α-1 acid glycoprotein inhibits insulin responses by glucose oxidation, protein synthesis and protein breakdown in mouse C2C12 myotubes.

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor 14C-glucose oxidation or to measure protein synthesis by incorporation of 3H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P0.05, n=6 trials), whereas incubation with both AGP and insulin reduced 3H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.

Regulation of fetuin A gene expression in the neonatal pig liver.

Fetuin A (also known as α2-Heremans-Schmid glycoprotein) is a protein primarily expressed by the liver and secreted into the blood. Previous studies have suggested that plasma concentrations of fetuin A are elevated with impaired growth rate in swine. The present study was designed to examine the relationship of porcine fetuin A with growth rate in the pig and to also elucidate the regulation of fetuin A expression by examining the hormonal and cytokine regulation of fetuin A mRNA abundance in hepatocytes prepared from suckling piglets. Quantitative real-time PCR assay was used to quantify the number of fetuin A mRNA molecules/molecule cyclophilin mRNA. Total RNA was isolated from liver of three different groups of pigs to assess changes in mRNA abundance of fetuin A: normal piglets at day 1, day 7 day 21 or 6 months of age (n=6 for each age); runt and control piglets at day 1 of age (n=4); slow growing and normal growing piglets at 21 days of age (n=8). Following birth, fetuin A gene expression increased from day 1 and 7 of age (P<0.05), and then declined at 21 days of age (P<0.05), with a much greater decline to 6 months of age (P<0.01). Fetuin A mRNA abundance was higher in runt pigs v. their normal birth weight littermates (P<0.05). Similarly, fetuin A gene expression was higher in livers of pigs that were born at a normal weight but that grew much slower than littermates with the same birth weight (P<0.05). Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h to permit examination of the influences of hormones, cytokines and redox modifiers on fetuin A mRNA abundance. Fetuin A gene expression was enhanced by glucagon, T3 and resveratrol (P<0.05). Growth hormone, cytokines (interleukin6, tumor necrosis factor-α) and antioxidants (N-acetylcysteine, quercertin) reduced fetuin A mRNA abundance (P<0.05). A role for fetuin A in postnatal development is suggested by the differences in fetuin A mRNA abundance between runt piglets or slow growing piglets and their normal growing sized littermates. The hepatocyte experiments suggest multiple hormones and cytokines may contribute to the regulation of fetuin A during early growth of the pig.

A sandwich ELISA for porcine alpha-1 acid glycoprotein (pAGP, ORM-1) and further demonstration of its use to evaluate growth potential in newborn pigs.

A simple, reproducible sandwich, ELISA was developed to measure porcine alpha-1 acid glycoprotein (pAGP, ORM-1) in pig plasma. Porcine AGP isolated from serum was purchased and a polyclonal antisera was prepared in rabbits using the whole pAGP molecule as immunogen. The antiserum was affinity purified, and a portion of the purified antibody fraction was labeled with horseradish peroxidase. Porcine AGP protein was used as a standard, whereas commercially available buffers and reagents were utilized throughout the assay. The assay was specific for pAGP, had a lower limit of detection of 3.2 ng/mL, and could be used to quantify pAGP in plasma or serum. Using this ELISA, we corroborated our previous findings obtained by RID assay, which demonstrated that the AGP concentration in newborn piglets is negatively associated with preweaning growth rate. The current data were obtained using piglets from a different geographical location and genetic background and showed that elevated AGP at birth was associated with reduced preweaning growth rate (P < 0.001, r = 0.433, n = 19 litters). In addition, litters with a greater average AGP at birth were at a growth disadvantage compared with litters with reduced average AGP plasma concentrations (P < 0.001, r = 0.708, n = 19 litters). Litter average plasma AGP was a better predictor of litter preweaning growth rate than average litter birth weight. The data represent further support for using perinatal AGP concentrations as a tool to identify potential slower growing pigs and as a plasma biomarker for predicting litter growth rate.

Predicting pre- and postoperative pain of endodontic origin in a southern Brazilian subpopulation: an electronic database study.

