Differences In Biological Actions Research Articles

Structure of focal adhesion kinase in healthy heart versus pathological cardiac hypertrophy: A modeling and simulation study

Focal adhesion kinase (FAK) is required for signaling in the heart. S910 phosphorylated FAK is known to cause pathological cardiac hypertrophy. The switching of FAK between its inactive (-i), activated (-a) and hyperactive (-h) state is controlled by phosphorylation. FAK consists of three domains, namely: FERM, Kinase, and FAT joined by linkers L1 and L2. The structural basis of FAK phosphorylation and signaling to the downstream pathways is not understood. In this work, we carried out homology modeling and domain assembly of full length human iFAK and aFAK. 100 ns classical molecular dynamic simulations were performed using AMBER14 and effect of S910 phosphorylation on FAK was investigated. The iFAK model superposed on a small angel X-ray scattering (SAXS) derived model with RMSD of 1.18 Å for 590 Cα atoms. aFAK showed S910 phosphorylation site in L2 shielded by FERM. S910 phosphorylation in hFAK led to its exposure accompanied by a large conformational change and exposing the previously buried Grb2 interaction site responsible for causing cardiac hypertrophy. The models of FAK are in agreement with diverse experimental data and observed differences in biological action. Understanding the structure activity relationships of FAK in response to phosphorylation is important for its future therapeutic modulation.

<i>In-Vitro</i> Determination of Biological and Anabolic Functions of Weak Androgen Dehydroepiandrosterone (DHEA) Using a Variety of Cell Lines

Dehydroepiandrosterone (DHEA) is a weak androgen and is shown to have anti-cancer, an- ti-atherogenic, anti-adipogenic and anti-inflammatory effects on mouse, rat and rabbit models. However, human clinical trials data did not support animal findings and were inconclusive. These systemic differences in biological actions between rodents and humans were attributed to the low level of DHEA in rodents. In order to further understand the differences in biological functions between rodents and humans, we resorted to an in-vitro approach involving mouse, rat and hu- man cell lines to assess DHEA biological and anabolic functions separately and independently without systemic influence. Results indicated that DHEA was effective on mouse and rat cell lines but not on human cell lines, as observed in in-vivo studies. In addition, our in-vitro study showed that DHEA was able to induce myogenesis in mouse mesenchymal cells revealing its anabolic func- tion, even though DHEA was considered as a weak androgen. This observation lent credence to the ban on DHEA by IOC medical commission, citing DHEA as an anabolic steroid. These in-vitro expe- riments suggested that the differences in biological actions of DHEA between rodents and humans existed not only in-vivo at the systemic level, but also in-vitro at the cellular level and thus paving the way to study the mechanism responsible for these differences at the cellular level itself.

The multifaceted functions of CXCL10 in cardiovascular disease.

C-X-C motif ligand 10 (CXCL10), or interferon-inducible protein-10, is a small chemokine belonging to the CXC chemokine family. Its members are responsible for leukocyte trafficking and act on tissue cells, like endothelial and vascular smooth muscle cells. CXCL10 is secreted by leukocytes and tissue cells and functions as a chemoattractant, mainly for lymphocytes. After binding to its receptor CXCR3, CXCL10 evokes a range of inflammatory responses: key features in cardiovascular disease (CVD). The role of CXCL10 in CVD has been extensively described, for example for atherosclerosis, aneurysm formation, and myocardial infarction. However, there seems to be a discrepancy between experimental and clinical settings. This discrepancy occurs from differences in biological actions between species (e.g. mice and human), which is dependent on CXCL10 signaling via different CXCR3 isoforms or CXCR3-independent signaling. This makes translation from experimental to clinical settings challenging. Furthermore, the overall consensus on the actions of CXCL10 in specific CVD models is not yet reached. The purpose of this review is to describe the functions of CXCL10 in different CVDs in both experimental and clinical settings and to highlight and discuss the possible discrepancies and translational difficulties. Furthermore, CXCL10 as a possible biomarker in CVD will be discussed.

Anabolic steroids, testosterone-precursors and virilizing androgens induce distinct activation profiles of androgen responsive promoter constructs

