Vessel development in compound buds of interspecific hybrid grape induced by artificial deacclimation treatments.

Grape compound buds adapt to subfreezing temperatures in winter by supercooling, but the supercooling ability is thought to be lost upon formation of xylem connections between canes and buds. It was reported that compound buds of the Vitis vinifera variety 'Chardonnay' lack xylem cells in mid-winter, and that vessels differentiate during deacclimation. However, the pattern of vessel formation in compound buds may differ in cold-hardy Vitis species and interspecific hybrid varieties grown in colder regions. We investigated vessel formation in compound buds of the interspecific hybrid variety 'Yamasachi', which were harvested in mid-winter, during artificial deacclimation treatments. Before these treatments, 'Yamasachi' buds had a high supercooling ability (approx. -30°C) and contained cells with characteristics of vessel elements, that is, secondary wall thickening and lignification, at the basal parts. However, the cells still contained organelles and did not have a hydraulic conductivity function. Xylem continuity between the canes and buds was established from day 7 of deacclimation at 20°C. The different pattern of seasonal vessel formation in compound buds of 'Yamasachi' from that of V. vinifera may reflect the rapid development traits of Vitis species growing in cold regions with short growing seasons.

Responses of the Plant Cell Wall to Sub-Zero Temperatures: A Brief Update.

Our general understanding of plant responses to sub-zero temperatures focuses on mechanisms that mitigate stress to the plasma membrane. The plant cell wall receives comparatively less attention, and questions surrounding its role in mitigating freezing injury remain unresolved. Despite recent molecular discoveries that provide insight into acclimation responses, the goal of reducing freezing injury in herbaceous and woody crops remains elusive. This is likely due to the complexity associated with adaptations to low temperatures. Understanding how leaf cell walls of herbaceous annuals promote tissue tolerance to ice does not necessarily lead to understanding how meristematic tissues are protected from freezing by tissue-level barriers formed by cell walls in overwintering tree buds. In this mini-review, we provide an overview of biological ice nucleation and explain how plants control the spatiotemporal location of ice formation. We discuss how sugars and pectin side chains alleviate adhesive injury that develops at sub-zero temperatures between the matrix polysaccharides and ice. The importance of site-specific cell-wall elasticity to promote tissue expansion for ice accommodation and control of porosity to impede ice growth and promote supercooling will be presented. How specific cold-induced proteins modify plant cell walls to mitigate freezing injury will also be discussed. The opinions presented in this report emphasize the importance of a plant's developmental physiology when characterizing mechanisms of freezing survival.

Freezing resistance and behavior of winter buds and canes of wine grapes cultivated in northern Japan.

In high-latitude regions, the cold hardiness of buds and canes of grapevine is important for budburst time and yield in the next season. The freezing resistance of buds and canes sampled from six wine grapes currently cultivated in Hokkaido, Japan, all of them grown from autumn to winter, was investigated. A significant difference between the cultivars in their freezing resistance was detected in the buds harvested in winter. In addition, outstanding differences in the lower temperature exotherms (LTE) related to the supercooling ability of tissue cells happened in the winter buds, and there is a close relationship between freezing resistance and LTE detected in the winter buds. This suggests that the supercooling ability of tissue cells in winter buds is strongly related to the freezing resistance. However, detailed electron microscopy exposed that the differences in freezing resistance among cultivars appeared in freezing behavior of leaf primordium rather than apical meristem. This indicated that as the water mobility from the bud apical meristem to the spaces around the cane phloem progressed, the slightly dehydrated cells improved the supercooling ability and increased the freezing resistance.

A single seed treatment mediated through reactive oxygen species increases germination, growth performance, and abiotic stress tolerance in Arabidopsis and rice.

