Spatiotemporally quantitative in vivo imaging of mitochondrial fatty acid ß-oxidation at cellular-level resolution in mice.

Mitochondrial fatty acid ß-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes ß-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(N-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution.NEW & NOTEWORTHY Fatty acid ß-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.

Maxacalcitol (22-Oxacalcitriol (OCT)) Retards Progression of Left Ventricular Hypertrophy with Renal Dysfunction Through Inhibition of Calcineurin-NFAT Activity.

PURPOSE: Left ventricular hypertrophy (LVH) is a cardiovascular complication highly prevalent in patients with chronic kidney disease (CKD). Previous studies analyzing 1α-hydroxylase or vitamin D receptor (Vdr) knockout mice revealed active vitamin D as a promising agent inhibiting LVH progression. Paricalcitol, an active vitamin D analog, failed to suppress the progression of LV mass index (LVMI) in pre-dialysis patients with CKD. As target genes of activated VDR differ depending on its agonists, we examined the effects of maxacalcitol (22-oxacalcitriol: OCT), a less calcemic active vitamin D analog, on LVH in hemodialysis patients and animal LVH models with renal insufficiency. METHODS: In retrospective cohort study, patients treated with OCT who underwent hemodialysis were enrolled. Using cardiac echocardiography, LV mass was evaluated by the area-length method. In animal study, angiotensin II (Ang II)-infused Wister rats with heminephrectomy or Ang II-stimulated neonatal rat ventricular myocytes (NRVM) were treated with OCT. RESULTS: OCT significantly inhibited the progression of LVMI in hemodialysis patients. In Ang II-infused heminephrectomized rats, OCT suppressed the progression of LVH in a blood pressure-independent manner. OCT also suppressed the activity of calcineurin in the left ventricle of model rats. Specifically, OCT reduced the protein levels of calcineurin A, but not the mRNA levels of Ppp3ca (calcineurin Aα). Luciferase assays showed that OCT increased the promoter activity of Fbxo32 (atrogin1), an E3 ubiquitin ligase targeting calcineurin A. Finally, OCT promoted ubiquitination and degradation of calcineurin A. CONCLUSION: Our works indicated that OCT retards progression of LVH through calcineurin-NFAT pathway, which reveal a novel aspect of OCT in attenuating pathological LVH.

Recurrent membranous nephropathy with a possible alteration in the etiology: a case report.

BACKGROUND: Phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type-1 domain-containing 7A (THSD7A) are the two major pathogenic antigens for membranous nephropathy (MN). It has been reported that THSD7A-associated MN has a higher prevalence of comorbid malignancy than PLA2R1-associated MN. Here we present a case of MN whose etiology might change from idiopathic to malignancy-associated MN during the patient's clinical course. CASE PRESENTATION: A 68-year-old man with nephrotic syndrome was diagnosed with MN by renal biopsy. Immunohistochemistry showed that the kidney specimen was negative for THSD7A. The first course of corticosteroid therapy achieved partial remission; however, nephrotic syndrome recurred 1 year later. Two years later, his abdominal echography revealed a urinary bladder tumor, but he did not wish to undergo additional diagnostic examinations. Because his proteinuria increased consecutively, corticosteroid therapy was resumed, but it failed to achieve remission. Another kidney biopsy was performed and revealed MN with positive staining for THSD7A. PLA2R1 staining levels were negative for both first and second biopsies. Because his bladder tumor had gradually enlarged, he agreed to undergo bladder tumor resection. Pathological examination indicated that the tumor was THDS7A-positive bladder cancer. Subsequently, his proteinuria decreased and remained in remission. CONCLUSIONS: This case suggests that the etiology of MN might be altered during the therapeutic course. Intensive screening for malignancy may be preferable in patients with unexpected recurrence of proteinuria and/or change in therapy response.

Lithocholic acid increases intestinal phosphate and calcium absorption in a vitamin D receptor dependent but transcellular pathway independent manner.

