Hydroxycinnamaldehyde-derived benzofuran components in lignins.

Lignin is an abundant polymer in plant secondary cell walls. Prototypical lignins derive from the polymerization of monolignols (hydroxycinnamyl alcohols), mainly coniferyl and sinapyl alcohol, via combinatorial radical coupling reactions and primarily via the endwise coupling of a monomer with the phenolic end of the growing polymer. Hydroxycinnamaldehyde units have long been recognized as minor components of lignins. In plants deficient in cinnamyl alcohol dehydrogenase, the last enzyme in the monolignol biosynthesis pathway that reduces hydroxycinnamaldehydes to monolignols, chain-incorporated aldehyde unit levels are elevated. The nature and relative levels of aldehyde components in lignins can be determined from their distinct and dispersed correlations in 2D 1H-13C-correlated nuclear magnetic resonance (NMR) spectra. We recently became aware of aldehyde NMR peaks, well resolved from others, that had been overlooked. NMR of isolated low-molecular-weight oligomers from biomimetic radical coupling reactions involving coniferaldehyde revealed that the correlation peaks belonged to hydroxycinnamaldehyde-derived benzofuran moieties. Coniferaldehyde 8-5-coupling initially produces the expected phenylcoumaran structures, but the derived phenolic radicals undergo preferential disproportionation rather than radical coupling to extend the growing polymer. As a result, the hydroxycinnamaldehyde-derived phenylcoumaran units are difficult to detect in lignins, but the benzofurans are now readily observed by their distinct and dispersed correlations in the aldehyde region of NMR spectra from any lignin or monolignol dehydrogenation polymer. Hydroxycinnamaldehydes that are coupled to coniferaldehyde can be distinguished from those coupled with a generic guaiacyl end-unit. These benzofuran peaks may now be annotated and reported and their structural ramifications further studied.

Preparation of intracellular proteins from a white-rot fungus surrounded by polysaccharide sheath and optimization of their two-dimensional electrophoresis for proteomic studies.

The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora, which produces large sheath amounts in the presence of a lignin-derived aromatic compound. Using this approach, we achieved thorough removal of sheath and cell disruption using beads and a solution with a high protein-solubilizing power, which enabled the efficient extraction of intracellular proteins from C. subvermispora surrounded by sheath. In addition, for proteomic analysis, we investigated whether these extracted proteins were compatible with two-dimensional electrophoresis. By efficiently concentrating on protein solubilization in the first dimension and using a stacking gel in the second dimension, we successfully obtained a high-resolution proteome map of C. subvermispora. We also used the same proteins for fluorescence two-dimensional difference gel electrophoresis to obtain the quantitative protein expression profiles. These steps demonstrated that two-dimensional electrophoresis-based proteomics can be used to clarify the composition of intracellular proteins from sheath-producing white-rot fungi.

Structural characterization of highly branched glucan sheath from Ceriporiopsis subvermispora.

Wood rotting basidiomycetes produce extracellular mucilaginous sheaths interfacing fungal hyphae and plant biomass. While the versatility of these fungal sheaths has been addressed, sheaths generated by selective white-rot fungi remain poorly understood. To fill this gap, the sheath produced by the basidiomycete Ceriporiopsis subvermispora, which degrades lignin while inflicting limited cellulose damage, was analyzed in this study. Fluorescence and transmission electron microscopy revealed that the sheath formed three days after inoculation into a beech wood slice on an agar plate and was embedded at the interface between fungal hyphae and wood cell walls. The sheath's chemical structure was evaluated from fungus cultures in a liquid medium containing [U-13C6]-d-glucose and beech wood slices. Compositional analysis, methylation analysis, and 13C NMR demonstrated that the sheath mainly consisted of a comb-like ß-1,6-glucopyranose residue-branched ß-1,3-glucan, which is advantageous to retain water and extracellular secondary metabolites.

Assessment of stress and autonomic nervous activity in Japanese female ambulance paramedics working 24-hour shifts.

