Descriptions of two new and one little known species of the genus Cacopsylla (Hemiptera: Psyllidae) on Sorbus japonica (Rosaceae) and an observation of their biology

Three species of the genus Cacopsylla were collected from Sorbus japonica in Japan. Two of them, Cacopsylla elegans and Cacopsylla sorbicoccinea, are described as new species based on adults and fifth (final) nymphal instars. The third species, Cacopsylla midoriae, is newly transferred from the genus Psylla. The adult of C. midoriae is redescribed and the nymph is described for the first time. A key to the species feeding on S. japonica (adults and nymphs) is provided. The three species were observed to coexist on the same tree of S. japonica in northern Kyushu. Such coexistence of psylloid congeners is uncommon in Japan.     

Hypomorphic phenotype of <Emphasis Type="Italic">Foxn1</Emphasis> gene-modified rats by CRISPR/Cas9 system

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.     

Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.     

Phenanthridin-6-one derivatives as the first class of non-steroidal pharmacological chaperones for Niemann-Pick disease type C1 protein

Niemann-Pick disease type C is a fatal, progressive neurodegenerative disease mostly caused by mutations in Nieamnn-Pick type C1 (NPC1), a late endosomal membrane protein that is essential for intracellular cholesterol transport. The most prevalent mutation, I1061T (Ile to Thr), interferes with the protein folding process. Consequently, mutated but intrinsically functional NPC1 proteins are prematurely degraded via proteasome, leading to loss of NPC1 function. Previously, we reported sterol derivatives as pharmacological chaperones for NPC1, and showed that these derivatives can normalize folding-defective phenotypes of I1061T NPC1 mutant by directly binding to, and stabilizing, the protein. Here, we report a series of compounds containing a phenanthridin-6-one scaffold as the first class of non-steroidal pharmacological chaperones for NPC1. We also examined their structure-activity relationships.     

Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′S,5′S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5′-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.     

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.     

Gene expression of a novel defensin antimicrobial peptide in the silkworm, Bombyx mori

cDNA encoding a novel defensin (BmDefensinB) was cloned from the fat body of the silkworm, Bombyx mori, and gene expression was analyzed. BmDefensinB showed typical structural characteristics of invertebrate defensins. Phylogenetic and bootstrap analyses indicated that it has no orthologs, whereas previously reported BmDefensinA is the ortholog of Spodoptera frugiperda (Sf)Spodoptericin. The BmDefensinB gene was expressed tissue-specifically in the fat body and was strongly activated by bacteria such as Escherichia coli and Bacillus subtilis, and by an entomopathogenic fungus Beauveria bassiana. In contrast, the BmDefensinA gene was expressed to a much lesser extent. Expression of the BmDefensinB gene was strongly stimulated by B. mori Rel proteins RelB and Relish, supporting the observation that this gene is activated by E. coli, B. subtilis, and B. bassiana. These results suggest that BmDefensinB gene expression is controlled through both the Toll and the Imd pathway, and that this gene plays an important role in B. mori immune reactions against infection by bacteria and fungi.     

Quantum chemical studies for oxidation of morpholine by Cytochrome P450

Since morpholine oxidation has recently been shown to involve Cytochrome P450, the study on its mechanism at molecular level using quantum chemical calculations for the model of cytochrome active site is reported here. The reaction pathway is investigated for two electronic states, the doublet and the quartet, by means of density functional theory. The results show that morpholine hydroxylation occurs through hydrogen atom abstraction and rebound mechanism. However, in the low spin state, the reaction is concerted and hydrogen atom abstraction yields directly ferric-hydroxy morpholine complex without a distinct rebound step while in quartet state the reaction is stepwise. The presence of nitrogen in a morpholine heterocycle is postulated to greatly facilitate hydrogen abstraction. The hydroxylated product undergoes intramolecular hydrogen atom transfer from hydroxy group to nitrogen, leading to the cleavage of the C-N bond and the formation of 2-(2-aminoethoxy) acetaldehyde. The cleavage of the C-N bond is indicated as the rate-determining step for the studied reaction. The assistance of explicit water molecule is shown to lower the energy barrier for the C-N bond cleavage in enzymatic environment whereas solvent effects mimicked by COSMO solvent model have minor influence on relative energies along the pathway.     

Growth responses of <Emphasis Type="Italic">Salix gracilistyla</Emphasis> cuttings to a range of substrate moisture and oxygen availability

The objective of this study is to determine the effects of substrate moisture and oxygen availability on growth traits of Salix gracilistyla Miquel, which colonizes gravel bars along rivers, the shoot growth schedule, biomass production, and resource allocation were examined under greenhouse conditions. We used four treatments representing a range of substrate moisture and oxygen availability: drought (D), flooding with standing water (FS), flooding with running water (FR), and control without drought or flooding (C). Cuttings in D stopped flushing and had low biomass production, reduced total leaf mass, and small leaves. Under anaerobic conditions, cuttings in FS stopped flushing and had low biomass production, small root biomass, low biomass allocation to roots, shallow roots, high biomass allocation to hypertrophied lenticels, and a few small, thick leaves. Under aerobic conditions, cuttings in FR showed continuous branch elongation and flushing, large biomass production, and large leaf biomass, similar to cuttings in C, in addition to low allocation to hypertrophied lenticels and many large leaves. The growth of cuttings was not inhibited by flooding of the roots throughout the experiment unless the conditions were anaerobic. Thus, cuttings respond to water stress under low moisture conditions by reducing the transpiration area and respond to flooding under low oxygen conditions by high allocation to hypertrophied lenticels and reduced transpiration area. Plasticity in the shoot growth schedule, biomass production, and resource allocation according to moisture conditions and the ability to develop hypertrophied lenticels upon flooding allow S. gracilistyla to colonize sites in which both desiccation and flooding occur.