Tissue-specific activity of the pro-opiomelanocortin gene promoter.

The pro-opiomelanocortin (POMC) gene is specifically expressed in corticotroph cells of the anterior pituitary. To define the POMC promoter sequences responsible for tissue-specific expression, we assessed POMC promoter activity by gene transfer into POMC-expressing pituitary tumor cells (AtT-20) and fibroblast L cells. The rat POMC promoter was only efficiently utilized and correctly transcribed in AtT-20 cells. 5'-End deletion analysis revealed two promoter regions required for activity in AtT-20 cells. When tested by fusion to a heterologous promoter, DNA fragments corresponding to both regions exhibited tissue-specific activity, suggesting the presence of at least two tissue-specific DNA sequence elements within the promoter. In summary, POMC promoter sequences from -480 to -34 base pairs appear sufficient to mimic the specificity of anterior pituitary expression.

Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription.

Glucocorticoids rapidly and specifically inhibit transcription of the pro-opiomelanocortin (POMC) gene in the anterior pituitary, thus offering a model for studying negative control of transcription in mammals. We have defined an element within the rat POMC gene 5'-flanking region that is required for glucocorticoid inhibition of POMC gene transcription in POMC-expressing pituitary tumor cells (AtT-20). This element contains an in vitro binding site for purified glucocorticoid receptor. Site-directed mutagenesis revealed that binding of the receptor to this site located at position base pair -63 is essential for glucocorticoid repression of transcription. Although related to the well-defined glucocorticoid response element (GRE) found in glucocorticoid-inducible genes, the DNA sequence of the POMC negative glucocorticoid response element (nGRE) differs significantly from the GRE consensus; this sequence divergence may result in different receptor-DNA interactions and may account at least in part for the opposite transcriptional properties of these elements. Hormone-dependent repression of POMC gene transcription may be due to binding of the receptor over a positive regulatory element of the promoter. Thus, repression may result from mutually exclusive binding of two DNA-binding proteins to overlapping DNA sequences.

Comparison of N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid in the regulation of retinoid receptor-mediated gene expression in human breast cancer cell lines.

The activities of N-(4-hydroxyphenyl)retinamide [(4-HPR), Fenretinide] and all-trans-retinoic acid (RA) were determined for (a) the inhibition of cell proliferation; (b) the activation of human retinoid receptor-mediated target gene expression; (c) the inhibition of estradiol- and progesterone-induced gene activation in breast cancer cell lines; and (d) the regulation of the expression of tumor suppressor retinoblastoma protein. Similar to RA, both 4-HPR and its active metabolite N-(4-methoxyphenyl)retinamide (4-MPR) effectively impeded the growth of MCF7 and T-47D human breast cancer cell lines, except that 4-HPR also inhibited the proliferation of RA-resistant BT-20 cells. However, when tested in human recombinant retinoic acid receptor (RAR-alpha, RAR-beta, and RAR-gamma)-induced reporter gene assays, RA was much more potent (>100-fold) than either 4-HPR or 4-MPR. 4-HPR induced transcriptional activation through all three RAR subtypes at 1-10microM, while RA showed comparable activity at 10-100microM. Despite the apparent weak interaction at the RAR level, 4-HPR was comparable to RA in the inhibition of both estrogen receptor- and progesterone receptor-mediated transcriptional activation in MCF7 and T-47D cells, respectively. Moreover, similar to RA, 4-HPR and 4-MPR caused marked up-regulation of tumor suppressor retinoblastoma protein in both MCF7 and T-47D cells. Since RA and 4-HPR showed comparable activity in the inhibition of estrogen recptor- and progesterone receptor-induced gene transcription and in the stimulation of retinoblastoma protein expression in MCF7 and T-47D cells, the reduced RAR activation by 4-HPR may result in the lack of hepatic toxicity and therefore the improved therapeutic efficacy relative to RA.

Differential regulation of human progesterone receptor A and B form-mediated trans-activation by phosphorylation.

