Enhanced North Pacific deep-ocean stratification by stronger intermediate water formation during Heinrich Stadial 1.

The deglacial history of CO2 release from the deep North Pacific remains unresolved. This is due to conflicting indications about subarctic Pacific ventilation changes based on various marine proxies, especially for Heinrich Stadial 1 (HS-1) when a rapid atmospheric CO2 rise occurs. Here, we use a complex Earth System Model to investigate the deglacial North Pacific overturning and its control on ocean stratification. Our results show an enhanced intermediate-to-deep ocean stratification coeval with intensified North Pacific Intermediate Water (NPIW) formation during HS-1, compared to the Last Glacial Maximum. The stronger NPIW formation causes lower salinities and higher temperatures at intermediate depths. By lowering NPIW densities, this enlarges vertical density gradient and thus enhances intermediate-to-deep ocean stratification during HS-1. Physically, this process prevents the North Pacific deep waters from a better communication with the upper oceans, thus prolongs the existing isolation of glacial Pacific abyssal carbons during HS-1.

Antiviral effect of hepatitis B virus S/C gene loci antisense locked nucleic acid on transgenic mice in vivo.

We investigated the effects of hepatitis B virus (HBV) S/C double gene loci antisense locked nucleic acid on replication and expression of HBV in hepatitis transgenic mice. HBV mice (N = 30) were randomly divided into five groups of six mice: 5% glucose solution control, empty liposome control, single-target S, single-target C, and dual-target SC groups. An antisense locked nucleic acid fragment was injected into the mice. Serum HBsAg, serum HBV DNA, HBV C-mRNA expression in liver tissue, HbsAg and HbcAg expression in hepatocytes, serum albumin, alanine transaminase (ALT), urea nitrogen, and creatinine were detected. Liver and kidney sections were examined for the effects of antisense locked nucleic acid. The expression of HBsAg was markedly inhibited; the inhibition rates of the S, C, and SC target groups were 36.63, 31.50, and 54.87%, respectively; the replication of HBV DNA was also inhibited: 23.97, 21.13, and 35.83%, respectively. After injection at 1, 3, and 5 days, the corresponding rates for HBsAg inhibition were 14.40, 25.61, and 31.33%, and for HBV DNA inhibition they were 11.04, 19.24, and 24.13%. Compared with the control group, the differences in serum albumin, ALT, urea nitrogen, and creatinine in each group were not statistically significant, and the number of HbsAg- and HBcAg-positive cells in the mouse liver was significantly reduced. The liver and kidney tissues were normal. The gene therapy had significant inhibitory effects on the replication and expression of HBV in transgenic mice, and double-gene targeting was better than single-gene targeting.

Blocking the expression of the hepatitis B virus S gene in hepatocellular carcinoma cell lines with an anti-gene locked nucleic acid in vitro.

The aim of this study was to investigate the effects of inhibition of the hepatitis B virus (HBV) S gene by polypurine region locked nucleic acid on viral replication in cells. We designed and synthesized a locked nucleic acid, phosphorothioate oligonucleotides, unmodified oligonucleotides, and unrelated control sequence for the hepatitis B virus S gene polypurine region. HepG2.2.15 cells were transfected by cationic liposome, and fluorescence quantitative polymerase chain reaction technology (PCR) and time-resolved fluoroimmunoassay technology was utilized to monitor the content of HBV DNA, HbsAg, and HBeAg at 2, 4, 6, 8 and 10 days post-transfection. The effects on cell metabolism were detected by four methyl thiazolyl tetrazolium assay. The locked nucleic acid had an obvious effect on HBV DNA replication and HBsAg and HBeAg expression in a dose and time dependent man-ner. The inhibition rates were 52.14, 57.48, and 29.63% after 6 days, respectively. The locked nucleic acid had no significant effect on cell metabolism. The HBV S gene polypurine region locked nucleic acid could effectively inhibit the replication of HBV in vitro, and could provide an effective target for the treatment of HBV and a theoretical and experimental basis for anti-gene therapy.

Enhanced angiotensin-converting enzyme 2 attenuates angiotensin II-induced collagen production via AT1 receptor-phosphoinositide 3-kinase-Akt pathway.