Phosphate/calcium homeostasis is crucial for health maintenance. Lithocholic acid, a bile acid produced by intestinal bacteria, is an agonist of vitamin D receptor. However, its effects on phosphate/calcium homeostasis remain unclear. Here, we demonstrated that lithocholic acid increases intestinal phosphate/calcium absorption in an enterocyte vitamin D receptor-dependent manner. Lithocholic acid was found to increase serum phosphate/calcium levels and thus to exacerbate vascular calcification in animals with chronic kidney disease. Lithocholic acid did not affect levels of intestinal sodium-dependent phosphate transport protein 2b, Pi transporter-1, -2, or transient receptor potential vanilloid subfamily member 6. Everted gut sac analyses demonstrated that lithocholic acid increased phosphate/calcium absorption in a transcellular pathway-independent manner. Lithocholic acid suppressed intestinal mucosal claudin 3 and occludin in wild-type mice, but not in vitamin D receptor knockout mice. Everted gut sacs of claudin 3 knockout mice showed an increased permeability for phosphate, but not calcium. In patients with chronic kidney disease, serum 1,25(OH)2 vitamin D levels are decreased, probably as an intrinsic adjustment to reduce phosphate/calcium burden. In contrast, serum and fecal lithocholic acid levels and fecal levels of bile acid 7α-dehydratase, a rate-limiting enzyme involved in lithocholic acid production, were not downregulated. The effects of lithocholic acid were eliminated by bile acid adsorptive resin in mice. Thus, lithocholic acid and claudin 3 may represent novel therapeutic targets for reducing phosphate burden.

[A Case of Neoadjuvant Chemotherapy with Modified FOLFOXIRI plus Bevacizumab for Transverse Colon Cancer with Invasion of the Gastric Antrum].

A 66-year-old male diagnosed with transverse colon cancer was admitted to our hospital. Computed tomography, colonoscopy, and esophagogastroduodenoscopy revealed locally advanced cancer with invasion of the gastric antrum. We staged the disease as cT4a, cN2, cM0, Stage ⅢB, with wild-type RAS expression. We performed an ileostomy prior to administering chemotherapy. The patient received 4 courses of modified FOLFOXIRI plus bevacizumab and 2 courses of FOLFIRI. The size of the tumor noticeably decreased after chemotherapy. The patient experienced grade 3 neutropenia, anorexia, and oral mucositis during chemotherapy. We performed a right hemicolectomy(D3), partial gastrectomy and ileum resection after administering neoadjuvant chemotherapy. The pathological stage of the disease was ypT2, ypN0, ypM0, ypStageⅠ, and the effect of the chemotherapy was Grade 1b. After the resection, he received mFOLFOX6 and CapeOX for 3 months as adjuvant chemotherapy. He remained cancer-free for 1 year and 3 months after the surgery. This result suggests that preoperative modified FOLFOXIRI plus bevacizumab chemotherapy is a useful regimen for the treatment of locally advanced colon cancer.

Hepatic phosphate uptake and subsequent nerve-mediated phosphaturia are crucial for phosphate homeostasis following portal vein passage of phosphate in rats.

Fibroblast growth factor 23, parathyroid hormone, and 1,25-dihydroxyvitamin D are critical in phosphate homeostasis. Despite these factors' importance, regulators of phosphaturia in the acute postprandial phase remain largely unknown. This study investigated the mechanism of acute phosphate regulation in the postprandial phase in rats. Duodenal administration of radiolabeled phosphate (32P) showed that 32P levels in the inferior vena cava (IVC) blood were lower than those in the portal vein (PV) blood. Serum phosphate concentration transiently increased 5 min after phosphate solution administration through IVC, while it was maintained after the administration through PV. Phosphate administration through both IVC and PV resulted in increased fractional excretion of phosphate (FEPi) at 10 min without elevation of the known circulating factors, but urinary phosphate excretion during the period was 8% of the dose. Experiments using 32P or partial hepatectomy showed that the liver was one of the phosphate reservoirs. The elevation of FEPi and suppression of sodium-phosphate cotransporter 2a in the kidney at 10 min was attenuated in rats with SCH23390, hepatic denervation, or renal denervation, thus indicating that the liver communicated with the kidney via the nervous system to promote phosphaturia. These results revealed previously unknown mechanisms for serum phosphate maintenance.

[A case of multiple liver metastases of gastric cancer successfully treated by combination chemotherapy of S-1 and paclitaxel].

A 54-year-old man underwent distal gastrectomy for early gastric cancer in September 2002. CT performed 6 months after the operation revealed liver metastases, and they were resected. Hepatic arterial infusion therapy of 5-FU was performed; however, multiple liver metastases appeared in October 2003. We added arterial infusion of CDDP to 5-FU, but liver metastases increased. We then applied a combination chemotherapy of S-1 and paclitaxel from February 2004. Subsequently, stable disease continued, and after 67 courses of S-1 plus paclitaxel, we changed the administration to S-1 alone from August 2009. After that, liver metastases did not increase, so we discontinued chemotherapy on August 2010, followed by observation. Progression of liver metastases has not been to date.

