Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases

Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment.     

A comparison of screening methods for antioxidant activity in seaweeds

The inhibition of lipid peroxidation and radical scavenging effects were studied to evaluate the antioxidant activity for extracts of 17 species of seaweed. The antioxidant effect was evaluated by determination of lipoxygenase activity and by α, α-diphenyl-β-picrylhydrazyl (DPPH) decolorization. Lipoxygenase activity was depressed in the presence of aqueous and ethanol extracts of 4 algal species; Sargassum species had the highest antioxidant activity of all the species examined. The ethanol extracts of one Sargassum species showed competitive inhibition with the substrate. The same species also showed radical scavenging activity in the DPPH decolorization test. Comparison of these results shows no relationship between enzyme inhibition and radical scavenging activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp. under mixotrophic conditions

Three species of microalgae able to produce eicosapentaenoic acid(EPA) were collected from brackish and sea water around Japan. The species were identified as Navicula saprophila, Rhodomonassalina and Nitzschia sp. EPA as a proportion of total fatty acids increased in the presence of acetic acid for Rhodomonas salina and Nitzschia sp. However, Navicula saprophila displayed the greatest productivity of EPA and the EPA content of its biomass was enhanced under mixotrophic conditions in the presence of acetic acid. This revised version was published online in August 2006 with corrections to the Cover Date.     

Monocyte Chemoattractant Protein-1/CCL2 Produced by Stromal Cells Promotes Lung Metastasis of 4T1 Murine Breast Cancer Cells

MCP-1/CCL2 plays an important role in the initiation and progression of cancer. Since tumor cells produce MCP-1, they are considered to be the main source of this chemokine. Here, we examined whether MCP-1 produced by non-tumor cells affects the growth and lung metastasis of 4T1 breast cancer cells by transplanting them into the mammary pad of WT or MCP-1^−/− mice. Primary tumors at the injected site grew similarly in both mice; however, lung metastases were markedly reduced in MCP-1^−/− mice, with significantly longer mouse survival. High levels of MCP-1 mRNA were detected in tumors growing in WT, but not MCP-1^−/− mice. Serum MCP-1 levels were increased in tumor-bearing WT, but not MCP-1^−/− mice. Transplantation of MCP-1^−/− bone marrow cells into WT mice did not alter the incidence of lung metastasis, whereas transplantation of WT bone marrow cells into MCP-1^−/− mice increased lung metastasis. The primary tumors of MCP-1^−/− mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of MCP-1, and intravenous injection of 4T1 cells producing a high level of MCP-1 resulted in increased tumor foci in the lung of WT and MCP-1^−/− mice. Thus, stromal cell-derived MCP-1 in the primary tumors promotes lung metastasis of 4T1 cells, but tumor cell-derived MCP-1 can also contribute once tumor cells enter the circulation. A greater understanding of the source and role of this chemokine may lead to novel strategies for cancer treatment.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice approximately 100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-gamma and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-gamma and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-gamma drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.