Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy

Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.     

A novel light‐dependent selection marker system in plants

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin‐degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring‐cleavage extradiol dioxygenase in the Exiguobacterium sp. 255‐15; however, unlike other extradiol dioxygenases, Mn²⁺and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light‐dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high‐density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.     

Guaiane- and aristolane-type sesquiterpenoids of Nardostachys chinensis roots

Two guaiane-type compounds, nardoguaianone J and K (1 and 2) and two aristolane-type compounds, kanshone F and G (3 and 4), were isolated from Nardostachys chinensis roots. The structures including the absolute configurations were elucidated by spectral means and by comparison of their CD spectra.     

Phylogenetic Relationships of Amaryllidaceae Based on matK Sequence Data

matK gene, which is located in the chloroplast genome and evolves more quickly than the rbcL gene. A total of 31 species representing 31 of the 59 genera in the family were examined in this study. We also used 21 species from another ten families of Asparagales, four species from three families of Liliales and Acorus as outgroups. We obtained partial sequences of matK with lengths of 1,109–1,148 bp, corresponding to positions 230 to 1,343 of the Oryza sativa matK gene. The pairwise percentage sequence divergence ranged from 0 to 19.1% for all the species examined except Acorus, and 0 to 4.6% within Amaryllidaceae. Two methods of phylogenetic analysis, the Maximum Parsimony and Neighbor-Joining methods, were used. The trees obtained from these two analyses were fundamentally consistent. In both trees, the Amaryllidaceae sensu Dahlgren et al. formed a well-supported monophyletic clade with 100% bootstrap support. Amaryllidaceae were included in the Asparagales; however, its phylogenetic position within the Asparagales was not clearly resolved. Judging from the NJ tree, Agapanthus might be a sister group of the Amaryllidaceae, although bootstrap support for this was low. Character-state mapping was used to infer a center of origin and the biogeographic history of Amaryllidaceae. The result supports the hypothesis that the family evolved in Africa and subsequently spread to other continents, further suggesting that South America is the center of secondary diversification. Received 6 January 1999/ Accepted in revised form 8 April 1999     

Two New Ergosterol Derivatives from the Basidiomycete Cortinarius glaucopus

Two new sterols 1 and 2 and five known ones 3 – 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1‐ and 2D‐NMR spectra and HR‐FABS‐MS. The relative configuration of 1 was firmly determined by comparison of the observed ¹H–¹H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6‐31 + G(d,p) level of theory, in CHCl₃ as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A ( 1 ) and B ( 2 ), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)‐5,6‐epoxy‐3,7,23‐trihydroxystrophast‐8‐en‐14‐one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)‐3,5‐dihydroxyergosta‐6,8(14),22‐trien‐15‐one, respectively. Moreover, the configuration of known strophasterol C ( 3 ) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A ( 1 ) and strophasterol C ( 3 ) represent the second finding in nature of steroids with the rare strophastane skeleton.     

Luxol Fast Blue Mbs and Phloxine; a Stain for Mitochondria

The applicability of Luxol fast blue MBS as a 0.1% solution in 0.05% acetic acid to the staining of mitochondria, first recognized in rat kidney by Shanklin and Nassar (Stain Techn., 34: 257-60. 1959), was confirmed in various organs (formalin-Zenker and Regaud's fixations; paraffin embedding) of the mouse and bullfrog. In liver cells and in the epithelium of renal tubules, mitochondria were stained green, selectively and clearly. The dark cells of the renal tubules and the middle piece of sperms in both animals were conspicuously demonstrated by their dense assemblages of green granules. The periodic acid-Schiff procedure proposed by Shanklin and Nassar as a counterstain was replaced by staining in 0.5% aqueous phloxine, 2-3 min; differentiation in 5% phosphotungstic acid, 2 min; and washing in water, 5 min. This simplified and accelerated the techique, and gave a better color contrast. Advantages of Luxol fast blue MBS and phloxine staining over traditional methods for mitochondria in paraffin sections are: durability of the stain, high specificity, simplicity of procedure, and constant result.     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.     

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction

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