Different androgens, e.g. virilizing androgens such as testosterone and its precursors as well as synthetic anabolic steroids, respectively, induce diverse biological effects. The molecular basis for this variety in biological actions, however, is not well understood. We hypothesized that this variability of actions may be due to steroid-specific target gene expression profiles following androgen receptor (AR)-activation. Therefore, we investigated androgen receptor dependent transactivation of three structurally different androgen responsive promoter constructs ((ARE) 2TATA-luc, MMTV-luc, GRE-OCT-luc) in co-transfected Chinese hamster ovary (CHO)-cells as an artificial model simulating different natural target genes. Three virilizing androgens (dihydrotestosterone, testosterone, methyltrienolone), three anabolic steroids (oxandrolone, stanozolol, nandrolone) and two testosterone-precursors of gonadal and adrenal origin (dehydroepiandrosterone, androstenedione) were used as ligands (0.001–100 nM). All steroids proved to be potent activators of the AR. Remarkably, anabolic steroids and testosterone-precursors showed characteristic promoter activation profiles distinct from virilizing androgens with significantly lower (ARE) 2TATA-luc activation. Hierarchical clustering based on similarity of activation profiles lead to a dendrogram with two major branches: first virilizing androgens, and second anabolics/testosterone-precursors. We conclude that steroid-specific differences in gene transcription profiles due to androgen receptor activation could contribute to differences in biological actions of androgens.

S(+)-ketamine, but not R(-)-ketamine, reduces postischemic adherence of neutrophils in the coronary system of isolated guinea pig hearts.

Polymorphonuclear neutrophils (PMN) play a crucial role in the initiation of reperfusion injury. In a previous study, we found that ketamine reduced the postischemic adherence of PMN to the intact coronary system of isolated guinea pig hearts. Because ketamine is a racemic mixture (1:1) of two optical enantiomers, we looked for possible differences in action between the stereoisomers. Seventy-six guinea pig hearts were perfused in the "Langendorff" mode under conditions of constant flow (5 mL/min) using modified Krebs-Henseleit buffer. After 15 min of global warm ischemia, freshly isolated human PMN (10(6)) were infused as a bolus into the coronary system during the second minute of reperfusion. PMN adhesion was expressed as the numeric difference between PMN recovered in the effluent and those applied. Series A hearts received 5 microM S(+), 5 microM R(-), or 10 microM racemic ketamine starting 20 min before ischemia and during reperfusion. In Series B hearts, 10 microM nitro-L-arginine, an inhibitor of NO synthase, was added to the perfusate. In Series C, PMN were preincubated for 15 min with 5 microM S(+)- or R(-)-ketamine. Coronary vascular leak was assessed by measuring the rate of formation of transudate on the epicardial surface. Ischemia/reperfusion without anesthetics increased coronary PMN adherence from 25.5% +/-2.3% (basal) to 35.3%+/-1.5% of the number applied. S(+)-ketamine reduced postischemic adherence in each series (A, 25.5%+/-5.1%; B, 22.5%+/-1.7%; C, 25.3%+/-7.7%), as did racemate (A, 26.4%+/-3.7%). Although 5 microM R(-)-ketamine had no effect on adhesion (A, 30.5%+/-6.7%; B, 34.3%+/-5.1%; C, 34.3%+/-4.3%), it significantly increased vascular leak in the presence of NOLAG. These findings indicate stereoselective differences in biological action between the two ketamine isomers: S(+)-ketamine inhibited PMN adherence, R(-)-ketamine worsened coronary vascular leak in reperfused isolated hearts. In this study, we demonstrated stereoselective differences in the biologic action of the two ketamine isomers in an animal model of myocardial ischemia. Polymorphonuclear neutrophil adherence to the coronary vasculature after ischemia was inhibited by S(+)-ketamine, whereas R(-)-ketamine increased coronary vascular fluid leak.

S(+)-Ketamine, but Not R(-)-Ketamine, Reduces Postischemic Adherence of Neutrophils in the Coronary System of Isolated Guinea Pig Hearts

Polymorphonuclear neutrophils (PMN) play a crucial role in the initiation of reperfusion injury.In a previous study, we found that ketamine reduced the postischemic adherence of PMN to the intact coronary system of isolated guinea pig hearts. Because ketamine is a racemic mixture (1:1) of two optical enantiomers, we looked for possible differences in action between the stereoisomers. Seventy-six guinea pig hearts were perfused in the "Langendorff" mode under conditions of constant flow (5 mL/min) using modified Krebs-Henseleit buffer. After 15 min of global warm ischemia, freshly isolated human PMN (106) were infused as a bolus into the coronary system during the second minute of reperfusion. PMN adhesion was expressed as the numeric difference between PMN recovered in the effluent and those applied. Series A hearts received 5 [micro sign]M S(+), 5 [micro sign]M R(-), or 10 [micro sign]M racemic ketamine starting 20 min before ischemia and during reperfusion. In Series B hearts, 10 [micro sign]M nitro-L-arginine, an inhibitor of NO synthase, was added to the perfusate. In Series C, PMN were pre-incubated for 15 min with 5 [micro sign]M S(+)- or R(-)-ketamine. Coronary vascular leak was assessed by measuring the rate of formation of transudate on the epicardial surface. Ischemia/reperfusion without anesthetics increased coronary PMN adherence from 25.5% +/- 2.3% (basal) to 35.3% +/- 1.5% of the number applied. S(+)-ketamine reduced postischemic adherence in each series (A, 25.5% +/- 5.1%; B, 22.5% +/- 1.7%; C, 25.3% +/- 7.7%), as did racemate (A, 26.4% +/- 3.7%). Although 5 [micro sign]M R(-)-ketamine had no effect on adhesion (A, 30.5% +/- 6.7%; B, 34.3% +/- 5.1%; C, 34.3% +/- 4.3%), it significantly increased vascular leak in the presence of NOLAG. These findings indicate stereoselective differences in biological action between the two ketamine isomers: S(+)-ketamine inhibited PMN adherence, R(-)-ketamine worsened coronary vascular leak in reperfused isolated hearts. Implications: In this study, we demonstrated stereoselective differences in the biologic action of the two ketamine isomers in an animal model of myocardial ischemia. Polymorphonuclear neutrophil adherence to the coronary vasculature after ischemia was inhibited by S(+)-ketamine, whereas R(-)-ketamine increased coronary vascular fluid leak. (Anesth Analg 1999;88:1017-24)

