Development and characterization of cell lines from heart, liver, spleen and head kidney of sea perch Lateolabrax japonicus

Five cell lines (LJHK, LJS, LJL, LJH-1 and LJH-2) were established from the head kidney, spleen, liver and heart of sea perch Lateolabrax japonicus . The cell lines LJHK, LJS, LJL, LJH-1 and LJH-2 were subcultured 46, 32, 32, 36 and 34 times in minimum essential medium (MEM) supplemented with foetal bovine serum (FBS), sea perch serum and 10 ng ml⁻¹ basic fibroblast growth factor (bFGF). Morphology of primary cultures and subcultures of the five cell lines were observed continuously by microscopy. The suitable temperature for growth was 18 to 30° C for all of these cell lines with the optimum growth at 24° C and a reduced growth rate <18° C. The optimum concentration of FBS was found to be 10% and addition of bFGF to the medium significantly increased the growth rate of the cells. The doubling time of LJS, LJH-1, LJL, LJH-2 and LJHK cells was determined to be 52·7, 54·9, 57, 58·7 and 66 h at a plating density of 1 × 10⁵ cells ml⁻¹ at 24° C, respectively. Chromosome analysis revealed that 42, 48, 38, 43 and 45% cells maintained normal diploid chromosome number (48) in the LJH-1, LJH-2, LJHK, LJL and LJS cell lines, respectively. The LJHK cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies. Furthermore, treatment of the LJHK cells with lipopolysaccharide led to increased expression of IL-1β, demonstrating that LJHK cells might be a valuable tool for studying the expression and function of immunomodulatory gene in fishes.     

The origin, initiation and development of axillary shoot meristems in Lotus japonicus

BACKGROUND AND AIMS: Lotus japonicus 'Gifu' develops multiple axillary shoots in the cotyledonary node region throughout the growth of the plant. The origin, initiation and development of these axillary meristems were investigated. METHODS: Morphological, histological and mRNA in situ analyses were done to characterize the ontogeny of cotyledonary axillary shoot meristems in Lotus. Morphological characterization of a putative Lotus shoot branching mutant (super-accessory branches) sac, is presented. KEY RESULTS: By using expression of an L. japonicus STM-like gene as a marker for meristematic tissues, it was demonstrated that groups of cells maintained in the meristematic state at the cotyledonary axil region coincide with the sites where additional axillary meristems (accessory meristems) form. A Lotus shoot branching mutant, sac, is a putative Lotus branching mutant characterized by increased proliferation of accessory shoots in all leaf axils including the cotyledons. CONCLUSION: In Lotus, axillary shoot meristems continually develop at the cotyledonary node region throughout the growth of the plant. These cotyledonary primary and accessory axillaries arise from the position of a meristematic zone of tissue at the cotyledonary node axil region.     

FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.     

A Set of Lotus japonicus Gifu x Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.     

刺参酸性粘多糖对诱发性肝癌大鼠的干预作用及免疫功能的影响

目的：通过建立诱发性大鼠肝癌动物模型,研究用刺参酸性粘多糖（SJAMP）对大鼠诱发性肝癌的干预作用及免疫功能的影响。方法：将雄性Wistar大鼠50只随机均分为5个组,正常对照组、模型组和3个SJAMP干预组（A组,B组和c组）,模型组和SJAMP干预组灌胃0．2％DEN生理盐水溶液以诱发肝癌,同时SJAMP干预组按照不同剂量（0．175μg／g,0．35μg／g,O．7μg/g）给予SJAMP,至16周处死大鼠,取血,无菌取脾、胸腺,计算脾指数、胸腺指数。比较各组〉3mm和〉5mm的结节数以及最大结节的体积,计算肿瘤抑制率。贴壁法纯化巨噬细胞,用中性红吞噬实验检测巨噬细胞吞噬功能,MTT法检测巨噬细胞杀伤功能。结果：SJAMP干预组〉3mm和〉5mm的结节数明显少于模型组,最大结节的平均体积明显小于模型组（P〈0．05）;与模型组相比,SJAMP干预组脾指数和胸腺指数明显升高（P〈0．05）,SJAMP干预组巨噬细胞吞噬能力和杀伤功能显著提高（P〈0．05）。结论：刺参酸性粘多糖对大鼠诱发性肝癌有明显的抑制作用;其机制可能是通过刺激免疫器官生长,增强机体的细胞免疫能力来实现的。     

