The introduction to Japan of the Titan barnacle,Megabalanus coccopoma (Darwin, 1854) (Cirripedia: Balanomorpha) and the role of shipping in its translocation

The Titan Acorn barnacle, Megabalanus coccopoma, a native of the tropical eastern Pacific, has become established in the western Atlantic (Brazil and the northern Gulf of Mexico to the Carolinas), northwestern Europe and the western Indian Ocean (Mauritius), and therefore its dispersal capabilities are well known. This study reports its introduction to Japan and confirms its occurrence in Australia. In an attempt to determine the source of this introduction, phylogeographic techniques, involving cytochrome c oxidase I sequences of various widely separate populations of M. rosa and M. volcano, were utilized. No significant genetic differentiation or haplotype patterns between widely separated populations of each of the three species were found. Lack of such differentiation indicates recent geographical isolation and thus negates a null hypothesis predicting that the occurrence of one of more of these species in Australia was natural.     

Genetic engineering of rotaviruses by reverse genetics

The rotavirus genome is composed of 11 gene segments of dsRNA. A recent breakthrough in the field of rotaviruses is the development of a reverse genetics system for generating recombinant rotaviruses possessing a gene segment derived from cloned cDNA. Although this approach is a helper virus‐driven system that is technically limited and gives low levels of recombinant viruses, it allows alteration of the rotavirus genome, thus contributing to our understanding of these medically important viruses. So far, this approach has successfully been applied to three of the 11 viral segments in our laboratory and others, and the efficiency of recovery of recombinant viruses has been improved. However, we are still waiting for the development of a helper virus‐free reverse genetics system for generating an infectious rotavirus entirely from cDNAs, as has been achieved for other members of the Reoviridae family.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

The Molten Globule of β2-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

The acid transition of β₂-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH 4 (25 °C). The pH 4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH 4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH 4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pK_a value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH 4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH 4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state → MG → burst-phase intermediate → native state.     

Chicken IL-6 is a heat-shock gene

The febrile response is elicited by pyrogenic cytokines including IL-6 in response to microorganism infections and diseases in vertebrates. Mammalian HSF1, which senses elevations in temperature, negatively regulates the response by suppressing pyrogenic cytokine expression. We here showed that HSF3, an avian ortholog of mammalian HSF1, directly binds to and activates IL-6 during heat shock in chicken cells. Other components of the febrile response mechanism, such as IL-1β and ATF3, were also differently regulated in mammalian and chicken cells. These results suggest that the febrile response is exacerbated by a feed-forward circuit composed of the HSF3-IL-6 pathway in birds.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Ribosomes in Dormant and Germinating Conidia of Aspergillus oryzae

Ribosomes were isolated from dormant and germinating conidia of Asp. oryzae No. 13. The ribosomes which consisted of 80 S were easily dissociated into 40 S and 60 S in low Mg^{+ +} buffer. Polyribosomes were not found in dormant conidia, but were found in germinating conidia. Ribosomes in Aspergillus fungi consisted of almost equal amount of RNA and protein, and the base compositions of RNA were alike, as compared as ribosomal RNA between dormant and germinating conidia.     

Rapid Gas Chromatographic Determination of Micro-quantities of N-Methyl Carbamates as their 2, 4-Dinitrophenyl Derivatives

A rapid and sensitive method is presented for the determination of micro-quantities (1 to 100 μg) of N-methyl carbamates as dinitrophenyl methylamine (DNP-MA) by electron capture gas liquid chromatography (GLC). The method described is characterized by rapidity and simplicity of procedure due to an improvement made in the present investigation, i.e., hydrolysis of an N-methyl carbamate and dinitrophenylation of the resulting methylamine with 2, 4-dinitro-1-fluorobenzene (DNFB) were accomplished simultaneously in a single reaction mixture. A novel approach was also made to eliminate unreacted DNFB by conversion to dinitrophenyl glycine (DNP-glycine).     

31P NMR Spectra of Alkalophilic Bacillus No. A-59

Some advanced cancer patients suffer from pungent sulfury malodor. To determine the chemical identity of the odorant, we performed gas chromatography-mass spectrometry-olfactometry analysis of volatiles from fungating cancer wounds. We identified the source of the characteristic smell as dimethyl trisulfide, a compound that is known to be emitted from some vegetables and microorganisms. Controlling the production of dimethyl trisulfide should improve quality of life of patients.     

Cloning and Expression of the Bacillus subtilis No. 313 γ-Cyclodextrin Forming CGTase Gene in Escherichia coli

All four stereoisomers of pyriculol were synthesized to assist in forming a correlation between their chemical structure and biological activity. The (R,E)-2-hydroxy-3-pentenal derivative was coupled with a lithium acetylide derivative to give a diastereomeric mixture of the acetylenic alcohol, which led to the antipode of pyriculol and its 3′-epimer. Similarly obtained were the natural pyriculol and its 3′-epimer from the (S)-isomer of this aldehyde.