[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

Genomic and phylogenetic traits of Staphylococcus phages S25-3 and S25-4 (family Myoviridae,genus Twort-like viruses)

The genomes of Staphylococcus phages S25-3 and S25-4 (family Myoviridae, genus Twort-like viruses) were sequenced and analyzed from an evolutionary perspective. The genome-based phylogeny and genome analyses of phages S25-3 and S25-4 showed that they had diverged evolutionarily from the majority of this viral genus based on the presence of mobile genetic elements, i.e., a putative transposase and the homing endonuclease I-MsaI. These results suggest that genetic elements such as transposases and homing endonucleases are likely to be involved with the evolution of some Twort-like phages, including phages S25-3 and S25-4.     

Life cycle of an endangered riffle beetle,Leptelmis gracilis Sharp (Coleoptera: Elmidae), in the Hiikawa River system,Shimane prefecture,Japan

We studied the life cycle of the riffle beetle Leptelmis gracilis Sharp, 1888 from a population located in a river stream with lotic environments. Samples were collected monthly, between September 2018 and August 2019, from a tributary of the Hiikawa River in Izumo city, Shimane Prefecture, Honshu, Japan. Larvae were separated into five distinct groups based on size. The first and last instar larval sizes were identified through rearing methods. From these results, we determined that the larval stage of this species consists of five instars. From the monthly evaluations, the number of individuals corresponding to each instar period suggested that last-instar larvae were dominant in May and that new adults emerged from July to August. Landing and pupation were estimated to occur from May to June. Monthly observations revealed that mature eggs were present in the female abdomen from April to June, as well as from August to September. It is, therefore, predicted that overwintering adults mainly oviposit from spring to early summer, while new adults oviposit from late summer to early autumn.     

Natural history of model organisms: The secret (group) life of Drosophila melanogaster larvae and why it matters to developmental ecology

1. Model organisms such as Drosophila melanogaster have been key tools for advancing our fundamental and applied knowledge in biological and biomedical sciences. However, model organisms have become intertwined with the idea of controlled and stable laboratory environments, and their natural history has been overlooked.
2. In holometabolous insects, lack of natural history information on larval ecology has precluded major advances in the field of developmental ecology, especially in terms of manipulations of population density early in life (i.e., larval density). This is because of relativistic and to some extent, arbitrary methodologies employed to manipulate larval densities in laboratory studies. As a result, these methodologies render comparisons between species impossible, precluding our understanding of macroevolutionary responses to population densities during development that can be derived from comparative studies.
3. We recently proposed a new conceptual framework to address this issue, and here, we provide the first natural history investigation of Drosophila melanogaster larval density under such framework. First, we characterized the distribution of larval densities in a wild population of D. melanogaster using rotting apples as breeding substrate in a suburban area in Sweden.
4. Next, we compiled the commonly used methodologies for manipulating larval densities in laboratory studies from the literature and found that the majority of laboratory studies identified did not manipulate larval densities below or above the densities observed in nature, suggesting that we have yet to study true life history and physiological responses to low and high population densities during D. melanogaster development.
5. This is, to our knowledge, the first direct natural history account of larval density in nature for this model organism. Our study paves the way for a more integrated view of organismal biology which re‐incorporates natural history of model organisms into hypothesis‐driven research in developmental ecology.

     

Effects of pH and Lactate on Hydrogen Sulfide Production by Oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H₂S), one of the major components of oral malodor. However, there is little information on the physiological properties of H₂S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H₂S production. Thus, in the present study, the H₂S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H₂S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H₂S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H₂S. H₂S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H₂S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H₂S, ammonia was produced in cell extracts of all the strains, indicating that H₂S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H₂S from l-cysteine and that their H₂S production can be regulated by oral environmental factors, namely, pH and lactate.     