OBJECTIVES: We studied the physical and mental conditions of 8 healthy young female ambulance paramedics working 24-hour shifts during their menstrual cycle, including assessment of cardiac autonomic nervous system activity by heart rate variability power spectral analysis. METHODS: The autonomic activity during the awake period of on- and off-duty days in the follicular, late luteal, and menstruation phases was measured. Questionnaires regarding fatigue and menstrual distress were administered and correlated with the autonomic profile. RESULTS: While degrees of fatigue significantly increased after work, the changes in autonomic activity during the awake period on on-duty days were not significantly different from those on off-duty days (LF/HF, p=0.123; HF/(HF+LF), p=0.153). As for the sleeping period, there were no significant differences. Although the Menstrual Distress Questionnaire (MDQ) revealed the presence of mild menstrual discomfort in the late luteal and menstruation phases, no significant difference was observed in the autonomic profile of the three menstrual cycle phases. No significant correlation was observed between the degree of menstrual distress and autonomic profile, though there was a significant correlation in the late luteal phase between degree of menstrual distress and fatigue after work (p<0.01). CONCLUSION: These results showed that, while subjects experienced menstrual discomfort and fatigue after work, their autonomic profile did not alter in the menstrual cycle. It is suggested that healthy young female ambulance paramedics may tolerate 24-hour shifts, though attention should be paid to subjective menstrual symptoms and fatigue.

Degradation and synthesis of ß-glucans by a Magnaporthe oryzae endotransglucosylase, a member of the glycoside hydrolase 7 family.

BACKGROUND: Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition. RESULTS: A novel endotransglucosylase catalyzes cleavage and transfer of ß-glucans and decreases the physical strength of plant cell walls. CONCLUSION: Endotransglucosylation causes depolymerization and polymerization of ß-glucans, depending on substrate molecular size. SIGNIFICANCE: Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-ß-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing ß-1,4-linkages, in which 1,3-1,4-ß-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into ß-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of ß-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-ß-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize ß-glucans, depending on the size of the acceptor substrate.

A comparative study of matrix- and nano-assisted laser desorption/ionisation time-of-flight mass spectrometry of isolated and synthetic lignin.

INTRODUCTION: Lignin is the second most abundant biopolymer next to cellulose. However, because of the complexity of the heterogeneous macromolecules, it is difficult to elucidate the polymeric structures of lignin by conventional analytical methods. OBJECTIVE: To obtain the detailed structures of lignin, we comparatively applied nano-assisted laser desorption/ionisation time-of-flight mass spectrometry (NALDI-TOF MS) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). METHODOLOGY: Synthetic lignin from coniferyl alcohol and an isolated lignin from Pinus densiflora were subjected to NALDI- and MALDI-TOF MS. RESULTS: We first obtained NALDI-TOF MS of synthetic and isolated lignin. Mass increments of 178 and 196 Da were observed in NALDI- and MALDI-TOF mass spectra of the synthetic and isolated lignin. The mass intervals indicated that radical coupling forming ß-O-4 bonds is the major pathway. Peaks in the low molecular mass region between m/z 500 and 800 were observed more extensively using NALDI-TOF MS than MALDI-TOF MS, which enabled detailed analysis of the interunit linkages in lignin. CONCLUSION: Owing to the ionisation profile differentiation from MALDI-TOF MS, NALDI-TOF MS is useful for the structural analysis of lignin.

Some embryological aspects of cholinergic innervation in the cardiovascular system--a close association with the subintestinal circulatory channel.

A series of our studies on the dog venous system revealed that cholinergic excitatory innervation was localized in a group of veins: the portal, mesenteric, and hepatic veins and the middle segment of the inferior vena cava. Our studies on pharmacological responsiveness of dog veins also revealed that they could be divided into two groups: the visceral and somatic parts, and the cholinergic excitatory innervation localized to the visceral part. Considering these results and some relevant literature, a hypothesis is proposed on the classification of muscles of the cardiovascular system and some embryological aspects of the parasympathetic cholinergic innervation in the circulatory system are discussed. The embryonic circulatory system of vertebrates can be divided into two parts: somatic and visceral. The body of an embryo is regarded as a double tube and vessels of the visceral part and the heart belong to the inner tube. The muscle of these vessels and the heart are derived from visceral mesoderm, either the coelomic epithelium or mesenchymal cells, in common with muscle of the digestive tube; and thus the parasympathetic cholinergic nerves innervating the muscle of the digestive tube also distribute to these vessels and the heart. The heart and vascular muscles in the visceral part are structures developed early in the course of evolution in invertebrates. Their primary function is to propel the body fluid, and the chief structure containing them is the subintestinal circulatory channel (ventral aorta - heart - subintestinal vein). They exhibit spontaneous, rhythmic activity, showing characteristics of a single unit muscle, and receive parasympathetic cholinergic innervation. On the other hand, the vascular muscles in the somatic part are endothelium-associated muscles developed anew in the vertebrate; do not contract spontaneously, being classified as a multiunit muscle; and lack parasympathetic cholinergic innervation.

