Ligands for fluorescence correlation spectroscopy on g protein-coupled receptors.

G protein-coupled receptors (GPCRs) comprise a large protein family of transmembrane receptors involved in many physiological processes. They are engaged in various transduction processes of extracellular signals into intracellular responses. Due to their involvement in numerous diseases they represent an important pharmacological target. Fluorescence correlation spectroscopy (FCS) poses a very sensitive analytical technique well-suited for the investigation of GPCRs. It is minimally invasive and operates on a single molecular level. It further provides detailed pharmacological information on receptor kinetics and quantities of activated receptors on the cell membrane. In addition, FCS allows distinguishing between different receptor states based on different diffusion time constants. In order to be applicable for FCS, the molecule of interest has to be fluorescently labeled. This review focuses on the physical requirements for dyes intended for FCS, their influence on the binding characteristics of coupled ligands and strategies to generate dye labeled ligands, exemplified on GPCR ligands.

[Heparin induced thrombocytopenia, left ventricular thrombus and cerebral embolism during an acute myocardial infarction]. / Heparininduzierte Thrombozytopenie, Ventrikelthrombus und zerebrale Embolie im Verlauf eines akuten Myokardinfarkts.

HISTORY AND CLINICAL FINDINGS: A 58-year-old male patient was admitted to the emergency room after reanimation in hypotensive shock, under sedation and respiration. INVESTIGATIONS: The cardiac laboratory markers were within normal limits. The ECG demonstrated a sinustachycardia with typical infarction signs. The echocardiogram showed a hypokinesia of the cardiac apex. DIAGNOSTICS, TREATMENT AND COURSE: Acute myocardial infarction was treated with a systemic rt-PA lysis. The course was complicated by: confirmed heparin-induced thrombocytopenia, left ventricular thrombus and a cerebral embolism. After multiple adaptations of the anticoagulation regimen, the patient was discharged in a good condition without major neurologic deficits. CONCLUSION: With a careful anticoagulation regimen it is possible to achieve a successful outcome in major complications such as heparin-induced thrombocytopenia, left ventricular thrombus and cerebral embolism.

[Changes in indications for surgical coronary revascularization 1985 to 1992]. / Wandel der Indikationsstellung zur operativen Koronarrevaskularisation in den Jahren 1985 bis 1992.

We analyzed the changing use of coronary artery bypass grafting in our institution during the years from 1985 to 1992. All clinical parameters indicating an increased perioperative risk for the surgical intervention increased during the study period (increased percentage of old patients, females, patients with severe coronary artery disease (high modified Gensini-index or triple-vessel disease), and left main stenoses). During a 2-year follow-up there was a constant proportion of patients with a good postoperative clinical result; perioperative mortality as well as global and cardiac 2-year mortality showed no significant changes. Despite an increased proportion of patients with higher perioperative risk the acute and long-term results of coronary artery bypass surgery in our study were quite satisfactory. This must be attributed to improvements in operative techniques, improved personal skills of the surgeons, and improvements in perioperative treatment and critical care.

Suppression of endotoxin mitogenicity of spleen cells by lipoxygenase inhibitors and its reversal by 13-hydroxyoctadecadienoic acid.

The effects of several inhibitors of lipoxygenases were investigated in murine spleen cell cultures activated with endotoxin (lipopolysaccharide). It was found that these inhibitors interfere with the proliferative response of the cultures. Indomethacin, a specific cyclooxygenase inhibitor, had no such effect. Endotoxin induced the synthesis of tumour necrosis factor alpha in spleen cells which was prevented by treatment with a lipoxygenase inhibitor. The inhibition of the mitogenic effect of endotoxin could be reversed by addition of 13-hydroxyoctadecadienoic acid. This was not the case with leukotriene B4 and C4 or 15-hydroxyeicosatetraenoic acid. In contrast, these substances had inhibitory effects on the mitogenicity of spleen cells. It is suggested that 13-hydroxyoctadecadienoic acid is involved in the development of the mitogenic reaction, possibly on the level of tumour necrosis factor alpha production of macrophages present in the cultures.

