Aldehyde Oxidase in Wild Type and abal Mutant Leaves of Nicotiana plumbaginifolia

Aldehyde oxidase (AO; EC 1.2.3.1 [EC] ) activity was measured in rosetteleaves of the wild type and aba1 mutant (1217) of Nicotianaplumbaginifolia. An activity band was detected in the extractof the wild type by staining after gel electrophoresis usingcinnamaldehyde as a substrate, but not in that of 1217. However,after treatment with Na₂S and dithionite, an AO-activity bandwas detected in the extract of 1217 at the same position asthat of the wild type extract. These results indicated that1217 had AO apoprotein but the last step of molybdenum cofactorbiosynthesis, from nitrate reductase form (dioxo form) to hydroxylaseform (desulfo form), was blocked. Since abal is known to beimpaired in ABA synthesis, we examined whether the leaf AO isan abscisic aldehyde (ABAld) oxidase. AO was purified from theleaves of wild type plants. After several steps of purificationusing cinnamaldehyde as a substrate which has a structure similarto ABAld, a partially purified enzyme preparation with a purificationfactor of about 160-fold was obtained. The apparent molecularmass of AO was estimated to be approximately 290 kDa by gelfiltration. The enzyme had a relatively wide substrate specificityfor aldehydes including ABAld. The possible involvement of NicotianaAO in ABA biosynthesis is discussed. (Received June 24, 1998; Accepted September 21, 1998)     

Cloning and Characterization of High-CO2-Specific cDNAs from a Marine Microalga, Chlorococcum littorale, and Effect of CO2 Concentration and Iron Deficiency on the Gene Expression

Two cDNA clones exclusively induced under an extremely high-CO₂concentration (20%) were isolated from Chlorococcum littoraleby differential screening and named HCR (high-CO₂ response)1 and 2, respectively. The amino acid sequence of the proteinencoded by HCR2 exhibited homology to the gp91-phox protein,a critical component of a human phagocyte oxidoreductase, andto the yeast ferric reductases, Saccharomyces cerevisiae FRE1and FRE2 and Schizosaccharomyces pombe Frpl. The induction ofboth HCR mRNAs required extremely high-CO₂ conditions and irondeficiency, being suppressed under air conditions and by ironsufficiency, suggesting that the expression of these two HCRgenes required extremely high-CO₂ conditions and iron deficiencyin combination. The HCR2 protein was detected in the membranefractions of cells grown under conditions which would favorthe induction of HCR2-mRNA and the protein level was loweredwhen the cells were transferred from iron deficient to 10 µMFeSO₄ conditions (with 20% CO²). (Received September 10, 1997; Accepted November 14, 1997)     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.     

3, 4-Dihydroxyphenylalanine is oxidized by phenoxyl radicals of hydroxycinnamic acid esters in leaves ofVicia faba L

Mechanisms of oxidation of 3,4-dihydroxyphenylalanine (dopa) in leaves ofVicia faba have not yet been elucidated in details. The author hypothesized its oxidation by radicals of hydroxycinnamic acid esters that were generated by a peroxidase-dependent reaction in vacuoles. The results obtained in this study were followings. 1) Vacuolar peroxidase isolated from the leaves oxidized dopa more slowly than 4-coumaric and caffeic acid esters isolated from the leaves. 2) The hydroxycinnamic acid esters enhanced peroxidase-dependent oxidation of dopa and dopa suppressed their oxidation. 3) Degree of the enhancement was roughly correlated with rates of the oxidation of hydroxycinnamic acid esters. 4) The hydroxycinnamic acid esters increased levels of dopa radical in the presence of peroxidase. 5) In protoplasts of mesophyll cells ofV. faba, hydrogen peroxide-induced oxidation of dopa was faster than that of 4-coumaric acid and caffeic acid esters. These results support the above hypothesis that dopa in vacuoles is oxidized by phenoxyl radicals of hydroxycinnamic acid esters that are generated by vacuolar peroxidase.     

