Regeneration of soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 μM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 μM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.     

茶园间作柑桔杨梅或吊瓜对叶蝉及蜘蛛类群数量和空间格局的影响

为评价茶园间作几种常见经济作物对重要害虫假眼小绿叶蝉及其主要天敌蜘蛛类群数量和空间格局的影响,遂选乌牛早品种纯茶园、乌牛早分别与柑桔、杨梅和吊瓜的间作茶园、以及安吉白茶与吊瓜间作茶园,2007年9月上旬—2008年12月下旬,每旬1次调查茶丛上、中、下层叶蝉和各种蜘蛛的数量。结果表明:(1)与纯茶园相比,间作茶园叶蝉种群数量和蜘蛛类群个体数量显著地增加,间作茶园蜘蛛种数显著地增加;(2)间作茶园茶丛上、中、下层叶蝉、蜘蛛个体数量分布明显区别于纯茶园茶丛上、中、下层叶蝉、蜘蛛个体数量分布;(3)茶丛上层的嫩梢是制作高档茶的原料,而纯茶园茶丛上层叶蝉虫口百分率为54.16%,间作茶园茶丛上层叶蝉虫口百分率皆减小,并且叶蝉高峰期间蜘蛛的跟随效应增强;(4)间作增加了经济收入并减少了防治次数。认为:(1)间作可在一定程度上调控叶蝉种群、蜘蛛类群的数量和空间格局;(2)间作可减轻叶蝉为害造成的产值损失,增强了茶园群落对于叶蝉的自然控制潜能。     

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.     

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.     

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H⁺ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca²⁺ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca²⁺ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca²⁺ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca²⁺ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.     

Proliferation of myofibroblasts in the stroma of renal oncocytoma

Objectives: Myofibroblasts are a vital component of stroma of many malignant neoplasms, but it is not yet established whether stromal myofibroblasts also exist in benign tumours such as oncocytoma of the kidney. Materials and methods: Histomorphological and immunohistochemical analysis of 16 renal oncocytomas diagnosed at Chang Gung Memorial Hospital, Taiwan, has been performed. Results: Renal oncocytomas were composed of oncocytes, large cells with granular eosinophilic cytoplasm, arranged mostly in sheets, in tubulocystic or combined pattern. Few oncocytes appeared to be undergoing proliferation or apoptosis. MIB‐1 and active caspase 3 indices were low, but higher in tumour than in surrounding non‐tumour parenchyma (MIB‐1: 0.93 ± 0.09 versus 0.46 ± 0.07, P < 0.001 and active caspase 3: 0.76 ± 0.08 versus 0.41 ± 0.09, P < 0.001). Wnt/β‐catenin signalling was not implicated in this neoplasm, as there was no loss of E‐cadherin membranous localization or expression of intranuclear β‐catenin in the cells. Clumps of oncocytes were stained with periodic acid Schiff and had collagen I‐, collagen III‐ and fibronectin‐positive, but desmin‐ and human caldesmon‐negative stromas. Importantly, α‐smooth muscle actin (SMA)‐immunostaining established the myofibroblastic nature of many of the stromal cells. Some of the myofibroblasts were also positive for MIB‐1, indicating a proliferative role for them in the stroma. Conclusions: Renal oncocytomas were composed of two independent compartments: benign oncocytes and pronounced fibrotic stroma, which consisted of proliferating myofibroblasts (SMA‐ and MIB‐1‐positive) which were associated with excessive deposition of extracellular matrix (periodic acid Schiff‐component, collagen I‐, collagen III‐ and fibronectin‐positive, and desmin‐ and human caldesmon‐negative).     

Effects of high temperature on the chlorophyll a fluorescence of Alhagi sparsifolia at the southern Taklamakan Desert

Climate change is expected to result in an increase in the frequency and magnitude of extreme weather events. Alhagi sparsifolia is an important factor for wind prevention and sand fixation in the forelands of the Taklamakan Desert. The effects of high temperature on desert plants remain widely unknown. In this work, chlorophyll a fluorescence induction kinetics were investigated at different time stresses of 5, 20, 40, and 60 min at temperature gradients of 38–44 °C at 2 °C intervals. A pronounced K-step was found, and the values of the maximum quantum yield for primary photochemistry, the quantum yield of electron transport, the density of reaction centers and the performance index on absorption basis were lowest after 60 min at 44 °C, thus indicating that the oxygen-evolving complex was damaged, the inactivated reaction centers increased, and the activity of the photosystem II (PSII) reaction center in leaves was seriously limited. Therefore, we suggest that under normal temperature (below 42 °C), the PSII of A. sparsifolia would be unaffected. When such temperature is maintained for 40 min, the activity of PSII would be limited, and when retained for 60 min, PSII may be severely damaged.     

Identification and characterization of lineage-specific genes in Populus trichocarpa

Species- or lineage-specific genes can facilitate studying the unique characteristics of biological processes. Updated genome sequences in Populus trichocarpa were screened against thirty newly sequenced or resequenced plant genomes to identify a set of species-specific genes (PtSS). Forty PtSS genes have been isolated with no similarity to any sequence outside the P. trichocarpa genome, therefore have no annotated functions. Protein motif, intron/exon features, subcellular localization and gene expression were analyzed in these PtSS genes. Results reflect their basic genic characters, expression analysis and primary function exploration might provide insight to their possible involvements in lineage specific biological process in woody plants.     

应用KV-CBCT分析鼻咽癌调强放射治疗的摆位误差

目的:应用KV-CBCT分析鼻咽癌调强放射治疗时的摆位误差,为鼻咽癌调强放射治疗计划设计时CTV外扩PTV边界的大小提供参考。方法:选取30例IMRT的鼻咽癌患者,治疗过程中每周一次应用KV-CBCT采集患者治疗前的CT图像,将所得图像与定位CT图像进行匹配,分别测定X、Y、Z轴三个方向的摆位误差。结果:30例患者共拍摄168次KV-CBCT,获得168组摆位误差结果,群体摆位误差分别为X轴-0.15±1.43 mm,Y轴0.20±1.58 mm,Z轴-0.21±1.65 mm;根据Van Herk公式计算得到各方向的CTV-PTV外放边界值X、Y、Z轴分别为3.1 mm、3.3 mm和3.4 mm。结论:应用KV-CBCT影像系统可实时测量摆位误差并在线进行纠正,减小摆位误差,为CTV-PTV外放边界提供参考。     

Computational identification of post‐translational modification‐based nuclear import regulations by characterizing nuclear localization signal‐import receptor interaction

The binding affinity between a nuclear localization signal (NLS) and its import receptor is closely related to corresponding nuclear import activity. PTM‐based modulation of the NLS binding affinity to the import receptor is one of the most understood mechanisms to regulate nuclear import of proteins. However, identification of such regulation mechanisms is challenging due to the difficulty of assessing the impact of PTM on corresponding nuclear import activities. In this study we proposed NIpredict, an effective algorithm to predict nuclear import activity given its NLS, in which molecular interaction energy components (MIECs) were used to characterize the NLS‐import receptor interaction, and the support vector regression machine (SVR) was used to learn the relationship between the characterized NLS‐import receptor interaction and the corresponding nuclear import activity. Our experiments showed that nuclear import activity change due to NLS change could be accurately predicted by the NIpredict algorithm. Based on NIpredict, we developed a systematic framework to identify potential PTM‐based nuclear import regulations for human and yeast nuclear proteins. Application of this approach has identified the potential nuclear import regulation mechanisms by phosphorylation of two nuclear proteins including SF1 and ORC6. Proteins 2014; 82:2783–2796. © 2014 Wiley Periodicals, Inc.