Change in the H2 photoproduction capability in a synchronously grown aerobic nitrogen-fixing cyanobacterium,Synechococcus sp. Miami BG 043511

Synchronously growing cells of nitrogen-fixing Synechococcus sp. Miami BG 043511 were harvested periodically and the capability for hydrogen photoproduction in closed vessels was measured under hydrogen production conditions. The capability for hydrogen photoproduction in cells was correlated with that of cellular carbohydrate content. Cells with a high carbohydrate content exhibited a high capacity for hydrogen production and those with low carbohydrate content exhibited low capacity for hydrogen production. Nitrogenase activity at the onset of incubation did not coincide with a capability for the cells to produce hydrogen during the subsequent incubation period. Interestingly, when cells with a high capacity for hydrogen photoaccumulation were incubated, alternate periods of hydrogen and oxygen accumulation were observed at 12 hour intervals. About 0.5 ml of hydrogen per ml of cell suspension was accumulated in flasks during the initial 12-h incubation period. These observations indicate that the use of synchronous culture can be one of the ways of provide materials suitable not only for basic studies but also for applied aspects of hydrogen photoproduction.     

Mitochondrial genome of the Komodo dragon: efficient sequencing method with reptile-oriented primers and novel gene rearrangements.

The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region.     

Laboratory maintenance of Eucyclops serrulatus (Copepoda: Cyclopoida).

A simple method for culturing a freshwater copepod Eucyclops serrulatus is described. A flagellate Chilomonas paramecium serves as the food organism. Starting from a single, egg-bearing female collected in a pond, the copepod proliferated for at least 10 generations apparently with no adverse changes. The stock culture has been maintained with minimal effort for at least 19 months. The method is expected to make the copepod useful in the laboratory as an intermediate host of various parasitic helminths.     

Assimilation and transport of nitrogen in rice II. 15N-labelled nitrate nitrogen

An investigation was made to study the assimilation and transportof ¹⁵N-labelled nitrate nitrogen in rice plant (Oryza sativaL.). Nitrogen from labelled nitrate at the end of plant feedingwas found mainly in nitrate form, and was more prevalent inroots, stem and leaf sheaths. The nitrite fraction had the nextlargest ¹⁵N enrichment. The ¹⁵NO₃ assimilation in the newlyemerged panicle was mainly in amide and amino acid. The ¹⁵N-incorporation at day 0 was greatest in amino acid andnitrate of roots and decreased towards the stem and leaves.Incorporation in these fractions considerably decreased fromday 0 to day 10. Probably most of the nitrogen from the nitratesource was transported from the roots to the shoot in nitrateand amino acid forms. A decrease of ¹⁵N-incorporation in the soluble N fraction andincrease in the insoluble N fraction from day 0 to day 10 inplant parts, particularly the blades, suggested that proteinsynthesis occurred mostly in young parts of the shoot duringthis period. The marked variation in ¹⁵N distribution in differentparts of the plant during the 10 days indicated that the nitrogenin roots and tillers was probably remobilized and transportedto other parts, particularly the upper leaf blades. Ammonium and nitrate nitrogen transport in rice plant are compared. (Received May 11, 1974; )     

Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink: statistical evidence for archosaurian affinity of turtles.

Turtles have highly specialized morphological characteristics, and their phylogenetic position has been under intensive debate. Previous molecular studies have not established a consistent and statistically well supported conclusion on this issue. In order to address this, complete mitochondrial DNA sequences were determined for the green turtle and the blue-tailed mole skink. These genomes possess an organization of genes which is typical of most other vertebrates, such as placental mammals, a frog, and bony fishes, but distinct from organizations of alligators and snakes. Molecular evolutionary rates of mitochondrial protein sequences appear to vary considerably among major reptilian lineages, with relatively rapid rates for snake and crocodilian lineages but slow rates for turtle and lizard lineages. In spite of this rate heterogeneity, phylogenetic analyses using amino acid sequences of 12 mitochondrial proteins reliably established the Archosauria (birds and crocodilians) and Lepidosauria (lizards and snakes) clades postulated from previous morphological studies. The phylogenetic analyses further suggested that turtles are a sister group of the archosaurs, and this untraditional relationship was provided with strong statistical evidence by both the bootstrap and the Kishino-Hasegawa tests. This is the first statistically significant molecular phylogeny on the placement of turtles relative to the archosaurs and lepidosaurs. It is therefore likely that turtles originated from a Permian-Triassic archosauromorph ancestor with two pairs of temporal fenestrae behind the skull orbit that were subsequently lost. The traditional classification of turtles in the Anapsida may thus need to be reconsidered.     

