5种光合细菌种间原生质体融合及优良农用融合子的筛选鉴定

处于对数生长期的光合细菌球形红假单胞菌(Rhodopseudomonassphaeroides)、沼泽红假单胞菌(Rhodopseudomonaspalustris)、嗜酸红假单胞菌(Rhodopseudomdnasacidophila)、深红红螺菌(Rhodospirarubrum)、万尼氏红微菌(Rhodomocrobiumvannielii),经溶菌酶(3mg/L)处理50min后,获得了它们的菌体形成的原生质体,其再生率分别为80%、71%、82%、61%、74%.取等量的亲本菌株在35%的PEG(MW6000)诱导下两两融合5min,共10种组合.其融合率为球×沼2.5×10-4、球×嗜2.1×10-4、球×深2.0×10-4、球×万2.1×10-4、沼×嗜2.8×10-4、沼×深2.4×10-4、沼×万2.6×10-4、嗜×深2.0×10-4、嗜×万2.3×10-4、深×万2.4×10-4.经影印法鉴定:形成的融合子可以分别生长于以相应的有机物为唯一碳源的培养基上,所有融合子体积均相当于两亲本株体积之和,融合子菌落形态特征介于两亲本株之间.从中随机挑选100个融合子,以辣椒苗作为靶标植物,从上述融合子中筛选到了1株具有显著促进作物生长、提高抗病性的融合子.     

Immobilization of cellulases on the reversibly soluble polymer Eudragit S‐100 for cotton treatment

Cellulases can penetrate into the fiber, causing tensile strength loss of the cellulosic fibers or fabrics. To minimize the tensile strength loss, we have immobilized cellulases on Eudragit S‐100. The characteristics of covalent Eudragit cellulase were evaluated using gel filtration analysis and UV spectra. Gel filtration analysis revealed that the cellulases were covalently bound to the polymer. Covalent Eudragit cellulase was loaded with the enzyme of about 40% and had a relative activity about 80% at a Eudragit S‐100 concentration of 15 g/L. When cellulase is bound to the polymer, the solubility profile becomes similar to the one of Eudragit. In addition, the effects of the enzyme on the cotton yarns and fabric using cellulases have been investigated. Native and immobilized cellulases caused improvements in whiteness and wrinkle recovery angle of the fabric in comparison to the control samples. The bending stiffness results show that native and immobilized cellulase treated cotton fabric has an improved softness than the control samples. It was found that using the immobilized cellulase reduced the weight and tensile strength, because the hydrolytic attack is only limited to the surfaces of cotton fibers.     

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.     

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

水杉愈伤组织诱导及植株再生

通过愈伤组织诱导器官发生途径,建立了水杉（Metasequoia glyptostroboides）的植株再生体系,探讨了不同外植体（种胚、幼叶切块、茎段、根段）和植物生长调节剂对不定芽直接再生和愈伤组织诱导器官发生的影响。结果表明：以种胚、无菌苗叶片、茎段和根作为外植体,在MS补加2,4-D、NAA和6-BA不同组合的培养基上都能诱导得到愈伤组织,其中种胚诱导愈伤组织效果最好,诱导率可达100%,茎诱导效果次之,诱导率为97.1%。诱导愈伤组织效果较好的培养基有：MS＋1.0mg·L-12,4-D＋0.5mg·L-16-BA、MS＋0.1mg·L-16-BA＋1.0mg·L-1NAA、MS＋0.5mg·L-16-BA＋1.0mg·L-1NAA、MS＋1.0mg·L-16-BA＋1.0mg·L-1NAA、MS＋0.5mg·L-16-BA＋2.0mg·L-1NAA、MS＋1.0mg·L-16-BA＋2.0mg·L-1NAA和MS＋0.5mg·L-12,4-D＋0.5mg·L-1NAA。以愈伤组织在MS培养基上植株再生效果最好,再生率为62.5%。     

Novel aminopeptidase N inhibitors derived from antineoplaston AS2–5 (Part I)

Overexpression of zinc-dependent metalloproteinase, aminopeptidase N (APN/CD13), is considered to be involved in the process of tumor invasion and metastasis. Herein we describe the synthesis and in vitro enzymatic inhibition assay of antineoplaston AS2–5 scaffold peptidomimetic compounds. The results demonstrated that most of these l-iso-glutamine derivatives displayed selective inhibitory activity against APN as compared with MMP-2, with IC₅₀ values in the micromole range. The structure–activity relationships were also briefly discussed.