Abstract
Unfixed tissue specimens most frequently are stored for long term research uses at either −80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects ...such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at −80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for −80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at −80° C compared to those stored in VPLN. Long term storage of human research tissues at −80° C provides at least the same quality of RNA and protein as storage in VPLN.
The six divisions of the Cooperative Human Tissue Network in the USA bank and distribute tens of thousands of tissue specimens to researchers annually. Major operational concerns include: maintaining ...tissue integrity, managing informatics, and protecting patient confidentiality. Increasing molecular genetics testing is also resulting in an increased demand for high-quality nucleic acids.
We previously described a series of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepine CCK-A agonists exemplified by compound 1 (GW 5823), which is the first reported binding selective CCK-A full agonist ...demonstrating oral efficacy in a rat feeding model. In this report we describe analogs of compound 1 designed to explore changes to the C3 and N1 pharmacophores and their effect on agonist activity and receptor selectivity. Agonist efficacy in this series was affected by stereoelectronic factors within the C3 moiety. Binding affinity for the CCK-A vs CCK-B receptor showed little dependence on the structure of the C3 moiety but was affected by the nature of the second substituent at C3. Structure−activity relationships at the N1-anilidoacetamide “trigger” moiety within the C3 indazole series were also investigated. Both agonist efficacy and binding affinity within this series were modulated by variation of substituents on the N1-anilidoacetamide moiety. Evaluation of several analogs in an in vivo mouse gallbladder emptying assay revealed compound 1 to be the most potent and efficacious of all the analogs tested. The pharmacokinetic and pharmacodynamic profile of 1 in rats is also discussed.
Numerous reports have implicated theY5 receptor as the ‘feeding’ receptor mediating the orexigenic action of neuropeptide Y (NPY). This notion is supported by the correlation between the in vitro ...functional and binding activities of different peptide agonists and their potent stimulation of food intake in rodents. We have discovered a series of small molecule heterocycles with high affinity, selectivity, and functional antagonism for Y5 receptors. Intraperitoneal (i.p.) administration of GW438014A into rodents, resulted in a potent reduction of NPY-induced and normal overnight food intake. Brain levels of GW438014A were detected well in excess of its binding IC
50 for up to 3 h post-dosing. Daily (i.p., BID, 10 mg/kg) administration of this compound to Zucker Fatty rats for a period of 4 days resulted in a marked decrease in the rate of weight gain and a reduction in fat mass. No effect on food intake was observed following oral administration of GW438014A (25–100 mg/kg), consistent with the poor oral bioavailability (<3%) and low brain levels observed.
We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key ...parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified.
Atypical antipsychotic drug (APD)-induced weight gain causes non-compliance, increasing the risk of relapse and medical complications.
In an animal model, we assessed body weights, food intake, body ...fat/lean body mass contents and blood serum levels of glucose and lipids in female rats treated with olanzapine (Experiment 1). Also, we investigated the effect of aripiprazole vs olanzapine treatment on weight gain (WG) and plasma prolactin secretion in two strains (Wistar and Sprague-Dawley) and in two housing conditions (singly and group housed; Experiment 2).
In Experiment 1, Wistar females received either vehicle or olanzapine (5.0 mg kg(-1), p.o.) twice daily for 14 days. In Experiment 2, female rats (Wistar or Sprague-Dawley), housed singly or in groups, received either vehicle, aripiprazole (2.0-8.0 mg kg(-1), p.o.), or olanzapine (1.0-10 mg kg(-1), p.o.) twice daily for 7 days. Body weights and food intake were assessed daily. Body composition and blood assays were analyzed at the end of the treatment.
WG induced by chronic olanzapine treatment was characterised by hyperphagia, increased body fat, and serum free fatty acid content and reduced lean tissue and serum glucose content. Subchronic aripiprazole treatment resulted in rapid and robust WG similar to those observed with olanzapine. In spite of similar effects on body weight, aripiprazole and olanzapine stimulated markedly different patterns of prolactin secretion. Body weight changes and prolactin secretion induced by these APDs were significantly modulated by housing and by strain.
Assessment of body weight in the present model may not have predictive validity, and other measures may be needed to differentiate between WG-inducing and weight-neutral drugs.
Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral ...cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are equipotent to CCK as anorectic agents in rats following intraperitoneal administration.
A series of 1,4-benzodiazepines,
N-1-substituted with an
N-isopropyl-
N-phenylacetamide moiety, was synthesized and screened for CCK-A agonist activity. In vitro agonist activity on isolated guinea ...pig gallbladder along with in vivo induction of satiety following intraperitoneal administration in a rat feeding assay was demonstrated.
A series of 1,4-benzodiazepines,
N-1-substituted with an
N-isopropyl-
N-phenylacetamide moiety, was synthesized and screened for CCK-A agonist activity. In vitro agonist activity on isolated guinea pig gallbladder along with in vivo induction of satiety following intraperitoneal administration in a rat feeding assay was demonstrated.
A series of modifications were made to the C-3 substituent of the 1,5-benzodiazepine CCK-A agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a C-3 quaternary ...carbon resulted in acetamide 6, which showed CCK-A receptor binding selectivity and sub-micromolar agonist activity in vitro. Benzodiazepine 6 was active in an in vivo mouse gallbladder emptying assay and represents a novel orally active, binding selective CCK-A agonist.
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