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Bromomethane(CH3Br) is an acutely toxic environmentalpollutant that contributes to ozone depletion. Molecular simulationcould be a valuable tool for studying its partitioning and transportin the environment if an accurate molecular model was available. Thegeneralized Amber force field (GAFF), OPLS (optimized potentials forliquid simulations) force field, and CHARMM general force field (CGenFF)were tested for their ability to model the physical properties ofliquid bromomethane. The OPLS force field was in fairly good agreementwith experiment, while CGenFF and GAFF were significantly in error.The Br Lennard-Jones parameters of the GAFF and CGenFF models werereparameterized, but their radial distribution functions still havesignificant deviations from those calculated by ab initio moleculardynamics (AIMD). A Drude polarizable force field for bromomethanewas parametrized with an off-center positively charged site to representthe C–Br σ-hole. This model is in good agreement withthe bulk physical properties and the AIMD RDFs. The modest solubilityof bromomethane was reproduced by this model, with dispersion interactionsbeing the dominant water–solute interaction. The water–soluteelectrostatic interactions are a smaller factor in solubility. Thismodel predicts bromomethane to have a 13 kJ mol–1surface excess potential at the water–vapor interface. [ABSTRACT FROM AUTHOR]

Thin films cast from binary blends of structurally homologous polystyrene-block-poly(2-vinylpyridine) polymers were used to obtain horizontal arrays of linear nanostructures which were visualized by metallizing the poly(2-vinylpyridine) blocks with a tetrachloroplatinate salt. By varying the blend compositions of the homologous block copolymers, fine control over the periodicity of lines was realized from ∼25 to 55 nm using a set of just 4 block copolymers. For neat block copolymers whose equilibrium structures are not horizontal cylinders, blending enabled cylindrical structures to form. The ordering in various films was studied by measurements of defect density, and it was found that in many cases blended films produced patterns of lower defect density than patterns formed from single component block copolymers. Annealing of the polymer films was carried out using a solvothermal microwave annealing technique able to rapidly produce few-defect films. Here the technique is adapted to use a household microwave oven (cost < $100) to rapidly induce self-assembly in under 2 min, enabling broad accessibility. [ABSTRACT FROM AUTHOR]

The synthesis and characterization of triphenylphosphinimine−arylimine bidentate ligands (aryl = 2,4,6-trimethylphenyl or 4-methylphenyl) along with their dichloropalladium(II) complexes are reported. These complexes form tridentate orthopalladated species upon heating in the presence of sodium acetate. The reverse reaction occurs upon addition of HCl·Et2O. The rate of orthopalladation and reprotonation is highly dependent on the steric bulk of the ligand. Related cationic species were also synthesized and characterized and found to be inert in the presence of 1-hexene and styrene. [ABSTRACT FROM AUTHOR]

Summary.  Using microdialysis, we have monitored extracellular levels of amino acids and related amines in the human liver at three stages of the transplantation procedure: donor retrieval, back table preparation and during 48 h post-implantation. By comparing the ratio of mean levels at the donor and back table stages, with the ratio between early (2–6 h) and late (43–48 h) post-reperfusion, these amines were classified into one of three groups. In one group, back table levels were markedly higher than during the donor stage, with levels declining over time post-reperfusion. A second group had much lower levels in the back table than during the donor phase, and post-reperfusion levels were either stable or increased over time. Concentrations of amino acids in the final group remained relatively constant at all stages. This study illustrates the value of microdialysis in providing organ-specific metabolic data that may indicate specific mechanisms of poor graft function. [ABSTRACT FROM AUTHOR]

One major obstacle in mammalian embryo culture has been unidentifiable biological contaminants in the media due to the inclusion of Bovine Serum Albumin (BSA) or Fetal Bovine Serum. The goal of this study was to remove BSA from culture media and develop chemically defined media based off the embryo''s biological and physiologic makeup. We evaluated the presence of message in various stages of porcine embryos and found that the message for the ionic glutamate receptor, N-Methyl-D-aspartic acid (NMDA) increased about 3-fold from oocyte to blastocyst. Thus, this study was conducted to determine if the addition of NMDA (0.5 mm) would improve development of embryos in an already chemically defined medium. Slaughterhouse derived ovaries were aspirated, cumulus–oocyte complexes were identified and then matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris buffered medium with 0.25 × 106mL-1frozen–thawed porcine semen for 5 h. Presumptive zygotes were then transferred to Porcine Zygote Medium with 0.3% BSA (PZM3) or 0.1% PVA (PZM4). After 28 h, cleaved embryos were selected and embryos were placed into treatment groups: (1) PZM3, (2) PZM4, or (3) PZM4 + 0.5 mmNMDA. Embryos were cultured in 5% CO2, 5%O2, 90% N2until Day 7. For this experiment the number of cleaved embryos cultured in each treatment group were 260 for group 1, 220 for group 2 and 300 for group 3. Percentage of development to blastocyst was determined and analyzed with SAS Proc GENMOD Procedure (a,b P< 0.05). The percentage developed to blastocyst was (1) 47.5% a, (2) 29.6% b, and (3) 36.1% a,b, respectively. Total cell number of the blastocysts was determined by using Hoechst nuclear stain and statistically analyzed by SAS Proc GENMOD Procedure. The average cell number for the treatment groups was (1) 25.8 a, (2) 19.6 b, and (3) 22.9 a,b, respectively. Culture without BSA significantly reduced development to blastocyst and total cell number; however, with the addition of 0.5 mmNMDA there was no significant difference from media containing BSA. This indicates that NMDA can be used to partially replace BSA to form a chemically defined media.Funded by a grant from the USDA NRI 2006-35203-17282. [ABSTRACT FROM AUTHOR]

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher''s exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitroand in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al.2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitroand in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nmand 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nmScriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P< 0.05). Percent cleaved was not different among four treatment groups. We used 500 nmScriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P< 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nmScriptaid treatment following activation increase both the in vitroand in vivodevelopment of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT.Funding from the National Institutes of Health National Center for Research Resources RR018877. [ABSTRACT FROM AUTHOR]

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 8.2% (169/208) and 62.1 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 5.8% (53/208) and 25.2 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 1.9 (NT, maximum was 58) and 16.8 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT. [ABSTRACT FROM AUTHOR]