Lipoxygenase inhibitors but not site specific 5-lipoxygenase blockers protect against endotoxic shock and inhibit production of tumor necrosis factor.

In the present study it was found that lipoxygenase inhibitors prevent LPS-, but not tumor necrosis factor alpha (TNF alpha)-evoked lethality. The specific 5-lipoxygenase inhibitors (MK-886, CGS-8515) were uneffective in endotoxin-induced shock. The 5-lipoxygenase inhibitors interfered with LTC4 formation in macrophages while they did not affect endotoxin induced TNF alpha-formation, neither in cell cultures nor in mice. The potency of other, less specific lipoxygenase blockers to suppress TNF alpha formation correlated quantitatively with their ability to interfere with 13-HODD synthesis. Based on the fact that a tight correlation exists between inhibition of TNF alpha synthesis and 13-HODD formation, this product might be important for TNF alpha formation.

The role of lipoxygenases in endotoxin-induced cytokine production.

The LPS induced synthesis of tumor necrosis factor in macrophage cultures, as determined in a fibroblast cytolysis assay was found to be effectively blocked by inhibitors of lipoxygenases. Likewise, the presence of tumor necrosis factor in serum of D-galactosamine sensitized mice after challenge with endotoxin was suppressed by the lipoxygenase inhibitors. From LPS-treated macrophages 13-hydroxylinoleic acid, a lipoxygenase product was isolated, which is significantly increased after LPS treatment of the cells, covalently bound to cellular constituents and was found to counterbalance the suppression of TNF-synthesis by a lipoxygenase inhibitor when added to macrophages exogenously.

Differential protective activities of site specific lipoxygenase inhibitors in endotoxic shock and production of tumor necrosis factor.

Lipoxygenase inhibitors have been shown to exert beneficial effects in experimental models of endotoxin shock. In the present study it was found that lipoxygenase inhibitors prevented LPS, but not tumor necrosis factor alpha (TNF alpha)-evoked leukopenia in mice. These inhibitors protected against endotoxin lethality but not against TNF alpha induced lethality. When the protective potency of the specific 5-lipoxygenase inhibitors (MK 886, CGS 81585) was tested in endotoxin-induced leukopenia and shock, they were found to be ineffective. Site specificity of the inhibitors was assessed by comparison of their effects on the formation of LTC4 and the conversion of linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODD) by macrophages. The 5-lipoxygenase inhibitors interfered with LTC4 formation in macrophages, however, they did not affect endotoxin-induced TNF alpha formation, neither in cell cultures nor in mice. The inhibitory strength of other, less specific lipoxygenase blockers to suppress TNF alpha formation correlated quantitatively with their ability to interfere with 13-HODD synthesis. From these findings it is concluded that lipoxygenase inhibitors interfere with endotoxic effects because they block TNF alpha formation. Since 5-lipoxygenase inhibitors neither prevented the formation of TNF alpha nor endotoxin leukopenia and lethality, it is suggested that a lipoxygenase product distinct from the leukotrienes is involved in TNF alpha synthesis. Based on the fact that a tight correlation exists between inhibition of TNF alpha synthesis and 13-HODD formation, activation of 15-lipoxygenase might be important for TNF alpha formation.

Material constants for a finite element model of the intervertebral disk with a fiber composite annulus.

A simple axisymmetric finite element model of a human spine segment containing two adjacent vertebrae and the intervening intervertebral disk was constructed. The model incorporated four substructures: one to represent each of the vertebral bodies, the annulus fibrosus, and the nucleus pulposus. A semi-analytic technique was used to maintain the computational economies of a two-dimensional analysis when nonaxisymmetric loads were imposed on the model. The annulus material was represented as a layered fiber-reinforced composite. This paper describes the selection of material constants to represent the anisotropic layers of the annulus. It shows that a single set of material constants can be chosen so that model predictions of gross disk behavior under compression, torsion, shear, and moment loading are in reasonable agreement with the mean and range of experimentally measured disk behaviors. It also examines the effects of varying annular material properties.