Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3′ ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.     

Crystal structures of carbohydrate recognition domain of blood dendritic cell antigen‐2 (BDCA2) reveal a common domain‐swapped dimer

We report on crystal structures of a carbohydrate recognition domain (CRD) of human C‐type lectin receptor blood dendritic cell antigen‐2 (BDCA2). Three different crystal forms were obtained at 1.8–2.3 Å resolution. In all three, the CRD has a basic C‐type lectin fold, but a long loop extends away from the core domain to form a domain‐swapped dimer. The structures of the dimers from the three different crystal forms superimpose well, indicating that domain swapping and dimer formation are energetically stable. The structure of the dimer is compared with other domain‐swapped proteins, and a possible regulation mechanism of BDCA2 is discussed. Proteins 2014; 82:1512–1518. © 2013 Wiley Periodicals, Inc.     

Interaction between the premotor processes of eye and hand movements: Possible mechanism underlying eye–hand coordination

Interaction between the execution process of eye movement and that of hand movement must be indispensable for eye–hand coordination. The present study investigated corticospinal excitability in the hand muscles during the premotor processes of eye and/or hand movement to elucidate interaction between these processes. Healthy humans performed a precued reaction task of eye and/or finger movement and motor-evoked potentials in the hand muscles were evoked in the reaction time. Leftward eye movement suppressed corticospinal excitability in the right abductor digiti minimi muscle only when little finger abduction was simultaneously executed. Corticospinal excitability in the first dorsal interosseous muscle was not suppressed by eye movement regardless of whether or not it was accompanied by finger movement. Suppression of corticospinal excitability in the hand muscles induced by eye movement in the premotor period depends on the direction of eye movement, the muscle tested, and the premotor process of the tested muscle. The suppression may reflect interaction between the motor process of eye movement and that of hand movement particularly active during eye–hand coordination tasks during which both processes proceed.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.     

Deep phylogeographical structure and parallel host range evolution in the leaf beetle Agelasa nigriceps

To understand the mechanisms behind the diversification of herbivorous insects through insect–plant interactions, it is important to know how the insects change their diet breadth in response to environmental changes. In this study, we investigated the phylogeographical pattern of the leaf beetle Agelasa nigriceps to infer the evolutionary history of its host range. While this beetle commonly uses Actinidia arguta (Actinidiaceae) as a host plant, it has been recorded recently on Pterostyrax hispidus (Styracaceae), which is now increasing in abundance at some localities in Japan due to the indirect effects of high population size of a mammalian herbivore. Considerable variation among populations in the ability of Ag. nigriceps to use P. hispidus suggests that P. hispidus is a newly acquired host plant for this beetle. Phylogenetic analyses using mitochondrial DNA sequences and amplified fragment length polymorphism (AFLP) revealed a high degree of phylogeographical structure in Ag. nigriceps throughout Japan, which is consistent with the hypothesis that several glacial refugia existed in the Japanese archipelago. In contrast, no genetic structure associated with the host plants was detected. Both the mitochondrial DNA and AFLP analyses showed that populations that can use P. hispidus are polyphyletic. These results and geographical variation in host use suggest that the host range expansion to a novel host, P. hispidus, is a very recent and possibly ongoing phenomenon and has occurred independently in several regions. Our study illustrates that the host range of herbivorous insects can evolve repeatedly in response to similar environmental changes.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

NCYM,a Cis-Antisense Gene of MYCN,Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.     

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma

Background

CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.

Methods

We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.

Results

Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.

Conclusion

These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.