Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

Development of DNA markers for identifying chrysanthemum cultivars generated by ion-beam irradiation

Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ (Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.     

Body size and temperature dependence of routine metabolic rate and critical oxygen concentration in larvae and juveniles of the round crucian carp Carassius auratus grandoculis Temminck & Schlegel 1846

Routine metabolic rate (RMR, mgO₂ g^?1 h^?1) and critical oxygen concentration (Pc, a hypoxia tolerance indicator, mgO₂ L^?1) were determined in larvae and juveniles of round crucian carp, Carassius auratus grandoculis Temminck & Schlegel 1846, by measuring oxygen consumption at 15°C, 20°C, and 30°C. In addition, the dependence of RMR and Pc on fish body weight (W, g) and temperature (T, °C) was examined to construct minimal mathematical models. RMR depended on W and showed smaller values in larger individuals. RMR was different among the three temperature conditions and showed higher values at higher temperatures. Pc was significantly related to W and was low in larger individuals; that is, larger individuals had a higher hypoxia tolerance. In contrast, Pc was independent of temperature, implying that seasonal temperature fluctuations do not cause seasonal disequilibrium of hypoxia tolerance in round crucian carp. The RMR and Pc models were RMR = 0.0674W^?0.193e^0.0562T and Pc = 1.35W^?0.107, respectively. The metabolic information clarified in this study is essential for habitat quality assessments and aquaculture management of this species.     

Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system

Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors.     

Vegetation and fire history with their implication for climatic change and fire events since the last deglacial in the Aso Valley, central Kyushu, southwestern Japan: new pollen and charcoal data

To better understand the response of forest vegetation to climate and fire regimes with reference to human activities over the last deglacial period in the Aso Caldera, central Kyushu, southwestern Japan, a 33.9 m long sediment core was examined in order to reconstruct the vegetational and fire history using pollen and charcoal analyses. The results show that a cool temperate broad-leaved deciduous forest, dominated by Quercus (deciduous oaks) with Carpinus and Fagus, prevailed in the Aso Valley from ca. 14.6 ka cal. b.p., indicating warming since the last glacial period. The landscape was presumably covered by a mosaic of deciduous Quercus forests and terrestrial Artemisia communities. Around 12.8–11.7 ka cal. b.p., Quercus dominated the forest and fires occurred frequently. Co-expansion of distinctive Ulmus–Zelkova and Celtis–Aphananthe forests coupled with a progressive retreat of Quercus in the early Holocene could reflect a strengthening of the East Asian summer monsoon under mild and humid climate conditions. Around 8 ka cal. b.p., significant increases in Cyclobalanopsis (evergreen oaks), Castanopsis/Castanea and Podocarpus indicate a further warming, in particular an increased winter temperature. Warm temperate lucidophyllous forests, dominated by Cyclobalanopsis, flourished after 7.3 ka cal. b.p., probably corresponding to the “Holocene Climatic Optimum” interval. Progressive expansion of Quercus at the expense of Cyclobalanopsis began around 6.4 ka cal. b.p. and paralleled an increase in charcoal until ca. 4.8 ka cal. b.p.; this could be evidence of fire disturbance induced by the early-middle Jomon people. The disturbed evergreen forest experienced a temporary recovery but then opened again from 3.6 ka cal. b.p. due to extensive fire deforestation, as suggested by the high charcoal levels during this time. Human exploitation and buckwheat (Fagopyrum) agriculture may have contributed to the opening of the forest, which allowed secondary forests (primarily Pinus and Quercus) and herbaceous communities (mainly Poaceae) to spread. These results are discussed in comparison with other high-resolution pollen data from western Japan to better elucidate the vegetation and fire history over the last deglacial in the Aso Caldera.     

Isolation of Allo-isoleucic Acid (α-Hydroxy-β-methylvaleric Acid) and β-Hydroxy-β-methylvaleric Acid from Turkish Tobacco Leaves

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.

The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.

After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.

The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. $u=114\times \frac{E_{t1}?E_{t2}}{t_2?t_1}$

Where E_t1 and E_t2 mean the optical density of the ethanol solution at 310 mμ prepared after t₁ and t₂ minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.

The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid.     

General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

Tunicamycin-induced inhibition of protein secretion into culture medium of Arabidopsis T87 suspension cells through mRNA degradation on the endoplasmic reticulum

The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.     

Three-dimensional structures of c-Kit-positive cellular networks in the guinea pig small intestine and colon

Cryosections and whole-mount preparations of the guinea pig small intestine and colon were single or double immunolabeled using the anti-c-Kit and protein gene product 9.5 antibodies. Immunolabeled specimens were observed under a confocal laser scanning microscope. The main findings of the present study are: (1) the distribution and profiles of three-dimensional structures of c-Kit-positive cellular networks in the small intestine and colon, and (2) the anatomical relations of c-Kit-positive cells to the enteric nerves in the layers. In the small intestine, c-Kit-positive cellular networks were observed at levels of the deep muscular plexus and myenteric plexus. The c-Kit-positive cellular networks ran along or overlay the nerve fibers at the deep muscular plexus, while they showed the reticular structures intermingled with the nerve elements at the myenteric plexus. In the colon, c-Kit-positive cellular networks were observed at levels of the submuscular plexus and myenteric plexus, and were further identified within the circular and longitudinal muscle layers as well as in the subserosal layer. In the circular muscle layer, c-Kit-positive cells surrounded the associated nerve fibers and extended several long processes toward the adjacent c-Kit-positive cells. The c-Kit-positive cellular networks within the longitudinal muscle layer as well as in the subserosal layer were not associated with the nerve fibers. In the layers of the intestinal wall with c-Kit-positive cells, the cellular networks of the interstitial cells were identified in ultrastructure. The characteristic profiles of c-Kit-positive cellular networks provide a morphological basis upon which to investigate the mechanisms regulating intestinal movement. Received: 14 July 1998 / Accepted: 2 September 1998