Recent reports support a protective role for angiotensin-converting enzyme 2 (ACE2) against glomerular diseases, especially by decreasing of extracellular matrix (ECM) proteins. However, the mechanism regulating this effect appears to be complex and poorly understood. Our aim was to investigate whether or not ACE2 ameliorates the profibrotic effects of Ang II-mediated, Akt-dependent pathways in the mouse mesangial cell line, MES-13.Gene transfer of ACE2 suppressed Ang II-activated Akt-phosphorylation, accompanied by a decreased level of collagen type I in cells. In addition, Ang II-induced collagen type I synthesis in MES-13s by activating the Ang II/AT-1R-PI3K pathway. This transactivation was dependent on cAMP/Epac but not on PKA. TGF-ßRI played a pivotal role in this signaling pathway inducing collagen deposition effects which could be reversed by ACE2 gene transfer in MES-13 cells. The results revealed that gene transfer of ACE2 regulated Ang II-mediated AT1R-TGFßRI-PI3K-Akt signaling and involved the synthesis of collagen. The beneficial effect of ACE2 overexpression appeared to result mainly from blocking phosphorylation of Akt in mesangial cells, suggesting that the ACE2 gene might be a novel therapeutic target for glomerular diseases.

Increased expression of phosphorylated Smad2 and Smad3 in the hippocampus of streptozotocin-induced diabetic rats.

Activation of the Smad signalling pathway has been implicated in the pathological process of diabetic associated complications. The current study was designed to see whether Smad signalling was activated in the hippocampus of streptozotocin-induced diabetic rats. Compared with vehicle-treated controls, immunoblot analysis of hippocampal extracts showed that phosphorylated Smad2 was upregulated at 8 weeks post streptozotocin induction (p<0.01), and phosphorylated Smad3 protein was upregulated at 4 and 8 weeks post streptozotocin induction (p<0.01) in streptozotocin-induced diabetic rats. In addition, immunofluorescence labelling assay showed that the percentage of pSmad2 immunoreactive astrocytes increased significantly in CA1, CA3 and dentate gyrus region (p<0.01), and pSmad3 immunoreactive astrocytes increased significantly in CA1 region (p<0.01) and in CA3 and dentate gyrus region (p<0.05) of the hippocampus in diabetic rats. These data indicate that Smad signalling is enhanced in hippocampal astrocytes of diabetic rats, and may thereby represent a clue to explore its exact role in the development of diabetic encephalopathy.

SSR marker and ITS cleaved amplified polymorphic sequence analysis of soybean x Glycine tomentella intersubgeneric derived lines.

Wild perennial Glycine species are an invaluable gene resource for the cultivated soybean [ Glycine max (L.) Merr., 2 n=40]. However, these wild species have been largely unexplored in soybean breeding programs because of their extremely low crossability with soybean and the need to employ in vitro embryo rescue methods to produce F(1) hybrids. The objective of this study was to develop molecular markers to identify gene introgression from G. tomentella, a wild perennial Glycine species, to soybean. A selection of 96 soybean simple sequence repeat (SSR) markers was evaluated for cross-specific amplification and polymorphism in G. tomentella. Thirty-two SSR markers (33%) revealed specific alleles for G. tomentella PI 483218 (2 n=78). These SSR markers were further examined with an amphidiploid line (2 n=118) and monosomic alien addition lines (MAALs), each with 2 n=40 chromosomes from soybean and one from G. tomentella. The results show that the use of SSR markers is a rapid and reliable method to detect G. tomentella chromosomes in MAALs. We also developed a cleaved amplification polymorphism sequence (CAPS) marker according to the sequences of internal transcribed spacer (ITS) regions in soybean and G. tomentella. Four MAALs that carry the ITS (rDNA) locus from G. tomentella were identified. The SSR and ITS-CAPS markers will greatly facilitate the introgression and characterization of gene transfer from G. tomentella to soybean.

Association of the yellow leaf (y10) mutant to soybean chromosome 3.

At least 19 single recessive gene yellow leaf mutants and one duplicate recessive gene mutant have been described in soybean. This study was conducted to associate a yellow leaf mutant, y10, with a specific soybean chromosome by using primary trisomics (2n = 41). Seven soybean primary trisomics were hybridized as female parent with genetic stock strain, T161, carrying y10. F(1) disomic and primary trisomic plants were identified cytologically. One disomic (control) and all primary trisomic plants were allowed to self-pollinate and F(2) populations were classified for green versus yellow leaf mutant. The F(2) population of Triplo 3 segregated in a 17:1 ratio, while a disomic (3:1) ratio was observed with Triplo 8-, 17-, 18-, and 20-derived F(2) populations, suggesting that the y10 locus is on chromosome 3. The y10 locus was examined with four simple sequence repeat (SSR) markers (Satt584, Sat_033, Satt387, and Satt022) from molecular linkage group (MLG) N and y10 was found linked with Satt022. Therefore we confirmed the association of MLG N with chromosome 3. The possible association of y10 with Triplo 16 and Triplo 19 are discussed.

