Early obesity leads to increases in hepatic arginase I and related systemic changes in nitric oxide and <Emphasis Type="SmallCaps">l</Emphasis>-arginine metabolism in mice

Obesity is a risk factor for vascular endothelial cell dysfunction characterized by low-grade, chronic inflammation. Increased levels of arginase I and concomitant decreases in l-arginine bioavailability are known to play a role in the pathogenesis of vascular endothelial cell dysfunction. In the present study, we focused on changes in the systemic expression of arginase I as well as l-arginine metabolism in the pre-disease state of early obesity prior to the onset of atherosclerosis. C57BL/6 mice were fed a control diet (CD; 10% fat) or high-fat diet (HFD; 60% fat) for 8 weeks. The mRNA expression of arginase I in the liver, adipose tissue, aorta, and muscle; protein expression of arginase I in the liver and plasma; and systemic levels of l-arginine bioavailability and NO₂ ^? were assessed. HFD-fed mice showed early obesity without severe disease symptoms. Arginase I mRNA and protein expression levels in the liver were significantly higher in HFD-fed obese mice than in CD-fed mice. Arginase I levels were slightly increased, whereas l-arginine levels were significantly reduced, and these changes were followed by reductions in NO₂ ^? levels. Furthermore, hepatic arginase I levels positively correlated with plasma arginase I levels and negatively correlated with l-arginine bioavailability in plasma. These results suggested that increases in the expression of hepatic arginase I and reductions in plasma l-arginine and NO₂ ^? levels might lead to vascular endothelial dysfunction in the pre-disease state of early obesity.     

DNA Gyrase of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> is characterized as Type II bacterial topoisomerase and its activity is differentially regulated by PprA in vitro

The multipartite genome of Deinococcus radiodurans forms toroidal structure. It encodes topoisomerase IB and both the subunits of DNA gyrase (DrGyr) while lacks other bacterial topoisomerases. Recently, PprA a pleiotropic protein involved in radiation resistance in D. radiodurans has been suggested for having roles in cell division and genome maintenance. In vivo interaction of PprA with topoisomerases has also been shown. DrGyr constituted from recombinant gyrase A and gyrase B subunits showed decatenation, relaxation and supercoiling activities. Wild type PprA stimulated DNA relaxation activity while inhibited supercoiling activity of DrGyr. Lysine133 to glutamic acid (K133E) and tryptophane183 to arginine (W183R) replacements resulted loss of DNA binding activity in PprA and that showed very little effect on DrGyr activities in vitro. Interestingly, wild type PprA and its K133E derivative continued interacting with GyrA in vivo while W183R, which formed relatively short oligomers did not interact with GyrA. The size of nucleoid in PprA mutant (1.9564 ± 0.324 µm) was significantly bigger than the wild type (1.6437 ± 0.345 µm). Thus, we showed that DrGyr confers all three activities of bacterial type IIA family DNA topoisomerases, which are differentially regulated by PprA, highlighting the significant role of PprA in DrGyr activity regulation and genome maintenance in D. radiodurans.     

Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation

Molecular and Cellular Biochemistry - Calcium ion (Ca2+) uptake into the mitochondrial matrix influences ATP production, Ca2+ homeostasis, and apoptosis regulation. Ca2+ uptake across the...     

Aluminum tolerance in a spermidine synthase-overexpressing transgenic European pear is correlated with the enhanced level of spermidine via alleviating oxidative status

Aluminum (Al) stress is a major cause of poor crop yields, particularly in those countries where acid soil predominate. To verify whether polyamine can confer Al tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. ‘Ballad’) line #32 overexpressing apple spermidine synthase (MdSPDS1) and the wild type (WT) were subjected to long-term stress for 30 μM AlCl₃. Based on net increment of shoot height (SHI) or fresh weight (FWI) and morphological changes upon the stress, the performance of line #32 was much better than that of WT. Although SPDS expression levels and spermidine (Spd) titers in line #32 were higher than those in WT, firmly due to the transgene (MdSPDS1) expression, no further induction of SPDS expression was observed from the long-term Al stress trial in both lines. While, Spd titers were considerably increased in both lines after the stress. The activities of superoxide dismutase (SOD) or glutathione reductase (GR) and the accumulation of proline or malondialdehyde (MDA) altered upon this stress toward a more favorable status for survival in the transgenic line #32 than in WT. These antioxidant parameters were closely related to Spd titer. Concentrations of calcium (Ca) and some co-factor metals of SOD in line #32 were diversely higher than that in WT after the stress. These evidences indicate that Spd is implicated in elevating of Al stress tolerance of the transgenic line #32 chiefly via ameliorating oxidative status as well as by affecting mineral element balance.     

