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Mammalian oocytes undergo a long‐term meiotic arrest that can last for almost the entire reproductive lifespan. This arrest occurs after DNA replication and is prolonged with age, which poses a challenge to oocytes in maintaining replication‐dependent chromosomal proteins required for the completion of meiosis. In this study, we show that chromosomal histones are reduced with age in mouse oocytes. Both types of histone H3 variants, replication‐dependent H3.1/H3.2 and replication‐independent H3.3, decrease with age. Aging‐associated histone reduction is associated with transcriptomic features that are caused by genetic depletion of histone H3.3. Neither the genetic reduction of chromosomal H3.1/H3.2 nor H3.3 accelerates the aging‐associated increase in premature chromosome separation that causes meiotic segregation errors. We suggest that aging‐associated reduction of chromosomal histones is linked to several transcriptomic abnormalities but does not significantly contribute to errors in meiotic chromosome segregation during the reproductive lifespan of mice. [ABSTRACT FROM AUTHOR]

A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the [CD44.sup.hi][CD62L.sup.lo][CXCR3.sup.lo] pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma., Introduction Allergic asthma is a chronic inflammatory airway disease characterized by airway eosinophilia, mucus hypersecretion, and airway hyperresponsiveness (1). Its incidence is steadily increasing, with an estimated 339 million people [...]

The role of electrostatic interactions and the effect of ionic cosolutes on amyloid formation are understood by investigating the effect of salts such as NaCl, NaI, NaClO4, and Na2SO4 on the amyloid fibril growth of beta2-microglobulin. The study conducted suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

Aims Accumulating evidence demonstrates that cardiomyocyte death contributes to the onset and progression of heart failure (HF) after myocardial injury. Recent studies revealed that immune/inflammatory reactions play important roles in cardiovascular diseases. However, it remains unclear whether immunosurveillance system, which eliminates cytopathic cells, including infected or malignant cancer cells, is involved in cardiomyocyte death, though cardiomyocytes are exposed to pathological stresses during post-infarct remodelling. The aim of this study is to clarify the pathophysiological significance of Natural Killer Group 2 member D (NKG2D)/NKG2D ligand (NKG2DL)-mediated cell death in HF after myocardial infarction (MI). Methods and results MI was generated by ligating left anterior descending artery in mice. The expression of NKG2D, NKG2DLs, especially Retinoic acid early induced transcript-1ɛ (Rae-1ɛ), perforin and granzyme B was concomitantly up-regulated after MI. Immunohistological analysis revealed that Rae-1 was expressed on the membranes of injured cardiomyocytes in the infarct and border area. The MI-induced increase of Rae-1 expression was suppressed in p53−/− mice and Rae-1 was induced by the overexpression of p53. We identified p53-binding sites in Rae-1ɛ gene promoter, by chromatin immunoprecipitation assay, indicating that Rae-1 expression was mediated partially through p53. Flow cytometric analysis indicated that NKG2D-expressing immune cells in the post-infarct myocardium were mainly γδT cells. The co-culture with γδT cells increased the frequency of apoptotic cells in the cultured cardiomyocytes. The blockade of NKG2D/NKG2DL interaction by intraperitoneal injection of anti-Rae-1ɛ antibody after MI reduced the frequency of apoptotic cardiomyocytes, accompanied by suppression of cardiac fibrosis, attenuating cardiac dysfunction. Finally, tamoxifen-inducible cardiomyocyte-specific Rae-1ɛ overexpressing mice exhibited the susceptibility to post-infarct remodelling with increased cardiomyocyte apoptosis and severer cardiac dysfunction. Conclusion The interaction between immune cells and cardiomyocytes via NKG2D/NKG2DL induces cardiomyocyte death, exacerbating cardiac remodelling after MI. The blockade of NKG2D/NKG2DL interaction could be a promising therapeutic strategy against HF. [ABSTRACT FROM AUTHOR]

β2-Microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although β2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, β2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-β2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-β2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the β2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-β2-m, implying a major structural difference between them. [ABSTRACT FROM AUTHOR]

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on β2-microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded β2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native β2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mM sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of β2-m, producing fibrils adapted to the respective pH. [ABSTRACT FROM AUTHOR]

β2-Microglobulin (β2-m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Recent studies have focused on the mechanism by which amyloid fibrils are formed under physiological conditions, which had been difficult to reproduce quantitatively. Yamamoto et al. (Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F. & Naiki, H. (2004) Biochemistry 43, 11075-11082) showed that a combination of seed fibrils prepared under acidic conditions and a low concentration of sodium dodecyl sulfate below its critical micelle concentration enabled extensive fibril formation at pH 7.0. Here, we found that repeated self-seeding at pH 7.0 with fibrils formed at the same pH causes a marked acceleration of growth, indicating the maturation of fibrils. The observed maturation can be simulated by assuming the existence of two types of fibrils with different growth rates. Importantly, some mutations of β2-m or the addition of a low concentration of urea, both destabilizing the native conformation, were not enough to extend the fibrils at pH 7.0, and a low concentration of sodium dodecyl sulfate (i.e. 0.5 mM) was essential. Thus, even though the first stage fibrils in patients are unstable and require stabilizing factors to remain at neutral pH, they can adapt to a neutral pH with repeated selfseeding, implying a mechanism of development of amyloid deposition after a long latent period in patients. [ABSTRACT FROM AUTHOR]

A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma. [ABSTRACT FROM AUTHOR]

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.

Beta2-microglobulin (beta2-m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Recent studies have focused on the mechanism by which amyloid fibrils are formed under physiological conditions, which had been difficult to reproduce quantitatively. Yamamoto et al. (Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F. & Naiki, H. (2004) Biochemistry 43, 11075-11082) showed that a combination of seed fibrils prepared under acidic conditions and a low concentration of sodium dodecyl sulfate below its critical micelle concentration enabled extensive fibril formation at pH 7.0. Here, we found that repeated self-seeding at pH 7.0 with fibrils formed at the same pH causes a marked acceleration of growth, indicating the maturation of fibrils. The observed maturation can be simulated by assuming the existence of two types of fibrils with different growth rates. Importantly, some mutations of beta2-m or the addition of a low concentration of urea, both destabilizing the native conformation, were not enough to extend the fibrils at pH 7.0, and a low concentration of sodium dodecyl sulfate (i.e. 0.5 mM) was essential. Thus, even though the first stage fibrils in patients are unstable and require stabilizing factors to remain at neutral pH, they can adapt to a neutral pH with repeated self-seeding, implying a mechanism of development of amyloid deposition after a long latent period in patients.

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.