AIM: To determine the prevalence of preoperative endodontic pain (PREP) and the incidence of postoperative endodontic pain (POEP), identifying the predictors of PREP and POEP in a southern Brazilian subpopulation, using clinical data from an electronic chart database (ECD). METHODOLOGY: This retrospective observational study included 563 consecutive individuals presenting for root canal treatment (RCT). Patients were treated by undergraduate and graduate students, following standard RCT protocols. Demographic, medical and dental variables were extracted from a pre-structured and standardized ECD. The main outcomes PREP and incident POEP were collected through a 0-10 numeric rating scale, dichotomized as none/mild (<4) or moderate/severe (≥4) pain. Predictive models calculating the prevalence ratios (PR) of PREP and the relative risks (RR) of incident POEP were carried out with Poisson regression analysis, estimating the relationship between clinical factors, PREP and incident POEP. RESULTS: Mean age at baseline was 49.2 ± 17.1 years, with 68.4% women. The prevalence and incidence of moderate/severe PREP and POEP were 44.4% and 3.8%, respectively. RCT intervention significantly reduced PREP (P < 0.001). Multivariate analysis revealed that group of teeth, location (mandibular teeth), pulpitis, necrotic pulp, preoperative swelling and periapical radiolucency were independently associated with moderate/severe PREP, whilst age ≥60 years and root canal retreatments were independent protective factors to PREP (P < 0.05). No demographic, medical or dental variables were associated with POEP, although molar teeth (RR = 4.23, 95%CI = 0.93-19.2, P = 0.056) had a borderline nonsignificant association. CONCLUSIONS: Moderate/severe PREP was independently associated with age, group of teeth, location, preoperative swelling, retreatments and pulp and periapical status. No demographic, medical or dental variable predicted moderate/severe POEP following RCT amongst this subpopulation.

α 1-acid glycoprotein inhibits lipogenesis in neonatal swine adipose tissue.

Serum α1-acid glycoprotein (AGP) is elevated during late gestation and at birth in the pig and rapidly declines postnatally. In contrast, the pig is born with minimal lipid stores in the adipose tissue, but rapidly accumulates lipid during the first week. The present study examined if AGP can affect adipose tissue metabolism in the neonatal pig. Isolated cell cultures or tissue explants were prepared from dorsal subcutaneous adipose tissue of preweaning piglets. Porcine AGP was used at concentrations of 0, 100, 1000 and 5000 ng/ml medium in 24 h incubations. AGP reduced the messenger RNA (mRNA) abundance of the lipogenic enzymes, malic enzyme (ME), fatty acid synthase and acetyl coA carboxylase by at least 40% (P<0.001). The activity of ME and citrate lyase were also reduced by AGP (P<0.05). Glucose oxidation was reduced by treatment with 5000 ng AGP/ml medium (P<0.05). The 14C-glucose incorporation into fatty acids was reduced by ~25% by AGP treatment for 24 h with 1000 ng AGP/ml medium (P<0.05). The decrease in glucose metabolism by AGP appears to function through an inhibition in insulin-mediated glucose oxidation and incorporation into fatty acids. This was supported by the analysis of the mRNA abundance for sterol regulatory element-binding protein (SREBP), carbohydrate regulatory element-binding protein (ChREBP) and insulin receptor substrate 1 (IRS1), which all demonstrated reductions of at least 23% in response to AGP treatment (P<0.05). These data demonstrate an overall suppression of lipogenesis due to AGP inhibition of lipogenic gene expression in vitro, which the metabolic data and SREBP, ChREBP and IRS1 gene expression analysis suggest is through an inhibition in insulin-mediated events. Second, these data suggest that AGP may contribute to limiting lipogenesis within adipose tissue during the perinatal period, as AGP levels are highest for any serum protein at birth.

Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture.

Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

Identification and characterization of a nuclear factor-κ B-p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture.

