Effects of calcium oxide treatment of dry and modified wet corn distillers grains plus solubles on growth performance, carcass characteristics, and apparent digestibility of feedlot steers.

The objectives of this study were to determine the effects of feeding dried corn distillers grains (DDGS) or modified wet corn distillers grains (MDGS) with or without CaO treatment to feedlot steers on 1) growth performance and carcass characteristics and 2) diet digestibility, pattern of intake, and meal distribution. In Exp. 1, steers (n = 139; average initial BW = 336 ± 75 kg) were used in a randomized complete block design. Treatments were arranged in a 2 × 2 factorial design, and pens were randomly allotted to 1 of the 4 dietary treatments (DM basis): 1) 50% DDGS untreated, 2) 48.8% DDGS treated with 1.2% CaO, 3) 50% MDGS untreated, or 4) 48.8% MDGS treated with 1.2% CaO. The remainder of the diet was corn husklage, dry rolled corn, and vitamin and mineral supplement. In Exp. 2, fistulated steers (n = 8; average initial BW = 540 ± 250 kg) were used in a replicated 4 × 4 Latin square design with the same dietary treatments as in Exp. 1. There was no interaction (P ≥ 0.14) between distillers grains plus solubles (DGS) and CaO inclusion for DMI, ADG, final BW, or USDA yield and quality grades. However, steers fed CaO-treated DGS had decreased (P < 0.01) DMI, regardless of DGS type. Because CaO treatment decreased DMI without affecting (P = 0.66) ADG, steers fed CaO-treated DGS had increased (P < 0.01) G:F compared to steers not fed CaO. The variation in DMI found in this experiment could be explained by differences in meal size and distribution. Steers fed CaO-treated DGS ate a similar (P = 0.36) number of meals but ate smaller (P < 0.01) meals. No effects (P ≥ 0.55) of CaO treatment or its interaction with DGS type were found for apparent total tract DM or NDF digestibility. However, steers fed MDGS had increased (P < 0.01) NDF digestibility compared to steers fed DDGS. In conclusion, CaO treatment of DGS improved feed efficiency when DGS-based diets were fed but did not improve digestibility.

Differential placental hormone gene expression during pregnancy in a transgenic mouse containing the human growth hormone/chorionic somatomammotropin locus.

The human (h) growth hormone/chorionic somatomammotropin (GH/CS) gene locus presents a unique model to gain insight into the molecular mechanisms that have allowed a closely related family of genes to be expressed in two distinct cell lineages/tissues: pituitary somatotrophs and placental syncytiotrophoblasts. However, studies of external factors that regulate gene expression have been somewhat limited by (i) a lack of human cell lines expressing endogenous GH or CS appropriately; and (ii) the fact that the GH/CS locus is unique to primates and thus does not exist in rodents. In the current study, a transgenic (171 h GH/CS-TG) mouse was generated containing the intact hGH/CS gene cluster and hGH locus control region (LCR) in a 171-kilobase DNA fragment. Pituitary and placental-specific expression of hGH/CS RNA was detected at embryonic day (E) 18.5. Immunostaining of hGH was seen in somatotrophs of the anterior pituitary beginning in late gestation. The presence of hCS protein was detected in the placental labyrinth in trophoblasts functionally analogous to the syncytiotrophoblast of the chorionic villi. This pattern of gene expression is consistent with the presence of essential components of the hGH/CS LCR. Transcript levels for hCS-A, hCS-B and placental hGH-variant increased in 171 hGH/CS-TG placenta during gestation (E11.5-E18.5), as previously observed in human placental development. Throughout gestation, hCS-A RNA levels were proportionately higher, accounting for 91% of total CS RNA by E18.5, comparable to term human placenta. Finally, the previous correlation between the transcription factor AP-2alpha and hCS RNA expression observed in developing primary human cytotrophoblast cultures, was extended to pregnancy in the 171 hGH/CS-TG mouse. The 171 hGH/CS-TG mouse thus provides a model to investigate hGH/CS gene expression, including in pregnancy.

Chronic pain and head injury following motor vehicle collisions: a double whammy or different sides of a coin.