Hormone-dependent phosphorylation of progesterone receptors (PRs) plays a functional role in their transcriptional activity. However, hormone-independent phosphorylation has also been shown to modulate the chicken PR-mediated trans-activation in the presence of phosphorylating agents. The present study was designed to investigate the effects of protein kinase A- and protein kinase C-mediated signal transduction pathways on the regulation of the activity of the two forms of human PR (hPRA and hPRB). Similar to chicken PR, hPR was activated by 8-bromo-cAMP (8-Br-cAMP) in the absence of ligand, whereas 8-Br-cAMP synergized with the progestin agonist R5020 to amplify hPRA- and hPRB-mediated reporter activity. Interestingly, the effect of 8-Br-cAMP was much more pronounced on hPRA-induced trans-activation than on hPRB. This differential regulation by 8-Br-cAMP could also be mimicked by okadaic acid. Both mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyl transferase and progesterone response element-thymidine kinase-chloramphenicol acetyl transferase showed a similar response to 8-Br-cAMP in the presence of R5020. Protein kinase C, on the other hand, did not discriminate between hPRA- and hPRB-mediated trans-activation. Unlike 8-Br-cAMP, phorbol 12-myristate 13-acetate did not cause marked ligand-independent trans-activation through either of the two receptor forms. RU486, an antagonist of progestin, preferentially blocked R5020-induced trans-activation compared to R5020 + 8-Br-cAMP synergism. As expected, H-89, a specific inhibitor of protein kinase A was more effective in inhibiting ligand-independent activity. Western analysis of transfected receptors suggested that 8-Br-cAMP and 8-Br-cAMP + R5020 but not R5020 alone down-regulated the level of hPRB in COS-1 cells. Only marginal modulation of hPRA levels was observed with R5020 treatment in the presence and absence of 8-Br-cAMP. These data suggest that R5020 and 8-Br-cAMP mediate PR-dependent transactivation through distinct pathways, and that phosphorylation can differentially regulate the activity of hPRA and hPRB forms, an observation which may be important for selective target gene activation in vivo by progestins.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase: a new class of antiviral agents.

We have been investigating a new class of antiviral compounds effective against herpes simplex virus (HSV) in vitro and in vivo. Antiviral activity results from inhibition of HSV ribonucleotide reductase (RR). The inhibitors are designed as mimics of the RR small subunit C-terminus, a region essential for RR subunit association and consequently enzymatic activity. Inhibition results from specific binding of the inhibitor to the HSV RR large subunit thereby preventing subunit association. This report details the structure--activity studies that lead to the indentification of BILD 1263, a potent inhibitor of HSV RR subunit association (IC50, 0.2 nM) that also inhibits the replication of HSV types 1 and 2 in cell culture (EC50, 3 and 4 microM) and reduces the severity of HSV-1-induced keratitis in a murine ocular model. The discovery of inhibitors with in vitro antiviral results from a combination of improving inhibitor potency in a RR binding assay and modifying inhibitor physicochemical properties. The importance and possible role of the new structural modifications introduced into this inhibitor series is discussed.

beta-Lactam derivatives as inhibitors of human cytomegalovirus protease.

The development of novel monobactam inhibitors of HCMV protease incorporating a carbon side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at the C-3 position of the beta-lactam ring gave an increase in enzymatic activity and in stability; however, a lack of selectivity against other serine proteases was noted. The use of both tri- and tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl substitution of the urea moiety was beneficial, especially when strong electron-withdrawing groups where attached at the para position. Modest antiviral activity was found in a plaque reduction assay.

Ureido-based peptidomimetic inhibitor of herpes simplex virus ribonucleotide reductase: an investigation of inhibitor bioactive conformation.

