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Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone pigments in Boraginaceae. The new fixation method combined glutaraldehyde containing malachite green, imidazole-osmium and p-phenylenediamine treatments, and cells were then observed with a transmission electron microscope. The method prevented the extraction of lipids, including shikonin derivatives, and improved the visualization of subcellular structures, especially the membrane system, when compared with that of conventional fixation. The improved quality of the transmission electron micrographs is because malachite green ionically binds to the plasma membrane, organelles and lipids and acts as a mordant for electron staining with osmium tetroxide. Imidazole promotes the reaction of osmium tetroxide, leading to enhanced electron staining. p-Phenylenediamine reduces osmium tetroxide bound to cellular materials and increases the electron density. This protocol requires only three additional reagents over conventional chemical fixation using glutaraldehyde and osmium tetroxide., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.)

The distribution of lignin, 8-5' and 8-8' linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5' linked lignin substructure), and no KM2 labeling (8-8' linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5' linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply., (© 2014 American Society of Plant Biologists. All rights reserved.)

Objective: The 7-year follow-up of the US oncology 9735 trial demonstrated the superiority of TC [docetaxel (DTX)/cyclophosphamide (CPA)] to doxorubicin/CPA therapy. To introduce TC therapy in Japan, the verification of the safety and tolerability is essential. We performed a collaborative prospective safety study with Okayama University to introduce TC therapy., Methods: The subjects were 53 patients aged from 33 to 67 years at intermediate risk based on the St Gallen risk classification who underwent radical surgery for primary breast cancer between August 2007 and December 2008. As post-operative adjuvant chemotherapy, four cycles of TC (DTX 75 mg/m(2) + CPA 600 mg/m(2)) were administered at 3-week intervals. Adverse events were evaluated based on National Cancer Institute-Common Terminology Criteria for Adverse Events ver. 3.0. The safety and completion rate were evaluated as the primary and secondary endpoints, respectively., Results: Regarding hematological toxicity, Grade (G) 4 neutropenia occurred in 71.7% and G3 in 26.4%. G3-4 leukopenia developed in 32.1% and 56.6%, respectively, G4 anemia in 1.9% and G1-2 anemia in 26.4%. Regarding non-hematological toxicity, systemic malaise, skin eruption, edema, myalgia, arthralgia and nausea were noted in most patients. The completion rate was 94.3%, dose reduction was necessary in 7.5% and granulocyte colony-stimulating factor (G-CSF) support was required in 17.0%. On comparison between patients aged 65 years or older and younger than 65 years, the completion rate, dose reduction and incidence of febrile neutropenia (FN) were higher in the elderly patients. G-CSF support was more often needed in this subgroup., Conclusions: TC therapy is tolerable for Japanese patients, but attention should be paid to the development of FN and neutropenia. The completion rate was lower in the elderly patients, showing that tolerability was not necessarily favorable.