Efficient production of androgenetic embryos by round spermatid injection

Mammalian androgenetic embryos can be produced by pronuclear exchange of fertilized oocytes or by dispermic in vitro fertilization of enucleated oocytes. Here, we report a new technique for producing mouse androgenetic embryos by injection of two round spermatid nuclei into oocytes, followed by female chromosome removal. We found that injection of round spermatids resulted in high rates of oocyte survival (88%). Androgenetic embryos thus produced developed into mid‐gestation fetuses at various rates, depending on the mouse strain used. All the fetuses examined maintained paternally specific genomic imprinting memories. This technique also enabled us to produce complete heterozygous F1 embryos by injecting two spermatids from different strains. The best rate of fetal survival (12% per embryos transferred) was obtained with C57BL/6 × DBA/2 androgenetic embryos. We also generated embryonic stem cell lines efficiently with the genotype of Mus musculus domesticus × M. m. molossinus. Thus, injection of two round spermatid nuclei followed by maternal enucleation is an effective alternative method of producing androgenetic embryos that consistently develop into blastocysts and mid‐gestation fetuses. genesis 47:155–160, 2009. © 2009 Wiley‐Liss, Inc.     

The Effect on Intracytoplasmic Sperm Injection Outcome of Genotype,Male Germ Cell Stage and Freeze-Thawing in Mice

Background

Intracytoplasmic sperm injection (ICSI) has been widely used to study the mechanisms of mammalian fertilization and to rescue male-factor infertility in humans and animals. However, very few systematic analyses have been conducted to define factors affecting the efficiency of ICSI. In this study, we undertook a large-scale series of ICSI experiments in mice to define the factors that might affect outcomes.

Methodology/Principal Findings

We used a 5×3×2 factorial design with the following factors: mouse genotype (ICR, C57BL/6, DBA/2, C3H/He, and 129/Sv strains), type of male germ cells (epididymal sperm, elongated or round spermatids), and their freeze–thawing treatment. The efficiencies (parameters) of each developmental step were analyzed by three-way ANOVA (significance level P<0.01). The type of male germ cells affected all the four parameters observed: oocyte survival after injection, cleavage of oocytes, implantation, and birth of offspring. Genotype affected the oocyte survival, cleavage and birth rates, whereas freeze–thawing had no effects on any of the parameters. There were significant genotype/cell type interactions for oocyte survival and cleavage, indicating that they were determined by a combination of strain and germ cell maturity. Multiple comparisons revealed that spermatozoa and elongated spermatids gave better implantation and birth rates than did round spermatids, while spermatozoa and elongated spermatozoa were indistinguishable in their ability to support embryonic development. The best overall efficiency (birth rate per oocytes injected) was obtained with frozen–thawed DBA/2 strain elongated spermatids (23.2±4.2%).

Conclusions/Significance

The present study provides the first comprehensive information on ICSI using the mouse as a model and will contribute to the efficient use of materials, time, and efforts in biomedical research and clinics involving ICSI.     

Role of IP-10/CXCL10 in the progression of pancreatitis-like injury in mice after murine retroviral infection

Exocrinopathy and pancreatitis-like injury were developed in C57BL/6 (B6) mice infected with LP-BM5 murine leukemia virus, which is known to induce murine acquired immunodeficiency syndrome (MAIDS). The role of chemokines, especially CXCL10/interferon (IFN)-gamma-inducible protein 10 (IP-10), a chemokine to attract CXCR3+ T helper 1-type CD4+ T cells, has not been investigated thoroughly in the pathogenesis of pancreatitis. B6 mice were inoculated intraperitoneally with LP-BM5 and then injected every week with either an antibody against IP-10 or a control antibody. Eight weeks after infection, we analyzed the effect of IP-10 neutralization. Anti-IP-10 antibody treatment did not change the generalized lymphadenopathy and hepatosplenomegaly of mice with MAIDS. The treatment significantly reduced the number of IP-10- and CXCR3-positive cells in the mesenteric lymph nodes (mLNs) but not the phenotypes and gross numbers of cells. In contrast, IP-10 neutralization reduced the number of mononuclear cells infiltrating into the pancreas. Anti-IP-10 antibody treatment did not change the numbers of IFN-gamma+ and IL10+ cells in the mLN but significantly reduced their numbers, especially IFN-gamma+ and IL-10+ CD4+ T cells and IFN-gamma+ Mac-1+ cells, in the pancreas. IP-10 neutralization ameliorated the pancreatic lesions of mice with MAIDS probably by blocking the cellular infiltration of CD4+ T cells and IFN-gamma+ Mac-1+ cells into the pancreas at least at 8 wk after infection, suggesting that IP-10 and these cells might play a key role in the development of chronic autoimmune pancreatitis.     

Peptidic HIV integrase inhibitors derived from HIV gene products: Structure–activity relationship studies

Structure–activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an α-helical conformation, Glu-Lys pairs were introduced into the i and i + 4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an α-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.     

Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

The C1b domain of protein kinase Cδ (PKCδ), a potent receptor for ligands such as diacylglycerol and phorbol esters, was synthesized by utilizing native chemical ligation. With this synthetic strategy, the domain was efficiently constructed and shown to have high affinity ligand binding and correct folding. The C1b domain has been utilized for the development of novel ligands for the control of phosphorylation by PKC family members. This strategy will pave the way for the efficient construction of C1b domains modified with fluorescent dyes, biotin, etc. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.