The effect of GABA on in vitro binding of two novel non-benzodiazepines, PK 8165 and CGS 8216, to benzodiazepine receptors in the rat brain

The effect of gamma-aminobutyric acid (GABA) on the binding of PK 8165, a quinoline derivative, and CGS 8216, a pyrazoloquinoline, was assessed in two different regions of the rat brain. PK 8165, a compound with reported anxiolytic properties, inhibited [3H]-propyl beta-carboline-3-carboxylate labeled receptors in the cerebellum with an IC50 of 844 nM and 370 nM in the absence and presence of micro M GABA, respectively. GABA (100 micro M) was less effective in the cerebral cortex, decreasing the IC50 value from 280 to 197 nM. In saturation isotherm studies with [3H]-CGS 8216, a benzodiazepine receptor antagonist, GABA (100 micro M) induced a small but significant reduction in the apparent affinity of [3H]-CGS 8216 for benzodiazepine receptors in the cerebral cortex but the Bmax was unchanged.     

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.     

Basal metabolism and dynamic activity of food in obesity

Metabolic rate of obese patients is reported, recorded to cellular active mass. The metabolic rate is higher in obese patients and the value is diucthy related to individual actual weight. The dinamic-specific action of proteins is normal.     

The effect of temperature on CL 218872 and propyl beta-carboline-3-carboxylate inhibition of [3H]-flunitrazepam binding in rat brain

The competitive inhibition of [³H]-flunitrazepam binding by CL 218872 and propyl beta-carboline-3-carboxylate (PCC), non-benzodiazepine compounds that show differential affinities for benzodiazepine (BZD) receptor subtypes, was studied in the rat cerebral cortex and hippocampus at different temperatures of incubation. The potency of both inhibitors was significantly greater at 0° than at 37°C. The magnitude of temperature induced enhancement of potency may correlate with the pharmacological efficacy of compounds that interact with BZD receptors. Hill slopes for CL 218872 shifted from 0.52 to 0.97 in the cerebral cortex when incubations were performed at 0° and 37°C, respectively. Hill values for PCC changed from 0.68 to 0.93 under similar temperature conditions. These observations suggest the presence of a homogenous population of benzodiazepine receptors at physiological temperatures or the inability of CL 218872 and PCC to distinguish between receptor subtypes at 37°C.     

Resolution and characterization of two forms of phosphoinositide-specific phospholipase C from bovine rod outer segments.

Two forms (I and II) of phospholipase C, specific for phosphatidyl inositol 4,5-bisphosphate, were resolved from bovine retinal rod outer segment (ROS) cytosol by DEAE-Sepharose column chromatography. The two isozymes showed reproducible differences in their catalytic properties in spite of similar substrate specificity and hydrolyzed specifically inositol 4,5-bisphosphate in a Ca(2+)-dependent fashion. In the presence of deoxycholate (DOC), pH optima were at 6.5 and 7.0 for phospholipase C I and II, respectively. Maximal phosphatidylinositol 4,5-bisphosphate hydrolysis rates were obtained at 10(-4) and 10(-5)M Ca2+ for phospholipase C I and II, respectively. Treatment with cAMP-dependent protein kinase did not alter either isozyme activity. Further purification steps were prevented by the extreme lability of the isozymes.     

Heterogeneity of natural killer cell subsets in NK-1.1+ and NK-1.1- inbred mouse strains and their progeny.

The 4LO3311 monoclonal antibody, a new NK-specific reagent recently produced in our laboratory, reacts with spleen cells of 11 mouse strains, most of which do not express the NK-1.1 alloantigen recognized by the PK136 mAb. Among positive strains, the susceptibility of spleen cells to the complement-dependent NK-inhibiting activity of the 4LO3311 mAb was variable but independent of the initial NK cell activity level of cells tested. This property was furthermore not modified after poly(I:C) stimulation. The susceptibility of spleen cells to the in vitro 4LO3311 mAb plus complement treatment is however influenced by the absolute number of 4LO3311+ cells as well as by the density of the corresponding alloantigen at the cell surface. Moreover, it was established that the strain-related variations observed also depended upon the relative size of the 4LO3311 cell subset within the lytic NK cell population. Indeed, when C3H (NK-1.1-4LO3311+) mice were inoculated with the 4LO3311 mAb, the lytic activity of their spleen cells was almost unaltered but 4LO3311-reactive cells were no longer detected in the spleen of treated animals and remaining NK cells were totally resistant to the in vitro 4LO3311 mAb plus complement treatment. These findings indicate that the 4LO3311 mAb identifies a subset rather than all NK cells, even in a NK-1.1- strain. Since a NK-1.1-unreactive cell subset was identified in NZB (NK-1.1+4LO3311-) mice inoculated with the PK136 mAb, the NK-1.1+ cell population is not necessarily responsible for all the splenic NK cell activity in all NK-1.1+ strains. In B6C3F1 hybrid mice, a relatively large subset of NK-1.1-4LO3311- cells was found in addition to those expressing the NK-1.1, the 4LO3311 alloantigen, or both. According to these results, NK cell heterogeneity should thus be taken as an evolving concept whose resolution appears more and more complex with the identification of new NK-specific reagents.     

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979