SOLiD SAGE sequencing shows differential gene expression in jejunal lymph node samples of resistant and susceptible red deer (Cervus elaphus) challenged with Mycobacterium avium subsp. paratuberculosis.

This study compared in vivo lymph node gene expression levels between six young red deer that were either relatively resistant (R) or susceptible (S) to paratuberculosis following experimental challenge with Mycobacterium avium subsp. paratuberculosis. Intestinal lymph nodes were biopsied at 4, 12 and 50 weeks post challenge (pc) and parallel changes in histopathology, immunology and bacterial load monitored. SOLiD SAGE (serial analysis of gene expression) next generation sequencing of biopsied lymph node samples generated a total of 373 million transcript tags 26-28bp in length after filtering. A total of 36,632 unique transcripts were identified and 14,325 of these were able to be annotated. The copy number of each transcript was counted, averaged and compared for R and S animals (R-S). P values and False Discovery Rates (FDR) were calculated for each transcript. Genes differentially upregulated ≥2 fold (FDR<0.5) totalled 9, 40 and 32 in R animals (+ values) and 23, 164 and 47 in S animals (- values) at weeks 4, 12, and 50pc, respectively. Transcripts displaying greatest differential expression between R and S animals at each time point were IFIT2 (189 fold) and S100A8 (-32.7 fold) at week 4, LRR1 (52.7 fold), SERPINF2 (-214.6 fold) at week 12 and CEACAM8 (84.6 fold), and STK31 (-129.5 fold) at week 50, respectively. All 9 genes significantly upregulated at week 4 in R animals relate specifically to host defence and all involve Type I interferon stimulated genes. By contrast genes upregulated in S animals at week 4, relate predominantly to inflammation, but also involve adaptive immune responses, mitochondrial function and apoptosis regulation. At week 12, the genes differentially upregulated in R animals are linked predominantly to regulation of adaptive immunity and mucosal immunity, while many of the genes in S animals are associated with pro-inflammatory interleukins involved with innate and adaptive immunity. These correlated with greater lesion severity and higher MAP numbers in lymph nodes of S animals. By week 50 the number of upregulated genes declined in both groups. A number of genes upregulated in R animals appear to be associated with host resistance and regulation of adaptive immunity, especially CEACAM8. Genes upregulated in S animals involve antigen presentation (ENDOD1) and gut associated immune pathology (HSH2D). In conclusion, gene expression in jejunal lymph nodes of resistant and susceptible deer infer that the resistant phenotype is associated with pathways of adaptive immunity, while susceptibility is linked with upregulated non-protective pro-inflammatory responses, following experimental MAP infection.

Actionable recommendations in the Bright Futures child health supervision guidelines.

BACKGROUND: With the growing use of electronic health record systems, there is a demand for an electronic version of the leading American pediatric preventive care guideline, Bright Futures. As computer implementation requires actionable recommendations, it is important to assess to what degree Bright Futures meets criteria for actionability. OBJECTIVES: We aimed to 1) determine the number of actionable recommendations in the current edition of Bright Futures and 2) to recommend a specific format for representing an important class of guidelines in a way that better facilitates computer implementation. METHODS: We consolidated all action statements in Bright Futures into recommendations. We then used two dimensions (decidability and executability) in the Guideline Implementability Appraisal v 2.0 (GLIA) to determine the actionability of the recommendations. Decidability means the recommendation states precisely under what conditions to perform those actions. Executability means actions are stated specifically, unambiguously and in sufficient detail. The results were presented in a figure titled Service Interval Diagram (SID), describing actionable recommendations, age intervals during which they are applicable, and how frequently they should occur in that interval. RESULTS: We consolidated 2161 action items into 245 recommendations and identified 52 that were actionable (21%). Almost exclusively, these recommendations addressed screening, such as newborn metabolic screening, or child safety, such as car seat use. A limited number (n=13) of recommendations for other areas of anticipatory guidance were also actionable. No recommendations on child discipline, family function or mental health met our criteria for actionability. The SID representing these recommendations is presented in a figure. CONCLUSION: Only a portion of the Bright Futures Guidelines meets criteria for actionability. Substantial work lies ahead to develop most recommendations for anticipatory guidance into a computer implementable format.

Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates.

Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.

Using expressed sequence tag databases to identify ovarian genes of interest.

GenBank contains 4879 expressed sequence tags (EST) derived from four non-normalized human ovarian cDNA libraries. Of these EST, 2646 are contributors to UniGene clusters and have UniGene numbers. The EST map to 1206 distinct UniGenes. A gene expression profile was established for the human ovary by identifying the abundance of each UniGene cluster and its corresponding annotation. The most highly expressed transcripts were for proteins associated with protein synthesis (ribosomal proteins, elongation factors, thymosins, etc.). However, there are also transcripts for genes of unknown function that are ovary-specific. This ovarian gene expression profile provides useful data for the design of DNA microarrays targeted at ovarian function and highlights novel sequences that warrant further investigation.

A set of 1542 mouse blastocyst and pre-blastocyst genes with well-matched human homologues.

GenBank contains 57,151 Expressed Sequence Tags (EST) derived from 11 preimplantation embryo mouse cDNA libraries ranging from the 2-cell embryo to the blastocyst. EST were matched to UniGene clusters to identify a composite set of 11,291 UniGenes. These 11,291 UniGenes were screened using HomoloGene to identify a subset of 3467 mouse UniGenes with matches in at least two other species, one of which was human. Of the 3467 matches, 1542 are for named human proteins. Four of the 11 preimplantation embryo libraries were for blastocysts and contain 22,307 EST. These blastocyst EST generate 5762 UniGenes, of which 2246 have matches in at least two other species. Of the 2246 matches, 1170 are for named human proteins. Comparison of the expression profile of the blastocyst set with a similarly derived set from the mouse oocyte identified a number of transcripts that are significantly up-regulated during preimplantation development. The set of named blastocyst and pre-blastocyst genes complements the similar set published recently for the mouse oocyte. They provide a database for identifying signalling pathways that may play a role in determining cell fate in preimplantation embryo development.

Gene expression profiling of human GV oocytes: an analysis of a profile obtained by Serial Analysis of Gene Expression (SAGE).

A gene expression profile of the human GV oocyte has recently been established by Serial Analysis of Gene Expression (SAGE). A significant number of the genes identified in this profile had not previously been associated with mammalian oocytes. We sought to confirm gene matches by RT-PCR amplification of candidate transcripts using mouse eggs. Attention focused on receptors, proteins involved in apoptosis, and cytoskeletal proteins. Two receptors found in the human catalogue, CCR6 and PAR3, were not found in mouse eggs, whereas myosin light chain, LLGL, beta-actin, 5HT receptor, bad, bak, DFF45, and Caspase homologue (cash) were. Individual SAGEtags can match more than one gene and, in some cases, more than ten. Examination of transcript sequences that generate multiple gene assignments identified a common denominator of short interspersed elements or Alu sequences. For reasons which are, as yet, unclear, the human GV oocyte SAGE catalogue contains relatively high abundances of SAGEtags in Alu sequences. This may reflect normal expression of Alu-containing genes in eggs or upregulated expression of Alu elements following stress. The degeneracy of gene matches in SAGE generated by Alu sequences makes independent confirmation of candidate genes essential.

A set of 840 mouse oocyte genes with well-matched human homologues.

GenBank contains 14 477 expressed sequence tags (EST) derived from mouse oocyte cDNA libraries: 3499 of these are from two unfertilized oocyte libraries and 10 978 are from two fertilized oocyte libraries. Gene expression profiles were obtained for these libraries by matching library EST to UniGene clusters. The 14 477 EST identified 4226 UNIGENES: These were screened using HomoloGene to identify 1386 homologous UniGene clusters in two other species with one of the matches being human. Within these human matches, 840 encoded named proteins, 223 encoded hypothetical proteins, and 323 encoded clustered EST. The set of named genes provides the first step in establishing a database of genes expressed in mouse oocytes and, by extension, human oocytes.

Meta-analysis of gene expression in mouse preimplantation embryo development.

