Production of Tiger Puffer <Emphasis Type="Italic">Takifugu rubripes</Emphasis> Offspring from Triploid Grass Puffer <Emphasis Type="Italic">Takifugu niphobles</Emphasis> Parents

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2–5 kg) much larger than average market size (0.6–1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.     

Rat embryonic stem cells create new era in development of genetically manipulated rat models

Embryonic stem (ES) cells are isolated from the inner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocysts leads to the creation of gene-targeted mice such as knock-in and knock-out mice; these gene-targeted mice contribute greatly to scientific development. Although the rat is considered a useful laboratory animal alongside the mouse, fewer gene-modified rats have been produced due to the lack of robust establishment methods for rat ES cells. A new method for establishing rat ES cells using signaling inhibitors was reported in 2008. By considering the characteristics of rat ES cells, recent research has made progress in improving conditions for the stable culture of rat ES cells in order to generate gene-modified rats efficiently. In this review, we summarize several advanced methods to maintain rat ES cells and generate gene-targeted rats.     

Site-directed affinity-labeling of delta opioid receptors by

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala², Leu⁵]-enkephalyl-Cys(Npys)⁶ and [-Ala²,Leu(CH₂SNpys)⁵]enkephalin, and dynorphin A analogues of [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide and [-Ala²,Cys(Npys)⁸]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide.     

Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells <Emphasis Type="Italic">in vitro</Emphasis>

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro, and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.     

Real‐time observation of flexible domain movements in CRISPR–Cas9

The CRISPR‐associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single‐guide RNA (sgRNA). Structural studies have revealed the multi‐domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single‐molecule FRET. We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo‐Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9‐catalyzed DNA cleavage.     

Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

Senescent cells accumulate in tissues during aging and are considered to underlie several aging‐associated phenotypes and diseases. We recently reported that the elimination of p19^ARF‐expressing senescent cells from lung tissue restored tissue function and gene expression in middle‐aged (12‐month‐old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19^ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19^ARF‐expressing cells prevented lung tissue from elastase‐induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema‐associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema.     

Efficient formation of vesicular stomatitis virus pseudotypes bearing the native forms of hepatitis C virus envelope proteins detected after sonication

Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in addition to acute hepatitis. The HCV genome encodes two envelope glycoproteins, E1 and E2. To investigate the role of E1 and E2 in HCV infection, we used a recombinant vesicular stomatitis virus (VSV), VSVdeltaG*, harboring the green fluorescent protein gene instead of the VSV G envelope protein gene. It was complemented with the native form of E1 and E2, or E1 or E2 alone, to make HCV pseudotypes VSVdeltaG*(HCV), VSVdeltaG*(E1), and VSVdeltaG*(E2). Neither E1 nor E2 expression was detected on the cell surface, as reported. Unlike previous reports, infectious activities of VSVdeltaG*(HCV), VSVdeltaG*(E1) and VSVdeltaG*(E2) pseudotypes were detected under conditions where VSV was completely neutralized by anti-VSV. We could enhance the infectious titers 100-fold by sonication upon virus harvest. Bovine lactoferrin efficiently inhibited infection by VSVdeltaG*(HCV) as well as VSVdeltaG*(E2), as the interaction between E2 and lactoferrin has been thought to contribute to the inhibition of HCV infectivity. VSVdeltaG*(HCV) infected many adherent cell lines, including hepatic cell lines, but not most hematopoietic cell lines. Treatment of cells with trypsin, tunicamycin, or sulfated polysaccharides before infection reduced the infectivity of VSVdeltaG*(HCV) by about 90%, suggesting that a cell surface protein(s) with sugar chains plays an important role in HCV infection. The VSV pseudotypes developed here would be useful for analyzing the early stages of HCV infection.     

Interleukin-1β induction of tissue inhibitor of metalloproteinase (TIMP-1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts

Elevated levels of tissue inhibitor of metalloproteases-1 (TIMP-1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂), in the control of TIMP-1 synthesis and expression in human synovial fibroblasts. Recombinant human IL-1β induced a time- and dose-dependent saturable response in terms of TIMP-1 mRNA expression (effective concentration for 50% maximal response, EC₅₀ = 31.5 ± 3.3 pg/ml) and protein synthesis (EC₅₀ = 30 ± 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H-7, staurosporine, and calphostin C, reversed the rhIL-1β induction of TIMP-1 mRNA. PGE₂ also inhibited rhIL-1β-stimulated TIMP-1 mRNA expression and protein secretion in a dose-dependent fashion. The concentration of PGE₂ necessary to block 50% of rhIL-1β-stimulated TIMP-1 secretion, IC₅₀, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE₂. The phorbol ester, PMA, up-regulated considerably the mRNA expression of TIMP-1 but had no effect on protein production. Calphostin C substantially reduced PMA-activated TIMP-1 expression. Staurosporine, calphostin C, H-7, and substances that elevate cellular levels of cAMP, like PGE₂, also reduced basal expression and synthesis of TIMP-1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP-1 expression and secretion in human synovial fibroblasts.     

Chla-specific productivity of picophytoplankton not higher than that of larger phytoplankton off the South Shetland Islands in summer

Size-fractionated chlorophyll a (Chla)-specific productivity (μgC μgChla ⁻¹ h⁻¹) was measured at 11 stations off the northern coast of the South Shetland Islands during summer. The Chla-specific productivity of the 2- to 10 or 10- to 330-μm fraction was highest at 100% and 23% light depths. The Chla-specific productivity of the 2- to 10-μm fraction was generally highest, and that of the <2 or 10- to 330-μm fraction was sometimes highest at 12% and 1% light depths. Temperature was less than 3°C within the euphotic zone at all stations. The hypothesis of Shiomoto et al., according to which Chla-specific productivity of picophytoplankton (<2 μm) is not significantly higher than that of larger phytoplankton (>2 μm) in water colder than 10°C, was supported on condition that light is not limited for larger phytoplankton. Received: 16 September 1997 / Accepted: 8 December 1997     

Oxygen isotope ratios between soil water and stem water of trees in pot experiments

Since the oxygen isotopic ratio of water extracted from stems reflects that of water taken up by roots, the stem water isotope ratio can be used to analyze the source of water for plant growth. However, it is known that the fractionation of isotopes during evaporation from the surface soil increases the isotope ratio in soil water drastically. In this study, it was experimentally confirmed that the stem water of Elaeocarpus sylvestris vs. ellipticus Hara seedlings is not isotopically similar to the water source in the case where evaporation from the soil occurs actively. However, since water in these plant bodies was replaced in about 2 days in the pot experiments, the 2-day-averaged values of the soil water isotope ratio approached the stem water isotope ratio. Thus, time-course samplings of the soil and stems, and measurements of the replacement time of water in the plant body (water volume in plant/transpiration rate) are recommended for correct interpretation of the isotopic signature of soil water and stem water.