Hepatic responses to proinflammatory signals are controlled by the activation of several transcription factors, including, nuclear factor-κ B (NF-κB). In this study, hepatocytes prepared from suckling pigs and maintained in serum-free monolayer culture were used to define a novel proinflammatory cytokine-specific NF-κB subunit modification. The immunoreactive p65 protein was detected by Western blot analysis at the appropriate molecular weight in the cytosol of control cultures and those incubated with tumor necrosis factor-α (TNF). However, in nuclei, the p65 antisera cross-reacted with a protein of approximately 38 kDa (termed p38) after TNF addition, which was not observed in the cytosol of control or cytokine-treated cells. Specifically, incubation with TNF also resulted in phosphorylation (P < 0.05) of the inhibitor complex protein (IκB), whereas incubation with other cytokines, IL-6, IL-17a, or oncostatin M was not associated with either phosphorylation of IκB or nuclear translocation of p65. Intracellular endothelial nitric oxide synthase was deceased (P < 0.05) and plasminogen activator inhibitor-1 secretion was increased (P < 0.05) after TNF incubation. The TNF-induced p38 protein was purified from hepatocyte nuclei by immunoprecipitation, concentrated by electrophoresis, and subsequently analyzed by mass spectrometry. Ten unique NF-κB p65 peptides were identified after digestion with trypsin and chymotrypsin; however, all were mapped to the N-terminus and within the first 310 amino acid residues of the intact p65 protein. Although low molecular weight immunoreactive p65 molecules were previously observed in various human and rodent systems, this is the first report to positively identify the p38 fragment within hepatocyte nuclei or after specific cytokine (TNF) induction.

Methyl-ß-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue.

This study was designed to determine whether methyl-ß-cyclodextrin (MCD) can substitute for albumin in incubation medium for neonatal swine adipose tissue explants, or whether MCD affects metabolism and cytokine expression. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21-day-old pigs. Explants were incubated in medium 199 supplemented with 25 mM HEPES, 1.0 nM insulin at 37°C. The medium also contained bovine serum albumin (BSA) or MCD at 0%, 0.05%, 0.1%, 0.2% or 0.3%. Tissue explants were treated with these media for 1 h and then switched to the same basal incubation medium containing 0.05% BSA. Explants were removed from basal medium at 2 or 8 h of incubation, and real-time PCR was performed to assess expression of tumor necrosis α (TNF) and interleukin 6 (IL6), acetyl CoA carboxylase (ACAC) and fatty acid synthase (FASN). Alternatively, rates of 14C-glucose oxidation and lipogenesis were monitored ± insulin (100 nM), following MCD treatment. Incubation with BSA had minimal effects on gene expression or adipose tissue metabolism, only producing a doubling in TNF mRNA abundance (P < 0.01). Treatment with MCD increased TNF mRNA abundance by eightfold (P < 0.009), whereas IL6 gene expression increased a 100-fold (P < 0.001) with a suppression in ACAC and FASN expression (P < 0.01). This was paralleled by MCD inhibition of insulin-stimulated glucose oxidation and lipogenesis (P < 0.001). Addition of a TNF antibody to the incubation medium alleviated this inhibition of insulin-stimulated glucose metabolism by ~30% (P < 0.05).

Twenty years of embryonic stem cell research in farm animals.

Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that an ESC can maintain an undifferentiated state indefinitely, self-renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the terminally differentiated cells of a conceptus. These attributes make the ESC an ideal source for genome editing for both agricultural and biomedical applications. Although, ESC lines have been successfully established from rodents and primates, authentic ungulate stem cell lines on the contrary are still not available. Outstanding issues including but not limited to differences in pluripotency characteristics among the existing ESC lines, pre-implantation embryo development, pluripotency pathways, and culture conditions plague our efforts to establish authentic ESC lines from farm animals. In this review, we highlight some of these issues and discuss how the recent derivation of induced pluripotent stem cells (iPSCs) might augur the establishment of robust authentic ESC lines from farm animals.

Iron dextran treatment does not induce serum protein carbonyls in the newborn pig.