Hydroxyl radical (•OH) is considered to be the most damaging among reactive oxygen species. Although afew studies have reported on its effects on growth and stress adaptation of plants, no detailed studies have been performed using •OH in germination and early seedling growth under abiotic stresses. Here we report a single seed treatment with •OH on germination and seedling growth of Arabidopsis and rice under non-stressed (ambient) and various abiotic-stressed conditions (chilling, high temperature, heat, and salinity). The treatment resulted in faster seed germination and early seedling growth under non-stressed conditions, and, interestingly, these effects were more prominent under abiotic stresses. In addition, Arabidopsis seedlings from treated seeds showed faster root growth and developed more lateral roots. These results show apositive and potential practical use for •OH in model and crop plants for direct seeding in the field, as well as improvement of tolerance against emerging stresses. Abbreviations: AUC: area under curve; MGT: mean germination time; t50: time to reach 50% germination; U7525: time for uniform germination from 25% to 75%; ROS: reactive oxygen species; GSI: germination speed index; SI: stress index; DI: dormancy index.

Bark cells and xylem cells in Japanese white birch twigs initiate deacclimation at different temperatures.

Appropriate timing of cold deacclimation is an important component of winter survival of perennial plants, such as trees, in temperate and boreal zones. Recently, concerns about predicted global climate change disturbing deacclimation timing have been increasing. The relationship between ambient temperatures and the manner by which cells' freezing resistance changes is essential for forecasting the timing of deacclimation. In this study, Japanese white birch twigs that underwent deacclimation treatment at a constant temperature of -2, 0, 4, 10, or 20 °C were separated into bark in which cells adapted to subfreezing temperatures by extracellular freezing and xylem in which cells adapted to subfreezing temperatures by deep supercooling, and the freezing resistance of cells in each tissue type was investigated by measuring percentage electrolyte leakage. Birch cells deacclimated in a different manner according to tissue type. Within 7 days under deacclimation treatment, xylem cells decreased their freezing resistance significantly at a high subfreezing temperature (-2 °C). In contrast, bark cells required a temperature of 10 or 20 °C for a detectable decrease in freezing resistance to occur within the same period. At a temperature lower than 0 °C, bark cells did not decrease their freezing resistance, even after 28 days of treatment. The difference in freezing behavior of cells might involve the difference in how deacclimation occurred in bark and xylem cells.

Mechanism of Overwintering in Trees.

Boreal trees possess very high freezing resistance, which is induced by short-day length and low temperatures, in order to survive severe subzero temperatures in winter. During autumn, cooperation of photoreceptors and circadian clock system perceiving photoperiod shortening results in growth cessation, dormancy development, and first induction of freezing resistance. The freezing resistance is further enhanced by subsequent low temperature during seasonal cold acclimation with concomitant changes in various morphological and physiological features including accumulation of sugars and late embryogenesis abundant proteins. The mechanism of adaptation to freezing temperatures differs depending on the type of tissue in boreal trees. For example, bark, cambium, and leaf cells tolerate freezing-induced dehydration by extracellular freezing, whereas xylem parenchyma cells avoid intracellular freezing by deep supercooling. In addition, dormant buds in some trees respond by extraorgan freezing. Boreal trees have evolved overwintering mechanisms such as dormancy and high freezing resistance in order to survive freezing temperatures in winter.

Supercooling-Promoting (Anti-ice Nucleation) Substances.

Studies on supercooling-promoting substances (SCPSs) are reviewed introducing name of chemicals, experimental conditions and the supercooling capability (SCC) in all, so far recognized, reported SCPSs and results of our original study are presented in order to totally show the functional properties of SCPSs which are known in the present state. Many kinds of substances have been identified as SCPSs that promote supercooling of aqueous solutions in a non-colligative manner by reducing the ice nucleation capability (INC) of ice nucleators (INs). The SCC as revealed by reduction of freezing temperature (°C) by SCPSs differs greatly depending on the INs. While no single SCPS that affects homogeneous ice nucleation to reduce ice nucleation point has been found, many SCPSs have been found to reduce freezing temperatures by heterogeneous ice nucleation with a large fluctuation of SCC depending on the kind of heterogeneous IN. Not only SCPSs increase the degree of SCC (°C), but also some SCPSs have additional SCC to stabilize a supercooling state for a long term to stabilize supercooling against strong mechanical disturbance and to reduce sublimation of ice crystals. The mechanisms underlying the diverse functions of SCPSs remain to be determined in future studies.

