The residual structure of acid-denatured β2-microglobulin is relevant to an ordered fibril morphology

β₂-Microglobulin (β2m) forms amyloid fibrils in vitro under acidic conditions. Under these conditions, the residual structure of acid-denatured β2m is relevant to seeding and fibril extension processes. Disulfide (SS) bond-oxidized β2m has been shown to form rigid, ordered fibrils, whereas SS bond-reduced β2m forms curvy, less-ordered fibrils. These findings suggest that the presence of an SS bond affects the residual structure of the monomer, which subsequently influences the fibril morphology. To clarify this process, we herein performed NMR experiments. The results obtained revealed that oxidized β2m contained a residual structure throughout the molecule, including the N- and C-termini, whereas the residual structure of the reduced form was localized and other regions had a random coil structure. The range of the residual structure in the oxidized form was wider than that of the fibril core. These results indicate that acid-denatured β2m has variable conformations. Most conformations in the ensemble cannot participate in fibril formation because their core residues are hidden by residual structures. However, when hydrophobic residues are exposed, polypeptides competently form an ordered fibril. This conformational selection phase may be needed for the ordered assembly of amyloid fibrils.     

Spawning season and size at sexual maturity of <Emphasis Type="Italic">Kyphosus bigibbus</Emphasis> (Kyphosidae) from northwest Kyushu,Japan

The reproductive characteristics of Kyphosus bigibbus were examined using individuals collected between June 2004 and February 2009 off Nagasaki Peninsula in northwest Kyushu, Japan. The spawning season and size at sexual maturity of this species were characterized based on a gonad index and histological examination of the gonads. The spawning season extends from June to October. This species is assumed to be an indeterminate, multiple-batch spawner. Females reached sexual maturity at larger size than males (fork length at 50% sexual maturity: males 284 mm, females 360 mm).     

Kinetic studies of the effect of a 17‐nucleotide difference in the 0.3‐kb region containing the R‐U5‐5′ leader sequence of Friend murine leukemia virus on viral gene expression

In addition to the env gene, a 0.3‐kb fragment containing the R‐U5‐5′ leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr‐MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.3‐kb fragment, and Rec5, carrying the 0.3‐kb fragment of the non‐neuropathogenic Fr‐MLV clone 57. These analyses suggested that the 0.3‐kb fragment influenced the expression level of the Env protein by regulating the amount of spliced env‐mRNA rather than the amount of total viral mRNA or viral production.     

Change in the viscoelastic properties of agarose gel by HAp precipitation by osteoblasts cultured in an agarose gel matrix

The viscoelastic properties of cell-seeded agarose gel were measured as a function of culture time. Because the seeded cells, MC3T3-E1, were osteoblast-like cells, the system can be regarded as a model osteogenesis system. For all specimens the characteristic stress relaxation curve of agarose gel was observed—a large relaxation up to 10⁴ s followed by a gel plateau, where the former was attributed to molecular motion of polymer chains between two adjacent cross-links of the gel and the latter to the elasticity of the gel network. The viscoelasticity was quantified by fitting stress relaxation data to an empirical equation. The relaxation time and its distribution did not change with culture time. The initial and equilibrium moduli, E ₀ and E _e, respectively, and relaxation strength, ΔE = E ₀ ? E _e, did not change up to day 15 of culture but changed significantly at day 18 of culture. The change in ΔE with culture period correlated well with that in E ₀. The changes in the mechanical properties of the cell-seeded agarose gel system were explained in terms of the function of MC3T3-E1 in precipitating calcium phosphate mineral particles. The precipitation was detected by alizarin red S staining of the system at day 9 of culture. The precipitated calcium phosphate was confirmed to be hydroxyapatite (HAp) and the amount of HAp increased monotonically with culturing time, both of which were observed by X-ray diffraction studies. The dependence of the modulus of the composite on mineral fraction is discussed. A simple model of mixing of the components based on the continuum material concept was not applicable, but a model considering percolation of mineral particles in a network chain with culture time was suitable to explain the observed results. The results may be particularly important for predicting the stiffness of functionally engineered bony tissue implanted in a fractured bone.     

