Density Gradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal antibodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two-phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mitochondria peaked at 41%, but Golgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. The 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrifugation. Victorin binding to 65- and 45-kD proteins was detected in all fractions in the continuous sucrose density gradients. The 65- and 45-kD proteins were both detected in purified plasma membranes, but only the 65-kD protein was detected in purified mitochondria. The subcellular location of VBPs was the same in sensitive and resistant oat cells.     

Molecular cloning of a homologue of dad-1 gene in citrus: distinctive expression during fruit development

A cDNA homologue to the human defender against apoptotic death gene (dad-1), which is involved in programmed cell death, was isolated from satsuma mandarin (Citrus unshiu Marc.) fruit. It (Citdad-1-1) was 345 bp long, with a deduced protein sequence of 115 amino acids. Southern hybridization suggests that dad-1-related sequences are present as a small gene family in the citrus genome. Expression of Citdad-1-1 was progressively down-regulated in leaves as they matured, but not in juice sac/segment epidermis (edible part) towards fruit ripening. The role of dad-1 during citrus development is also discussed.     

Apoptosis of mouse embryonic stem cells induced by single cell suspension

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts. Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS. Cellular activity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restoration. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.     

Degradation of Mono-chlorinated Dibenzo-p-Dioxins by Janibacter sp. Strain YA Isolated from River Sediment

Strain YA was newly isolated from an enrichment culture of river sediment and was identified as Janibacter sp. It was able to utilize dibenzofuran as the sole source of carbon and energy. Strain YA degraded > 90% of 1-chloro-dibenzo-p-dioxin (1-CDD) and > 80% of 2-chloro-dibenzo-p-dioxin in 18 hours with each initial concentration at 40 mg/L. A novel metabolite, 2-chloro-2′,6-dihydroxydiphenylether, was observed in 1-CDD degradation. From the metabolites detected by gas chromatography–mass spectrometry, strain YA was supposed to have at least two types of oxidation pathways in 1-CDD degradation.     

Approaches to repigmentation of vitiligo skin: new treatment with ultrasonic abrasion,seed-grafting and psoralen plus ultraviolet A therapy

Vitiligo vulgaris is a common disease throughout the world although its pathogenesis is not yet known. The most frequent treatment used for vitiligo is PUVA (psoralen plus ultraviolet A) and topical steroids but against stable refractory vitiligo, various other surgical techniques have been developed such as autografting, epidermal grafting with suction blisters, epithelial sheet grafting, and transplantation of cultured melanocytes. We have discovered a new method using ultrasonic abrasion, seed-grafting and PUVA therapy. The ultrasonic surgical aspirator abrades only the epidermis of recipient sites. This easily and safely removes only the epidermis, even on spotty lesions or intricate regions which are difficult to remove using a conventional motor-driven grinder or liquid nitrogen. Epidermal seed-grafting can cover more area than sheet-grafting, and subsequent PUVA treatment can enlarge the area of pigmentation with coalescence of adjacent grafts. In this article, we provide a general overview of the current surgical therapies including our method for treating stable refractory vitiligo.     

A zinc-binding citrus protein metallothionein can act as a plant defense factor by controlling host-selective ACR-toxin production

Metallothionein is a small cysteine-rich protein known to have a metal-binding function. We isolated three different lengths of rough lemon cDNAs encoding a metallothionein (RlemMT1, RlemMT2 and RlemMT3), and only RlemMT1-recombinant protein had zinc-binding activity. Appropriate concentration of zinc is an essential micronutrient for living organisms, while excess zinc is toxic. Zinc also stimulates the production of host-selective ACR-toxin for citrus leaf spot pathogen of Alternaria alternata rough lemon pathotype. Trapping of zinc by RlemMT1-recombinant protein or by a zinc-scavenging agent in the culture medium caused suppression of ACR-toxin production by the fungus. Since ACR-toxin is the disease determinant for A. alternata rough lemon pathotype, addition of RlemMT1 to the inoculum suspension led to a significant decrease in symptoms on rough lemon leaves as a result of reduced ACR-toxin production from the zinc trap around infection sites. RlemMT1-overexpression mutant of A. alternata rough lemon pathotype also produced less ACR-toxin and reduced virulence on rough lemon. This suppression was caused by an interruption of zinc absorption by cells from the trapping of the mineral by RlemMT1 and an excess supplement of ZnSO₄ restored toxin production and pathogenicity. Based on these results, we propose that zinc adsorbents including metallothionein likely can act as a plant defense factor by controlling toxin biosynthesis via inhibition of zinc absorption by the pathogen.     