刺参“腐皮综合症”致病菌LNUB415的分离及防治的初步研究

从大连海域患“腐皮综合症”的养殖刺参肠道中分离纯化到1株优势细菌LNUB415,经人工回接感染试验确定其对刺参具有较强的致病性.经形态学、生理生化和16S rDNA序列分析,该菌被初步鉴定为蜡样芽胞杆菌(Bacillus cereus).从多种具有免疫增强作用的中草药中筛选到抑菌效果最好的黄连,可使刺参死亡率降低30％.     

What can metazoan parasites reveal about the taxonomy of Scomber japonicus Houttuyn in the coast of South America and Madeira Islands?

The metazoan parasites of four populations of the chub mackerel Scomber japonicus were analysed from two localities in the Atlantic Ocean (Madeira Islands, Portugal, and Rio de Janeiro, Brazil) and two localities in the Pacific Ocean (Callao, Peru, and Antofagasta, Chile), collected during 2002 and 2003. A total of 373 fish specimens were studied and 34 metazoan parasite species were obtained. Parasites identified from the populations of chub mackerel studied could be separated into three categories: parasites with a wide distribution, present in the Pacific and Atlantic, parasites proper of the Pacific Ocean and parasites proper of the Atlantic Ocean. The analyses of some highly specific parasites of the genus Scomber ( i.e. monogeneans of the genus Kuhnia and didymozoid digeneans) strongly suggest the need for a revision of the taxonomic status of chub mackerels from the Atlantic and Pacific coast of America. The results demonstrated the usefulness of parasites as adequate tools to clarify the taxonomic status of their hosts.     

Flexibility of first brood production in a claustral ant, Camponotus japonicus (Hymenoptera: Formicidae)

The numbers of first-batch eggs and workers (minims) in a carpenter ant Camponotus japonicus are not fixed but are regulated in response to various internal and external factors of founding queens. The initial body weight of founding queens was positively correlated with the number of minims. The amount of queen weight loss during the founding period was highly correlated with the number of minims. The numbers of first-batch eggs and minims for artificially fed queens were significantly greater than those for control group queens and for unfed queens, respectively. The number of eggs laid by a queen increased when eggs deposited in the early stage of founding were removed. In contrast, artificial addition of alien eggs reduced the number of eggs laid by a queen. The adaptive implications of this flexibility in the production of the first-batch brood in C. japonicus are discussed. Received: December 18, 2000 / Accepted: March 19, 2001     

Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Loop‐mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species

Invasive Aedes mosquito species (Diptera: Culicidae) are of public health concern in Europe because they are either recognized or potential vectors of pathogens. Loop‐mediated isothermal amplification (LAMP) is a rapid and simple method for amplifying DNA with high specificity and efficiency, with the technique having potential for application in the field, including in high‐throughput format. Specific LAMP assays based on rDNA internal transcribed spacers 1 or 2 sequences, considering intraspecies variability at these loci, were developed for Aedes aegypti, Aedes albopictus, Aedes japonicus, Aedes koreicus and the indigenous Aedes geniculatus. No such assays could be developed for Aedes atropalpus and Aedes triseriatus because both loci were too short to serve as target. The assays rely on the clearly visible colour change from violet to sky blue after successful amplification. Sensitivity of egg detection was confirmed with ratios of up to one mosquito egg in 99 other eggs. Simple sample preparation of adults or eggs by mechanical homogenization in water required an additional heat treatment or centrifugation step to avoid non‐specific colour changes. Thus, further technical improvements are needed to render these assays truly field‐applicable, which would greatly facilitate surveillance of these invasive mosquito species and allow for prompt implementation of control measures.