连接与切断处理对土壤氮素资源异质环境中结缕草主匍匐茎的影响效应

通过实验检验了克隆植物结缕草在异质生境条件下的生理整合作用。结缕草被栽植于4种类型的“生境”内(它们的总体土壤氮素资源水平呈现梯度变化),并同时对匍匐茎的节间实施连接与切断2种处理。实验结果表明,呈连接状态的结缕草克隆的平均复合节数量、平均匍匐茎长度和生物量在各个生境类型间并未表现出明显的差异。但主匍匐茎实施切断处理后的结缕草克隆的上述各项指标均显著减小。生长于贫瘠土壤斑块上的复合节平均根系生物量和根/茎比均高于肥沃土壤斑块。这一特征与其它典型的克隆植物明显不同。结缕草克隆在保持连接的情况下,土壤氮素对节间长度和比节间长度的伸长生长以及分蘖的形成具有促进作用。实验过程中的节间切断处理抑制了节间的进一步伸长但提高了比节间长度。保持结缕草克隆整合并尽量避免严重干扰显得十分重要,特别是在结缕草早期生长阶段。     

Multiphase characterization of wild Vigna associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant Bradyrhizobium strains having high symbiotic compatibility with soybean and mungbean

ABSTRACT

Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with V. riukiuensis grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to Bradyrhizobium japonicum, B. elkanii and B. jicamae family. However, analysis of symbiotic nifH and nodD1 genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with B. elkanii, growth was observed at 40°C, which exceed the highest record for B. elkanii to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with B. diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V. riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than B. diazoefficiens USDA110. In addition, six isolates attained higher soybean biomass production compared with B. diazoefficiens USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29% higher soybean biomass production than B. diazoefficiens USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics.     

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.     

Establishment of a patient-derived xenograft mouse model of pleomorphic leiomyosarcoma

Soft tissue sarcomas are difficult to treat using chemotherapy owing to a current deficiency in candidate drugs for specific targets. Screening candidate compounds and analyzing therapeutic targets in sarcomas is insufficient, given the lack of an appropriate human sarcoma animal model to accurately evaluate their efficacy, as well as the lack of an adequate technical protocol for efficient transplantation and engraftment of sarcoma specimens in patient-derived xenograft (PDX) models. Accordingly, in this study, we sought to identify the optimal type of sarcoma and develop a protocol for generating a PDX model. We characterized a PDX mouse model using histopathological and immunohistochemical analyses to determine whether it would show pathological characteristics similar to those of human sarcomas. We achieved engraftment of one of the 10 transplanted sarcoma specimens, the xenografted tumor of which exhibited massive proliferation. Histologically, the engrafted sarcoma foci resembled a primary tumor of pleomorphic leiomyosarcoma and maintained their histological structure in all passages. Moreover, immunohistochemical analysis revealed the expression of specific markers of differentiation to smooth muscle, which is consistent with the features of leiomyosarcoma. We thus demonstrated that our pleomorphic leiomyosarcoma PDX mouse model mimics at least one aspect of human sarcomas, and we believe that this model will facilitate the development of novel therapies for sarcomas.     

Preservation of quail blastoderm cells in liquid nitrogen.

1. Quail blastoderm cells isolated from the yolk were preserved in liquid nitrogen. 2. Frozen-thawed blastoderm cells of the quail were viable and survived in vitro. By injecting the frozen-thawed cells into chick embryos, quail-chick chimaeras were produced.     

Physiologic effects of administration of interleukin 1 beta in cows.

The immunomodulating polypeptide interleukin 1 beta (IL-1 beta) has been shown to be homologous to osteoclast-activating factor and is capable of stimulating increased osteoclastic bone resorption. This effect prompted an investigation into the potential use of IL-1 beta for prevention of parturient paresis, a disease of dairy cows characterized by hypocalcemia and poor osteoclastic resorption of bone. Six nonpregnant cows were treated with a high dosage of IL-1 beta (166 ng/kg of body weight) every 8 hours for 4 days. The IL-1 beta treatment significantly (P < 0.05) increased urinary hydroxyproline excretion, an index of osteoclast activity, indicating that bone calcium resorption might be stimulated by IL-1 beta treatment of cows. However, IL-1 beta treatment also caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis. The acute, but transient, effect of IL-1 beta treatment was to cause a decrease in plasma calcium and phosphorus concentrations. The pleiotropic effects of IL-1 beta administration negated the positive effects on osteoclastic bone resorption, and indicates that this cytokine may be of minimal benefit for prevention of parturient paresis.     