Dosing Time‐Dependent Effect of Raloxifene on Plasma Fibrinogen Concentration in Ovariectomized Rats

The chronopharmacological effect of raloxifene, a selective estrogen‐receptor modulator, was evaluated by repeated dosing of ovariectomized rats. Bilateral ovariectomy or sham operation was performed at age 12 wks, and animals were kept in rooms with a 12 h light‐12 h dark cycle. Raloxifene (3 mg/kg, once daily for 10 wks) or vehicle was given repeatedly at either 2 h after lights‐on (2 HALO) or 14 h after lights‐on (14 HALO). Plasma fibrinogen concentration at the end of the study was reduced by the drug, and the reduction was significantly prominent in rats in whom the drug was dosed at 2 HALO rather than 14 HALO. Femur bone density decreased, and urinary excretion of deoxypyridinoline, an index of bone resorption capacity of osteoclasts, increased in ovariectomized animals at the end of the study. Treatment with raloxifene ameliorated these changes in a dosing time‐independent manner. Serum calcium, ALT, and total protein concentrations at the end of the study also did not differ acccording to treatment regime, which indicates that protein synthesis and liver function may not contribute to the effects. This is the first study to determine dosing time‐dependent changes in the efficacy of raloxifene in an animal model of osteoporosis. Because fibrinogen concentration is reported to be a marker of cardiovascular events, consideration of dosing time of raloxifene may be important to obtain a better cardioprotective effect of this medication when it is prescribed to postmenopausal women with osteoporosis.     

The substrate binding cavity of particulate methane monooxygenase from Methylosinus trichosporium OB3b expresses high enantioselectivity for n-butane and n-pentane oxidation to 2-alcohol

The particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b oxidized n-butane and n-pentane and mainly produced (R)-2-butanol and (R)-2-pentanol that comprised 78 and 89% of the product, respectively, indicating that the pro-R hydrogen of the 2-position carbon of n-butane and n-pentane is oriented toward a catalytic site within the substrate binding site of pMMO. The protein cavity adjacent to the catalytic center for pMMO has optimum volume for recognizing n-butane and n-pentane for enantioselective hydroxylation.     

A monoamine in the gustatory cell of the frog's taste organ

Summary Fluorescence histochemistry reveals that in the frog's taste organ a yellow fluorescence is regularly observed at the most basal region of the sensory epithelium. The fluorescence has a strong intensity, but it fades rapidly upon the UV-irradiation. The peak of the emission spectrum is at 520 m. Following reserpine treatment the yellow fluorescence is markedly reduced, but not depleted completely. From these characteristics the monoamine fluorescence is regarded as representing 5-HT (serotonin).The ultrastructural study on sensory epithelia shows that the terminal portions of gustatory cell processes are localized at the basal region. These portions are filled with dense cored vesicles (700–1000 Å in diameter) and frequently opposed with nerve fibers penetrating into the epithelium. The gustatory cell processes are also interposed between the terminal portions or nerve fibers. The cytoplasm of the gustatory cell process is characterized by many mitochondria, fine filaments and glycogen particles, but contains few cored vesicles. The distribution of terminal portions of gustatory cell processes seems to correspond fairly well to that of the monoamine fluorescence observed discontinuously along the basal lamina. Accordingly it is concluded that the fluorigenic monoamine is localized in the cored vesicles of the gustatory cell.These results were reported in a preliminary form to the October, 1974 meeting of the Japan Society of Histochemistry and Cytochemistry.The authors gratefully acknowledge the support and helpful advice of Prof. Dr. T. Kanaseki.