Identification of dual ligands targeting angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-γ by core hopping of telmisartan

It has been reported previously that some angiotensin II receptor blockers not only antagonize angiotensin II type 1 receptor (AT₁R), but also exert stimulation in peroxisome proliferator-activated receptor γ (PPARγ) partial activation, among which telmisartan displays the best. Telmisartan has been tested as a bifunctional ligand with antihypertensive and hypoglycemic activity. Aiming at more potent leads with selective AT₁R antagonism and PPARγ partial agonism, the three parts of telmisartan including the distal benzimidazole ring, the biphenyl moiety, and the carboxylic acid group experienced modification by core hopping method in our study. The central benzimidazole ring, however, remained intact considering its great affinity toward AT₁R and PPARγ. We utilized computational techniques for the sake of details on the binding interactions and conformational stability. Standard precision docking analysis and absorption, distribution, metabolism, excretion, and toxicity prediction received 10 molecules with higher Glide scores, similar interactions, and improved pharmacokinetic profiles compared to telmisartan. Comp#91 with highest scores for AT₁R (?11.92 kcal/mol) and PPARγ (?13.88 kcal/mol) exhibited excellent binding modes and pharmacokinetic parameters. Molecular dynamics trajectories on best docking pose of comp#91 confirmed the docking results and verified the conformational stability with both receptors throughout the course of 20-ns simulations. Thus, comp#91 could be identified as a promising lead in the development of dual AT₁R antagonist and PPARγ partial agonist against hypertension and type 2 diabetes.     

MicroRNA-199a-5p aggravates angiotensin II–induced vascular smooth muscle cell senescence by targeting Sirtuin-1 in abdominal aortic aneurysm

Vascular smooth muscle cells (VSMCs) senescence contributes to abdominal aortic aneurysm (AAA) formation although the underlying mechanisms remain unclear. This study aimed to investigate the role of miR-199a-5p in regulating VSMC senescence in AAA. VSMC senescence was determined by a senescence-associated β-galactosidase (SA-β-gal) assay. RT-PCR and Western blotting were performed to measure miRNA and protein level, respectively. The generation of reactive oxygen species (ROS) was evaluated by H2DCFDA staining. Dual-luciferase reporter assay was used to validate the target gene of miR-199a-5p. VSMCs exhibited increased senescence in AAA tissue relative to healthy aortic tissue from control donors. Compared with VSMCs isolated from control donors (control-VSMCs), those derived from patients with AAA (AAA-VSMCs) exhibited increased cellular senescence and ROS production. Angiotensin II (Ang II) induced VSMC senescence by promoting ROS generation. The level of miR-199a-5p expression was upregulated in the plasma from AAA patients and Ang II–treated VSMCs. Mechanistically, Ang II treatment significantly elevated miR-199a-5p level, thereby stimulating ROS generation by repressing Sirt1 and consequent VSMC senescence. Nevertheless, Ang II–induced VSMC senescence was partially attenuated by a miR-199a-5p inhibitor or Sirt1 activator. Our study revealed that miR-199a-5p aggravates Ang II–induced VSMC senescence by targeting Sirt1 and that miR-199a-5p is a potential therapeutic target for AAA.     

The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.     

MicroRNA-451a attenuates angiotensin II–induced cardiac fibrosis and inflammation by directly targeting T-box1

Journal of Physiology and Biochemistry - Hypertension or angiotensin II (Ang II) induces cardiac inflammation and fibrosis, thus contributing to cardiac remodeling. MicroRNAs (miRNAs) are...     

How does stress affect human being—a molecular dynamic simulation study on cortisol and its glucocorticoid receptor

Stress can be either positive or negative to human beings. Under stressful conditions, the mental and physical conditions of human can be affected. There exists certain relation between stress and illness. The cortisol and other glucocorticoids bind to the same receptor, which is called glucocorticoid receptor. Some evidences indicated that cortisol molecule binding to its glucocorticoid receptor was necessary for the stress response. Up to now, the structure–function relationships between cortisol molecule and its glucocorticoid receptor have not been deliberated from the atomic-level. In order to get a detailed understanding of the structure–function relationships between the cortisol molecule and glucocorticoids receptor, we have carried out molecular dynamic (MD) simulations on glucocorticoid receptor (Apo system) and cortisol with its glucocorticoid receptor complex (HCY system). On the basis of molecular dynamic simulations, a couple of key residues were identified, which were crucial for the binding of cortisol molecule. The results of binding free energy calculations are in good agreement with the experiment data. Our research gives clear insights from atomic-level into the structural–functional aspects of cortisol molecule and its glucocorticoid receptor, and also provides valuable information for the design of drug which can treat stress related illnesses.     

