Molecular cloning and expression of two novel beta-N-acetylglucosaminidases from silkworm Bombyx mori

Beta-N-acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.     

Superoxide anion radical scavenging activities of herbs and pastures in northern Japan determined using electron spin resonance spectrometry

Free radicals are not only destructive to the living cells but also reduce the quality of animal products through oxidation. As a result the superoxide anion radical (O2-), one of the most destructive reactive oxygen species, is a matter of concern for the animal scientists as well as feed manufacturers to ensure the quality of product to reach consumers demand. The superoxide anion radical scavenging activities (SOSA) of water and MeOH extracts of 2 herbs and 9 pasture samples collected from lowland and highland swards were determined against a 5,5-dimethyl-1-pyroline-N-oxide-O2-spin adduct based on a hypoxanthine-xanthine oxidase reaction using electron spin resonance spectrometry. Both the water and MeOH extracted SOSA differed among the herbs and pastures. Species and altitudinal variations were observed between extraction methods. The herbs were higher in both water and MeOH extracted SOSA than the pastures except for water extracts of one pasture, white clover (Trifolium repens L.). Among the pastures, quackgrass (Agrophyron repens L.) showed higher SOSA in both the MeOH and water extracts, and timothy (Phleum pretense L.) showed higher MeOH extracted SOSA. It is apparent that the kind and amount of antioxidants differ among herbs and pastures. Animal health and quality of animal products could be improved by adequate selection and combining of herbs and pastures having higher SOSA.     

Clinical Features and Mortality of COVID-19-Associated Mucormycosis: A Systematic Review and Meta-Analysis

The recent increase of COVID-19-associated mucormycosis (CAM) has been commanding global attention. However, basic epidemiologic characteristics have not firmly been established. In this systematic review and meta-analysis, we sought to determine the clinical manifestations, potential risk factors, and outcomes of CAM. Observational studies reporting CAM were searched with PubMed and EMBASE databases in January 2022. We collected data on comorbidities and treatment for COVID-19, and performed a one-group meta-analysis on the frequency of orbital exenteration procedure and mortality of CAM using a random-effect model. Fifty-one observational studies, including a total of 2,312 patients with proven CAM, were identified. Among the 51 studies, 37 were conducted in India, 8 in Egypt, and 6 in other countries. The most common comorbidity was diabetes mellitus (82%). While 57% required oxygenation, 77% received systemic corticosteroids. Among CAM, 97% were rhino-orbital-cerebral (ROCM), and 2.7% were pulmonary mucormycosis. Usual presentations were headache (54%), periorbital swelling/pain (53%), facial swelling/pain (43%), ophthalmoplegia (42%), proptosis (41%), and nasal discharge/congestion (36%). Regarding the outcomes, orbital exenteration was performed in 17% (95% CI: 12–21%, I²?=?83%) of the COVID-19-associated ROCM patients. The mortality of CAM was 29% (95% CI; 22–36%, I²?=?92%). In conclusion, this systematic review and meta-analysis indicated that the most prevalent type of CAM was ROCM, and most CAM patients had diabetes mellitus and received systemic glucocorticoids. Clinicians in the endemic areas should have a high index of suspicion for this invasive fungal complication of COVID-19 when a diabetic patient who received high-dose systemic glucocorticoids developed rhino-orbital symptoms.

     

Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9^ΔC-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9^ΔC-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

A novel β-loop scaffold of phage-displayed peptides for highly specific affinities

Loop peptides stabilized by two β-strands were used as a scaffold for a phage displayed peptide library. Affinity-based screening for insulin provided peptides, which showed affinity constants of 10(5) M(-1) order for insulin over 100 times greater than their affinity for the structurally similar insulin-like growth factor 1. The results suggested that the scaffold offers a powerful tool for generating and screening peptides as ligands for drugs and biologics.     

Symbiotic rhizobium and nitric oxide induce gene expression of non-symbiotic hemoglobin in Lotus japonicus

We characterized the expression profiles of LjHb1 and LjHb2, non-symbiotic hemoglobin (non-sym-Hb) genes of Lotus japonicus. Although LjHb1 and LjHb2 showed 77% homology in their cDNA sequences, LjHb2 is located in a unique position in the phylogenetic tree of plant Hbs. The 5'-upstream regions of both genes contain the motif AAAGGG at a position similar to that in promoters of other non-sym-Hb genes. Expression profiles obtained by using quantitative RT-PCR showed that LjHb1 and LjHb2 were expressed in all tissues of mature plants, and expression was enhanced in mature root nodules. LjHb1 was strongly induced under both hypoxic and cold conditions, and by the application of nitric oxide (NO) donor, whereas LjHb2 was induced only by the application of sucrose. LjHb1 was also induced transiently by the inoculation with the symbiotic rhizobium Mesorhizobium loti MAFF303099. Observations using fluorescence microscopy revealed the induction of LjHb1 expression corresponded to the generation of NO. These results suggest that non-sym-Hb and NO have important roles in stress adaptation and in the early stage of legume-rhizobium symbiosis.     

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.     

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins

Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.     

An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9

Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.