Mitogenomic perspectives into iguanid phylogeny and biogeography: Gondwanan vicariance for the origin of Madagascan oplurines

Complete or nearly complete nucleotide sequences of mitochondrial genomes (mtDNAs) were determined from eight species which, together with previous mtDNA data for two other taxa, cover most subfamilies of Iguanidae sensu lato. These iguanid mtDNAs were found to be rather conservative with respect to gene arrangements and molecular evolutionary rates, which contrasts with mtDNAs of Acrodonta (Agamidae and Chamaeleonidae) in which several gene rearrangements and highly accelerated molecular evolutionary rates have been known. Phylogenetic analyses consistently suggested the earliest shoot-off of a Malagasy subfamily Oplurinae and an affinity of Polychrotinae and Tropidurinae sensu stricto. However, even with the ample molecular characters derived from complete mtDNA sequences, phylogenetic relationships between iguanid subfamilies were poorly resolved in general, presumably due to the rapid ancient cladogenesis. Divergence time estimation without assuming the molecular clock suggested the Late Triassic/Early Jurassic divergence of Iguanidae from acrodonts and the Middle/Late Jurassic divergence of Oplurinae from the other iguanids. Together with geological and paleontological evidence, these results led us to propose Gondwanan vicariance for the origin of Malagasy oplurines without invoking a land bridge connection between South America/Antarctica and drifting Madagascar/India.     

The historical biogeography of the freshwater knifefishes using mitogenomic approaches: A Mesozoic origin of the Asian notopterids (Actinopterygii: Osteoglossomorpha)

The continental distributions of freshwater fishes in the family Notopteridae (Osteoglossomorpha) across Africa, India, and Southeast Asia constitute a long standing and enigmatic problem of freshwater biogeography. The migrational pathway of the Asian notopterids has been discussed in light of two competing schemes: the first posits recent transcontinental dispersal while the second relies on distributions being shaped by ancient vicariance associated with plate-tectonic events. In this study, we determined complete mitochondrial DNA sequences from 10 osteoglossomorph fishes to estimate phylogenetic relationships using partitioned Bayesian and maximum likelihood methods and divergence dates of the family Notopteridae with a partitioned Bayesian approach. We used six species representing the major lineages of the Notopteridae and seven species from the remaining osteoglossomorph families. Fourteen more-derived teleosts, nine basal actinopterygians, two coelacanths, and one shark were used as outgroups. Phylogenetic analyses indicated that the African and Asian notopterids formed a sister group to each other and that these notopterids were a sister to a clade comprising two African families (Mormyridae and Gymnarchidae). Estimated divergence time between the African and Asian notopterids dated back to the early Cretaceous when India–Madagascar separated from the African part of Gondwanaland. Thus, estimated time of divergence based on the molecular evidence is at odds with the recent dispersal model. It can be reconciled with the geological and paleontological evidence to support the vicariance model in which the Asian notopterids diverged from the African notopterids in Gondwanaland and migrated into Eurasia on the Indian subcontinent from the Cretaceous to the Tertiary. However, we could not exclude an alternative explanation that the African and Asian notopterids diverged in Pangea before its complete separation into Laurasia and Gondwanaland, to which these two lineages were later confined, respectively.     

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80 °C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50 °C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4 °C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.     

Interaction of epicatechin gallate with phospholipid membranes as revealed by solid-state NMR spectroscopy

Epicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [13C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by 13C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state 31P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state 13C NMR analysis under static conditions, the carbonyl carbon signal of the [13C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular-interatomic distance between the labeled carbonyl carbon of [13C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1 ? by 13C-(31)P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation-π interaction between the galloyl ring and quaternary amine of the phospholipid head-group.