Increased rate of force development of elbow flexors by antagonist conditioning contraction.

The effects of isometric antagonist conditioning contraction (ACC) at various durations and intensities on the contractile force, electromyographic (EMG) amplitude, and their rates of rise of elbow flexor muscles were examined in healthy participants. In particular, we focused on the change in the maximum rate of initial force development of agonists (dFagonist/dt(max)), which was evaluated by subtracting antagonist force decaying from apparent initial force development. While the ACC caused no statistically significant effect on the average force during elbow flexion, dFagonist/dt(max) was significantly increased by the ACC of short durations (1-2s) and large intensities. Similarly, the ACC did not affect the root mean square EMG amplitude of biceps brachii during elbow flexion, but significantly increased the maximum rate of rise of the absolute EMG amplitude (dE/dt(max)). These results suggested that facilitating effects of the ACC could be observed in the initial phase of agonist action in healthy participants, and ACC of shorter durations might be more effective. The increased dE/dt(max) suggested that increased neural activities might contribute to the antagonist conditioned facilitation of force development.

Twisted flavors and tribimaximal neutrino mixing.

A new framework for handling flavor symmetry breaking in the neutrino sector is discussed where the source of symmetry breaking is traced to the global property of right-handed neutrinos in extra-dimensional space. Light neutrino phenomenology has rich and robust predictions such as the tribimaximal form of generation mixing, controlled mass spectrum, and no need of flavor mixing couplings in the theory.

Structure-activity relationship studies on niphimycin, a guanidylpolyol macrolide antibiotic. Part 1: The role of the N-methyl-N''-alkylguanidinium moiety.

Several N-methyl-N''-alkylguanidinium derivatives were synthesized and used as simplified analogues of niphimycin (NM), a guanidylpolyol macrolide, in structure-activity relationship studies. The C16-alkylated derivative exerted fungicidal activity by directly damaging the fungal plasma membrane and inducing oxidative stress in a manner similar to niphimycin. These results indicate that the N-methyl-N''-alkylguanidinium moiety is required for antifungal activity by NM.

Decreased expression of glial cell line-derived neurotrophic factor signaling in rat models of neuropathic pain.

1. In an attempt to clarify whether glial cell line-derived neurotrophic factor (GDNF), a survival factor for subpopulations of primary afferent neurons, is involved in the states of neuropathic pain, we observed changes in the expressions of GDNF and its signal-transducing receptor Ret after nerve injury in two rat models of neuropathic pain. 2. In the rats treated with sciatic nerve ligation (chronic constrictive injury (CCI) model) or spinal nerve ligation at L5 (SNL model), the thresholds of paw withdrawal in response to mechanical or heat stimuli began to decrease on the injured side within the first week after the operation and the decreases in the thresholds persisted for more than 2 weeks. 3. In CCI-treated rats, the GDNF contents in L4 and L5 dorsal root ganglia (DRGs) on the injured side were markedly decreased at day 7 after the operation and stayed at low levels at day 14. In SNL-treated rats, comparable reductions of GDNF levels in L4 and L5 DRGs on the injured side were observed at 14 postoperative days. 4. Significant decreases of the percentages of DRG neurons expressing Ret were also observed at L4 DRGs in CCI-treated rats at 7 and 14 postoperative days and in SNL-treated rats at 14 days. 5. In CCI- or SNL-treated rats, continuous intrathecal administration of GDNF (12 microg day-1) using an osmotic pump suppressed the increased sensitivities to nociceptive stimuli to control levels. 6. The present results suggested that the dysfunction of GDNF signaling in the nociceptive afferent system may contribute to the development and/or maintenance of neuropathic pain states.