Glomerulonephritis Caused by Bartonella spp. Infective Endocarditis: The Difficulty and Importance of Differentiation from Anti-neutrophil Cytoplasmic Antibody-related Rapidly Progressive Glomerulonephritis.

A 65-year-old man with valvular disorder presented to his physician because of widespread purpura in both lower extremities. Blood tests showed elevated serum creatinine levels and proteinase 3-anti-neutrophil cytoplasmic antibody (ANCA) with hematuria, suggesting ANCA-related rapidly progressive glomerulonephritis (RPGN). Although multiple blood cultures were negative, transthoracic echocardiography revealed warts in the valves, and a renal biopsy also showed findings of glomerular infiltration by mononuclear leukocytes and C3 deposition in the glomeruli, suggesting infection-related glomerulonephritis. Later, Bartonella antibody turned positive. Antimicrobial treatment improved the purpura and renal function without any recurrence. ANCA-positive RPGN requires the exclusion of infective endocarditis, especially that induced by Bartonella spp.

Single cell RNA sequencing uncovers cellular developmental sequences and novel potential intercellular communications in embryonic kidney.

Kidney development requires the coordinated growth and differentiation of multiple cells. Despite recent single cell profiles in nephrogenesis research, tools for data analysis are rapidly developing, and offer an opportunity to gain additional insight into kidney development. In this study, single-cell RNA sequencing data obtained from embryonic mouse kidney were re-analyzed. Manifold learning based on partition-based graph-abstraction coordinated cells, reflecting their expected lineage relationships. Consequently, the coordination in combination with ForceAtlas2 enabled the inference of parietal epithelial cells of Bowman's capsule and the inference of cells involved in the developmental process from the S-shaped body to each nephron segment. RNA velocity suggested developmental sequences of proximal tubules and podocytes. In combination with a Markov chain algorithm, RNA velocity suggested the self-renewal processes of nephron progenitors. NicheNet analyses suggested that not only cells belonging to ureteric bud and stroma, but also endothelial cells, macrophages, and pericytes may contribute to the differentiation of cells from nephron progenitors. Organ culture of embryonic mouse kidney demonstrated that nerve growth factor, one of the nephrogenesis-related factors inferred by NicheNet, contributed to mitochondrial biogenesis in developing distal tubules. These approaches suggested previously unrecognized aspects of the underlying mechanisms for kidney development.

Electrocardiogram findings at the initiation of hemodialysis and types of subsequent cardiovascular events.

The prognostic value of electrocardiograms (ECGs) has been reported in predialysis patients but not in incident hemodialysis patients with overhydration and electrolyte disturbances, both of which potentially affect ECG results. We performed a retrospective multicenter cohort study involving incident hemodialysis patients and examined whether ECG parameters immediately before hemodialysis initiation can predict subsequent cardiovascular disease (CVD) using Cox proportional hazards models. We explored potential effect modifications by several electrolytes on the predictive power of ECG abnormalities. Among the 618 enrolled patients, 16%, 10%, 46%, and 22% showed a PR interval ≥ 200 ms, QRS interval ≥120 ms, QTc interval ≥ 450/460 ms (male/female), and left ventricular hypertrophy (LVH) by voltage criteria, respectively. Over a median 3-year follow-up, 19% and 16% of the patients developed atherosclerotic and nonatherosclerotic CVD, respectively. The Cox regression model results revealed that the sum of the number of abnormalities in PR, QRS, and QT intervals was a significant risk factor for nonatherosclerotic CVD (hazard ratios (HRs) [95% confidence interval (CI)]: 1.58 [1.24-2.01] per number of abnormalities). The predictive value of LVH for atherosclerotic CVD was attenuated over time. At up to 36 months, although the proportional hazards assumption was met, LVH was significantly associated with atherosclerotic CVD (HR [95% CI]: 1.89 [1.15-3.11]). The adjusted HR was particularly high (HR [95% CI]: 4.02 [1.68-9.60]) among patients who were in the lowest tertile of serum magnesium levels (P for interaction = 0.04). PR, QRS, and QT prolongation additively predicted nonatherosclerotic CVD, while LVH predicted atherosclerotic CVD in the short term.