Oxidation of serum proteins can lead to carbonyl formation that alters their function and is often associated with stress-related diseases. As it is recommended that all pigs reared in modern production facilities be given supplemental iron at birth to prevent anemia, and metals can catalyze the carbonylation of proteins, the primary objective of this study was to determine whether standard iron dextran treatment was associated with enhanced serum protein oxidation in newborn piglets. Piglets were treated with 100 mg of iron dextran intramuscularly either on the day of birth, or on the third day after birth. Blood samples were collected from piglets 48 or 96 h after treatment and serum was harvested. For quantification, serum protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and analyzed spectrophotometrically. To identify and determine relative distribution of carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by two-dimensional polyacrylamide gel electrophoresis, stained with avidin-fluorescein and identified by mass spectrometry. The standard iron dextran treatment was associated with no increase in total oxidized proteins if given either on the first or third day of life. In addition, with a few noted exceptions, the overall distribution and identification of oxidized proteins were similar between control and iron dextran-treated pigs. These results indicate that while iron dextran treatment is associated with a marked increase in circulating iron, it does not appear to specifically induce the oxidation of serum proteins.

Oviductal expression of avidin, avidin-related protein-2, and progesterone receptor in turkey hens in relation to sperm storage: effects of oviduct tissue type, sperm presence, and turkey line.

The sperm storage tubules (SST) of the turkey hen, which are located in the uterovaginal junction (UVJ) of the oviduct, maintain viable sperm for up to 10 wk after a single insemination. The mechanisms of this in vivo sperm storage are poorly understood. Our objective was to evaluate mRNA and protein expression of avidin and 2 avidin-associated factors, avidin-related protein-2 (AVR2) and progesterone receptor, in the oviducts of 2 different lines to determine the extent to which they were sperm responsive and tissue specific. At 38 wk of age, Hybrid Grade Maker and Converter turkey hens were artificially inseminated with diluted semen (AI) or were sham-inseminated with extender alone (SI). Forty-eight hours after insemination, total RNA was extracted from the UVJ epithelium (containing SST) and vaginal epithelium (VGE) of SI and AI hens. Real time-polymerase chain reaction data showed a clear tissue region-specific effect on gene expression in the turkey hen oviduct, with much greater (P < 0.0001) expression in the UVJ compared with VGE region for avidin and AVR2 mRNA in both lines and for progesterone receptor mRNA in the Converter line. In contrast to real-time PCR data, in situ hybridization of SI and AI tissues showed that the presence of sperm increased avidin mRNA in the SST and UVJ surface epithelium in the Converter hens. Immunohistochemistry confirmed the presence of avidin protein in the epithelium of the UVJ in both lines; however, whereas avidin protein was localized in the SST of SI-Grade Maker hens, this protein was not detected in the SST of Converter hens. The upregulation of avidin and AVR2 mRNA within the sperm storage region indicates the involvement of avidin, and perhaps avidin analogs, in the sustained storage of sperm in the SST, possibly through the binding of biotin to avidin. The absence of avidin protein in the SST and VGE of Converter hens in the presence of increased mRNA may indicate a rapid turnover of protein.

The effect of intrauterine growth retardation on the expression of developmental factors in porcine placenta subsequent to the initiation of placentation.

Intrauterine growth retardation (IUGR) hinders fetal growth and postnatal development in swine; however the etiology of IUGR is essentially unknown. Expression of fourteen candidate genes associated with placental development or IUGR was examined in gestational day 50 (gd50) control and IUGR fetus whole placental tissue or areolae by real-time PCR. Endothelial nitric oxide synthase (ENOS) mRNA expression was elevated in gd50 IUGR placenta and areola compared to gd50 control. Since ENOS could modulate vascular tone and angiogenesis via nitric oxide production, data suggest that the increase in IUGR may be an adaptive response to poor perfusion to maintain pregnancy.

Theoretical Study of the Reaction Mechanism of Streptomyces coelicolor Type II Dehydroquinase.

