Plant regeneration in <Emphasis Type="Italic">Actinidia polygama</Emphasis> Miq. by leaf,stem, and petiole culture with zeatin,and from stem-derived calli on low-sucrose medium

We examined the effect of zeatin on the formation of shoot buds from explants and callus tissues derived from stem segments of Actinidia polygama Miq. (matatabi or silver vine). Stem and petiole segments cultured on combined broad-leaved tree medium and woody plant medium (BW medium) supplemented with zeatin for 40 days formed many shoot buds. Callus tissues derived from the stem segments and subcultured on BW medium supplemented with 6-benzyladenine and 1-naphthaleneacetic acid formed shoot buds when the medium contained 13.7µM zeatin. BW medium containing low concentrations of sucrose strongly induced the formation of shoot buds from the callus.     

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

Therapeutic effects of vitamin E supplementation in 4 dogs with poor semen quality and low superoxide dismutase activity in seminal plasma

Four dogs with poor semen quality, low seminal plasma superoxide dismutase (SOD) activity and low blood plasma testosterone (T) levels were orally administered one vitamin E tablet containing 50 mg α-tocopheryl acetate per dog daily for 4 weeks. The mean values of semen quality were temporarily improved after the start of vitamin E treatment and the values of 4, and 5 weeks after that were significantly different from those before the treatment (P<0.05–0.001). The mean blood plasma T and seminal plasma SOD activity values slightly increased in the 4 dogs after the treatment. The results of the present study indicate that poor semen quality in dogs with low seminal plasma SOD can be improved by vitamin E treatment.     

Effects of various selective phosphodiesterase inhibitors on carbachol-induced contraction and cyclic nucleotide contents in the guinea pig gall bladder

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), and zaprinast (type 5), inhibited CCh-induced contractions in a concentration-dependent manner. The rank order of potency for the gall bladder was Ro20-1724 > vinpocetine > EHNA > milrinone > zaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 muM), vinpocetine, milrinone, and Ro20-1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20-1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta.     

Age estimation of Antarctic minke whales <Emphasis Type="Italic">Balaenoptera bonaerensis</Emphasis> based on aspartic acid racemization technique

Counting of the growth layers in the earplugs is the most accepted technique for determining chronological age of Antarctic minke whales; however, unreadable growth layers form in some individuals, especially in young animals. In this study, aspartic acid racemization (AAR) technique was developed for estimating ages in this species with the aim of complementing the age estimated using earplugs. To validate the technique and to determine the specific coefficients for age estimation, the ratio of d and l-enantiomers of aspartic acid (Asp D/L) in lens of 18 whales and 20 fetuses were analyzed and compared with earplug-based age estimates. The equation for age estimation by AAR in this species was as follows: Log_e{[1 + (Asp D/L)_act]/[1 ? (Asp D/L)_act]} = 2.30 × 10^?3 × earplug age (year) + 0.0201 (p < 0.001, r ²  = 0.918). There is a strong correlation between the age estimates by AAR and earplugs. This study was successful in developing the AAR technique for the Antarctic minke whale, and the application of this technique can complement the age estimation of this species based on earplug readings, especially for young animals with unreadable earplugs.     

Within- and between-site variations in leaf longevity in hinoki cypress (Chamaecyparis obtusa) plantations in southwestern Japan

We investigated mean leaf retention time in order to elucidate the factors affecting regional and local variations in stand-level leaf longevity in hinoki cypress (Chamaecyparis obtusa) plantations. Our study sites consisted of six stands at a low elevation (320–370 m a.s.l.) and 12 stands at a high elevation (850–970 m a.s.l.) in southwestern Japan. We also used published data on leaf longevity in stands at various elevations to clarify the regional-scale variations in leaf longevity and their relationships to environmental factors. At the regional scale, leaf longevity increased with increasing elevation and with decreasing air temperature, growing season length, and Kira’s warmth index across sites. Similar relationships were obtained for the variation in leaf mass. At the local scale, leaf longevity did not show a clear relationship with topographic position, soil water content, or soil C/N ratio. Contrary to our expectation, leaf longevity was negatively correlated with the leaf C/N ratio at both study sites, although the significance level was marginal. This indicates greater leaf longevity with better leaf nutrient status. Our results suggest that responses of leaf longevity in hinoki cypress stands to environmental factors would be more prominent at the regional scale than at the local scale, although large variation was detected at the local scale. Air temperature and growing season length appear to be the main drivers of the variation in leaf longevity at the regional scale, whereas the causal factors are unclear at the local scale.     

Effects of hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder

To elucidate the dependence of aerobic energy metabolism and utilization of glucose in contraction of urinary bladder smooth muscle, we investigated the changes in the reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determined the phosphocreatine (PCr) and ATP contents of the porcine urinary bladder during contractions induced by high K⁺ or carbachol (CCh) and with and without hypoxia (achieved by bubbling N₂ instead of O₂) or in a glucose-free condition. Hyperosmotic addition of 65 mM KCl (H-65K⁺) and 1 µM CCh induced a phasic contraction followed by a tonic contraction. A glucose-free physiological salt solution (PSS) did not change the subsequent contractile responses to H-65K⁺ and CCh. However, hypoxia significantly attenuated H-65K⁺- and CCh-induced contraction. H-65K⁺ and CCh induced a sustained increase in PNred fluorescence, representing glycolysis activity. Hypoxia enhanced H-65K⁺- and CCh-induced increases in PNred fluorescence, whereas glucose-free PSS decreased these increases, significantly. In the presence of H-65K⁺, hypoxia decreased the PCr and ATP contents; however, the glucose-free PSS did not change the PCr contents. In conclusion, we demonstrated that high K⁺- and CCh-induced contractions depend on aerobic metabolism and that an endogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSS in the porcine urinary bladder.     

An expanded palette of genetically encoded Ca²⁺ indicators

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.     

Isolation of Encephalitozoon cuniculi using primary tissue culture techniques from a rabbit in a colony showing encephalitozoonosis

Encephalitozoon spores were isolated in a primary tissue culture of the kidneys from an encephalitozoonosis-suspected rabbit in a municipal zoo in Hokkaido. The isolated spores were morphologically characteristic of microsporidial ones in chromotrope stain, and proven to be E. cuniculi by a polymerase chain reaction (PCR) with a species-specific primer set and by direct DNA sequencing of the PCR products.