Quantitative Analyses of Foot Processes, Mitochondria, and Basement Membranes by Structured Illumination Microscopy Using Elastica-Masson- and Periodic-Acid-Schiff-Stained Kidney Sections.

INTRODUCTION: Foot process effacement and mitochondrial fission associate with kidney disease pathogenesis. Electron microscopy is the gold-standard method for their visualization, but the observable area of electron microscopy is smaller than light microscopy. It is important to develop alternative ways to quantitatively evaluate these microstructural changes because the lesion site of renal diseases can be focal. METHODS: We analyzed elastica-Masson trichrome (EMT) and periodic acid-Schiff (PAS) stained kidney sections using structured illumination microscopy (SIM). RESULTS: EMT staining revealed three-dimensional (3D) structures of foot process, whereas ponceau xylidine acid fuchsin azophloxine solution induced fluorescence. Conversion of foot process images into their constituent frequencies by Fourier transform showed that the concentric square of (1/4)2-(1/16)2 in the power spectra (PS) included information for normal periodic structures of foot processes. Foot process integrity, assessed by PS, negatively correlated with proteinuria. EMT-stained sections revealed fragmented mitochondria in mice with mitochondrial injuries and patients with tubulointerstitial nephritis; Fourier transform quantified associated mitochondrial injury. Quantified mitochondrial damage in patients with immunoglobulin A (IgA) nephropathy predicted a decline in estimated glomerular filtration rate (eGFR) after kidney biopsy but did not correlate with eGFR at biopsy. PAS-stained sections, excited by a 640 nm laser, combined with the coefficient of variation values, quantified subtle changes in the basement membranes of patients with membranous nephropathy stage I. CONCLUSIONS: Kidney microstructures are quantified from sections prepared in clinical practice using SIM.

Dietary casein, egg albumin, and branched-chain amino acids attenuate phosphate-induced renal tubulointerstitial injury in rats.

Dietary phosphate intake is closely correlated with protein intake. However, the effects of the latter on phosphate-induced organ injuries remain uncertain. Herein, we investigated the effects of low (10.8%), moderate (23.0%), and high (35.2%) dietary casein and egg albumin administration on phosphate-induced organ injuries in rats. The moderate and high casein levels suppressed renal tubulointerstitial fibrosis and maintained mitochondrial integrity in the kidney. The serum creatinine levels were suppressed only in the high casein group. Phosphate-induced muscle weakness was also ameliorated by high dietary casein. The urinary and fecal phosphate levels in the early experiment stage showed that dietary casein did not affect phosphate absorption from the intestine. High dietary egg albumin showed similar kidney protective effects, while the egg albumin effects on muscle weakness were only marginally significant. As the plasma branched-chain amino acid levels were elevated in casein- and egg albumin-fed rats, we analyzed their effects. Dietary supplementation of 10% branched-chain amino acids suppressed phosphate-induced kidney injury and muscle weakness. Although dietary protein restriction is recommended in cases of chronic kidney disease, our findings indicate that the dietary casein, egg albumin, and branched-chain amino acid effects might be reconsidered in the era of a phosphate-enriched diet.

Hidden Hypocalcemia as a Risk Factor for Cardiovascular Events and All-Cause Mortality among Patients Undergoing Incident Hemodialysis.

Lower corrected calcium (cCa) levels are associated with a better prognosis among incident dialysis patients. However, cCa frequently overestimates ionized calcium (iCa) levels. The prognostic importance of the true calcium status defined by iCa remains to be revealed. We conducted a retrospective cohort study of incident hemodialysis patients. We collected data of iCa levels immediately before the first dialysis. We divided patients into three categories: apparent hypocalcemia (low iCa; <1.15 mmol/L and low cCa; <8.4 mg/dL), hidden hypocalcemia (low iCa despite normal or high cCa), and normocalcemia (normal iCa). The primary outcome was the composite of all-cause death and cardiovascular diseases after hospital discharge. Among the enrolled 332 patients, 75% of the patients showed true hypocalcemia, defined as iCa <1.15 mmol/L, 61% of whom showed hidden hypocalcemia. In multivariate Cox models including other potential risk factors, true hypocalcemia was a significant risk factor (hazard ratio [HR], 2.34; 95% confidence interval [CI], 1.03-5.34), whereas hypocalcemia defined as corrected calcium <8.4 mg/dL was not. Furthermore, hidden hypocalcemia was significantly associated with an increased risk of the outcome compared with normocalcemia (HR, 2.56; 95% CI, 1.11-5.94), while apparent hypocalcemia was not. Patients with hidden hypocalcemia were less likely to receive interventions to correct hypocalcemia, such as increased doses of active vitamin D or administration of calcium carbonate, than patients with apparent hypocalcemia (odds ratio, 0.45; 95% CI, 0.23-0.89). Hidden hypocalcemia was a strong predictor of death and cardiovascular events, suggesting the importance of measuring iCa.