Selection of light or darkness, locomotor, and stridulatory activities in the cricket, Gryllus bimaculatus Degeer (Orthoptera: Gryllidae)

Male crickets, Gryllus bimaculatus Degeer, turned lights on or off in a chamber by a seesaw device: (1) during a 12 h, and (2) during a 24 h day. The crickets in (1) and the last-instar nymph in (2) turned the lights on and off at irregular intervals and duration. The selection rate for darkness was greater than that for light by an average exceeding 80%. The locomotor activity of the nymph in (1) was arrhythmic. In (1) the adults stridulated and were active in continuous darkness during the 12 h, while, in contrast in (2) they turned on the light and stridulated without switching the light off. The locomotor and stridulatory activities of the adult crickets in (2) were free-running. These activities resulted in a free-running rhythm of selection for light or darkness in (2). Under the conditions of the present experiments, the circadian pacemaker functioned in the same way in light and dark cycles as in constant light conditions.     

Morphological responses of four species of cyclomorphic Daphnia to a short-term exposure to the insecticide carbaryl

Four species of cyclomorphic Daphnia (D.pulex, D.galeata mendotae,D.retrocurva, D.lumholtzi) were exposed to the insecticide carbarylfor a short term (8–4 h) from the final embryonic stageto the first instar. Daphnia pulex formed neckteeth, and theremaining three Daphnia species developed high helmets and longtailspines. The results suggest that the development of suchprotuberant structures (anti-predator morphologies) in responseto the insecticide exposure is a general phenomenon in Daphnia,and that stimuli on the nervous system of Daphnia may inducethe morphological changes, which originally evolved as a responseto predator kairomone. Two clones of D.pulcx were examined anda clone which was more sensitive to the predator Chaobonts kairomonethan another developed more marked neckteeth in response tocarbaryl, suggesting that the sensitivity in morphological responseto the insecticide may be related to the sensitivity to thekairomone. ¹Present and permanent address: Regional Environment Division,National Institute for Environmental Studies Onogawa, Tsukuba,Ibaraki 305, Japan     

Micropropagation of garlic through in vitro bulblet formation

We developed a novel micropropagation method for garlic (Allium sativum L.) by the combination of initial shoot-tip culture, shoot multiplication and in vitro bulblet formation. Garlic shoot-tips were cultured on LS medium containing 1 M indole-3-acetic acid (IAA) and 1 M 6-benzyladenine (BA) to regenerate proliferative shoots. These shoot-tips produced multiple shoots when transferred to modified LS medium containing 5 M 1-naphthaleneacetic acid (NAA) and 10 M BA, and cultured at 20°C under 12-h light conditions. Higher ratios of KNO₃/NH₄Cl in the media promoted multiple shoot formation, together with suppressing vitrification of these shoots. The proliferated shoots of early maturing cultivars produced bulblets by culture on LS growth regulator-free medium at 25°C under 16-h light. On the other hand, the late maturing cultivar, Howaito-roppen, formed bulblets after a low temperature treatment of the proliferated shoots for 6 months followed by culture on LS medium containing 6 to 12% sucrose for two months. The dormancy of the bulblets of cv. Howaito-roppen was broken by successive treatments at a high (35°C), a middle (20°C), and then a low (5°C) temperature.Abbreviations IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - LS Linsmaier and Skoog macro- and microelements     

滑膜肉瘤动物模型的分子细胞遗传学研究

用1例日本滑膜肉瘤（SS）患者的瘤组织标本接种裸鼠,获得了肿瘤生长。亲本肿瘤与裸鼠肿瘤在病理组织形态上存在一定差异,但两者的免疫组织化学特征相同。染色体分析表明,SS细胞株存在染色体数目和结构异常,患者外周血淋巴细胞染色体核型为正常女性,46,XX。 ＳＳ细胞株巴氏小体检出率低于对照,其正常X染色体比易位X染色体晚复制17小时。DNA印迹实验表明,SS DNA存在D2S3座位等位片段丢失,D1S57,D17S5和D13S30座位基因的部分缺失,但无DXS7,DXS14,和D2S44座位基因改变。     

Fungal succession on pine needles in Germany

The mycofloral succession on the needles ofPinus sylvestris was investigated in Tübingen, southwest Germany. Dead needles attached to the branches (D-type), those caught on branches (C-type) and three types of fallen needles, i.e., freshly fallen (L-type), slightly discolored (OL-type) and almost black needles (F-type) were examined for their fungal flora. Common primary saprophytes were rich on the dead needles on the tree, and on the L-type needles. They were replaced by successive species that contained the well-known species preferring pine needles as their substratum, such asVerticicladium trifidum orSympodiella acicola. Their ecological niches in pine leaf litter and their distribution patterns from a biogeographical viewpoint were discussed.