Increased neutrophil-to-lymphocyte ratio is associated with disease-specific mortality in patients with penile cancer.

BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR), a simple marker of the systemic inflammatory response, has been demonstrated to correlate with patient outcomes for various solid malignancies. We investigated the utility of the pretreatment NLR as a prognosticator in patients who presented with penile cancer. METHODS: A total of 41 patients who underwent complete blood count with differential and subsequent radical penectomy from 1988 to 2014 were analyzed. We assessed the correlation between the NLR and the prognosis of penile cancer. RESULTS: The median and mean (± SD) NLRs in 41 penile cancer patients were 3.42 and 5.03 ± 4.99, respectively. Based on the area under receiver operator characteristic curve, the cut-off value of NLR was determined to be 2.82. Patients with a high NLR (≥2.82) showed a significantly poorer cancer-specific survival (p = 0.023) than those with a low NLR. CONCLUSIONS: The pretreatment NLR may function as a biomarker that precisely predicts the prognosis in patients with penile cancer.

Freeze-thaw stress: effects of temperature on hydraulic conductivity and ultrasonic activity in ten woody angiosperms.

Freeze-thaw events can affect plant hydraulics by inducing embolism. This study analyzed the effect of temperature during the freezing process on hydraulic conductivity and ultrasonic emissions (UE). Stems of 10 angiosperms were dehydrated to a water potential at 12% percentage loss of hydraulic conductivity (PLC) and exposed to freeze-thaw cycles. The minimal temperature of the frost cycle correlated positively with induced PLC, whereby species with wider conduits (hydraulic diameter) showed higher freeze-thaw-induced PLC. Ultrasonic activity started with the onset of freezing and increased with decreasing subzero temperatures, whereas no UE were recorded during thawing. The temperature at which 50% of UE were reached varied between -9.1°C and -31.0°C across species. These findings indicate that temperatures during freezing are of relevance for bubble formation and air seeding. We suggest that species-specific cavitation thresholds are reached during freezing due to the temperature-dependent decrease of water potential in the ice, while bubble expansion and the resulting PLC occur during thawing. UE analysis can be used to monitor the cavitation process and estimate freeze-thaw-induced PLC.

Characteristics of ultrasonic acoustic emissions from walnut branches during freeze-thaw-induced embolism formation.

Ultrasonic acoustic emission (UAE) methods have been applied for the detection of freeze-thaw-induced embolism formation in water conduits of tree species. Until now, however, the exact source(s) of UAE has not been identified especially in angiosperm species, in which xylem tissues are composed of diverse types of cells. In this study, UAE was recorded from excised branches of walnut (Juglans regia cv. Franquette) during freeze-thaw cycles, and attempts were made to characterize UAEs generated by cavitation events leading to embolism formation according to their properties. During freeze-thaw cycles, a large number of UAEs were generated from the sample segments. However, the cumulative numbers of total UAE during freeze-thawing were not correlated with the percentage loss of hydraulic conductivity after thawing, suggesting that the sources of UAE were not only cavitation leading to embolism formation in vessels. Among the UAEs, cumulative numbers of UAEs with absolute energy >10.0 fJ strongly correlated with the increase in percentage loss of hydraulic conductivity. The high absolute energy of the UAEs might reflect the formation of large bubbles in the large lumen of vessels. Therefore, UAEs generated by cavitation events in vessels during freeze-thawing might be distinguished from other signals according to their magnitudes of absolute energy. On the other hand, the freezing of xylem parenchyma cells was followed by a certain number of UAEs. These results indicate the possibility that UAE methods can be applied to the detection of both freeze-thaw-induced embolism and supercooling breakdown in parenchyma cells in xylem.

[Investigation of prognostic factors for occult inguinal lymph node metastasis in penile cancer patients with no palpable or visibly enlarged inguinal lymph node (cN0)].