Irreversibility of the interaction of human growth hormone with its receptor and analysis of irreversible reactions in radioreceptor assays—Theoretical considerations

Kinetic studies of the binding and dissociation of [¹²⁵I]-human growth hormone to rabbit liver and mammary gland membrane receptors have showed that the binding of [¹²⁵I]-human growth hormone was largely irreversible to liver membrane receptors and completely to the solubilised mammary gland receptor. As Scatchard analysis assumes complete reversibility of the hormone-receptor interaction the validity of estimates of affinity and capacity of receptors derived by this analysis may be questionable. Theoretical considerations show that in unimolecular irreversible interactions of hormone and receptor, a nonlinear (concave) or a linear Scatchard plot can be obtained. In linear Scatchard plots the capacity of the receptor obtained by extrapolation represents an overestimation of true capacity. This overestimation correlates with the value of the intercept in the Scatchard plot.     

Characterization of a cytosolic estrogen receptor and its up-regulation by 17β-estradiol and the xenoestrogen 4-tert-octylphenol in the liver of eelpout (Zoarces viviparus)

Estrogen binding activity was revealed in the cytosolic fraction of hepatic extracts from adult male and female eelpout (Zoarces viviparus). The binding moiety was characterized by a single class of high affinity binding sites (K_d=0.59±0.05 nM in males and 1.06±0.10 nM in females). The affinity was significantly higher in males. Binding sites were satiable and binding capacity was significantly elevated in vitellogenic females (2.92±0.28 pmol/g) compared to males (1.67±0.11 pmol/g). The binding was specific to known estrogens but not to other tested steroids. The binding moiety was able to bind to DNA–cellulose and was extractable by high salt concentrations. A time-course study of estrogen binding activity in liver cytosol and of vitellogenin (Vtg) in plasma, after intraperitoneal (i.p.) injections of 17β-estradiol (E₂) in male eelpout, was carried out. It was shown that both are inducible by E₂. Estrogen binding activity was significantly elevated 48 h and Vtg 72 h after E₂ treatment. The binding moiety was hereafter designated as a cytosolic estrogen receptor (ER). The estrogenicity of 4-tert-octylphenol (OP) was evaluated by measuring ER and Vtg after i.p. treatment. OP-treatment increased both receptor levels and Vtg concentrations in male fish, indicating that OP acts as an estrogen in male eelpout.     

High glucose and interleukin-1β downregulate interleukin-1 type I receptor (IL-1RI) in retinal endothelial cells by enhancing its degradation by a lysosome-dependent mechanism

Diabetic retinopathy has been considered a low-grade chronic inflammatory disease. The production of interleukin-1β (IL-1β) in the retina is increased, and this finding has been correlated with an increase in blood-retinal barrier permeability, suggesting that IL-1β might have an important role in the pathogenesis of diabetic retinopathy. However, in this context, no attention has been given to interleukin-1 type I receptor (IL-1RI), which is the receptor responsible for IL-1β triggered effects. Therefore, we investigated the effect of high glucose and IL-1β on the IL-1RI regulation in retinal endothelial cells. A time-dependent downregulation of IL-1RI protein levels was detected in retinal endothelial cells exposed (1–24 h) to high glucose, mannitol or IL-1β. Long-term exposure (7 days) to high glucose or mannitol also decreased IL-1RI protein content. IL-1RI downregulation was due to its activation by IL-1β, since it was inhibited by the presence of anti-IL-1RI or anti-IL-1β antibodies. Moreover, IL-1RI downregulation was prevented by lysosome inhibitors, chloroquine and ammonium chloride, but not by proteasome inhibitors, MG132 and lactacystin. We also found that IL-1RI translocates to the nucleus after high glucose or IL-1β treatment. In conclusion, our results indicate that high glucose, probably due to osmotic stress, and IL-1β downregulate IL-1RI in retinal endothelial cells. The downregulation of IL-1RI is triggered by its activation and is due, at least partially, to lysosomal degradation. High glucose and IL-1β also enhance the translocation of IL-1RI to the nucleus.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots—a soil zymography analysis

Aims

Hotspots of enzyme activity in soil strongly depend on carbon inputs such as rhizodeposits and root detritus. In this study, we compare the effect of living and dead Lupinus polyphyllus L. roots on the small-scale distribution of cellulase, chitinase and phosphatase activity in soil.

Methods

Soil zymography, a novel in situ method, was used to analyze extracellular cellulase, chitinase and phosphatase activity in the presence of i. living L. polyphyllus roots prior to shoot cutting and ii. dead/dying roots 10, 20 and 30 days after shoot cutting.

Results

After shoot cutting, cellulase and chitinase activities increased and were highest at the root tips. The areas of high cellulase and phosphatase activity extend up to 55 mm away from the root. Moreover, we observed microhotspots of cellulose, chitinase, and phosphatase activity up to 60 mm away from the next living root. The number and activity of microhotspots of chitinase activity was maximal 10 days after shoot cutting.

