Discovery of brain penetrant, soluble, pyrazole amide EP1 receptor antagonists

We describe the discovery of a series of pyrazole amide EP₁ receptor antagonists with good aqueous solubility and CNS penetration. In order to achieve solubility we investigated the incorporation of a basic group in the region of the molecule previously occupied by a carboxylic acid, which was known to be a key element of the pharmacophore. This study led to the identification of compounds such as 4h, 4j and 10b which demonstrated brain-to-blood ratios of 0.8:1–2.0:1 in addition to good solubility and metabolic stability.     

Connective Tissue Growth Factor (CTGF/CCN2) enhances lactogenic differentiation of mammary epithelial cells via integrin-mediated cell adhesion

Background  

Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program.     

Plant Essential Oils Synergize and Antagonize Toxicity of Different Conventional Insecticides against Myzus persicae (Hemiptera: Aphididae)

Plant-derived products can play an important role in pest management programs. Essential oils from Lavandula angustifolia (lavender) and Thymus vulgaris (thyme) and their main constituents, linalool and thymol, respectively, were evaluated for insecticidal activity and synergistic action in combination with insecticides against green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae). The essential oils and their main constituents exerted similar insecticidal activity when aphids were exposed by direct sprays, but were non-toxic by exposure to treated leaf discs. In synergism experiments, the toxicity of imidacloprid was synergized 16- to 20-fold by L. angustifolia and T. vulgaris essential oils, but far less synergism occurred with linalool and thymol, indicating that secondary constituents of the oils were probably responsible for the observed synergism. In contrast to results with imidacloprid, the insecticidal activity of spirotetramat was antagonized by L. angustifolia and T. vulgaris essential oils, and linalool and thymol. Our results demonstrate the potential of plant essential oils as synergists of insecticides, but show that antagonistic action against certain insecticides may occur.     

Inhibition of γ-aminobutyric acid release from rat cerebral cortex slices by barbiturate anesthesia

Amobarbital and pentobarbital anesthesia inhibited the potassium-stimulated, Ca-dependent release of -aminobutyric acid (GABA) from rat cerebral cortex slices during incubation in vitro. Inhibition of GABA release was not found when slices were prepared from rats shortly after they awakened from amobarbital anesthesia. Phenobarbital anesthesia did not affect the release of GABA.     

Validation and extension of an empirical Bayes method for SNP calling on Affymetrix microarrays

Multiple algorithms have been developed for the purpose of calling single nucleotide polymorphisms (SNPs) from Affymetrix microarrays. We extend and validate the algorithm CRLMM, which incorporates HapMap information within an empirical Bayes framework. We find CRLMM to be more accurate than the Affymetrix default programs (BRLMM and Birdseed). Also, we tie our call confidence metric to percent accuracy. We intend that our validation datasets and methods, refered to as SNPaffycomp, serve as standard benchmarks for future SNP calling algorithms.     

Cloning and Expression of Guanylin from the European eel (Anguilla anguilla)

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5' spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory "yellow" and the more sexually mature, migratory "silver" eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.     

Modulation of soluble ovarian adenosine 3',5'-monophosphate-dependent protein kinase activity during prepubertal development in the rat. II. Evaluation of the catalytic subunit inhibitor activity

In a previous report on the ontogeny of the ovarian adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity during prepubertal development of the rat, we concluded that the 4-fold decline in cAMP-dependent protein kinase activity observed in ovaries of 21- to 23-day-old rats was due to the presence of a heat-labile inhibitor in the ovarian extracts (Hunzicker-Dunn et al., 1984). We developed an assay for this ovarian kinase inhibitor activity that was based on the observation that ovarian cytosol added to an exogenous catalytic subunit of cAMP-dependent protein kinase caused a time-dependent and ovarian cytosol protein concentration-dependent inhibition of exogenous catalytic subunit phosphotransferase activity. The present studies were conducted to evaluate the basis for this catalytic subunit inhibitor present in soluble rat ovarian extracts of prepubertal-aged rats. This inhibitor activity was absent from cytosol extracts of rat corpora lutea, rat liver, rabbit follicles, and rabbit corpora lutea. Inhibitor activity present in rat ovarian cytosol was not attributable to insufficient levels of the phosphorylation substrate Kemptide. Inhibitor activity was also not related to the presence of the large amount of catalytic subunit-free regulatory subunit of the cAMP-dependent protein kinase present in ovarian extracts of late juvenile-aged rats. Inhibitor activity, however, did correlate with an endogenous adenosine triphosphatase (ATPase) activity that reduced assay ATP concentrations below levels needed to accurately measure phosphotransferase activity, despite the presence of sodium fluoride (an ATPase inhibitor) and ATP concentrations 5- to 15-fold greater than the Km of the kinase for ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous calcium oscillations during diastole in the whole heart: the influence of ryanodine reception function and gap junction coupling

Triggered arrhythmias due to spontaneous cytoplasmic calcium oscillations occur in a variety of disease conditions; however, their cellular mechanisms in tissue are not clear. We hypothesize that spontaneous calcium oscillations in the whole heart are due to calcium release from the sarcoplasmic reticulum and are facilitated by calcium diffusion through gap junctions. Optical mapping of cytoplasmic calcium from Langendorff perfused guinea pig hearts (n = 10) was performed using oxygenated Tyrode's solution (in mM): 140 NaCl, 0.7 MgCl, 4.5 KCl, 5.5 dextrose, 5 HEPES, and 5.5 CaCl? (pH 7.45, 34°C). Rapid pacing was used to induce diastolic calcium oscillations. In all preparations, pacing-induced multicellular diastolic calcium oscillations (m-SCR) occurred across most of the mapping field, at all pacing rates tested. Ryanodine (1 μM) eliminated all m-SCR activity. Low-dose caffeine (1 mM) increased m-SCR amplitude (+10.4 ± 4.4%, P < 0.05) and decreased m-SCR time-to-peak (-17.4 ± 6.7%, P < 0.05) and its temporal synchronization (i.e., range) across the mapping field (-26.9 ± 17.1%, P < 0.05). Surprisingly, carbenoxolone increased the amplitude of m-SCR activity (+14.8 ± 4.1%, P < 0.05) and decreased m-SCR time-to-peak (-11.3 ± 9.6%, P < 0.01) and its synchronization (-37.0 ± 19.1%, P < 0.05), similar to caffeine. In isolated myocytes, carbenoxolone (50 μM) had no effect on the frequency of aftercontractions, suggesting the effect of cell-to-cell uncoupling on m-SCR activity is tissue specific. Therefore, in the whole heart, overt m-SCR activity caused by calcium release from the SR can be induced over a broad range of pacing rates. Enhanced ryanodine receptor open probability and, surprisingly, decreased cell-to-cell coupling increased the amplitude and temporal synchronization of spontaneous calcium release in tissue.