We investigated the incidence of inguinal lymph node metastasis in 66 penile cancer patients with no palpable or visibly enlarged inguinal lymph nodes (cN0). Median follow up interval was 35. 7 months. During follow up, 14 patients (21.2%) had inguinal lymph node metastasis and 6 patients died of cancer. Five-year disease-free survival was 77.3%. Univariate analysis demonstrated that local tumor staging (T), differentiation, lymphovascular invasion (LVI), and tumor infiltration pattern (INF, Yamamoto-Kohama grading system) were associated with the incidence of inguinal lymphnode metastasis. Multivariate analysis demonstrated that only LVI was associated with incidence of inguinal lymph node metastasis (p = 0.008, Hazard ratio 17.947). According to EAU risk classification, the incidence of inguinal lymph node metastasis in the low-risk group, intermediate risk group and high-risk group was 12.0, 17.6 and 55.6%, respectively. In conclusion, LVI is an independent prognostic factor for inguinal lymph node metastasis in cN0 penile cancer. Furthermore, EAU risk classification is valid judging from our cases and in incidence of inguinal lymph node metastasis. Either sentinel node biopsy or inguinal lymph node dissection is recommended in high and intermediate risk patient.

[Additional administration of dutasteride in patients with benign prostatic hyperplasia who did not respond sufficiently to α1-adrenoceptor antagonist : investigation of clinical factors affecting the therapeutic effect of dutasteride].

We performed additional administration of dutasteride in patients who did not respond sufficiently to α1-adrenoceptor antagonist treatment for lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) (LUTS/BPH). Among 76 registered patients, efficacy was analyzed in 58 patients. International Prostate Symptom Score (IPSS), subscores for voiding and storage symptoms and quality of life (QOL) on the IPSS, and Overactive Bladder Symptom Score (OABSS) were all significantly improved from the third month of administration compared to the time of initiating additional administration of dutasteride. Additional administration of dutasteride also significantly reduced prostate volume, and residual urine with the exception of the sixth month after administration. Age at initiation of administration and voiding symptom subscore on the IPSS were clinical factors affecting the therapeutic effects of dutasteride. The rate of improvement with treatment decreased with increasing age at initiation of dutasteride administration, and increased as voiding symptom subscore on the IPSS increased. Therefore, additional administration of dutasteride appears useful for cases of LUTS/BPH in which a sufficient response is not achieved with α1-adrenoceptor antagonist treatment. Because patients who have severe voiding symptoms or begin dutasteride at an early age may be expected to respond particularly well to dutasteride in terms of clinical efficacy, they were considered to be suitable targets for additional administration.

[Neoadjuvant and adjuvant chemotherapy with paclitaxel, cisplatin and 5-fluorouracil for advanced penile cancer: a case report].

54-year-old male was introduced to our hospital in January 2012 for surgical treatment and chemotherapy. The pathological examination revealed well differentiated squamous cell carcinoma of the penis. Computed tomography and magnetic resonance imaging indicated right inguinal and pelvic lymphadenopathy. We diagnosed the tumor to be unresectable radically and administered paclitaxel, cisplatin and 5-fluorouracil (TPF) as neoadjuvant chemotherapy. After 3 courses of chemotherapy, the size of lymphadenopathy had become small enough to allow curative surgical treatment. Partial penectomy and lymph node dissection were performed after neoadjuvant chemotherapy. For 12 months after this radical treatment, the patient has been healthy with no local resurrence and no distant metastatic lesion. TPF chemotherapy for unresectable nodal metastasis from squamous cell carcinoma of penis was suggested to be effective.

Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees.

The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.

Genetics of chilling response at early growth stage in rice: a recessive gene for tolerance and importance of acclimation.

