The Genome Landscape of the African Green Monkey Kidney-Derived Vero Cell Line

Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells.     

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10 nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37 °C, but were almost completely denatured around 60 °C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.     

(125)I]-N-[(3-azido-5-iodo)benzyl]dantrolene and [(125)I]-N-[[3-iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl]dantrolene: photoaffinity probes specific for the physiological Ca(2+) release from sarcoplasmic reticulum of skeletal muscle

In order to capture and identify key molecules that regulate the release of Ca(2+) from the sarcoplasmic reticulum (SR) of skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a highly selective inhibitory effect on physiological Ca(2+) release (PCR) without affecting Ca(2+)-induced Ca(2+) release (CICR). Successful realization of the sharp discrimination between PCR and CICR has led to the creation of [(125)I]GIF-0082 and [(125)I]GIF-0276, which were synthesized by substituting a stannyl group with (125)I in the corresponding phenylstannane precursors.     

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.     

Depletion of Selenoprotein GPx4 in Spermatocytes Causes Male Infertility in Mice

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674–683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.     

Detection of a quantitative trait locus controlling carbon isotope discrimination and its contribution to stomatal conductance in japonica rice

Increasing leaf photosynthesis offers a possible way to improve yield potential in rice (Oryza sativa L.). Carbon isotope discrimination (Δ¹³C) has potential as an indirect selection criterion. In this study, we searched for quantitative trait loci (QTLs) controlling Δ¹³C, and assessed their association with leaf photosynthesis. Substitution mapping by using chromosome segment substitution lines (CSSLs), that carry segments from the indica cultivar Kasalath in the genetic background of the japonica cultivar Koshihikari, identified genomic regions affecting Δ¹³C on chromosomes (Chr.) 2, 3, 6, 7, and 12. One of the CSSLs, SL208, in which most regions on Chr. 3 were substituted with Kasalath segments, showed higher leaf stomatal conductance for CO₂ (g _s) and Δ¹³C than Koshihikari during the vegetative stage although leaf photosynthetic rate did not differ between them. These results suggest an association between Δ¹³C and g _s. To test this association, we performed a QTL analysis for Δ¹³C at vegetative and heading stages in an F₂ population derived from a cross between SL208 and Koshihikari. The results confirmed a QTL controlling Δ¹³C on the long arm of Chr. 3. By using a near-isogenic line specific to Hd6, we ruled out the possibility that variation in Δ¹³C was generated through the pleiotropic effect of heading date.     

An experimental approach to study the biosynthesis of brominated metabolites by the red algal genus Laurencia

The production of labeled brominated metabolites with radioactive ⁸²Br in Laurencia species was investigated as part of a study of the biosynthesis of halogenated metabolites from species belonging to the red algal genus Laurencia (Rhodomelaceae, Ceramiales). Radiobromide [⁸²Br], thin-layer chromatography (TLC), and TLC–autoradioluminography (ARLG) were used. When cultured in artificial seawater medium (ASP₁₂NTA including Na⁸²Br) under 16:8 h light:dark (LD) illumination cycles for 24 h, each of the strains of Laurencia, Laurencia japonensis Abe et Masuda, Laurencia nipponica Yamada (laurencin-producing race and laureatin-producing race), and Laurencia okamurae Yamada, produced species- (or race-) specific ⁸²Br-containing metabolites. In the case of the laurencin-producing race of L. nipponica, laurencin and deacetyllaurencin were found to be produced in approximately 1:1 ratio, though laurencin is the major metabolite in the wild sample. Furthermore, when cultured in the dark, the production rates of brominated metabolites in Laurencia spp. were found to be diminished. The present study strongly indicates that the use of radiobromine [⁸²Br] in combination with the TLC–ARLG method is an effective approach for investigating the biosynthesis of brominated metabolites in Laurencia.     

Radiation transfer and heat budget during the ice season in Lake Pääjärvi, Finland

Lake Pääjärvi, a boreal Finnish lake, was investigated in winter for weather conditions, structure and thickness of ice and snow, solar radiation, and under-ice current and temperature. Heat budget of Lake Pääjärvi in January–March was governed by terrestrial radiation losses of 20–35 W m^?2 recompensed by ice growth of 0.5–1.0 cm day^?1. In April, snow melted, albedo decreased from 0.8 to <0.1, and the mean ice melt rate was 1.5 cm day^?1. Internal melting and surface melting were about equal. The mean turbulent heat loss was small. The heat flux from the water to ice was about 5 W m^?2 in winter, increasing to 12 W m^?2 in the melting season. The light attenuation coefficient was 1.1 m^?1 for the congelation ice (black ice) in winter, compared with 1.5 m^?1 for the lake water, and it was up to 3 m^?1 for candled congelation ice in spring, and about 10 m^?1 for superimposed ice (white ice) and snow. Gas bubbles were the main factor that reduced the transparency of ice. The radiation penetrating the ice heated the water body causing convective currents and horizontal heat transfer. This increased the temperature of the water body to about 3°C before the ice break-up. After the snow had melted, the euphotic depth (the depth of 1% surface irradiance) was estimated as 2.0 m, only two-thirds that in summer.