Two aquaporins,SIP1;1 and PIP1;2, mediate water transport for pollen hydration in the Arabidopsis pistil

Pollination is the crucial initial step that brings together the male and female gametophytes, and occurs at the surface of the stigmatic papilla cell in Arabidopsis thaliana. After pollen recognition, pollen hydration is initiated as a second critical step to activate desiccated mature pollen grains for germination, and thus water transport from pistil to pollen is essential for this process. In this study, we report a novel aquaporin-mediated water transport process in the papilla cell as a control mechanism for pollen hydration. Coupled with a time-series imaging analysis of pollination and a reverse genetic analysis using T-DNA insertion Arabidopsis mutants, we found that two aquaporins, the ER-bound SIP1;1 and the plasma membrane-bound PIP1;2, are key players in water transport from papilla cell to pollen during pollination. In wild type plant, hydration speed reached its maximal value within 5 min after pollination, remained high until 10–15 min. In contrast, sip1;1 and pip1;2 mutants showed no rapid increase of hydration speed, but instead a moderate increase during ∼25 min after pollination. Pollen of sip1;1 and pip1;2 mutants had normal viability without any functional defects for pollination, indicating that decelerated pollen hydration is due to a functional defect on the female side in sip1;1 and pip1;2 mutants. In addition, sip1;1 pip1;2 double knockout mutant showed a similar impairment of pollen hydration to individual single mutants, suggesting that their coordinated regulation is critical for proper water transport, in terms of speed and amount, in the pistil to accomplish successful pollen hydration.     

Loss of EGF‐dependent cell proliferation ability on radioresistant cell HepG2‐8960‐R

Acquired radioresistance of cancer cells interferes with radiotherapy and increases the probability of cancer recurrence. HepG2‐8960‐R, which is one of several clinically relevant radioresistant (CRR) cell lines, has a high tolerance to the repeated clinically relevant doses of X‐ray radiation. In this study, HepG2‐8960‐R had slightly lower cell proliferation ability than HepG2 in the presence of FBS. In particular, epidermal growth factor (EGF) hardly enhanced cell proliferation and DNA synthesis in HepG2‐8960‐R. Additionally, EGF could not induce the activation of Erk1/2, because the expression of EGF receptor (EGFR) protein decreased in HepG2‐8960‐R in accordance with the methylation of the EGFR promoter region. Therefore, cetuximab did not inhibit HepG2‐8960‐R cell proliferation. Our study showed that HepG2‐8960‐R had radioresistant and cetuximab‐resistant abilities. Copyright © 2015 John Wiley & Sons, Ltd.     

Formulas for intrinsic noise evaluation in oscillatory genetic networks

The linear noise approximation is a useful method for stochastic noise evaluations in genetic regulatory networks, where the covariance equation described as a Lyapunov equation plays a central role. We discuss the linear noise approximation method for evaluations of an intrinsic noise in autonomously oscillatory genetic networks; in such oscillatory networks, the covariance equation becomes a periodic differential equation that provides generally an unbounded covariance matrix, so that the standard method of noise evaluation based on the covariance matrix cannot be adopted directly. In this paper, we develop a new method of noise evaluation in oscillatory genetic networks; first, we investigate structural properties, e.g., orbital stability and periodicity, of the solutions to the covariance equation given as a periodic Lyapunov differential equation by using the Floquet-Lyapunov theory, and propose a global measure for evaluating stochastic amplitude fluctuations on the periodic trajectory; we also derive an evaluation formula for the period fluctuation. Finally, we apply our method to a model of circadian oscillations based on negative auto-regulation of gene expression, and show validity of our method by comparing the evaluation results with stochastic simulations.