Chronic pain and head injury are common and burdensome sequelae of motor vehicle collisions. The aim of this study was to compare differences in physical injury and functional impairment, psychological distress and pain coping in head injured and non-head injured chronic pain persons subsequent to motor vehicle collisions. Two groups of 54 participants matched in terms of age, gender, and years of formal education underwent a psychological-legal assessment. As part of the assessment, participants completed the Multidimensional Pain Inventory, Sickness Impact Profile, Minnesota Multiphasic Personality Inventory-2, and Coping Strategies Questionnaires. Select scales from questionnaires were combined and underwent multivariate analyses of covariance to test the effects of pain sites at the time of psychological-legal assessment (low, high) and head injury status (head injured and non-head injured chronic pain). Overall, some differences between the two groups were noted but the results did not strongly support the hypothesis that head injured chronic pain participants have a greater physical or psychological burden than non-head injured chronic pain participants. The results suggest the import of assessing and managing pain sites and pain severity in persons injured in motor vehicle collisions.

Molecular cloning and functional characterization of MCH2, a novel human MCH receptor.

Melanin-concentrating hormone (MCH) is involved in the regulation of feeding and energy homeostasis. Recently, a 353-amino acid splice variant form of the human orphan receptor SLC-1 () (hereafter referred to as MCH(1)) was identified as an MCH receptor. This report describes the cloning and functional characterization of a novel second human MCH receptor, which we designate MCH(2), initially identified in a genomic survey sequence as being homologous to MCH(1) receptors. Using this sequence, a full-length cDNA was generated with an open reading frame of 1023 base pairs, encoding a polypeptide of 340 amino acids, with 38% identity to MCH(1) and with many of the structural features conserved in G protein-coupled receptors. This newly discovered receptor belongs to class 1 (rhodopsin-like) of the G protein-coupled receptor superfamily. HEK293 cells transfected with MCH(2) receptors responded to nanomolar concentrations of MCH with an increase in intracellular Ca(2+) levels and increased cellular extrusion of protons. In addition, fluorescently labeled MCH bound with nanomolar affinity to these cells. The tissue localization of MCH(2) receptor mRNA, as determined by quantitative reverse transcription-polymerase chain reaction, was similar to that of MCH(1) in that both receptors are expressed predominantly in the brain. The discovery of a novel MCH receptor represents a new potential drug target and will allow the further elucidation of MCH-mediated responses.

Distribution analysis of human two pore domain potassium channels in tissues of the central nervous system and periphery.

Potassium channels are amongst the most heterogeneous class of ion channels known and are responsible for mediating a diverse range of biological functions. The most recently described family of K+ channels, the 'two pore-domain family', contain four membrane spanning domains and two pore-forming domains, suggesting that two channel subunits associate to form a functional K+ pore. Several sub-families of the two pore domain potassium channel family have been described, including the weakly inward rectifying K+ channel (TWIK), the acid-sensitive K+ channel (TASK), the TWIK-related K+ channel (TREK) and the TWIK-related arachidonic acid stimulated K+ channel (TRAAK). However, comparison of the mRNA expression of these channels has been difficult due to the differences in methods used and the species studied. In the present study, we used a single technique, TaqMan semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), to investigate the mRNA distribution of all currently known two pore potassium channels in human central nervous system (CNS) and peripheral tissues. TWIK-1 and the TWIK-1-like channel KCNK7 were predominantly expressed in the CNS, in contrast to TWIK-2 which was preferentially expressed in peripheral tissues such as pancreas, stomach, spleen and uterus. TASK-1 was expressed in the CNS and some peripheral tissues, whereas TASK-2 was exclusively expressed in the periphery except for mRNA expression observed in dorsal root ganglion and spinal cord. In addition, mRNA expression of the recently identified TASK-3, was almost completely exclusive to cerebellum with little or no mRNA detected in any other tissues. TREK-1 and TRAAK mRNA expression was predominantly CNS specific in contrast to the closely related TREK-2, which was expressed in both CNS and peripheral tissues. Studying the mRNA expression profiles of known two pore domain K+ channels will aid in the understanding of the biological roles of these channels. Furthermore, identification of common areas of expression may help identify which channels, if any, associate to form heteromeric K+ channel complexes.

Cloning and characterization of human NTT5 and v7-3: two orphan transporters of the Na+/Cl- -dependent neurotransmitter transporter gene family.

Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.

Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel.