We have been investigating peptidomimetic inhibitors of herpes simplex virus (HSV) ribonucleotide reductase (RR). These inhibitors bind to the HSV RR large subunit and consequently prevent subunit association and subsequent enzymatic activity. This report introduces a new series of compounds that contain an extra nitrogen (a ureido function) at the inhibitor N-terminus. This nitrogen improves inhibitor binding potency 50-fold over our first published inhibitor series. Evidence supports that this improvement in potency results from a new hydrogen-bonding contact between the inhibitor and the RR large subunit. This report also provides evidence for the bioactive conformation around two important amino acid residues contained in our inhibitors. A tert-butyl group, which contributes 100-fold to inhibitor potency but does not directly bind to the large subunit, favors an extended beta-strand conformation that is prevalent in solution and in the bound state. More significantly, the bioactive conformation around a pyrrolidine-modified asparagine residue, which contributes over 30 000-fold to inhibitor potency, is elucidated through a series of conformationally restricted analogues.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase with improved in vivo antiviral activity.

We have been investigating the potential of a new class of antiviral compounds. These peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus (HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replication of viral DNA. The compounds disclosed in this paper build on our previously published work. Structure-activity studies reveal beneficial modifications that result in improved antiviral potency in cell culture in a murine ocular model of HSV-induced keratitis. These modifications include a stereochemically defined (2,6-dimethylcyclohexyl)amino N-terminus, two ketomethylene amide bond isosteres, and a (1-ethylneopentyl)amino C-terminus. These three modifications led to the preparation of BILD 1351, our most potent antiherpetic agent containing a ureido N-terminus. Incorporation of the C-terminal modification into our inhibitor series based on a (phenylpropionyl)valine N-terminus provided BILD 1357, a significantly more potent antiviral compound than our previously published best compound, BILD 1263.

Peptidomimetic inhibitors of the human cytomegalovirus protease.

The development of peptidomimetic inhibitors of the human cytomegalovirus (HCMV) protease showing sub-micromolar potency in an enzymatic assay is described. Selective substitution of the amino acid residues of these inhibitors led to the identification of tripeptide inhibitors showing improvements in inhibitor potency of 27-fold relative to inhibitor 39 based upon the natural tetrapeptide sequence. Small side chains at P1 were well tolerated by this enzyme, a fact consistent with previous observations. The S2 binding pocket of HCMV protease was very permissive, tolerating lipophilic and basic residues. The substitutions tried at P3 indicated that a small increase in inhibitor potency could be realized by the substitution of a tert-leucine residue for valine. Substitutions of the N-terminal capping group did not significantly affect inhibitor potency. Pentafluoroethyl ketones, alpha,alpha-difluoro-beta-keto amides, phosphonates and alpha-keto amides were all effective substitutions for the activated carbonyl component and gave inhibitors which were selective for HCMV protease. A slight increase in potency was observed by lengthening the P1' residue of the alpha-keto amide series of inhibitors. This position also tolerated a variety of groups making this a potential site for future modifications which could modulate the physicochemical properties of these molecules.

The NF-kappa B inhibitor, tepoxalin, suppresses surface expression of the cell adhesion molecules CD62E, CD11b/CD18 and CD106.

Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.

Clinical and biochemical heterogeneity in females of a large pedigree with ornithine transcarbamylase deficiency due to the R141Q mutation.

A large family with ornithine transcarbamylase deficiency due to mutation R141Q was ascertained through a propositus who presented with acute neonatal hyperammonemic coma. Of 13 females at risk, 11 were evaluated clinically and had laboratory studies performed. Seven were found to be heterozygous for the mutation. Of these seven, five had chronic clinical symptoms and two were asymptomatic. None of the heterozygotes had elevated plasma ammonia on random testing. Of the five symptomatic females, three had markedly elevated plasma glutamine levels on random testing, while two had levels in the upper range of normal. Plasma citrulline and arginine levels were somewhat lower in the symptomatic individuals but still within the normal range. Five heterozygotes who were tested had either spontaneous orotic aciduria or elevated orotic acid following ingestion of allopurinol, whereas one unaffected female and one unaffected male had normal allopurinol tests. A higher than expected proportion of female heterozygous for the R141Q mutation were clinically and biochemically symptomatic but remained undiagnosed for many years. Plasma glutamine determination and allopurinol testing should be performed in females who present with a combination of relatively non-specific symptoms detailed in this report.

