Adhesive cell cultivation on polymer particle having grafted epoxy polymer chain

In this study, we synthesized a new cell immobilization support having poly(glycidyl methacrylate) as a graft polymer chain and used this support for cell cultivation. Base polymer particle was synthesized by suspension polymerization and epoxy polymer chain was extended from particle surface on graft polymerization. Produced polymer particles had broad particle size distribution ranging from 20 to 1000 μm and the degree of polymerization of grafted polymer chain was ranged from 500 to 1000. The effects of various factors, such as grafted polymer chain length and its surface density, composition of base polymer network and graft polymer chain, on the cell growth of murine fibroblast cell line (MS-5 cell) on polymer particle were studied. This polymer particle could cultivate not only fibroblast cell line but also epidermal cell line (HeLa cell), osteoblast cell line (MC3T3E1 cell), and chondrocyte cell line (ch-8 cell) on its surface. Growth rate is almost the same as that of cells using poly(styrene) tissue culture dish. To apply this cell cultivation system for examination of cell co-culture, HeLa cell immobilized on 100 μm of polymer particle was successfully co-cultured with MS-5 cell immobilized on 300 μm of polymer particle for four weeks.     

Arginine controls heat-induced cluster-cluster aggregation of lysozyme at around the isoelectric point

The process of protein aggregation has attracted a great deal of research attention, as aggregates are first of all a nuisance to preparation of high quality protein and secondly used as novel materials. In the latter case, the process of protein aggregation needs to be controlled. Here, we show how arginine (Arg) regulates the process of heat-induced protein aggregation. Dynamic light scattering and transmission electron microscopy revealed that heat-induced aggregation of lysozyme at around the isoelectric point occurred in a two-step process: formation of start aggregates, followed by further growth mediated by their sticking with diffusion-limited cluster-cluster aggregation. In the presence of Arg, the diffusion-limited regime changed to reaction-limited cluster-cluster aggregation. The data indicated that the solution additives that coexisted with proteins would affect the property of the formed product, such as morphology and mechanic strength.     

Increase of Circulating CD11b+Gr1+ cells and Recruitment into the Synovium in Osteoarthritic Mice with Hyperlipidemia

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F₂ animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b⁺Gr1⁺ cells in spleens and peripheral blood was increased, and CD11b⁺Gr1⁺ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b⁺Gr1⁺ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b⁺Gr1⁺ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F₂ mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b⁺Gr1⁺ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b⁺Gr1⁺ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.     

Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.     

Polarization-based brightness discrimination in the foraging butterfly, Papilio xuthus

The human eye is insensitive to the angular direction of the light e-vector, but several animal species have the ability to discriminate differently polarized lights. How the polarization is detected is often unclear, however. Egg-laying Papilio butterflies have been shown to see false colours when presented with differently polarized lights. Here we asked whether this also holds in foraging butterflies. After training individuals to feed on nectar in front of an unpolarized spectral light, we carried out three dual-choice tests, where the discrimination of (i) the spectral content, (ii) the light intensity, and (iii) the e-vector orientation were investigated. In the first test, the butterflies selected the trained spectrum irrespective of its intensity, and in the second test they chose the light with the higher intensity. The result of the e-vector discrimination test was very similar to that of the second test, suggesting that foraging butterflies discriminate differently polarized lights as differing in brightness rather than as differing in colour. Papilio butterflies are clearly able to use at least two modes of polarization vision depending on the behavioural context.     

Characterization of the NikA histidine kinase implicated in the phosphorelay signal transduction of Aspergillus nidulans, with special reference to fungicide responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.     

The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.     

Resistance profiles of novel electrostatically constrained HIV-1 fusion inhibitors

Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.