We have isolated, by degenerate PCR, a complementary DNA encoding a novel two pore domain potassium channel. This is the 7th functional member of the human tandem pore domain potassium channel family to be reported. It has an open reading frame of 1.125 kb and encodes a 374 amino acid protein which shows 62% identity to the human TASK-1 gene: identity to other human members of the family is 31-35% at the amino acid level. We believe this gene to be human TASK-3, the ortholog of the recently reported rat TASK-3 gene: amino acid identity between the two is 74%. 'Taqman' mRNA analysis demonstrated a very specific tissue distribution pattern, showing human TASK-3 mRNA to be localised largely in the cerebellum, in contrast rat TASK-3 was reported to be widely distributed. We have shown by radiation hybrid mapping that human TASK-3 can be assigned to chromosome 8q24.3. Human TASK-3 was demonstrated to endow Xenopus oocytes with a negative resting membrane potential through the presence of a large K(+) selective conductance. TASK-3 is inhibited by extracellular acidosis with a mid-point of inhibition around pH 6. 5, supporting the predictions from the sequence data that this is a third human TASK (TWIK-related acid sensitive K(+) channel) gene.

Neurocognitive performance and emotional status in chronic pain patients.

The following study examined the association between neurocognitive performance and emotional status in chronic pain patients. Seventy-three chronic pain patients recruited consecutively from services in a general medical hospital completed a battery of 10 neurocognitive measures and the Symptom Checklist-90-Revised (SCL-90-R; a gross measure of emotional distress). Cluster analytic procedures were used to identify a three-cluster group solution based on the SCL-90-R. Results indicate that subjects highest in emotional distress experienced more neurocognitive difficulties in intellectual functioning, immediate and delayed recall of verbal and nonverbal material, abstract thinking and problem solving, and cognitive efficiency than subjects lowest in emotional distress. The differences in neurocognitive functioning among the three cluster groups were not confounded by any differences on a number of background variables. These results suggest that level of emotional distress is associated with difficulties in a range of neurocognitive domains and have implications for the assessment and management of chronic pain patients.

Identification of a placental-specific enhancer in the rat placental lactogen II gene that contains binding sites for members of the Ets and AP-1 (activator protein 1) families of transcription factors.

We previously identified a 3-kb proximal 5'-flanking region of the rat placental lactogen (rPLII) gene that is important for reporter gene transcription in the rat trophoblast cell line, Rcho, and targets expression to the placentas of transgenic mice. In our current studies we have used further deletion analysis and transfection studies in Rcho and GC cells to map more precisely the locations of regulatory elements involved in this placental expression. We show that sequences between - 1435 and -765 are necessary for minimal expression in Rcho cells and that there are negative regulatory elements between -3031 to -2838 and -1729 to -1435. Most importantly, we have identified a fragment between -1793 to -1729 that is essential for expression levels characteristic of the complete 3-kb 5'-region. When linked to the herpes simplex thymidine kinase minimal promoter, this fragment acts as an enhancing element in Rcho but not GC cells. Deoxyribonuclease I (DNAse I) protection and electrophoretic mobility shift assays with nuclear extracts and in vitro translated proteins identify binding sites for members of the activator protein-1 (AP-1) and Ets families of transcription factors. Site-directed mutagenesis of the individual AP-1- and Ets-binding sites leads to a partial loss of the enhancing activity; a double AP-1/Ets mutation leads to a complete loss of activity, demonstrating the functional importance of these sites. By these criteria, putative GATA-binding sites located within the enhancing fragment are not active. These new data suggest an important role for this enhancing fragment in rPLII placental giant cell expression and are the first to implicate a member of the Ets family in the regulation of this gene family.

Efficacy of omeprazole in functional dyspepsia: double-blind, randomized, placebo-controlled trials (the Bond and Opera studies).

BACKGROUND: The efficacy of H2-receptor antagonists in functional dyspepsia is equivocal and the therapeutic place of proton pump inhibitors in functional dyspepsia is unknown. AIM: To evaluate the efficacy of proton pump inhibitor therapy in functional dyspepsia. METHODS: Patients (n = 1262) with a clinical diagnosis of functional dyspepsia (persistent or recurrent epigastric pain or discomfort for at least 1 month and a normal upper gastrointestinal endoscopy) were randomized to receive omeprazole 20 mg, 10 mg or identical placebo, for 4 weeks. Symptoms were assessed using validated measures. Helicobacter pylori status was determined pre-entry by a 13C-urea breath test. RESULTS: On an intention-to-treat analysis (n=1248), complete symptom relief was observed in 38% on omeprazole 20 mg, compared with 36% on omeprazole 10 mg and 28% on placebo (P = 0.002 and 0.02, respectively). Among those with ulcer-like and reflux-like dyspepsia, complete symptom relief was achieved in 40% and 54% on omeprazole 20 mg, and 35% and 45% on omeprazole 10 mg, respectively, compared with 27% and 23% on placebo (all P < 0.05, except omeprazole 10 mg in ulcer-like dyspepsia, P = 0.08). There was no significant benefit of omeprazole over placebo in dysmotility-like dyspepsia. Symptom relief was similar in H. pylori-positive and negative cases. CONCLUSIONS: Omeprazole is modestly superior to placebo in functional dyspepsia at standard (20 mg) and low doses (10 mg) but not in patients with dysmotility-like dyspepsia.