Identifying indicator species for bryophyte conservation in fragmented forests

It is important to conserve bryophyte diversity in fragmented forests, due to the vulnerability of this group to environmental change. In this study, the utility of bryophyte functional groups (taxonomic classes, substrate classes, and life-forms) was examined as indicators for planning urban area conservation of bryophyte diversity. The study sites comprised 27 fragmented forests in Kyoto City, Japan. Using linear regression models, it was found that the species richness of almost all functional groups was positively correlated with patch size. Furthermore, two types of bryophyte life-form (fans and thalloid mats) were significantly correlated with environmental factors considered important for conservation planning. The species richness of fan bryophytes was positively correlated with the presence of natural forest patches and was negatively correlated with distance from mountainous areas. Similarly, the species richness of thalloid mat bryophytes was negatively correlated with maintenance practices. These results may be explained by the vulnerability of these two bryophyte groups to environmentally caused drought stress, accompanied by decreasing patch size, maintenance practices, disturbance, and/or loss of natural vegetation. Considering that drought stress represents a major threat to bryophyte diversity in fragmented forests, the species richness of hygrophilous life-forms (e.g., fans and thalloid mats) may be used as an indicator of fragmented forests that are less affected by drought stress, and these species should be preferentially conserved to maintain high levels of bryophyte diversity.     

Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity

Troponin C was removed almost completely from the porcine cardiac myofibrils by the same extraction procedure using CDTA as that previously reported for the rabbit skeletal myofibrils (Morimoto, S. & Ohtsuki, I. (1987) J. Biochem. 101, 291-301), and the effects of substitution of troponin C in cardiac myofibrils with rabbit skeletal troponin C or bovine brain calmodulin were examined. While the ATPase activity of intact cardiac myofibrils or cardiac troponin C-reconstituted cardiac myofibrils was activated at only a little higher concentration of Sr2+ than Ca2+, the skeletal troponin C-substituted cardiac myofibrils, as well as intact rabbit skeletal myofibrils, required more than 10 times higher concentration of Sr2+ than Ca2+ for activation of the myofibrillar ATPase activity. However, the concentrations of Ca2+ and Sr2+ required for the activation of the ATPase activity of the skeletal troponin C-substituted cardiac myofibrils were both about 5 times higher than those of intact skeletal myofibrils. The skeletal troponin C-substituted cardiac myofibrils, as well as intact skeletal myofibrils, also showed higher cooperativity in the Ca2+-activation of the ATPase activity than intact or cardiac troponin C-reconstituted cardiac myofibrils. The ATPase activity of calmodulin-substituted cardiac myofibrils was activated at a several times lower concentration of Ca2+ or Sr2+ than that of calmodulin-substituted skeletal myofibrils, while the ratios of the concentration of Sr2+ to Ca2+ required for activation were almost the same in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual distribution of floating seaweeds in the East China Sea in the early spring of 2012

Floating seaweeds play important ecological roles in offshore waters. Recently, large amounts of rafting seaweed have been observed in the East China Sea. In early spring, juveniles of commercially important fish such as yellowtail accompany these seaweed rafts. Because the spatial distributions of seaweed rafts in the spring are poorly understood, research cruises were undertaken to investigate them in 2010, 2011, and 2012. Floating seaweed samples collected from the East China Sea during the three surveys contained only Sargassum horneri. In 2010 and 2011, seaweed rafts were distributed only in the continental shelf and the Kuroshio Front because they had become trapped in the convergence zone of the Kuroshio Front. However, in 2012, seaweed was also distributed in the Kuroshio Current and its outer waters, and massive strandings of seaweed rafts were observed on the northern coast of Taiwan and on Tarama Island in the Ryukyu Archipelago. Environmental data (wind, currents, and sea surface height) were compared among the surveys of 2010, 2011, and 2012. Two factors are speculated to have caused the unusual distribution in 2012. First, a continuous strong north wind produced an Ekman drift current that transported seaweed southwestward to the continental shelf and eventually stranded seaweed rafts on the coast of Taiwan. Second, an anticyclonic eddy covering northeast Taiwan and the Kuroshio Current west of Taiwan generated a geostrophic current that crossed the Kuroshio Current and transported the rafts to the Kuroshio Current and its outer waters. Such unusual seaweed distributions may influence the distribution of fauna accompanying the rafts.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.