In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.     

Identification of myosin II as a binding protein to the PH domain of protein kinase B

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.     

Inspection of frass ejection could decrease the risk of white-spotted longicorn beetle Anoplophora malasiaca (Coleoptera: Cerambycidae) infestation of Japanese pine bonsais to negligible levels

Applied Entomology and Zoology - To evaluate the infection risk of Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae) in two species of Japanese pine bonsais (Japanese black pine and...     

Functional decline of sweet taste sensitivity of colobine monkeys

For many primates, sweet taste is palatable and is an indicator that the food contains carbohydrates, such as sugars and starches, as energy sources. However, we have found that Asian colobine monkeys (lutungs and langurs) have low sensitivity to various natural sugars. Sweet tastes are recognized when compounds bind to the sweet taste receptor TAS1R2/TAS1R3 in the oral cavity; accordingly, we conducted a functional assay using a heterologous expression system to evaluate the responses of Javan lutung (Trachypithecus auratus) TAS1R2/TAS1R3 to various natural sugars. We found that Javan lutung TAS1R2/TAS1R3 did not respond to natural sugars such as sucrose and maltose. We also conducted a behavioral experiment using the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) by measuring the consumption of sugar-flavored jellies. Consistent with the functional assay results for TAS1R2/TAS1R3, these Asian colobine monkeys showed no preference for sucrose or maltose jellies. These results demonstrate that sweet taste sensitivity to natural sugars is low in Asian colobine monkeys, and this may be related to the specific feeding habits of colobine monkeys.     

Copper Oxide Nanomaterials Derived from Zanthoxylum armatum DC. and Berberis lycium Royle Plant Species: Characterization,Assessment of Free Radical Scavenging and Antibacterial Activity

Copper oxide nanomaterials were synthesized by a facile sustainable biological method using two plant species (Zanthoxylum armatum DC. and Berberis lycium Royle ). The formation of materials was confirmed by FT‐IR, ATR, UV‐visible, XRD, TEM, SEM, EDX, TGA and PL. The antibacterial activity was evaluated by agar well diffusion method to ascertain the efficacy of plant species extract and extract derived copper oxide nanomaterials against six Gram‐positive bacteria namely Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Corynebacterium diphtheriae, Corynebacterium xerosis, Bacillus cereus and four Gram‐negative bacteria such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris against the standard drug, Ciprofloxacin for Gram‐positive and Gentamicin for Gram‐negative bacteria, respectively. In both cases, copper oxide nanomaterials were found to be sensitive in all the bacterial species. Sensitivity of copper oxide nanomaterials shows an be higher as compared to plant species extract against different bacteria. Scavenging activity of plant extracts along with nanomaterials have been accessed using previously reported protocols employing ascorbic acid as standard. Scavenging activity of copper oxide nanomaterials shows an increase with increase in concentration. The biological activity (bactericidal and scavenging efficiency) of plant derived copper oxide nanomaterials revealed that these materials can be used as potent antimicrobial agent and DPPH scavengers in industrial as well as pharmacological fields.     

Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle–eye–brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of α-DG, and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^?/? muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1^?/? satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^?/? myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of α-DG is important for maintenance of the proliferative activity of satellite cells in vivo.     

Polymorphism of axillary bud galls induced by Rhopalomyia longitubifex (Diptera: Cecidomyiidae) on Artemisia princeps and A. montana (Asteraceae) in Japan and Korea, with designation of new synonyms

In the past, Rhopalomyia longitubifex, Rhopalomyia shinjii, and Rhopalomyia sp. (Diptera: Cecidomyiidae) have been regarded as independent species based on differences in the sizes and shapes of axillary bud galls induced on Artemisia montana (Asteraceae) in Japan and A. princeps in Japan and Korea. However, comparison of morphological features and molecular sequencing data indicate that these Rhopalomyia gall midges are identical and that the differences in gall shape are polymorphisms, although the measurements of gall height and diameter overlap slightly. This finding suggests that although galls have frequently been regarded as extensions of the phenotype of a species, differences in gall shape may not always be reliable for identifying gall‐inducing cecidomyiids. The older name, R. longitubifex, is applied to these gall midges, and the names that were applied to this species on later occasions are revised or synonymized. The mature and immature stages of R. longitubifex are redescribed and information on the distribution, host range, and gall size of this species is provided. We also discuss the role of gall polymorphism in the early stages of speciation.     

Endogenous Ligand for GPR120, Docosahexaenoic Acid,Exerts Benign Metabolic Effects on the Skeletal Muscles via AMP-activated Protein Kinase Pathway

Docosahexaenoic acid (DHA) is an endogenous ligand of G protein-coupled receptor 120 (GPR120). However, the mechanisms underlying DHA action are poorly understood. In this study, DHA stimulated glucose uptake in the skeletal muscles in an AMP-activated protein kinase (AMPK)-dependent manner. GPR120-mediated increase in intracellular Ca²⁺ was critical for DHA-mediated AMPK phosphorylation and glucose uptake. In addition, DHA stimulated GLUT4 translocation AMPK-dependently. Inhibition of AMPK and Ca²⁺/calmodulin-dependent protein kinase kinase blocked DHA-induced glucose uptake. DHA and GW9508, a GPR120 agonist, increased GPR120 expression. DHA-mediated glucose uptake was not observed in GPR120 knockdown conditions. DHA increased AMPK phosphorylation, glucose uptake, and intracellular Ca²⁺ concentration in primary cultured myoblasts. Taken together, these results indicated that the beneficial metabolic role of DHA was attributed to its ability to regulate glucose via the GPR120-mediated AMPK pathway in the skeletal muscles.     

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.     

Successful methods for transplanting fragments of Acropora formosa and Acropora hyacinthus

In order to establish a successful method for the transplantation of branching and tabular coral fragments, we tested the effects of orientations of attachment, seasons of transplantation, and size of fragments on survival, growth, and spawning using Acropora formosa and A. hyacinthus. Vertically attached, large-sized fragments of A. formosa showed 98–100% survival rate after 18 months. The fragments transplanted in August exhibited better survival than those transplanted in November. The larger fragments had the higher percentage of spawning. The fragments that spawned had lower growth rate, while those resorbed the oocytes carried at the time of transplantation showed higher growth rate, suggesting the trade-off between growth and reproduction. Half of the fragments spawned 1 month earlier than the donor colonies. Only the vertically attached fragments of A. hyacinthus fused to the substratum, and those transplanted in February showed 100% survival rate after 14 months, indicating that this species is well suited for transplantation.