Laparoscopic excision of a retroperitoneal completely isolated enteric duplication cyst in an adult male: A case report and review of literature.

INTRODUCTION: Duplication cysts are very rare congenital malformations in adults. They are lined by gastrointestinal mucosa, connect to the digestive tract, and share smooth muscular layers and a common blood supply. In rare cases, duplication cysts are completely isolated from the digestive tract and have a proper blood supply. Completely isolated duplication cysts in the retroperitoneum are unusual so it is hard to diagnose them without a surgical resection. PRESENTATION OF CASE: A 19-year-old male presented at our emergency department with sharp abdominal pain. Contrast-enhanced computed tomography detected a 5-cm multilocular cystic mass located in the retroperitoneum, caudal to the pancreatic body. The cystic mass was safely resected with laparoscopic surgery without any complication. The final pathological diagnosis was an epithelium-lined duplication cyst in the retroperitoneal space. There was no evidence of malignancy in the duplication cyst. Intracystic bleeding was assumed to be the cause of the abdominal pain. DISCUSSION: The most common differential diagnoses of retroperitoneal cystic masses are pseudocysts related to pancreatitis, cysts from surrounding structures, and neoplasms. In this case, the cystic mass was diagnosed as completely isolated duplication cyst after surgical resection. It is very rarely observed in adults, but it should be listed on differential diagnoses because it has some possibility of malignancy. CONCLUSION: A completely isolated duplication cyst is very rare but noteworthy because there is some possibility of malignancy, ulcerative bleeding, and perforation. A surgical resection is recommended for diagnostic treatment. Laparoscopic surgery is favorable for intraoperative inspection and it is minimally invasive.

Abdominal compartment syndrome during hip arthroscopy for an acetabular fracture: a case report.

BACKGROUND: We encountered a case of abdominal compartment syndrome during hip arthroscopic surgery, caused by the irrigation fluid flowing into the peritoneal cavity. CASE PRESENTATION: A 47-year-old male patient with the acetabulum fracture underwent open reduction and internal fixation with hip arthroscopy. Hypothermia, increased airway pressure (under volume-controlled ventilation) and oliguria were observed during the operation, and arterial blood gas analysis showed decreased oxygenation and metabolic acidosis. Abdominal distention was observed, and a postoperative CT revealed accumulation of a large volume of irrigation fluid in the peritoneal cavity and retroperitoneum. The patient was diagnosed as having abdominal compartment syndrome and treated by percutaneous peritoneal drainage. His subsequent course was uneventful, and he was discharged 8 weeks after the operation. Intraperitoneal extravasation of irrigation fluid may occur during hip arthroscopic surgery, and is more likely to occur in the presence of an injury. CONCLUSION: Anesthesiologists should be aware of the possible occurrence of the abdominal compartment syndrome during hip arthroscopic surgery and ensure that it is detected early.

Preparation and evaluation of lignosulfonates as a dispersant for gypsum paste from acid hydrolysis lignin.

In order to effectively utilize a by-product of the acid saccharification process of woody materials, the chemical conversion of guaiacyl sulfuric acid lignin (SAL), one of the acid hydrolysis lignins, into water-soluble sulfonated products with high dispersibitity was investigated. At first, SAL was phenolated (P-SAL) to enhance the solubility and reactivity. Lignosulfonates were prepared from P-SAL by three methods of hydroxymethylation followed by neutral sulfonation (two-step method), sulfomethylation (one-step method) and arylsulfonation. Surprisingly, all prepared lignosulfonates possessed 30 to 70% higher dispersibility for gypsum paste than the commercial lignosulfonate. Evaluation of the preparations for gypsum paste suggested that the higher molecular weights and sulfur contents of the preparations increased their dispersibility.

Interleukin-12-gene transduction makes DCs from tumor-bearing mice an effective inducer of tumor-specific immunity in a peritoneal dissemination model.