The reaction mechanism of a type II dehydroquinase (DHQase) from Streptomyces coelicolor was investigated using molecular dynamics simulation and density functional theory (DFT) calculations. DHQase catalyzes the elimination of a water molecule from dehydroquinate (DHQ), a key step in the biosynthesis of aromatic amino acids in bacteria, fungi, and plants. In the DFT calculations, 10 models, containing up to 230 atoms, were used to investigate different proposals for the reaction mechanism, suggested on the basis of crystal structures and kinetic data. Probing the flexibility of the active site, molecular dynamics simulation reveals that deprotonated Tyr28 can act as the base that catalyzes the first reaction step, the proton abstraction of the pro-S proton at C2 of DHQ, and formation of the enolate intermediate. The computed barrier for the first transition state (TS1), 13-15 kcal/mol, is only slightly affected by the active site model used and is in good agreement with the corresponding experimental barrier of 13.4 kcal/mol for the rate-determining step. The previously proposed enol form of the intermediate is found to be significantly higher in energy than the enolate form and is thus thermodynamically not competitive. In the second and final reaction step, protonation of the hydroxyl group at C1 by His106 followed by water elimination, there is a substantial buildup of dipole moment due to the net transfer of a proton from His106 to Tyr28. A barrier for the second transition state (TS2) that fits well with the corresponding experimental barrier could only be found if the buildup of dipole moment is at least partly compensated during the second reaction step. We speculate that this could be facilitated by regeneration of the Tyr28 anion or by proton transfer to the vicinity of His106 before TS2 is reached. A revised mechanism for type II DHQase is discussed in light of the results of the present calculations.

Enantioseparation of omeprazole and its metabolite 5-hydroxyomeprazole using open tubular capillary electrochromatography with immobilized avidin as chiral selector.

The present paper demonstrates the enantiomeric separation of omeprazole and its metabolite 5-hydroxyomeprazole performed with open tubular capillary electrochromatography (OT-CEC). The protein avidin was used as the chiral selector. Avidin was immobilized by a Schiffs base type of reaction where the protein was via glutaraldehyde covalently bonded to the amino-modified wall of a fused-silica capillary, 50 microm i.d. Both racemates were baseline resolved. Resolution was 1.9 and 2.3, respectively, using ammonium acetate buffer, pH 5.8, 5% methanol, with UV-detection. These values of resolution using OT-CEC are higher than earlier published results regarding chiral separation of omeprazole and 5-hydroxyomeprazole on packed CEC. The number of theoretical plates also indicated good separation efficiency.

Conceptus development during blastocyst elongation in lines of pigs selected for increased uterine capacity or ovulation rate.

Lines of pigs selected for increased uterine capacity have improved conceptus survival, whereas pigs selected for increased ovulation rate have decreased conceptus survival relative to an unselected control line. The objective of this study was to evaluate conceptus development during blastocyst elongation as a potential contributing factor to differences in conceptus survival rate among these pig lines. Conceptuses were recovered from pregnant control, uterine capacity, and ovulation rate line gilts at d 10 and 12 of gestation. At d 10 of gestation, conceptus morphologic diversity was assessed by comparing within-litter average conceptus diameter and the standard deviation of conceptus diameters. At d 12 of gestation, conceptus morphologic diversity was assessed by comparing blastocyst populations obtained from individual gilts. Real-time PCR analyses for transcripts involved in steroidogenesis, cellular differentiation, and immune responsiveness were performed on spherical, ovoid, and filamentous conceptuses recovered from these selection lines. Uterine flushings were also assayed for total protein and estradiol-17beta at d 10 and 12 of gestation. Morphological data were analyzed using ANOVA with the fixed effects of line, farrowing season, and their interactions. Conceptus mortality, uterine flushing, and real-time PCR data were analyzed using ANOVA with the fixed effects of line, day or blastocyst morphology, farrowing season, and their interactions. Conceptus mortality, measured as the ratio of conceptus recovery to ovulation rate, was not different between the lines on d 10 and 12 of gestation. There were no significant line effects for conceptus morphologic diversity at d 10 and 12 of gestation. Expression of transcripts associated with steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and aromatase), cellular differentiation (cytokeratin-18 and vimentin), and immune responsiveness (interleukin-1beta) in spherical, ovoid, and filamentous conceptuses was not different between the lines. Furthermore, protein and estradiol-17beta in uterine flushings at d 10 and 12 of gestation were not different between the selection lines. These findings indicate limited, if any, deviations between these lines of pigs in conceptus development during blastocyst elongation and suggest that mechanisms involved in generating line differences in survival rate likely are manifested later in gestation.

Theoretical study of the reduction of nitric oxide in an A-type flavoprotein.

