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.     

Quality control of cultured tissues requires tools for quantitative analyses of heterogeneous features developed in manufacturing process

Tissue engineering and related technology have attracted a great deal of medical attention as promising fields for curing defective tissues in vivo. Nowadays, many companies have been established for supplying the reconstructed grafts of cultured tissues for transplantation. The manufacturing processes generally deals with the handlings of starter cells offered by patients (or donors) as raw materials to cultured tissues as products, requiring the construction of novel ex vivo methodologies based on principles different from conventional processes for chemical and pharmaceutical productions. In addition, the raw materials have heterogeneity depending on the state of patients and location of cell harvests, and the products possess spatial cell distribution in the three dimensional structure. These features request a unique strategy in manufacturing process accompanied with the quality control for raw materials and products. This review article describes the contribution of tissue bankers and biochemical engineers to the quality control of cultured tissues during manufacturing, introducing the advances in methodologies to evaluate spatial heterogeneity of cells (or aggregates) and matrices in cultured tissues. Presented at the 1st International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 16–20, 2007 at Vale de Santarem, Portugal.     

Placenta‐specific gene activation and inactivation using integrase‐defective lentiviral vectors with the Cre/LoxP system

Transgenic and knockout studies have advanced our understanding of the genetic control of embryonic development over the past decades. However, interpretation of the phenotype of mutant mice is potentially complicated, since the commonly used knockout approach modifies both the fetal and placental genome. To circumvent this problem, we previously developed a placenta‐specific gene manipulation system by lentiviral vector transduction of embryos at the blastocyst stage. In the present study, by combination with the Cre/LoxP system, we successfully demonstrate placenta‐specific gene activation and inactivation in EGFP reporter mice and Ets2 floxed mice, respectively. Transient expression using integrase‐defective lentiviral (IDLV) vectors diminished the toxic effect of Cre expression and solved the dilemma of mosaic recombination with lower concentrations and toxic effects with higher concentrations of Cre recombinase. We also show that placenta‐specific Ets2 disruption causes embryonic lethality and reconfirmed the critical role of Ets2 during placentation. This technology facilitates both gain and loss of gene function analyses in placental development during pregnancy. Since IDLV vectors can efficiently transduce a variety of cell types similarly to wild‐type vectors, our IDLV‐Cre strategy is potentially useful for a wide range of applications. genesis 47:793–798, 2009. © 2009 Wiley‐Liss, Inc.     

Dihydro-alpha-lipoic acid has more potent cytotoxicity than alpha-lipoic acid

Alpha-lipoic acid has been shown to possess cancer-cell-killing activity via activation of the apoptosis pathway. In this study, the cytotoxic activities of alpha-lipoic and dihydro-alpha-lipoic acid were compared in HL-60 cells. The cell-killing activity of dihydro-alpha-lipoic acid was higher than that of alpha-lipoic acid. Both alpha-lipoic and dihydro-alpha-lipoic acid induced caspase-3 cleavage and internucleosomal DNA fragmentation in treated cells. On the other hand, apparent necrotic or late-stage apoptotic cell populations could be detected in dihydro-alpha-lipoic acid cells but not in those treated with alpha-lipoic acid. Moreover, dihydro-alpha-lipoic acid, but not alpha-lipoic acid, induced marked mitochondrial permeability transition. Antioxidants could not prevent dihydro-alpha-lipoic- or alpha-lipoic-acid-induced cell death. In addition, dihydro-alpha-lipoic and alpha-lipoic acid did not up-regulate cellular reactive oxygen level. These results indicated that dihydro-alpha-lipoic acid exerts more potent cytotoxicity than alpha-lipoic acid through different cytotoxic actions.     

A at Single Nucleotide Polymorphism-358 Is Required for G at -420 to Confer the Highest Plasma Resistin in the General Japanese Population

Insulin resistance is a feature of type 2 diabetes. Resistin, secreted from adipocytes, causes insulin resistance in mice. We previously reported that the G/G genotype of single nucleotide polymorphism (SNP) at −420 (rs1862513) in the human resistin gene (RETN) increased susceptibility to type 2 diabetes by enhancing its promoter activity. Plasma resistin was highest in Japanese subjects with G/G genotype, followed by C/G, and C/C. In this study, we cross-sectionally analyzed plasma resistin and SNPs in the RETN region in 2,019 community-dwelling Japanese subjects. Plasma resistin was associated with SNP-638 (rs34861192), SNP-537 (rs34124816), SNP-420, SNP-358 (rs3219175), SNP+299 (rs3745367), and SNP+1263 (rs3745369) (P<10⁻¹³ in all cases). SNP-638, SNP -420, SNP-358, and SNP+157 were in the same linkage disequilibrium (LD) block. SNP-358 and SNP-638 were nearly in complete LD (r² = 0.98), and were tightly correlated with SNP-420 (r² = 0.50, and 0.51, respectively). The correlation between either SNP-358 (or SNP-638) or SNP-420 and plasma resistin appeared to be strong (risk alleles for high plasma resistin; A at SNP-358, r² = 0.5224, P = 4.94×10⁻³²⁴; G at SNP-420, r² = 0.2616, P = 1.71×10⁻¹³³). In haplotypes determined by SNP-420 and SNP-358, the estimated frequencies for C-G, G-A, and G-G were 0.6700, 0.2005, and 0.1284, respectively, and C-A was rare (0.0011), suggesting that subjects with A at −358, generally had G at −420. This G-A haplotype conferred the highest plasma resistin (8.24 ng/ml difference/allele compared to C-G, P<0.0001). In THP-1 cells, the RETN promoter with the G-A haplotype showed the highest activity. Nuclear proteins specifically recognized one base difference at SNP-358, but not at SNP-638. Therefore, A at -358 is required for G at −420 to confer the highest plasma resistin in the general Japanese population. In Caucasians, the association between SNP-420 and plasma resistin is not strong, and A at −358 may not exist, suggesting that SNP-358 could explain this ethnic difference.     