Mammalian preimplantation development is characterized by a number of major events. These potentially involve significant but transient changes in early embryonic gene expression. We have undertaken a meta-analysis of gene expression in mouse preimplantation development using a set of 71 346 expressed sequence tags (EST) derived from 15 non-normalized cDNA libraries. These libraries span seven stages of development from the unfertilized oocyte to the blastocyst stage. EST were clustered using UNIGENE: The 71 346 EST identified 11 483 separate genes, of which 1585 are not found elsewhere in the mouse. Aggregate sets of EST for each of the seven stages were analysed for differences in gene expression using Fisher's exact test. This analysis identified 109 genes that were differentially expressed. Some of these genes were associated with degradation of transcripts at the 1-cell stage whereas other genes underwent increased expression at the blastocyst stage. The set of 11 483 genes identified in mouse preimplantation embryo development provides the starting point for the design of DNA microarrays targeted at early mammalian embryogenesis. By anchoring the analysis of mouse preimplantation development in UniGene, it will be possible to identify homologous genes that are likely to be involved in human preimplantation embryo development.

Synthesis and biological activity of phenoxyphenyl oxamic acid derivatives related to L-thyronine.

The synthesis of substituted phenoxyphenyl oxamic acid derivatives related to L-thyronine (L-T3) is described. The in vitro and in vivo cholesterol lowering and cardiovascular effects of these compounds are presented and discussed.

Anilides of (R)-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

The optimization of a series of anilide derivatives of (R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase (PDHK) is described that started from N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1 (IC(50) = 35 +/- 1.4 microM). It was found that small electron-withdrawing groups on the ortho position of the anilide, i.e., chloro, acetyl, or bromo, increased potency 20-40-fold. The oral bioavailability of the compounds in this series is optimal (as measured by AUC) when the anilide is substituted at the 4-position with an electron-withdrawing group (i.e., carboxyl, carboxyamide, and sulfoxyamide). N-(2-Chloro-4-isobutylsulfamoylphenyl)-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionamide (10a) inhibits PDHK in the primary enzymatic assay with an IC(50) of 13 +/- 1.5 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts, lowers blood lactate levels significantly 2.5 and 5 h after oral doses as low as 30 micromol/kg, and increases the ex vivo activity of PDH in muscle, kidney, liver, and heart tissues. However, in contrast to sodium dichloroacetate (DCA), these PDHK inhibitors did not lower blood glucose levels. Nevertheless, they are effective at increasing the utilization and disposal of lactate and could be of utility to ameliorate conditions of inappropriate blood lactate elevation.

Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy.

BACKGROUND AND METHODS: Although potent antiretroviral therapy can control infection with human immunodeficiency virus type 1 (HIV-1), a long-lived reservoir of infectious virus persists in CD4+ T cells. We investigated this viral reservoir by measuring the levels of cell-associated viral DNA and messenger RNA (mRNA) that are essential for HIV-1 replication. Approximately every 6 months, we obtained samples of peripheral-blood mononuclear cells from five men with long-standing HIV-1 infection who had had undetectable levels of plasma HIV-1 RNA for 20 months or more during treatment with potent antiretroviral drugs. RESULTS: Before treatment, plasma levels of HIV-1 RNA correlated with the levels of cell-associated unintegrated HIV-1 DNA and unspliced viral mRNA. After treatment, plasma levels of HIV-1 RNA fell by more than 2.7 log to undetectable levels. The decrease in cell-associated integrated and unintegrated HIV-1 DNA and mRNA occurred in two phases. The first phase occurred during the initial 500 days of treatment and was characterized by substantial decreases in the levels of DNA and mRNA, but not to undetectable levels. The concentrations of cell-associated unintegrated viral DNA, integrated proviral DNA, and unspliced viral mRNA decreased by 1.25 to 1.46 log. The second phase occurred during the subsequent 300 days or more of treatment and was characterized by a plateau in the levels of HIV-1 DNA and unspliced mRNA. After an initial rapid decline, the ratio of unspliced to multiply spliced viral mRNA (a measure of active viral transcription) stabilized and remained greater than zero at each measurement. CONCLUSIONS: Despite treatment with potent antiretroviral drugs and the suppression of plasma HIV-1 RNA to undetectable levels for 20 months or more, HIV-1 transcription persists in peripheral-blood mononuclear cells. Unless the quasi-steady state levels of HIV DNA and mRNA eventually disappear with longer periods of therapy, these findings suggest that HIV-1 infection cannot be eradicated with current treatments.