Conclusions

The study showed that young root detritus stimulates enzyme activities stronger than living roots. Soil zymography allowed identification of microhotspots of enzyme activity up to several cm away from living and dying roots, which most likely were caused by arbuscular mycorrhizal fungi.     

Application of liquid chromatography–turbo ion spray tandem mass spectrometry for quantitative analysis of a potent motilin receptor agonist,EM574, and its metabolites in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the simultaneous determination of a new potent motilin receptor agonist as erythromycin derivative, EM574 (erythromycin derivative), and its three metabolites, M-IV, M-V and M-VI, in human plasma was developed. The internal standards (I.S.s) used were deuterated EM574, M-IV and M-V. For the quantitation of M-VI, deuterated M-V was used. The analytes and I.S. were extracted from plasma samples with diethyl ether at neutral pH. A turbo ion spray interface was used as the ion source of LC–MS–MS, and the analysis was performed in the selected reaction monitoring mode. The lower quantitation limits for all the analytes were 0.05 ng/ml when 0.2 ml of plasma was used, and the standard curves were linear in the range 0.05 to 20 ng/ml. The method was precise; the intra- and inter-day precisions of the method were not more than 19.8% for all the analytes. The accuracy of the method was good with the deviations between added and calculated concentrations of each analyte being typically within ±11.2%.     

Regression analysis of yield stability is strongly affected by companion test varieties and locations — examples from a study of Nordic barley lines

The suitability of regression analysis for studying the phenotypic stability of grain yield was investigated using a collection of 220 Nordic barley lines. Linear regression explained 26–52% of the genotype x environment (GE) interactions in different groupings of the material. The regression coefficient, b _i , measures the yield response of the i-th genotype to improved environmental conditions. Deviations from regression, S _di ² , have been used to estimate Tai's stability parameter, _i , which is a measure of the phenotypic yield stability in the agronomic sense. Repeatability of b _i , _i , and grain yield was studied by means of correlations between estimates obtained in each experimental year. Yield had the highest repeatability, with correlations between years ranging from 0.57 to 0.85. In this study, regression coefficients and _i -values were not repeatable, i.e. genotypes reacted differentially to the yearly climatic variations. Six-rowed (6r) barleys had higher responsiveness, but lower mean yields, than two-rowed (2r) barleys. This is partly due to the history of selection of 6r-barleys, which mainly originate from regions with low potential yield levels, i.e. Finland and Norway. In general, responsiveness and stability were not correlated with yield. The highest-yielding lines had b _i 1. The response pattern of the different types of barleys used in this study show that responsiveness can be changed by recombination.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.     

Simultaneous determination of taxol and its metabolites in microsomal samples by a simple thin-layer chromatography radioactivity assay — inhibitory effect of NK-104, a new inhibitor of HMG-CoA reductase

The inhibitory effect of NK-104, a potent inhibitor of HMG-CoA reductase, on taxol metabolism was examined using radio-TLC. This method is described for in vitro measurement of taxol metabolites as an alternative to the commonly used HPLC assay. After incubation of ¹⁴C-taxol with human liver microsomes, the supernatants were developed using a solvent system consisting of toluene–acetone–formic acid (60:39:1, v/v) and quantified with a bioimaging analyzer. The described method provides a valuable tool for the simultaneous determination of unchanged taxol and its major metabolites. There was no inhibitory effect of NK-104 on CYP-mediated metabolism of taxol in human liver microsomes.     

Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation — FH469→Stop

DNA from 40 unrelated familial hypercholesterolemia (FH) heterozygotes were subjected to analyses of single-strand conformation polymorphisms (SSCPs) of exon 10 of the low density lipoprotein receptor (LDLR) gene. Four different SSCP patterns were observed. The underlying mutations were characterized by DNA sequencing. Three of the patterns represented the three genotypes of a recently described sense mutation in codon 450. A method based upon the polymerase chain reaction (PCR) was developed to analyze this mutation. The frequencies of the wild-type (G at nucleotide 1413) and mutant (A at nucleotide 1413) alleles were 0.56 and 0.44, respectively. The fourth pattern was found in only one FH heterozygote and was caused by heterozygosity at nucleotide 1469 (G/A). Nucleotide 1469 is the second base of codon 469_Trp(TGG). The GA mutation changes this codon into the amber stop codon, and is referred to as FH_469Stop. The mutant receptor consists of the amino terminal 468 amino acids. Because the truncated receptor has lost the membrane-spanning domain, it will not be anchored in the cell membrane. FH_469Stop destroys an AvaII restriction site, and this characteristic was used to develop a PCR method to establish its frequency in Norwegian FH subjects. Two out of 204 (1%) unrelated FH heterozygotes possessed the mutation.     

Structural analysis of a polysaccharide from<Emphasis Type="Italic">Chlorella kessleri</Emphasis> by means of gas chromatography—mass spectrometry of its saccharide alditols

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated fromChlorella kessleri.¹H- and¹³C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to thel ord series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.     

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of S?o Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.