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.     

Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using φ29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, “Magnetospirillum magneticum AMB-1,” whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.Magnetotactic bacteria synthesize nanosized intracellular magnetic particles, also referred to as magnetosomes, by accumulating iron ions from the environment. Since the first report on the identification of magnetotactic bacteria (2), the morphological and phylogenetic diversity of these organisms has been observed in various aquatic environments (12, 25, 27, 30). However, bacterial strains available in pure culture are currently limited to a few genera. Desulfovibrio magneticus strain RS-1 is the only isolate of magnetotactic bacteria that is classified among the Deltaproteobacteria (13, 23), while Magnetospirillum spp., marine magnetic vibrio strain MV-1, and “Magnetococcus strain MC-1” are phylogenetically affiliated within the Alphaproteobacteria group (24, 27). This limitation is mainly because not much is known about their metabolic requirements, culturing conditions, and obligate coculture requirements.Isolation and enrichment of magnetotactic bacteria are generally conducted by applying a magnetic field to a container containing a sediment sample from the environment. The capillary racetrack method is a highly selective enrichment technique that separates magnetotactic bacteria from other contaminants (31). The magnetic separation method that involves the use of a large glass apparatus is efficient and suitable for analyzing samples containing more than 100 ml of sediment and water (12, 16). These techniques have been applied to investigate community structure and phylogenetic diversity of uncultured magnetotactic bacteria in the environment based on 16S rRNA analyses (3, 7, 26, 29). In a recent study, DNA isolation enabling gene cloning was examined by magnetically collecting a large number of magnetotactic cells from environmental samples, and two gene fragments, probably containing parts of magnetosome islands (MAIs) derived from magnetotactic bacteria of the Alphaproteobacteria, were identified (12). However, this approach allows only for sequence gene information to be obtained from a heterogeneous bacterial community in the sample.Multiple displacement amplification (MDA) can generate microgram quantities of high-quality DNA sample from a few femtograms of DNA template (5, 6). We previously revealed that MDA is a powerful tool for whole-genome amplification from the metagenome of an uncultured bacterial community (32). Studies have been conducted to determine the efficacy of MDA for analyzing genomic DNA preparations from a limited number of bacterial cells (14, 17, 21, 22, 28). Complete genomic sequencing of an uncultured gut symbiont in termites has been achieved using MDA products amplified from approximately 1,000 cells (9). Partial genome sequencing using MDA products from a single uncultured cell has also been reported (17, 22). Such targeted genome analyses using MDA products from a single cell or genetically identical microorganisms is advantageous because it allows the assignment of individual genes to the corresponding microorganisms.In this study, an improved genome preparation method involving racetrack purification and flow cytometry followed by MDA was investigated by using a small number of uncultured magnetotactic bacteria. This method can be used for the identification of new genes from rare magnetotactic bacteria in environmental samples.     

FGF receptor gene expression and its regulation by FGF signaling during early zebrafish development

The expression of all four fgfr genes was extensively examined throughout early embryogenesis of the zebrafish (Danio rerio). fgfr1 alone was expressed maternally throughout the blastoderm, and then zygotically in the anterior neural plate and presomitic mesoderm. fgfr4 expression was first detected in late blastulae and was gradually restricted to the brain. fgfr2 and fgfr3 expression were initiated in early and late gastrulae, respectively; fgfr2 was expressed in the anterior neural plate and somitic mesoderm, whereas fgfr3 was activated in the axial mesoderm and then in the midbrain and somitic mesoderm. During somitogenesis, each of these fgfr genes was expressed in a characteristic manner in the brain. Using an FGF signal inhibitor, dominant-negative FGF receptors and fgf8.1/fgf8a mutants, we found that fgfr expression is directly or indirectly regulated by FGF signaling during epiboly and at the end of somitogenesis, revealing the presence of an autoregulatory mechanism.     

Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

Cell–cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.     

Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to verotoxins

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 microg of VT-2 or 1.0 microg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.