Virus resistance in the toxic bloom-forming dinoflagellate Heterocapsa circularisquama to single-stranded RNA virus infection

HcRNAV is the only known cultured dinoflagellate-infecting RNA virus. Lysis of its host dinoflagellate Heterocapsa circularisquama caused by HcRNAV is followed by apparent cell regrowth. Here we investigate the mechanism supporting the survival phenomenon. The proportion of normal cells with intact nucleus decreased to ∼8% by 3 days post infection, and then, increased to > 90% at 15 days post infection. There were abnormal cells lacking an intact nucleus, and this was followed by propagation of virus-resistant survivor cells. The proportion of HcRNAV-resistant cells in three different subcultures and temporal fluctuations were compared: a clonal H. circularisquama culture without virus inoculation (virus-sensitive, VS), a surviving isolate from the HcRNAV-inoculated Culture-VS incubated in autoclaved medium (virus-resistant, VR) and a portion of Culture-VR incubated with HcRNAV (VR incubated with virus, VR + V). The proportion of HcRNAV-resistant cells in Culture-VS was 0% and in Culture-VR + V was > 94% during the experiment; and Culture-VR fluctuated from 4% to 71%. Hence, the virus resistance was assumed to be reversible. Using Northern hybridization, viral genome accumulation was not detected in Culture-VR + V cells either inoculated with HcRNAV or transfected with HcRNAV-genome; thus, intracellular viral RNA replication was assumed to be interrupted in the virus-resistant cells.     

An efficient quantitation method of next-generation sequencing libraries by using MiSeq sequencer

Library quantitation is a critical step to obtain high data output in Illumina HiSeq sequencers. Here, we introduce a library quantitation method that uses the Illumina MiSeq sequencer designated as quantitative MiSeq (qMiSeq). In this procedure, 96 dual-index libraries, including control samples, are denatured, pooled in equal volume, and sequenced by MiSeq. We found that relative concentration of each library can be determined based on the observed index ratio and can be used to determine HiSeq run condition for each library. Thus, qMiSeq provides an efficient way to quantitate a large number of libraries at a time.     

Preparation and evaluation of an enzyme which degrades yeast cell walls

Summary A method for the production and preparation of an enzyme which degrades yeast cell walls from a species of aRhizoctonia (tentatively identified asR. solani) was established on a commercial scale. The production of crude enzyme powder, having a lytic activity of 100 units/mg, in batches of 80 kg is feasible.The enzyme preparation was evaluated for industrial use. When yeast cells were treated with this enzyme, the digestibility of feed yeast was improved 1.4–2 fold in vitro; the efficiency of a mechanical disintegrator in extracting cellular substances was increased 35–50%; the release of soluble glucans having widely varying degrees of polymerization was induced; the extraction of cellular protein by alkali was facilitated 2–3 fold; an 80% release of cell-bound invertase was induced and 2–3 times more yeast extract could be prepared.Studies on Fungal Enzymes Active in Hydrolysing Yeast Cell Wall (VII)     

Crystal Structure of Zebrafish Hatching Enzyme 1 from the Zebrafish Danio rerio

Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 Å resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3₁₀-helices, three α-helices, and two β-sheets. The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1′ contributes the most and that the residues at P4-P2′ also contribute to the recognition of the major substrate peptide by ZHE1, whereas GluP3′ and the hydrophobic residues at P4-P2, P2′, and P5′ contribute significantly to the recognition of the minor substrate peptide by ZHE1. Molecular models of these two substrate peptides bound to ZHE1 have been built based on the crystal structure of a transition-state analog inhibitor bound to astacin. In substrate-recognition models, the AspP1′ in the major substrate peptide forms a salt bridge with Arg182 of ZHE1, while the GluP3′ in the minor substrate peptide instead forms a salt bridge with Arg182. Thus, these two substrate peptides would be differently recognized by ZHE1. The shapes and electrostatic potentials of the substrate-binding clefts of ZHE1 and the structurally similar proteins astacin and bone morphogenetic protein 1 are significantly dissimilar due to different side chains, which would confer their distinctive substrate preferences.     

Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution

A biased mutation-assembling method—that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Virtual cooperativity in myoglobin oxygen saturation curve in skeletal muscle <Emphasis Type="Italic">in vivo</Emphasis>

Background

Myoglobin (Mb) is the simplest monomeric hemoprotein and its physicochemical properties including reversible oxygen (O₂)binding in aqueous solution are well known. Unexpectedly, however, its physiological role in intact muscle has not yet been established in spite of the fact that the role of the more complex tetrameric hemoprotein, hemoglobin (Hb), in red cells is well established. Here, I report my new findings on an overlooked property of skeletal Mb.

Methods

I directly observed the oxygenation of Mb in perfused rat skeletal muscle under various states of tissue respiration. A computer-controlled rapid scanning spectrophotometer was used to measure the oxygenation of Mb in the transmission mode. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500–650 nm were recorded when O₂ uptake in the hindlimb muscle reached a constant value after every stepwise change in the O₂ concentration of the buffer.

Results

The O₂ dissociation curve (ODC) of Mb, when the effluent buffer O₂ pressure was used as the abscissa, was of a sigmoid shape under normal and increased respiratory conditions whereas it was of rectangular hyperbolic shape under a suppressed respiratory condition. The dissociation curve was shifted toward the right and became more sigmoid with an increase in tissue respiration activity. These observations indicate that an increase in O₂ demand in tissues makes the O₂ saturation of Mb more sensitive to O₂ pressure change in the capillaries and enhances the Mb-mediated O₂ transfer from Hb to cytochrome oxidase (Cyt. aa₃), especially under heavy O₂ demands.

Conclusion

The virtual cooperativity and O₂ demand-dependent shifts of the ODC may provide a basis for explaining why Mb has been preserved as monomer during molecular evolution.

     

Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence

Spermidine synthase (SPDS) catalyzes transfer of the propylamine group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine to yield methylthioadenosine (MTA) and spermidine. SPDS plays a regulatory role in cell proliferation and differentiation. This article describes the development of a high-throughput SPDS activity assay using homogeneous time-resolved fluorescence (HTRF) based on energy transfer from europium cryptate as a donor to crosslinked allophycocyanin (XL665) as an acceptor. First a highly specific anti-MTA monoclonal antibody, MTA-7H8, was generated, and then a competitive immunoassay for MTA determination was developed using europium cryptate-labeled MTA-7H8 and XL665-labeled MTA. In our homogeneous immunoassay, the percentage molar cross-reactivity of dcSAM with MTA-7H8 was 0.01% and the detection limit of MTA was 2.6 pmol/well. Our HTRF assay uses only one assay plate in which both enzyme reaction and MTA determination can be done successively. Therefore, our method can enable automatic screening of SPDS inhibitors from large numbers of samples.