Characterization of chemical defenses in ranid tadpoles against a fish predator

Tadpoles of some ranid species appear to possess chemical defenses against fish predators, but the chemicals have not been characterized. Here, we evaluated the vulnerability of three Japanese anuran tadpole species (Glandirana rugosa, Pelophylax nigromaculatus, and Hyla japonica) to a fish (Gnathopogon elongatus elongatus) and analyzed the defensive chemicals extracted from the unpalatable tadpoles. Additionally, we examined the defensive behavior of unpalatable tadpoles in response to fish chemical cues. The fish rejected both G. rugosa (83%) and P. nigromaculatus (48%), but not H. japonica (0%). Many of the rejected tadpoles survived (60–80%). Possible defensive chemicals were extracted by methanol from the skin of G. rugosa, but were not identifiable by gas chromatography–mass spectrometry because of small quantities. The chemicals have high polarity and non-volatility. When exposed to fish chemical cues, P. nigromaculatus decreased activity presumably as a defensive behavior, but G. rugosa did not. We demonstrated the presence of chemical defenses in at least two of these species and revealed that G. rugosa releases more effective or greater amounts of defense chemicals than P. nigromaculatus with respect to this fish predator. The increased efficacy of chemical defenses may correlate with decreasing defensive behavior.     

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.     

Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K _D = [1.7 ± 0.2] × 10⁻¹⁰ M) and IgG3 (K _D = [1.1 ± 0.2] × 10⁻¹⁰ M) were similar to those of glycosylated rhFcγRI.     

Surveillance of fish species composition using environmental DNA

Prompt and accurate methods for assessing the species composition of given areas are indispensable in addressing the rapid loss of biodiversity. Here, we propose a method for the surveillance of fish species composition in freshwater using environmental DNA as species markers. First, the applicability of the method was demonstrated through aquarium experiments. DNA was extracted from 120?ml aquarium water, and the degenerated primers targeting the fish mitochondrial cytochrome b gene were used for amplification. PCR-amplified fragments were analysed by random cloning, and all species reared in the aquarium were detected. Next, this method was applied to natural freshwater environments. Water samples were collected from three sites in the Yura River, Japan; DNA was concentrated from 2?l of environmental water, and then amplified and cloned. Up to four species of fish were detected by sequencing 47 randomly selected clones from a single water sample. Overall, the results were consistent with previous knowledge of fish habitat utilisation. Using this method, the surveillance of fish species composition can be conducted less laboriously than with traditional methods.     

A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC₅₀) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.     

Association between urinary calcium excretion level and mortality in inhabitants of the Jinzu River basin area of Japan

A follow-up study on 5442 inhabitants (2699 men, 2743 women) was conducted to determine the association between urinary Ca excretion level and mortality of the general population of three different areas of the Jinzu River basin area, namely, non-Jinzu River, mixed, and the Jinzu River water systems, over a period of 6127 d. More than 98% of the subjects were followed completely in each area. In comparison with the low- and high-Ca-excretion groups (cutoff values; 25.1 mg/dL in men, 20.4 mg/dL in women), the mortality rates per 1000 person-years and standardized mortality ratios (SMRs) tended to be greater in the low-Ca-excretion groups than in the high-Ca-excretion groups in both sexes for each of the three areas. Moreover, Cox’s hazard ratios in men and women of the three areas exhibited negative values, 0.99 except for men of the mixed water system. These values were statistically significant in both sexes for the Jinzu River water system and in women for the non-Jinzu River water system. We conclude that the life-span becomes shorter as urinary Ca excretion levels become lower.