Binding proteins on synaptic membranes for certain phospholipases A2 with presynaptic toxicity.

Many steps in the process of neurotransmitter release are vulnerable to various neurotoxins. Some of these presynaptic toxins exhibit phospholipase A2 (PLA2) activity. These neurotoxic PLA2s (or PLA2 neurotoxins) are members of a group of extracellular (or secreted) PLA2 proteins found in most if not all animals. Besides phospholipid metabolism, these PLA2s exhibit a variety of biological effects, including host defense, neurotoxicity (presynaptic and/or postsynaptic), myotoxicity, and alteration of coagulation, which may or may not be related to hydrolysis of phospholipids. Despite large differences in biological actions, the PLA2 chains of these proteins show high degrees of homology in the primary, secondary and possibly tertiary structures. A small number of these proteins, mostly isolated from the venoms of a number of snakes, act primarily at the presynaptic level to cause synaptic blockade by inhibiting the release of neurotransmitters, though most of them also produce postsynaptic toxicity and other effects. These presynaptic PLA2 toxins may be classified into three classes. The toxins differ in their subunit structures, but in every case, at least one subunit is an active PLA2 with M.W. of 12,000 to 16,000. Each toxin in the first class is a single-chained protein. In the second class, a toxin may comprise 2 to 4 homologous subunits associated noncovalently.

Effects of MSH on food intake, body weight and coat color of the yellow obese mouse

Viable yellow obese mice (A vy/a) were treated for 10 days with 5, 15, 50 or 150 ug/d of either alpha-MSH or desacetyl-MSH. The half-maximal effect on weight gain occurred with a dose of 5 ug/d for desacetyl-MSH and at a 30 fold higher level of 150 ug/d for alpha-MSH. In contrast, the half-maximal stimulation of eumelanin production by alpha-MSH occurred at 15 ug/d and with desacetyl-MSH at 150 ug/d, a 10-fold increase. Desacetyl-MSH produced a dose-related increase in the weight of muscle, as well as white and brown adipose tissue. Desacetyl-MSH and alpha-MSH both increased plasma corticosterone concentrations, but desacetyl-MSH was more potent. In a 2×2 factorial designed study, body weight was significantly increased in viable yellow mice only by treatment with desacetyl-MSH but in the lean animals, both alpha-MSH and desacetyl-MSH increased body weight. Food intake was significantly different between genotypes, and was stimulated by desacetyl-MSH. These studies demonstrate potent differences in biological actions on food intake, body weight, and a variety of organ weights between acetylated and desacetylated forms of MSH.

Pituitary gonadotropic hormone from a chondrostean fish, starred sturgeon ( Acipenser stellatus Pall.) : IV. Differences in biological action of isoforms and absence of sex-specific forms

The specific activities of four subfractions of the starred sturgeon ( Acipenser stellatus Pall.) pituitary gonadotropin (aci-GTH-A, -B, -C, -D) were compared using several test systems. The ratios of two of them (A and D) that contained the isoforms differing by the values of isoelectric point were shown to be different, especially when in vitro oocyte maturation and spermiation tests were used. These differences were not due to the presence in one of these preparations of the component affecting spermiation rather than oocyte maturation. The qualitative differences in the biological action of aci-GTH-A and -D were also revealed by the comparison of dose-response curves using toad oocyte in vitro maturation. Finally, as the spectra of aci-GTH isoforms were practically identical when female or male individual pituitary extracts were submitted to isoelectric focusing, we concluded that the structural and functional heterogeneity of aci-GTH was related neither to sex nor to genetic intrapopulation polymorphism.