Analysis of tissue- and hormone-specific regulation of the human prolactin-inducible protein/gross cystic disease fluid protein-15 gene in transgenic mice.

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (PIP/GCDFP-15) gene is expressed in more than 90% of human breast cancer biopsies but not in the normal mammary gland. However, it is expressed in several normal human apocrine glands such as the lacrimal and salivary glands. In human breast cancer cell lines, the gene is regulated by a number of hormones including androgen and prolactin. It is not known whether gene expression in normal tissues is under similar hormonal control. To understand the mechanisms by which hormone- and tissue-specific expression of the human PIP/GCDFP-15 gene are regulated in vivo, we generated transgenic mice using a 13.7 kb genomic DNA fragment containing the entire 7 kb human gene, together with 2.9 kilobases of 5' and 3.8 kilobases of 3' flanking sequences. The human PIP/GCDFP-15 transgene was found to be expressed in both the lacrimal and salivary glands but was not expressed in the mammary glands of transgenic mice. This tissue-specific pattern of the transgene expression in the mouse was very similar to that of the endogenous human PIP/GCDFP-15 gene, and to the endogenous mouse,gene. In the mouse salivary glands, the transgene expression was highest in the parotid, considerably less in the submaxillary (submandibular) and absent in the sublingual glands. In the mouse lacrimal gland, as in the human breast cancer cell lines, the human PIP/GCDFP-15 transgene was also up-regulated by androgen. These studies demonstrate that the human gene with its 6.3 kb flanking sequences is able to confer gene expression in vivo in a tissue-specific and hormone-responsive manner.

The effects of epidermal growth factor/transforming growth factor alpha on the expression of placental lactogen I and II mRNAs in a rat choriocarcinoma cell line.

Pregnancy in the rat and mouse is characterized by a developmental switch in expression at midgestation between the related placental lactogens I and II (PLI, PLII) suggesting that these proteins have important but different roles. The factors that control this differential gene expression are poorly understood. In this paper we describe experiments which investigate the effects of EGF and TGFalpha on rPLI and rPLII mRNA levels in the rat choriocarcinoma cell line, Rcho. This cell line has been widely used as a model system for studying genes expressed in the rodent placental giant cell. We find that in these cultures both growth factors produce a two fold increase in endogenous rPLI mRNA levels, and a two fold decrease in rPLII mRNA levels. Using DNA transfection assays we have tested the effects of TGFalpha on the expression of hybrid rPLI and rPLII 5'flanking/luciferase reporter constructs in Rcho cells. The transfected rPLI/luciferase reporter constructs produce a similar two fold increase in reporter expression to that seen for the endogenous mRNA, suggesting that EGF/TGFalpha positively regulate rPLI mRNA levels by effects on gene transcription. Unlike the endogenous gene, however, the rPLII/ luciferase constructs show a five fold increase in rPLII-directed luciferase expression in the presence of TGFalpha. The effect of EGF/TGFalpha on placental rPLII mRNA levels appears to involve negative response element(s) located elsewhere in the gene to those regions tested.

Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer.

