Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii

A gene (ST1218) encoding a d-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35 kDa and was highly thermostable. It preferably acted as an NAD-dependent d-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.     

Hepatitis C virus genotype 1a growth and induction of autophagy

We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.     

Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS-3

Leucine dehydrogenase ( -leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) was purified to homogeneity from the crude extract of an alkaliphilic halophile, Natronobacterium magadii MS-3, with a yield of 16%. The enzyme had a molecular mass of about 330 kDa and consisted of six subunits identical in molecular mass (55 kDa). The enzyme required a high concentration of salt for stability and activity. It retained the full activity after heating at 50 °C for 1 h and about 50% activity after being kept at 30 °C for 2 months in the presence of 2.5 M NaCl. The enzyme required NAD⁺ as a coenzyme and showed maximum activity in the presence of more than 3 M salt, as CsCl, RbCl, NaCl, or KCl. In addition to -leucine, -valine and -isoleucine were also good substrates in the oxidative deamination. In the reductive amination, 2-keto analogs of branched-chain amino acids were substrates. The Michaelis constants were 0.69 mM for -leucine, 0.48 mM for NAD⁺, 4.0 mM for 2-ketoisocaproate, 220 mM for ammonia, and 0.02 mM for NADH in the presence of 4 M NaCl. The K_m for -leucine depended on the concentration of salt and increased with decreasing salt concentration. The N. magadii enzyme was unique in its halophilicity among leucine dehydrogenases studied so far.     

The receptor-like cytoplasmic kinase BSR1 mediates chitin-induced defense signaling in rice cells

Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H₂O₂ production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.     

Mitochondrial phylogenetics and evolution of mysticete whales

The phylogenetic relationships among baleen whales (Order: Cetacea) remain uncertain despite extensive research in cetacean molecular phylogenetics and a potential morphological sample size of over 2 million animals harvested. Questions remain regarding the number of species and the monophyly of genera, as well as higher order relationships. Here, we approach mysticete phylogeny with complete mitochondrial genome sequence analysis. We determined complete mtDNA sequences of 10 extant Mysticeti species, inferred their phylogenetic relationships, and estimated node divergence times. The mtDNA sequence analysis concurs with previous molecular studies in the ordering of the principal branches, with Balaenidae (right whales) as sister to all other mysticetes base, followed by Neobalaenidae (pygmy right whale), Eschrichtiidae (gray whale), and finally Balaenopteridae (rorquals + humpback whale). The mtDNA analysis further suggests that four lineages exist within the clade of Eschrichtiidae + Balaenopteridae, including a sister relationship between the humpback and fin whales, and a monophyletic group formed by the blue, sei, and Bryde's whales, each of which represents a newly recognized phylogenetic relationship in Mysticeti. We also estimated the divergence times of all extant mysticete species, accounting for evolutionary rate heterogeneity among lineages. When the mtDNA divergence estimates are compared with the mysticete fossil record, several lineages have molecular divergence estimates strikingly older than indicated by paleontological data. We suggest this discrepancy reflects both a large amount of ancestral polymorphism and long generation times of ancestral baleen whale populations.     

Competition for spawning sites between two gobiid fishes, Bathygobius fuscus and Eviota abax, derived by alteration of mating tactics of the former

Interspecific competition for spawning sites between two gobiid fishes, Bathygobius fuscus and Eviota abax, was studied on a rocky shore. Large males (LM: 55–80mm in standard length: SL) and small males (SM: 34–52mm SL) of B. fuscus acted as nest holders and sneakers, respectively, in the early spawning season (July). The sympatric male E. abax (22–33mm SL) was smaller than SM B. fuscus, and synchronically used rocky holes much smaller than those of LM B. fuscus. In this season, aggressions between the two species were rarely observed. In late season (August), as the number of the LM decreased, the SM converted their tactics to nest holding, occupying nests of a sizes similar to those of E. abax. Eviota abax males were dispossessed by SM and shifted their nest sites to cavities between cobbles and sandy bottom, which might be poorer nest sites than rocky holes.     

Music recognition by Japanese children with cochlear implants

Congenitally deaf Japanese children with cochlear implants were tested on their recognition of theme songs from television programs that they watched regularly. The children, who were 4-9 years of age, attempted to identify each song from a closed set of alternatives. Their song identification ability was examined in the context of the original commercial recordings (vocal plus instrumental), the original versions without the words (i.e., karaoke versions), and flute versions of the melody. The children succeeded in identifying the music only from the original versions, and their performance was related to their music listening habits. Children gave favorable appraisals of the music even when they were unable to recognize it. Further research is needed to find means of enhancing cochlear implants users' perception and appreciation of music.