Characterisation of the chicken Cu,Zn superoxide dismutase gene.

A PCR product was generated from embryonic chicken spinal cord cDNA using primers designed to conserved regions of the human and bovine amino and carboxyl-terminal coding sequences of the Cu,Zn superoxide dismutase (SOD1, EC 1.15.1.1) gene. DNA sequencing confirmed this product to be the chicken homologue of the SOD1 gene. This sequence was compared to SOD1 from bovine, human and Xenopus laevis. Important structural features of SOD1 are shown to be conserved in the chicken gene.

Consumption of sugars.

The mean percent of energy from total sugars minus lactose is 18% in the United States, according to data from the 1987-1988 US Department of Agriculture Nationwide Food Consumption Survey. When sugars intake is distributed among food pyramid groupings, the primary contributor is the "others" group (39%). The relationship between sugars intakes and micronutrients was age and sex dependent. Consumers of high amounts of sugars do not necessarily have poorer quality diets. In the European Union, the mean percent energy from all sugars is 15.2%. The top five sources of sugar contributed 68% of sugar intake but only 11% of fat intake (UK data). Although sugars intake varies among these major developed regions, the consistent inverse relation between fat and sugars intake and the scarcity of individuals achieving dietary guidelines raises serious questions regarding current dietary recommendations.

Synthesis and structure-activity relationships of oxamic acid and acetic acid derivatives related to L-thyronine.

Aryloxamic acids 7 and 23, (arylamino)acetic acids 29, arylpropionic acids 33, arylthioacetic acids 37, and (aryloxy)acetic acid 41 related to L-triiodothyronine (L-T3) were prepared and tested in vitro for binding to the rat liver nuclear L-T3 receptor and the rat membrane L-T3 receptor. The structure-activity relationships for these compounds are described, with 7f, 23a, 29c, 33a, 37b, and 41 showing excellent potency (IC50's of 0.19, 0.16, 1.1, 0.11, 3.5, and 0.10 nM, respectively) to the nuclear receptor and significantly lower binding affinity to the membrane receptor (IC50's > 5 microM). Some of these compounds, especially in the oxamic acid series 7 and 23, showed an unprecedented potency for methyl-substituted derivatives such as 7f and 23a. Compounds 7f and 23a showed good lipid lowering effects in rats with ED50's of 20 and 5 micrograms/kg po, respectively, and a lack of cardiac side effects in rats at doses as high as 10 and 25 mg/kg po, respectively.

Effects of metalloprotease inhibitors on the conversion of proendothelin-1 to endothelin-1.

The IC50 values of phosphoramidon, CGS 25015, CGS 26129, thiorphan and benazeprilat for inhibition of endothelin converting enzyme partially purified from porcine aortic endothelial cells were 3.5, 18, 58, > 100 and > 100 microM, respectively. A similar rank order of potency was observed for inhibition of the proendothelin-1 (proET-1) -induced pressor response in the rat where phosphoramidon, CGS 25015, CGS 26129, thiorphan and benazeprilat at 30 mg/kg i.v. produced 65, 57, 27, 12, and 0% inhibition, respectively. A slightly different rank order of potency was obtained in the proET-induced contraction of porcine coronary arteries where IC50 values of < 10, 10-30, 10-30, 30-100 and 30-100 microM were exhibited by CGS 25015, CGS 26129, phosphoramidon, thiorphan and benazeprilat, respectively. These data indicate that the endothelin converting enzymes in the three systems studied are similar, except that phosphoramidon is a slightly more potent inhibitor in the in vitro assay and the in vivo pressor test than in the smooth muscle contraction assay.

Heterocyclic lactam derivatives as dual angiotensin converting enzyme and neutral endopeptidase 24.11 inhibitors.