Probasin (PB) gene product is prostate-specific, epithelial cell in origin, and androgen-regulated. A large 12-kb promoter fragment of the PB gene (LPB) was linked to the simian virus 40 (SV40) large T antigen (Tag) deletion mutant (that removes the expression of the small t antigen) to deliver consistently high levels of transgene expression to the transgenic mouse prostate. Seven male founders, their male offspring, and all the male offspring from two female founders developed at least prostatic epithelial cell hyperplasia by 10 weeks of age, indicating that the incidence of transformation was 100%. Tumorigenesis in the LPB-Tag animals progressed in a manner similar to that observed in the human prostate. Initially, multifocal proliferating lesions were detected in the prostatic epithelium, which continued to progress into hyperplasia involving the entire epithelium and then low-grade dysplasia. Reactive stromal proliferation was induced and continued to develop throughout the progression to high-grade dysplasia, carcinoma in situ, and adenocarcinoma. Immunohistochemical studies indicated that most stromal cells stained positively for both androgen receptor and smooth muscle alpha-actin, suggesting that stromal overgrowth largely represented mesenchymal cells that had differentiated into smooth muscle cells. Epithelial cell transformation was accompanied by the down-regulation of differentiated function, as suggested by the loss of dorsolateral prostate-specific secretory proteins. Tumor growth was regarded as androgen-dependent because tumors regressed in animals castrated at 11 weeks of age, and androgen treatment restored both epithelial/stromal cell ratio and tumor growth. Furthermore, small populations of prostatic epithelial cells in castrated animals continued to proliferate, suggesting the potential for androgen-independent growth. Although prostatic metastasis to other organs was not observed, local invasion was detected. In summary, the LPB-Tag animal model is unique in that it is the only model generated with the Tag alone, thereby eliminating any influences of the small t antigen on prostate tumor formation. Moreover, this model undergoes molecular changes similar to those found in human prostate including: (a) the multi-focal nature of tumorigenesis, (b) the progressive histopathologic changes from low- to high-grade dysplasia similar to human prostatic intraepithelial neoplasia, (c) stimulation of reactive stromal proliferation, and (d) the androgen-dependent growth of the primary tumor. Thus, the LPB-Tag prostate tumor model will be useful for studying the sequential mechanisms underlying the development of multistep tumorigenesis.

Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer.

Probasin (PB) gene product is prostate-specific, epithelial cell in origin, and androgen-regulated. A large 12-kb promoter fragment of the PB gene (LPB) was linked to the simian virus 40 (SV40) large T antigen (Tag) deletion mutant (that removes the expression of the small t antigen) to deliver consistently high levels of transgene expression to the transgenic mouse prostate. Seven male founders, their male offspring, and all the male offspring from two female founders developed at least prostatic epithelial cell hyperplasia by 10 weeks of age, indicating that the incidence of transformation was 100%. Tumorigenesis in the LPB-Tag animals progressed in a manner similar to that observed in the human prostate. Initially, multifocal proliferating lesions were detected in the prostatic epithelium, which continued to progress into hyperplasia involving the entire epithelium and then low-grade dysplasia. Reactive stromal proliferation was induced and continued to develop throughout the progression to high-grade dysplasia, carcinoma in situ, and adenocarcinoma. Immunohistochemical studies indicated that most stromal cells stained positively for both androgen receptor and smooth muscle alpha-actin, suggesting that stromal overgrowth largely represented mesenchymal cells that had differentiated into smooth muscle cells. Epithelial cell transformation was accompanied by the down-regulation of differentiated function, as suggested by the loss of dorsolateral prostate-specific secretory proteins. Tumor growth was regarded as androgen-dependent because tumors regressed in animals castrated at 11 weeks of age, and androgen treatment restored both epithelial/stromal cell ratio and tumor growth. Furthermore, small populations of prostatic epithelial cells in castrated animals continued to proliferate, suggesting the potential for androgen-independent growth. Although prostatic metastasis to other organs was not observed, local invasion was detected. In summary, the LPB-Tag animal model is unique in that it is the only model generated with the Tag alone, thereby eliminating any influences of the small t antigen on prostate tumor formation. Moreover, this model undergoes molecular changes similar to those found in human prostate including: (a) the multi-focal nature of tumorigenesis, (b) the progressive histopathologic changes from low- to high-grade dysplasia similar to human prostatic intraepithelial neoplasia, (c) stimulation of reactive stromal proliferation, and (d) the androgen-dependent growth of the primary tumor. Thus, the LPB-Tag prostate tumor model will be useful for studying the sequential mechanisms underlying the development of multistep tumorigenesis.

Rat placental lactogen II gene: characterization of gene structure and placental-specific expression.

