Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis

Background and Aims

The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys) and secrete substantial amounts of L-Glutamate (L-Glu) via the transport system Xc- (4F2hc+xCT), and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes.

Methods

We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM), and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0–300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha.

Results

The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19), the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio.

Conclusions

A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.     

Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins

The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase alpha and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected.     

Zebrafish Polycomb group gene ph2alpha is required for epiboly and tailbud formation acting downstream of FGF signaling

We analyzed Polycomb group gene ph2alpha functionally in zebrafish embryos by a gene knock-down procedure using morpholino antisense oligos. Inhibition of ph2alpha message translation resulted in abnormal epibolic movements as well as a thick tailbud or incomplete covering of the yolk plug. At the 24hpf stage, morphants had short trunks and tails, phenotypes similar to those with disturbances in FGF signaling. Accordingly, we looked at the effects of ph2alpha expression upstream and downstream of the FGF pathway. Treatment with SU5402, an inhibitor of Fgfrs, or injection of dominant-negative Fgfr1 DNA markedly reduced ph2alpha expression in the tailbud. In addition, cells expressing mRNAs for no tail, spadetail, myoD, and papc, which are involved in FGF-related development of posterior mesoderm, were distributed abnormally. Collectively, the data argue that ph2alpha is required for epiboly and tailbud formation, acting downstream of the FGF signaling pathway.     

Elucidation of the speciation history of three sister species of crown-of-thorns starfish (Acanthaster spp.) based on genomic analysis

The crown-of-thorns starfish (COTS) is a coral predator that is widely distributed in Indo-Pacific Oceans. A previous phylogenetic study using partial mitochondrial sequences suggested that COTS had diverged into four distinct species, but a nuclear genome-based analysis to confirm this was not conducted. To address this, COTS species nuclear genome sequences were analysed here, sequencing Northern Indian Ocean (NIO) and Red Sea (RS) species genomes for the first time, followed by a comparative analysis with the Pacific Ocean (PO) species. Phylogenetic analysis and ADMIXTURE analysis revealed clear divergences between the three COTS species. Furthermore, within the PO species, the phylogenetic position of the Hawaiian sample was further away from the other Pacific-derived samples than expected based on the mitochondrial data, suggesting that it may be a PO subspecies. The pairwise sequentially Markovian coalescent model showed that the trajectories of the population size diverged by region during the Mid-Pleistocene transition when the sea-level was dramatically decreased, strongly suggesting that the three COTS species experienced allopatric speciation. Analysis of the orthologues indicated that there were remarkable genes with species-specific positive selection in the genomes of the PO and RS species, which suggested that there may be local adaptations in the COTS species.     

Chain length effects of oligo(L-lysine)-shelled dendrimers on interaction with DNA

The interaction of novel oligo(L-lysine)-shelled dendrimers (G3-PLL) with DNA was studied by means of circular dichroism spectroscopy, dynamic light scattering, and melting behavior of double-stranded DNA. G3-PLLs having various oligo(L-lysine) (PLL) segment (n = 5-40) were successfully synthesized by graft-polymerization of L-lysine NCA initiated with amino groups at the 3rd-generation poly(amidoamine) dendrimer surface. The ionization property of the newly prepared G3-PLLs were first examined. The evaluated pK(a) values (8.3-8.5) for G3-PLLs were found to be significantly lower than those (9.2-9.4) for the corresponding linear PLLs, probably due to alignment of PLL segments on the three-dimensional core surface. The binding experiments of G3-PLLs to DNA showed that double-stranded DNAs were fairly strongly bound to G3-PLLs primarily through electrostatic interactions. In addition, G3-PLLs served as a DNA cross-linker. A longer PLL-containing G3-PLL was found to interact with DNA more effectively than a shorter one.     

Rho2 is a target of the farnesyltransferase Cpp1 and acts upstream of Pmk1 mitogen-activated protein kinase signaling in fission yeast

We have previously demonstrated that knockout of the calcineurin gene or inhibition of calcineurin activity by immunosuppressants resulted in hypersensitivity to Cl- in fission yeast. We also demonstrated that knockout of the components of the Pmk1 mitogen-activated protein kinase (MAPK) pathway, such as Pmk1 or Pek1 complemented the hypersensitivity to Cl-. Using this interaction between calcineurin and Pmk1 MAPK, here we developed a genetic screen that aims to identify new regulators of the Pmk1 signaling and isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vic1-1, carried a missense mutation in the cpp1+ gene encoding a beta subunit of the protein farnesyltransferase, which caused an amino acid substitution of aspartate 155 of Cpp1 to asparagine (Cpp1(D155N)). Analysis of the mutant strain revealed that Rho2 is a novel target of Cpp1. Moreover, Cpp1 and Rho2 act upstream of Pck2-Pmk1 MAPK signaling pathway, thereby resulting in the vic phenotype upon their mutations. Interestingly, compared with other substrates of Cpp1, defects of Rho2 function were more phenotypically manifested by the Cpp1(D155N) mutation. Together, our results demonstrate that Cpp1 is a key component of the Pck2-Pmk1 signaling through the spatial control of the small GTPase Rho2.     

Patterns of sheath elongation, cell proliferation, and manganese(II) oxidation in Leptothrix cholodnii

Leptothrix cholodnii is a Mn(II)-oxidizing and sheath-forming member of the class β-Proteobacteria. Its sheath is a microtube-like filament that contains a chain of cells. From a chemical perspective, the sheath can be described as a supermolecule composed of a cysteine-rich polymeric glycoconjugate, called thiopeptidoglycan. However, the mechanism that controls the increase in sheath length is unknown. In this study, we attempted to detect sheath elongation through microscopic examination by using conventional reagents. Selective fluorescent labeling of preexisting or newly formed regions of the sheath was accomplished using combinations of biotin-conjugated maleimide, propionate-conjugated maleimide, and a fluorescent antibiotin antibody. Epifluorescence microscopy indicated that the sheath elongates at the terminal regions. On the bases of this observation, we assumed that the newly secreted thiopeptidoglycan molecules are integrated into the preexisting sheath at its terminal ends. Successive phase-contrast microscopy revealed that all cells proliferate at nearly the same rate regardless of their positions within the sheath. Mn(II) oxidation in microcultures was also examined with respect to cultivation time. Results suggested that the deposition of Mn oxides is notable in the aged regions. The combined data reveal the spatiotemporal relationships among sheath elongation, cell proliferation, and Mn oxide deposition in L. cholodnii.