The mechanism for the reduction of nitric oxide to nitrous oxide and water in an A-type flavoprotein (FprA) in Moorella thermoacetica, which has been proposed to be a scavenging type of nitric oxide reductase, has been investigated using density functional theory (B3LYP). A dinitrosyl complex, [{FeNO}(7)](2), has previously been proposed to be a key intermediate in the NO reduction catalyzed by FprA. The electrons and protons involved in the reduction were suggested to "super-reduce" the dinitrosyl intermediate to [{FeNO}(8)](2) or the corresponding diprotonated form, [{FeNO(H)}(8)](2). In this type of mechanism the electron and/or proton transfers will be a part of the rate-determining step. In the present study, on the other hand, a reaction mechanism is suggested in which N(2)O can be formed before the protons and electrons enter the catalytic cycle. One of the irons in the diiron center is used to stabilize the formation of a hyponitrite dianion, instead of binding a second NO. Cleaving the N-O bond in the hyponitrite dianion intermediate is the rate-determining step in the proposed reaction mechanism. The barrier of 16.5 kcal mol(-1) is in good agreement with the barrier height of the experimental rate-determining step of 14.8 kcal mol(-1). The energetics of some intermediates in the "super-reduction" mechanism and the mechanism proceeding via a hyponitrite dianion are compared, favoring the latter. It is also discussed how to experimentally discriminate between the two mechanisms.

Design and implementation of a point-of-care computerized system for drug therapy in Stockholm metropolitan health region--Bridging the gap between knowledge and practice.

INTRODUCTION: Stockholm County Council is the largest health care provider in Sweden with an annual budget of US$ 5 billion and catering the needs of a metropolitan population of 2 million people. About 10% of health care costs are used on drugs. In 1996 Stockholm County Council decided to address the main problems associated with the process and the quality of drug prescribing. METHODS: A multiyear strategy was designed, including the establishment of a strong evidence-based organisation, Drug and Therapeutics Committees and editorial resources to adapt information to the IT-media and the development of the IT-architecture. The development and implementation of computerized tools such as a physician drug order entry system including decision support, a drug information website and electronic transmission of prescriptions were started in 1996. RESULTS: The implementation was slow at the point-of-care units. It took about 6 years before the implementation process gained speed. In September 2005 almost 1000 doctors could use the decision support system for prescribing drugs and more than 70% of all prescriptions were transmitted electronically in our region. CONCLUSIONS: The work with the strategy has shown that improvements in drug use can be accomplished by providing access to simple, rapid and safe electronic tools, but the information provided has to be associated with well-recognized regional and national expert organisations.

Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates.

Embryonic stem (ES) cell lines provide an invaluable research tool for genetic engineering, developmental biology and disease models. These cells can be maintained indefinitely in culture and yet maintain competence to produce all the cells within a fetus. While mouse ES cell lines were first established over two decades ago and primate ES cells in the 1990 s, validated ES cell lines have yet to be established in ungulates. Why competent, pluripotent ES cells can be established from certain strains of mice and from primates, and not from cows, sheep, goats or pigs is an on-going topic of interest to animal reproduction scientists. The identification of appropriate stem cell markers, functional cytokine pathways, and key pluripotency-maintaining factors along with the release of more comprehensive bovine and porcine genomes, provide encouragement for establishment of ungulate ES cell lines in the near future.

Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression.

In cultured cumulus oocyte complexes (COC), FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and to identify candidate mRNAs involved. Experiment I: murine COC were cultured 4 h in the presence of FSH with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82 +/- 7%). When DRB was added at 0, 5, or 10 min after culture initiation, oocyte maturation was blocked (17 +/- 7, 14 +/- 6, and 21 +/- 6% GVBD, respectively). When DRB was added after 15, 20, or 30 min, progressively more COC underwent GVBD (37 +/- 6, 39 +/- 6, and 66 +/- 6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a threefold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P < or = 0.01 and P

Reduction of nitric oxide in bacterial nitric oxide reductase--a theoretical model study.

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b(3)-Fe(B) active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe(B) consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu(B) in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b(3) and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe(B) (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe(B) the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b(3) taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe(B) will form a mu-oxo bridge to heme b(3) in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable mu-oxo bridge between heme b(3) and Fe(B) is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe(B) in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.