Simultaneous stereoinversion and isomerization at the Asp-4 residue in βB2-crystallin from the aged human eye lenses

The lens proteins are composed of α-, β-, and γ-crystallins that interact with each other to maintain the transparency and refractive power of the lens. Because the lens crystallins are long-lived proteins, they undergo various post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation. In βB2-crystallin, which is the most abundant β-crystallin, the deamidation of asparagine and glutamine residues has been reported. Here, we found that the aspartyl (Asp) residue at position 4 of βB2-crystallin in the lenses of elderly human individuals undergoes a significant degree of inversion and isomerization to the biologically uncommon residue D-β-Asp. Surprisingly, the D/L ratio of β-Asp at position 4 in βB2-crystallin from elderly donors (67-77 year old) was 0.88-3.21. A D/L ratio of amino acids greater than 1.0 is defined as an inversion of configuration from the L- to D-form, rather than a racemization. These extremely high D/L ratios are equivalent to those of Asp-58 and Asp-151 (D/L ratio: 3.1 for Asp-58 and 5.7 for Asp-151) in αA-crystallin from elderly donors (~80 year old) as reported previously. Initially, we identified specific Asp residues in the β-crystallin family of proteins that undergo a high degree of inversion. These results show that the isomerization and inversion of Asp residues occurs both in the α- and β-crystallins of the lens. Inversion of these Asp residues directly affects the higher order structure of the protein. Hence, this modification may change crystallin-crystallin interactions and disrupt the function of crystallins in the lens.     

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.     

Development of sequential multiple comparison procedure for dose response test

We propose a multiple comparison procedure to identify the minimum effective dose level by sequentially comparing each dose level with the zero dose level in the dose finding test. If we can find the minimum effective dose level at an early stage in the sequential test, it is possible to terminate the procedure in the dose finding test after a few group observations up to the dose level. Thus, the procedure is viable from an economical point of view when high costs are involved in obtaining the observations. In the procedure, we present an integral formula to determine the critical values for satisfying a predefined type I familywise error rate. Furthermore, we show how to determine the required sample size in order to guarantee the power of the test in the procedure. In practice, we compare the power of the test and the required sample size for various configurations of the population means in simulation studies and adopt our sequential procedure to the dose response test in a case study.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Assessment of the Annual Additional Effective Doses amongst Minamisoma Children during the Second Year after the Fukushima Daiichi Nuclear Power Plant Disaster

An assessment of the external and internal radiation exposure levels, which includes calculation of effective doses from chronic radiation exposure and assessment of long-term radiation-related health risks, has become mandatory for residents living near the nuclear power plant in Fukushima, Japan. Data for all primary and secondary children in Minamisoma who participated in both external and internal screening programs were employed to assess the annual additional effective dose acquired due to the Fukushima Daiichi nuclear power plant disaster. In total, 881 children took part in both internal and external radiation exposure screening programs between 1^st April 2012 to 31^st March 2013. The level of additional effective doses ranged from 0.025 to 3.49 mSv/year with the median of 0.70 mSv/year. While 99.7% of the children (n = 878) were not detected with internal contamination, 90.3% of the additional effective doses was the result of external radiation exposure. This finding is relatively consistent with the doses estimated by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). The present study showed that the level of annual additional effective doses among children in Minamisoma has been low, even after the inter-individual differences were taken into account. The dose from internal radiation exposure was negligible presumably due to the success of contaminated food control.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Melanocortins Contribute to Sequential Differentiation and Enucleation of Human Erythroblasts via Melanocortin Receptors 1, 2 and 5

In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.