Rat placental lactogen II (rPLII) was the first described member of the rat PRL-like placental gene family in which nine novel proteins have now been identified. In this article, we present data on the isolation and characterization of the rPLII gene. Two genomic clones, GC I (18.5 kb) and GC II (9.4 kb), were isolated from an EMBL3 Sprague-Dawley rat liver genomic DNA library. GC I, which was used for further analysis, contains the entire coding region and extensive 5' and 3' flanking information. The rPLII gene, estimated to be 5.4 kb in size, has the same five-exon and four-intron structure and identical intron/exon splice sites and types as the rPRL gene. A major transcription start site 58 bp upstream of the initiator methionine codon and several minor sites 1-3 bp 5' and 3' of this site were identified by primer extension of day 18 placental messenger RNA. The rPLII gene has been localized to chromosome 17, using a series of hybrid cell lines derived from mouse hepatoma cells (MWTG3) and adult rat hepatocytes; this is the same chromosome designation as the PRL gene itself and other cloned placental members of this gene family. Luciferase reporter constructs containing 5' flanking DNA sequences were tested in transient transfection assays in the rat choriocarcinoma cell line, Rcho, and the rat pituitary GC cell line. Both a 4.5- and 3-kb 5' flanking sequence supported luciferase expression in the Rcho but not the GC cells. A 765-bp fragment showed no activity in either cell type. Transient transgenic mice, generated with the 3-kb 5' rPLII/luciferase construct, expressed varying amounts of luciferase expression in the placenta.

SB-216641 and BRL-15572--compounds to pharmacologically discriminate h5-HT1B and h5-HT1D receptors.

Despite only modest homology between h5-HT1B and h5-HT1D receptor amino acid sequences, these receptors display a remarkably similar pharmacology. To date there are few compounds which discriminate between these receptor subtypes and those with some degree of selectivity, such as ketanserin, have greater affinity for other 5-HT receptor subtypes. We now report on two compounds, SB-216641 (N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide) and BRL-15572 3-[4-(3-chlorophenyl) piperazin-1-yl]-1,1-diphenyl-2-propanol), which display high affinity and selectivity for h5-HT1B and h5-HT1D receptors, respectively. In receptor binding studies on human receptors expressed in CHO cells, SB-216641 has high affinity (pKi = 9.0) for h5-HT1B receptors and has 25-fold lower affinity at h5-HT1D receptors. In contrast, BRL-15572 has 60-fold higher affinity for h5-HT1D (pKi = 7.9) than 5-HT1B receptors. Similar affinities for these compounds were determined on native tissue 5-HT1B receptors in guinea-pig striatum. Functional activities of SB-216641 and BRL-15572 were measured in a [35S]GTPgammaS binding assay and in a cAMP accumulation assay on recombinant h5-HT1B and h5-HT1D receptors. Both compounds were partial agonists in these high receptor expression systems, with potencies and selectivities which correlated with their receptor binding affinities. In the cAMP accumulation assay, results from pK(B) measurements on the compounds again correlated with receptor binding affinities (SB-216641, pK(B) = 9.3 and 7.3; BRL-15572, pK(B) = <6 and 7.1, for h5-HT1B and h5-HT1D receptors respectively). These compounds will be useful pharmacological agents to characterise 5-HT1B and 5-HT1D receptor mediated responses.

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors.

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT1B receptor ligand, and of BRL-15572, a preferential h5-HT1D receptor ligand, on the presynaptic h5-HT1B and h5-HT1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT1D heteroreceptors in the human atrium. The brain preparations, preincubated with [3H]serotonin ([3H]5-HT), and the segments of atrial appendages, preincubated with [3H]noradrenaline, were superfused with modified Krebs' solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K+ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2 microM; concentration 154 times higher than its Ki at h5-HT1D receptors) but was antagonized by SB-216641 (0.1 microM; concentration 100 times higher than its Ki at h5-HT1B receptors; apparent pA2 8.45). SB-216641 (0.1 microM) by itself facilitated, whereas BRL-15572 (2 microM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1 microM) also facilitated, and BRL-15572 (2 microM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K+-evoked tritium overflow. This response was unaffected by BRL-15572 (300 nM) but antagonized by SB-216641 (15 nM; drug concentrations 23 and 15 times higher than their Ki at h5-HT1D and h5-HT1B receptors, respectively). Both drugs, given alone, did not modify the K+-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300 nM; 23 times Ki at h5-HT1D receptors) but not by SB-216641 (30 nM; 30 times Ki at h5-HT1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT1B and 5-HT1D receptors in functional studies.

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice.

BACKGROUND: Androgen regulation and prostate-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (-426 to +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing. METHODS: To enhance transgene expression, a large fragment of the PB promoter (LPB, -11,500 to +28 bp) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines. RESULTS: As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line 1, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of CAT increasing from 3 to 40,936 dpm/min/mg protein. Upon sexual maturation and elevated serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with androgens returned LPB-CAT expression to precastration levels. In contrast, treatment with glucocorticoids had no significant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes. CONCLUSIONS: This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific expression and greatly increase levels of transgene expression induced by androgens and zinc.

The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice.

An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.