Dendritic cells (DCs) from cancer patients, as well as tumor-infiltrating DCs, are reported to have suppressed immunostimulatory capacity. One of the major problems in the clinical use of DCs for treating tumors is that the DCs must be autologous ones obtained from patients. Compared with normal DCs (nDCs), flow-cytometric analysis and allogeneic mixed lymphocyte reaction (MLR) have revealed lower expression of the costimulatory molecules and suppressed T-cell-stimulatory activity in DCs derived from tumor-bearing mice (tDCs) despite of culture. We reported previously that the interleukin-12 (IL-12)-gene-transduced nDCs inhibited tumor growth due to induced tumor-specific Th1 and cytotoxic T cells (CTLs) in a murine established subcutaneous tumor model. In the present study, we examined whether tDCs could induce immune responses against tumors after IL-12-gene transduction in an established peritoneal dissemination model. The intraperitoneal injection of IL-12-gene-transduced tDCs resulted in prolonged survival of some treated mice (log-rank test; P=0.001) and tumor-specific Th1 and CTL activity. The injection of IL-12-gene-transduced nDCs prolonged the survival of all treated mice (P<0.0001) and elicited tumor-specific immunity, which were better than those of IL-12-gene-transduced tDCs. Taken together, DC modification of IL-12-gene transduction is an effective and promising approach for cancer therapy even when immunosuppressive tDCs are employed.

Immunohistochemical localization of agatharesinol, a heartwood norlignan, in Cryptomeria japonica.

Localization of a heartwood norlignan, agatharesinol, in Sugi (Japanese cedar, Cryptomeria japonica D. Don, Taxodiaceae) was investigated by immunohistochemistry. Immuno light microscopy showed that the contents of ray parenchyma cells were immunostained in heartwood but not in sapwood. The staining of the heartwood tissue was competitively inhibited by agatharesinol but not by other Sugi heartwood extractives, and was, furthermore, markedly reduced by pre-extraction of the tissue with MeOH. These results indicated that the staining can be ascribed to the immunolabeling of agatharesinol in situ. The accumulations over the inner surface of some tracheid cell walls adjacent to the ray parenchyma cells were also immunolabeled, while the contents in axial parenchyma cells were not. In conclusion, agatharesinol was localized in the ray parenchyma cells in Sugi heartwood, and differences between the chemical structure of the contents of ray and axial parenchyma cells were also suggested.

Evidence for the biosynthetic pathway from sinapic acid to syringyl lignin using labeled sinapic acid with stable isotope at both methoxy groups in Robinia pseudoacacia and Nerium indicum.

A tracer experiment using synthesized labeled lignin precursors was designed to confirm the actual biosynthetic pathway for syringyl lignin. Tetradeuteroferulic acid-[8-D, 3-OCD(3)] and heptadeuterosinapic acid-[8-D, 3,5-OCD(3)] were synthesized and fed to shoots of robinia (Robinia pseudoacacia) and oleander (Nerium indicum) trees. The incorporation of each labeled precursor into lignin was traced by gas chromatography-mass spectrometry. The synthesized sinapic acid, in which both methoxy groups were labeled, was useful in monitoring the conversion of sinapic acid into syringyl lignin. When heptadeuterosinapic acid was fed, syringyl units containing seven deuterium labels were detected. The results of this study support the traditionally accepted pathway that sinapic acid is converted to sinapyl alcohol via sinapoyl-CoA in robinia and oleander.

The behavior of deuterium-labeled monolignol and monolignol glucosides in lignin biosynthesis in angiosperms.

To examine the behavior of monolignol and monolignol glucosides in lignin biosynthesis, pentadeutero[9-D(2), 3-OCD(3)]coniferyl alcohol and pentadeutero[9-D(2), 3-OCD(3)]coniferin were synthesized and fed to growing Eucalyptus camaldulensis and Magnolia kobus. The differences in the incorporation patterns of these labeled precursors were studied using gas chromatography-mass spectrometry (GC-MS). Both precursors were incorporated into lignin, but the labeled coniferyl alcohol was incorporated more directly, resulting in a high proportion of pentadeutero-labeled guaiacyl and syringyl units in newly formed xylem, while labeled coniferin tended to be incorporated in lignin as tetradeutero units, especially in syringyl lignin in both trees. However, the incorporation efficiencies of the precursors into syringyl lignin were higher in Magnolia than in Eucalyptus, and the ratios of tetradeutero to pentadeutero in guaiacyl lignin were lower in Magnolia than in Eucalyptus when the trees were fed coniferin.