A complementary DNA (cDNA) encoding a serotonin receptor with 51% sequence identity to the 5HT‐1C subtype was isolated from a rat brain cDNA library by homology screening. Transient expression of the cloned cDNA in mammalian cells was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes from transfected cells showed high‐affinity binding of the serotonin antagonists spiperone, ketanserin and mianserin, low affinity for haloperidol (a dopamine D2 receptor antagonist), 8‐OH‐DPAT as well as MDL‐72222 and no detectable binding of [3H]serotonin. This profile is consonant with the 5HT‐2 subtype of serotonin receptors. In agreement with this assignment, serotonin increased the intracellular Ca2+ concentration and activated phosphoinositide hydrolysis in transfected mammalian cells. The agonist also elicited a current flow, blocked by spiperone, in Xenopus oocytes injected with in vitro synthesized RNA containing the cloned nucleotide sequences.

The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies. Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein. Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM. Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48. These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity.

No abstract

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.