Histological evidence of lunar-synchronized ovarian development and spawning in the spiny rabbitfish Siganus spinus (Linnaeus) around the Ryukyus

ABSTRACT: This study clarifies the annual reproductive cycle and the lunar-synchronized spawning of the spiny rabbitfish ( Siganus spinus ) that inhabit the Okinawan waters. Annual and weekly changes in the gonadosomatic index (GSI) and the histological features of the ovaries were checked. Gonadosomatic index was high during the months of May to July, and yolk-laden oocytes were observed in the ovaries from March to July. Some of the ovaries collected during June and July contained oocytes at maturation stage or ovulatory follicles. These results suggest that the spiny rabbitfish undergo active vitellogenesis and spawning from May to July. During the reproductive season (May to July), collection of fish according to the lunar phase revealed that a high GSI occurred around the time of the new moon. Cyclic oocyte development with peaks around the time of the same moon phase was also observed, suggesting that, in Okinawan waters, this species is a lunar-synchronized spawner and spawns three times.     

Lingual squamous cell carcinoma in a California sea lion (Zalophus californianus).

A 28-yr-old female California sea lion (Zalophus californianus) in a commercial aquarium developed an ulcerated lingual tumor and died. Necropsy revealed a moderately differentiated squamous cell carcinoma. Immunohistochemical staining and electron microscopic studies revealed that the tumor cells were strongly positive with anti-keratin-cytokeratin antibody and had abundant tonofibrils and desmosomes. The neoplasm had metastasized to a mandibular lymph node.     

Analyses of the regulatory mechanism of porcine WEE1B: the phosphorylation sites of porcine WEE1B and mouse WEE1B are different

WEE1B, an oocyte-specific kinase, phosphorylates the CDC2 inhibitory site and maintains the meiotic arrest of oocytes at the first meiotic prophase in several mammalian species. However, the molecular mechanisms controlling WEE1B activity have not been fully examined in species other than mice. In the present study, we analyzed the regulation mechanisms of porcine WEE1B (pWEE1B), focusing on the cAMP-dependent protein kinase (PKA) phosphorylation site and intracellular localization. As the PKA phosphorylation site in mouse WEE1B (mWEE1B) was not conserved in pWEE1B, we predicted that four serine residues would be phosphorylatable by PKA in pWEE1B (Ser77, Ser118, Ser133 and Ser149) and constructed FLAG-tagged replaced-pWEE1Bs, in which each of the PKA-phosphorylatable serines was mutated into a non-phosphorylatable alanine. We injected one of their mRNAs into porcine immature oocytes and found that the Ser77-replaced pWEE1B lost the WEE1B function, whereas the wild-type and other replaced-pWEE1Bs could maintain the meiotic arrest of oocytes. Next, the localization of pWEE1B was examined by immunohistochemistry, and exclusive nuclear localization was revealed in the fully grown oocytes. We generated a nuclear localization signal (NLS)-deleted pWEE1B (ΔNLS-pWEE1B) and then overexpressed it in porcine immature oocytes. We found that ΔNLS-pWEE1B was distributed uniformly in the cytoplasm and could not maintain the meiotic arrest of porcine oocytes. These results suggest that pWEE1B is activated after phosphorylation of the Ser77 residue, which is different from the phosphorylation site that activates mWEE1B; that pWEE1B is localized in the nucleus; and that the nuclear localization is essential for its function.     

Preimplantation-embryo-specific cell-cycle regulation is attributable to a low expression of retinoblastoma protein rather than its phosphorylation

Mammalian preimplantation embryos enter the S phase immediately after the end of the M phase; their cell cycle lacks a substantial G1 phase. Previously, we suggested that the absence of the G1 phase was attributable to a loss of retinoblastoma protein (RB) function, which is required for suppression of S phase entrance and that this loss of RB function in turn was attributable to the low RB expression level during preimplantation development in mouse embryos. The present study aimed to examine whether or not RB inhibition by CDK4/6-cyclin D-dependent phosphorylation is involved in the loss of RB function in preimplantation mouse embryos by the expression of p16(INK4a), a potent endogenous inhibitor of CDK4/6-cyclin D. First, the decrease in RB expression between the four-cell and morula stages was confirmed in in vivo-derived mouse embryos. We then examined the efficiency of the p16(INK4a) expression vector in inhibiting RB phosphorylation and cell cycle progression using NIH-3T3 cells and obtained gradual RB dephosphorylation and a significantly lower proliferation rate in p16(INK4a)-transfected cells than in control cells. This indicated the successful p16(INK4a) effects on cell-cycle progression by the vector used. On the other hand, the development rate of mouse embryos injected with the p16(INK4a) expression vector was the same as that of the control embryos, although p16(INK4a) expression was detected at mRNA and protein levels in the former group but not in the control group. These results suggest that RB phosphorylation is not involved in RB dysfunction or in the lack of a G1 phase in mouse embryos and that the decrease in RB expression is important for preimplantation-embryo-specific cell-cycle regulation. Moreover, the present study indicates the similarity between preimplantation embryos and cancer cells, which p16(INK4a) expression does not arrest at the G1 phase.