A series of 13- and 14-membered ring lactam derivatives 9a,b, 10, 11, and 12a-c was prepared from L-cysteine. Compounds 9a,b and 12a,b were tested in vitro for inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) inhibition. The structure-activity profile of the series is discussed. Compound 9b, a 13-membered ring macrocyclic lactam, had an NEP IC50 of 18 nM and an ACEIC50 of 12 nM in vitro and showed dual plasma inhibition after intravenous or oral administration.

Substituted chromenes as potent, orally active 5-lipoxygenase inhibitors.

A series of chromene derivatives was synthesized and evaluated for their in vitro and ex vivo 5-lipoxygenase (5-LO) inhibitory activity. These compounds were prepared by condensation of appropriate salicyl aldehydes with alpha, beta-unsaturated carbonyl compounds, followed by transformation to the corresponding hydroxamic acids or N-hydroxyureas. Placement of phenoxy or p-fluorophenoxy substituents at the 6 position of the chromene ring led to a dramatic increase in the in vitro potency as demonstrated by the guinea pig PMN 5-LO assay. Chromene hydroxamic acids, in general, behaved poorly in the ex vivo dog model. On the other hand, replacement of the hydroxamic acid function with N-hydroxyurea yielded potent and long-lasting 5-LO inhibitors in the dog model. In most cases, the oral efficacy of the chromene N-hydroxyureas correlated very well with their in vitro activity. Compounds 43 (CGS 23885) and 55 (CGS 24891) are among the most potent inhibitors prepared, showing IC50 values of 48 and 51 nM, respectively. The values for the duration of action (DA) for compounds 43 and 55 are 21 and 20 h, respectively, following intravenous (i.v.) administration of 1.0 mg/kg. In the oral (po) experiments, 43 and 55 have DA's of 14 and 15 h, respectively, at a 1.0 mg/kg dose. In both iv and po experiments, 43 and 55 showed sustained maximal inhibition (> 95%) at earlier time points. The oral ED50 values of 43 and 55 in the ex vivo dog model are 0.23 and 0.23 mg/kg, respectively, at 6.0 h, and 2.37 and 1.63 mg/kg, respectively, at 24 h. Compound 43, which inhibits sheep seminal vesicle cyclooxygenase (CO) with an IC50 value of 36 microM, was shown to be a selective 5-lipoxygenase inhibitor in the ex vivo study. These compounds compare favorably with zileuton (A-64077) in all the parameters examined.

A game plan for regional marketing.

Should your company consider taking a niche-marketing approach to fragmented national markets? Regional marketing is not for every firm or for every product or service. Here are the pluses to consider, as well as the pitfalls that must be overcome, before wading into regional waters.

Inhibition of human phospholipase C by aurothiomalate and (triethylphosphine) gold complexes.

The effect of several gold complexes on the activity of phospholipase C from human blood platelets was studied in vitro. Aurothiomalate and auranofin--2 agents used for the chrysotherapy of rheumatoid arthritis--, gold chloride, (triethylphosphine)gold chloride, and 5 newly synthesized (triethylphosphine)gold complexes dose-dependently inhibited the enzyme with IC50 values between 0.8 mumol/l ((triethylphosphine)gold chloride) and over 100 mumol/l (auranofin). None of the compounds affected phospholipase A2 from human polymorphonuclear leukocytes at concentrations up to 100 mumol/l. Inhibition of phospholipase C by (triethylphosphine)gold chloride, aurothiomalate, and compound 3 was not significantly different at substrate concentrations of 20 and 100 mumol/l phosphatidylinositol, suggesting that these gold complexes do not inhibit phospholipase C by competing with the substrate. After confirming the Ca2+ dependence of the human platelet phospholipase C by demonstrating inhibition by the Ca2+ chelators EDTA and EGTA--but no inhibition by the Zn2+ chelator 1,10-phenanthroline--the inhibition of the enzyme by (triethylphosphine)gold chloride, aurothiomalate, and compound 3 was studied at 3 different concentrations of Ca2+ in the range of 0.2 to 2 mmol/l. Inhibition by (triethylphosphine)gold chloride was not affected by changes of Ca2+, whereas inhibition by aurothiomalate and compound 3 was markedly suppressed by increasing the Ca2+ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)