peritoneal mesothelial cells (PMCs). We previously reported preventive effects of implanting human epithelial-like PMCs (P-Epi) for mouse PF caused by mechanical peritoneum scrapings. In the present study, we analysed the preventive effects of culture supernatant of P-Epi in PF. Concentrated culture supernatant of P-Epi or human fibroblast-like PMCs (P-Fibro) or vehicles was injected into nude mice that had undergone mechanical scraping of the parietal and visceral peritoneum, and thickness and amount of adhesions were analysed. Although increased peritoneal adhesions and peritoneum thickening were observed in the vehicle-injected positive control group compared to the sham operation group, fewer number of adhesions and less thickness were observed in the mice treated with culture supernatant of P-Epi, but not P-Fibro, compared to the vehicle-injected positive controls. Immunofluorescent analysis revealed that the expression of extracellular matrix, type I collagen and fibronectin, was lower in the mice treated with culture supernatant of P-Epi than in the vehicle-injected positive controls. In addition, exosomes from P-Epi significantly reduced transforming growth factor-ß (TGF-ß)-induced expressions of type I collagen and fibronectin in 3T3 fibroblast cells. Collectively, culture supernatant of P-Epi has preventive effects on PF, thus cell therapy is not necessarily required. Further exploration of substances secreted by P-Epi and their protective mechanisms could lead to the development of therapeutic strategies to limit PF.

Role of Semaphorin 3A in Kidney Development and Diseases.

Kidney diseases are worldwide public health problems affecting millions of people. However, there are still limited therapeutic options against kidney diseases. Semaphorin 3A (SEMA3A) is a secreted and membrane-associated protein, which regulates diverse functions, including immune regulation, cell survival, migration and angiogenesis, thus involving in the several pathogeneses of diseases, including eyes and neurons, as well as kidneys. SEMA3A is expressed in podocytes and tubular cells in the normal adult kidney, and recent evidence has revealed that excess SEMA3A expression and the subsequent signaling pathway aggravate kidney injury in a variety of kidney diseases, including nephrotic syndrome, diabetic nephropathy, acute kidney injury, and chronic kidney disease. In addition, several reports have demonstrated that the inhibition of SEMA3A ameliorated kidney injury via a reduction in cell apoptosis, fibrosis and inflammation; thus, SEMA3A may be a potential therapeutic target for kidney diseases. In this review article, we summarized the current knowledge regarding the role of SEMA3A in kidney pathophysiology and their potential use in kidney diseases.

MicroRNAs as Biomarkers and Therapeutic Targets for Acute Kidney Injury.

Acute kidney injury (AKI) is a clinical syndrome where a rapid decrease in kidney function and/or urine output is observed, which may result in the imbalance of water, electrolytes and acid base. It is associated with poor prognosis and prolonged hospitalization. Therefore, an early diagnosis and treatment to avoid the severe AKI stage are important. While several biomarkers, such as urinary L-FABP and NGAL, can be clinically useful, there is still no gold standard for the early detection of AKI and there are limited therapeutic options against AKI. miRNAs are non-coding and single-stranded RNAs that silence their target genes in the post-transcriptional process and are involved in a wide range of biological processes. Recent accumulated evidence has revealed that miRNAs may be potential biomarkers and therapeutic targets for AKI. In this review article, we summarize the current knowledge about miRNAs as promising biomarkers and potential therapeutic targets for AKI, as well as the challenges in their clinical use.

Microscopic distribution of alkaloids in freeze-fixed stems of Phellodendron amurense.

Introduction: Phellodendron amurense Rupr. contains rich alkaloids, which have been extensively applied in clinical treatments for their various biological activities. However, detailed microscopic distribution and roles of such alkaloids in P. amurense stem still need to be clarified. Methods: In this study, the distribution of eight alkaloids in the transverse surface of freeze-fixed P. amurense stems in fall and summer has been visualized by cryo-time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM), which was found in living tissues with relative contents of different alkaloids varying with the position. In addition, the contents of these alkaloids quantified by high-performance liquid chromatography (HPLC) analysis suggested the seasonal variation from fall to the following summer. Results and discussion: Distribution of eight alkaloids in the freeze-fixed stems of P. amurense from fall and summer seasons has been visualized and assigned into specific living tissues, with relative contents varying in different positions with seasons, which suggested their possible roles in the physiological processes of the plant itself or plant responding to changes in the surrounding conditions. Conclusion: This study provided a significant basis for further discussion of the genes or enzymes involved in these processes, which will contribute to investigating biosynthetic pathways and specific in planta roles of alkaloids.