Novel intracellular signaling function of prostaglandin H synthase-1 in NF-kappa B activation.

Many potent nonsteroidal antiinflammatory drugs (NSAIDs) exert their effects by inhibiting the cyclooxygenase activity of prostaglandin H synthase-1 (PGHS1, thus disrupting prostaglandin biosynthesis. However, these drugs do not block the activation of NF-kappa B, an inducible transcription factor which regulates numerous inflammation-related genes. Here we demonstrate that PGHS1 peroxidase, a NSAID-insensitive activity of PGHS1, mediates NF-kappa B activation through an intracellular reactive oxygen signaling pathway. Overexpression of PGHS1 strongly potentiated NF-kappa B activation by phorbol esters and dramatically elevated the generation of intracellular reactive oxygen species (ROS) in response to low concentrations of t-butyl peroxide. Both functions were dependent on PGHS1 peroxidase activity and could be suppressed by the potent antioxidant pyrrolidine dithiocarbamate. In contrast, elimination of PGHS1 cyclooxygenase activity by NSAIDs or site-directed mutagenesis failed to block ROS production or NF-kappa B activation. Thus, PGHS1 peroxidase serves an intracellular signaling function leading to NF-kappa B activation, separable from its role in prostaglandin synthesis.

Potent beta-lactam inhibitors of human cytomegalovirus protease.

A series of novel monobactam inhibitors of human cytomegalovirus (HCMV) protease has been described that possess a heterocyclic thiomethyl side chain at C-4. Changes to the heterocycle did not significantly change the inhibitory activity of these compounds in an enzymatic assay, although improvements in solubility and cell culture activity were noted. A number of permutations between C-4 substitutions and N-1 derivatives led to the identification of several beta-lactams with antiviral activity in a plaque reduction assay. N-methyl thiotetrazole-containing compounds were found to be the most potent inhibitors in the enzymatic assay.

A comparative study of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes in foods.

Nineteen laboratories across Canada took part in a comparative study of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes in foods and environmental samples. The results show that the enrichment period of the FDA method can be shortened from 7 to 2 days without substantially reducing the number of positive samples. With a limited number of samples, the USDA method proved to be slightly more efficient in isolating L. monocytogenes than the FDA method. Fraser broth, in principle, proved to be useful as a screening tool but is not very selective. Oxford agar and lithium chloride-phenylethanol-moxalactam medium were better than modified McBride's agar in isolating this microorganism.

A comparison study of the EF-18 agar/Hydrophobic Grid Membrane Filter (HGMF) method and the enzyme linked antibody (ELA)/HGMF method to the HPB standard method in the isolation of Salmonella.

As part of a comparative and collaborative study of rapid methods for the detection of Salmonella and the standard Health Protection Branch (HPB) method, six Federal and Provincial Laboratories compared the EF-18 agar/Hydrophobic Grid Membrane Filters (HGMF) method to the standard HPB method. Two Federal Laboratories also compared the enzyme linked antibody (ELA)/HGMF method (which is a further development of the EF-18 agar/HGMF method) to the standard method. During this study the false-negative rates ranged from 0% to 15% for the standard HPB method, from 5.88% to 43.5% for the EF-18 agar/HGMF method, and from 0% to 10.5% for the ELA/HGMF method. The EF-18 agar/HGMF method did not compare well with the standard HPB method due to the number of false-negatives. Problems with this method resulted from the inability to isolate colonies of Salmonella on the HGMF due to the small colony size, abnormal colony coloration, and overgrowth by competitors. The ELA/HGMF method, however, was shown to be comparable to the standard HPB method. The main advantages of this method are that the antibody-staining step is independent of colony coloration and carbohydrate utilization on the plating media; the ability to detect some unusual strains of Salmonella irrespective of their atypical reactions on the media; and the ELA staining can indicate the presence of Salmonella even when the HGMF is overgrown by competitors. Also, cultural confirmation can proceed simultaneously yet independently of the ELA staining procedure. The data presented here indicate that this method is worth further study.

