Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.     

The effect of light fluence rate in photodynamic therapy of normal rat brain.

This paper reports the effect of incident light fluence rate on the depth to which necrotic lesions are produced by photodynamic therapy (PDT) in the brains of normal Fisher rats. The rats were injected intraperitoneally with Photofrin (12.5 mg kg-1) 48 h prior to PDT with a fixed incident fluence of 35 J cm-2. The treatment was performed at 10, 50, 100, and 200 mW cm-2 and also in a periodic manner (30 s "on" at 100 mW cm-2, 30 s "off"). The depth to which necrosis occurred was determined 24 h after treatment by microscopic examination of tissue sections. No differences were found in the depth to which necrosis was produced by any of the five irradiation schedules. This finding is discussed in the context of other published dose-rate experiments.     

Pulse-modulated photoacoustic measurements reveal strong gas-uptake component at high CO2-concentrations

The effect of high CO₂-concentration on photoacoustic signals from tobacco leaves is studied by means of a recently developed pulse modulation method which provides simultaneous information on photothermal and photobaric components in the millisecond time domain. High CO₂-concentrations are found to induce large gas-uptake signals. Simultaneous measurements of chlorophyll fluorescence suggest that the uptake signals are correlated with energy-dependent fluorescence quenching. Very similar CO₂-concentration dependencies are found in the absence and presence of methylviologen, which is known to catalyze O₂-reduction, and in the presence of glyceraldehyde, which blocks Calvin cycle and photorespiration. It is suggested that the CO₂-enhanced uptake signal is likely to reflect O₂-uptake in the Mehler reaction. However, it is not ruled out that also rapid CO₂-solubilisation or CO₂-binding caused by light-induced stroma alkalisation are involved. Strong uptake is also induced when the CO₂-concentration in the closed photoacoustic chamber increases due to dark-respiration. The consequences of these findings with respect to the interpretation of photoacoustic data (e.g., low-light effect) and to the regulatory role of O₂-dependent electron flow are discussed.     

Competitive binding assays for high-affinity binders in the presence of endogenous ligands: application to biotin-binding proteins

Endogenous ligands complicate radioligand-binding assays of high-affinity binding proteins by obscuring binding sites or by diluting the labeled ligand. We have developed a mathematical model for such systems where radioligand and endogenous ligand are structurally identical. Data which relate radioligand binding at equilibrium as a function of sample volume can be plotted such that the concentrations of endogenous ligand and binder are graphically determined; however, a more precise determination may be done by nonlinear regression with the aid of a microcomputer. The method is demonstrated for the assay of biotin-binding proteins in the presence of a range of endogenous biotin concentrations below and above that required to saturate the binding sites.     

The effect of experimental hyperthyroidism on renal and adrenal weight increase in mice.

The mouse kidneys are enlarged after the administration of thyroxine and this influence is not mediated through androgens. The administration of thyroxine increased the weight of the adrenals and the level of plasma corticosterone. Besides the direct effect of the thyroid hormones on the kidney, our findings indicate that the excess of triiodothyronine and thyroxine stimulates the activity of adrenals indirectly and evokes hyperadrenocorticism which could be related to the action of adrenal steroids on kidney function and kidney growth. In accordance with the above mentioned hypothesis it has been shown that aminoglutethimide, a potent blocker of adrenal steroidogenesis, decreases the level of plasma corticosterone and inhibits the enlargement of the kidney in hyperthyroid mice in spite of the high serum thyroxine values.     

Characterization and expression of the human leukocyte-common antigen (CD45) gene contained in yeast artificial chromosomes.