Assignment of molecular linkage groups to soybean chromosomes by primary trisomics.

Gene-linkage groups (classical linkage groups, CLGs; molecular linkage groups, MLGs) and chromosome relationship in soybean [ Glycine max (L.) Merr., 2n = 40] is not yet established. However, primary trisomics provide an invaluable cytogenetic tool to associate genes and linkage groups to specific chromosomes. We have assigned 11 MLGs to soybean chromosomes by using primary trisomics (2 x + 1 = 41) and SSR markers. Primary trisomics were hybridized with Glycine soja Sieb. and Zucc. (2n = 40) in the greenhouse, F(1) plants with 2n = 40 and 41 were identified cytologically and 41 chromosome plants were selfed. A deviation from the 1:2:1 ratio in the F(2) population suggests a marker is associated with a chromosome. Of the possible 220 combinations involving 20 MLGs and 11 primary trisomics, 151 combinations were examined. The relationships between soybean chromosomes and MLGs are: 1 = D1a+q, 3 = N, 5 = A1, 8 = A2, 9 = K, 13 = F, 14 = C1, 17 = D2, 18 = G, 19 = L and 20 = I. This study sets the stage to establish relationship between nine remaining MLGs with the other genetically unidentified nine primary trisomics. The association of CLGs with the soybean chromosomes will be discussed.

Skeletonization of ribbon-like shapes based on regularity and singularity analyses.

A major problem with traditional skeletonization algorithms is that their results do not always conform to human perceptions since they often contain unwanted artifacts. This paper presents an indirect skeletonization method to reduce these artifacts. The method is based on analyzing regularities and singularities of shapes. A shape is first partitioned into a set of triangles using the constrained Delaunay triangulation technique. Then, regular and singular regions of the shape are identified from the partitioning. Finally, singular regions are stabilized to produce a better result. Experiments show that skeletons obtained from the proposed method closely resemble human perceptions of the underlying shapes.

[Soybean germplasm diversity and genetic variance detected by microsatellite markers].

Microsatellite or Simple Sequence Repeats (SSR) marker is a new type of molecular marker developed recently, which can be used in genotype identification, pedigree analysis, and estimation of genetic distance. Using 5 pairs of SSR primer, 21 polymorphic fragments were obtained in 15 soybean germplasm. The number of alleles at each SSR locus is from 3 to 6, and the range of gene diversity is 0.439-0.668. In addition, analysis of genetic distance was also performed in this study. Pedigree analysis showed that mutation occurred for individuals in the F8 population of RIL after multigeneration meiosis, which was caused because of the changes in the number of repeat unit, to a limited extent.

Estradiol stimulates growth hormone production in female goldfish.

The effects of estradiol (E2) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E2, (100 micrograms/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E2 increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E2-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E2 production. The present results show that E2 can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish.

Structure-function analysis of the p35 subunit of mouse interleukin 12.

Mouse IL-12 acts on both mouse and human cells; human IL-12 acts only on human cells. This species specificity is determined by the p35 subunit of the IL-12 heterodimer. Since mouse and human p35 sequences are 60% identical, the determinants for the species specificity most likely residue in the nonhomologous sequences of mouse p35. To identify the regions on the p35 subunit interacting with the mouse IL-12 receptor, we constructed a series of chimeric mouse-human p35 molecules by replacing mouse sequences with the nonhomologous human counterparts. An IL-12 heterodimer containing a mouse-human p35 chimera with five residues changed in three discontinuous sites had drastically reduced (750-3000-fold) bioactivities on mouse cells. However, the competitive binding activity of the same mutant IL-12 heterodimer on mouse cells was only reduced 30-fold relative to wild-type IL-12. These findings therefore suggest that 1) the mouse p35 subunit participates in both receptor binding and signaling, 2) the mutations introduced into p35 affect signaling to a much greater extent than receptor binding, and 3) the five residues identified on p35 are required for interacting with the mouse, but not with the human IL-12 receptor and as such contribute extensively to the observed species specificity of IL-12.