Reproductive health and work: different experiences.

Quebec and Denmark passed legislation in 1981 that aims to protect pregnant women and their unborn children from health hazards in the workplace. Finland passed similar legislation in 1991. While these measures have much in common, they are applied in very different ways. Approximately 1% of pregnant working women benefit from a "special maternity leave" in Denmark, compared with 0.1% in Finland, while in Quebec, nearly 40% of working women benefit from preventive reassignment measures during their pregnancies. In this article, we will analyze the reasons for these disparities between rates of utilization in Finland, Denmark, and Quebec in light of the Quebec experience over the last 16 years.

D-lactate dehydrogenase. Substrate specificity and use as a catalyst in the synthesis of homochiral 2-hydroxy acids.

This note compares the substrate specificity of D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) to that of L-lactate dehydrogenase (L-LDH, EC 1.1.1.27), illustrates three procedures that use D-LDH in synthesis and two methods for recycling NADH, and provides experimental details illustrating the use of D-LDH in organic synthesis.

Prior red blood cell transfusions in cancer patients increase the risk of subsequent transfusions with or without recombinant human erythropoietin management.

Cancer patients often receive transfusions when their hemoglobin concentration falls to dangerously low levels due to chemotherapy or due to the disease itself. The availability of recombinant human erythropoietin (rHuEPO) has significantly reduced transfusion frequencies in cancer patients. However, the predictability of transfusions prior to the use of rHuEPO for future transfusions has not been evaluated. Data from five randomized, double-blind, placebo-controlled trials in cancer patients receiving chemotherapy and epoetin alfa were utilized to calculate the relative risk of subsequent transfusions in patients who were pretransfused. A meta-analysis with patient-level data was used to assess predictors of transfusion. Baseline data from an open-label study were used to compare quality-of-life (QOL) parameters between previously transfused and transfusion-naive patients. The mean relative risks (RR) of exposure to additional transfusion for pretransfused patients on placebo or epoetin alfa were 2.14 (95% confidence interval [CI]: 1.73, 2.65) and 2.51 (95% CI: 1.92, 3.27), respectively, compared with nontransfused patients. Data from the meta-analysis of patients on epoetin alfa showed that pretransfusion was the most significant predictor for subsequent transfusions (parameter estimate = -1.2628, p < 0.0001 from Logistic Regression Analysis). While epoetin alfa was similarly effective in reducing transfusion risks for patients with or without pretransfusions (compared with placebo), those who were pretransfused were more than twice as likely to be subsequently transfused, compared with those not pretransfused. QOL was significantly worse for pretransfused patients than for nontransfused patients, as measured by the Functional Assessment of Cancer Therapy -Anemia and the Linear Analogue Scale Assessment QOL instruments. The results suggest that transfusions prior to epoetin alfa therapy increase the risk of future transfusions, and early treatment with epoetin alfa might reduce the risk of subsequent transfusions.

[The prevalence of malocclusion problems and orthodontic treatment needs in 13 and 14-year old Quebec school children in 1983-1984]. / La prévalence des problèmes de malocclusion et les besoins de traitements orthodontiques des écoliers québécois de 13 et 14 ans en 1983-1984.

This article details the problems related to occlusion of 13-14 year old Quebec students. The data was derived from a province wide probability sample of 1201 children in 1983-84 using Granger's Orthodontic Treatment Priority Index (TPI). The principal conclusion are: 32% of the children are in Angle's class II; 18% have an overjet of 5 mm and over; 50% have one or more teeth in minor or major displacement; treatment is mandatory or highly desirable for 13.7% and only 2.9% of the students are under treatment. The results are compared to 6 surveys of the same nature.