Low-temperature adaptation in rice is mediated by the ability of a genotype to tolerate chilling temperatures. A genetic locus on chromosome 11 was analysed for chilling tolerance at the plumule stage in rice. The tolerant allele of A58, a japonica landrace in Japan, was inherited as a recessive gene (ctp-1A58), whereas the susceptible alleles from wild rice (Ctp-1W107) and modern variety (Ctp-1HY) were the dominant genes. Another recessive tolerant allele (ctp-1Silewah) was found in a tropical japonica variety (Silewah). Fine-mapping revealed that a candidate gene for the ctp-1 locus encoded a protein similar to the nucleotide-binding domain and leucine-rich repeat (NLR) protein, in which frameshift mutation by a 73 bp-deletion might confer chilling tolerance in ctp-1A58. Analysis of near-isogenic lines demonstrated that ctp-1A58 imparted tolerance effects only at severe chilling temperatures of 0.5 °C and 2 °C, both at plumule and seedling stages. Chilling acclimation treatments at a wide range of temperatures (8 °C-16 °C) for 72 h concealed the susceptible phenotype of Ctp-1W107 and Ctp-1HY. Furthermore, short-term acclimation treatment of 12 h at 8 °C was enough to be fully acclimated. These results suggest that the NLR gene induces a susceptible response upon exposure to severe chilling stress, however, another interacting gene(s) for acclimation response could suppress the maladaptive phenotype caused by the Ctp-1 allele. This study provides new insights for the adaptation and breeding of rice in a low-temperature environment.

Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in Cercidiphyllum japonicum.

Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2',5-tri-O-galloyl-α,ß-D-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-ß-D-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-ß-D-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5-4.5°C, at a concentration of 1 mg mL⁻¹. These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.

Bidirectional fluorescence properties of pyrene-based peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist.

Based on X-ray crystallographic analysis of a peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist complexed with human PPARs ligand binding domain (LBD), we previously reported the design and synthesis of a pyrene-based fluorescent PPARα/δ co-agonist 2. Here, we found that the fluorescence intensity of 2 increased upon binding to hPPARα-LBD, in a manner dependent upon the concentration of the LBD. But, surprisingly, the fluorescence intensity of 2 decreased concentration-dependently upon binding to hPPRδ-LBD. Site-directed mutagenesis of the two hPPAR subtypes clearly indicated that Trp264 of hPPARδ-LBD, located between H2' helix and H3 helix (omega loop), is critical for the concentration-dependent decrease in fluorescence intensity, which is suggested to be due to fluorescence resonance energy transfer (FRET) from the pyrene moiety of bound 2 to the nearby side-chain indole moiety of Trp264 in the hPPARδ-LBD.

Design, synthesis and in vitro evaluation of a series of α-substituted phenylpropanoic acid PPARγ agonists to further investigate the stereochemistry-activity relationship.

We previously demonstrated that the α-benzylphenylpropanoic acid-type PPARγ-selective agonist 6 exhibited a reversed stereochemistry-activity relationship, that is, the (R)-enantiomer is a more potent PPARγ agonist than the (S)-enantiomer, compared with structurally similar α-ethylphenylpropanoic acid-type PPAR agonists. Here, we designed, synthesized and evaluated the optically active α-cyclohexylmethylphenylpropanoic acid derivatives 7 and α-phenethylphenylpropanoic acid derivatives 8, respectively. Interestingly, α-cyclohexylmethyl derivatives showed reversal of the stereochemistry-activity relationship [i.e., (R) more potent than (S)], like α-benzyl derivatives, whereas α-phenethyl derivatives showed the 'normal' relationship [(S) more potent than (R)]. These results suggested that the presence of a branched carbon atom at the ß-position with respect to the carboxyl group is a critical determinant of the reversed stereochemistry-activity relationship.

Freezing activities of flavonoids in solutions containing different ice nucleators.

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 µL droplets at 0.2 °C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT(50) between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4-4.2 °C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 °C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 °C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 °C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 °C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 °C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.

Structure-based design, synthesis, and nonalcoholic steatohepatitis (NASH)-preventive effect of phenylpropanoic acid peroxisome proliferator-activated receptor (PPAR) α-selective agonists.

A series of α-ethylphenylpropanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) α-selective agonists, based on our PPARα/δ dual agonist 3 as a lead compound. Structure-activity relationship studies clearly indicated that the steric bulkiness and position of the distal hydrophobic tail part are critical for PPARα agonistic activity and PPARα selectivity, as had been predicted from a molecular-modeling study. A representative compound blocked the progression of nonalcoholic steatohepatitis (NASH) in an animal model.