The leukocyte-common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed uniquely by cells of hematopoietic origin. There are multiple isoforms of CD45 that are generated by the variable use of three exons (exons 4-6). The use of the variable exons results in changes near the amino-terminus of the mature glycoprotein. The gene is located on chromosome 1 for both human and mouse in a region that is homologous between these two species. This conserved linkage group contains a number of genes of immunological interest, such as the genes for complement regulatory proteins and the FCG2 receptor. Yeast artificial chromosomes provide a vector system in which large fragments of foreign DNA can be isolated and are suited to long-range physical mapping. To this end, three yeast artificial chromosomes containing the human CD45 gene have been isolated and characterized. They overlap to span 475 kb, establishing the largest physical map for DNA within the conserved linkage group. The CD45 gene is entirely encoded within one yeast artificial chromosome clone as determined by mapping with cDNA probes. A mouse B cell line transfected with this YAC clone expressed the low-molecular-weight isoform of the protein into the cell surface. The size of the human CD45 gene was determined to be approximately 120 +/- 10 kb.     

Calmodulin and cyclic nucleotide phosphodiesterase activities in erythrocytes from normal and schizophrenic subjects

Calmodulin and cyclic nucleotide phosphodiesterase activities were measured in hemolysates prepared from 18 normal and 17 schizophrenic subjects. No significant difference between groups was found for either activity. The results suggest that calmodulin is present in normal amounts in patients with schizophrenia. This is compatible with the idea that the interaction of calmodulin with antipsychotic agents is structurally non-specific.     

Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells

The association and dissociation mechanisms of lectin membrane receptor microclustering on HeLa cells have been studied by measuring resonance energy transfer between fluoresceinated and rhodaminated lentil lectin. Compounds known to affect membrane receptor mobility, such as Ca²⁺ ions, methylamine, cytochalasin D and nocodazole, did not modify the association kinetics nor the maximal energy transfer values at 4 and 37 °C. Dissociation of the membrane receptor microclusters was followed by measuring the temporal decrease in energy transfer values at 4 °C after preincubation for different time intervals at 37 °C. The rate of dissociation of the lectin receptors decreased in the presence of Ca²⁺ ions (10⁻³ M) and after cross-linking with anti-lectin antibodies. An increase was observed in the presence of cytochalasin D (10⁻⁶ M) and, to a lesser extent, of methylamine (10⁻² M). When cytochalasin D and methylamine were combined at subliminal concentrations, a partial synergistic effect was observed. Nocodazole (10⁻⁶ M) had no effect. The results suggest that the association of lectin membrane receptors in microclusters is mediated only by physicochemical parameters. Ca²⁺ ions, cytochalasin D (microfilaments) and methylamine (transglutaminase)-sensitive components appear, however, to play an important role in the stabilization of the receptor microclusters.     

Reversible uncoupling of energy transfer between phycobilins and chlorophyll in Anacystis nidulans. Light stimulation of cold-induced phycobilisome detachment

Phycobilin fluorescence of Anacystis nidulans grown at 28°C increases substantially upon cooling below 10°C. A maximal increase is found around ?5°C and amounts to 300%, with almost complete reversibility upon re-warming. Illumination with actinic light leads to considerable stimulation of the cold-induced phycobilin fluorescence increase. Analysis of the light stimulation phenomenon reveals: (1) Actinic illumination shifts the fluorescence-temperature characteristic by about 3°C upwards on the T-axis. At temperatures below 5°C the light stimulating effect becomes smaller again and fluorescence-temperature characteristics measured at high and low light intensity converge around ?5°C. (2) In the 13-8°C region a large (up to 100%) light-induced phycobilin fluorescence increase is observed, while only negligible changes occur in the dark. (3) 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea (DCMU) as well as uncouplers inhibit the light stimulation, which hence depends on coupled electron transport.In agreement with previous work (Schreiber, U. (1979) FEBS Lett. 107, 4–9) it is concluded that illumination enhances cold-induced phycobilisome detachment by increasing the net negative charge at the outer surface of the thylakoid membrane. The possible role of a fluid → ordered transition of membrane lipids (Murata, N. and Fork, D.C. (1975) Plant Physiol. 56, 791–796) is discussed.