The ubiquitin ligase Nedd4 mediates oxidized low-density lipoprotein-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low-density lipoprotein (LDL) is proatherogenic and induces smooth muscle cell apoptosis, which contributes to atherosclerotic plaque destabilization. We showed previously that oxidized LDL downregulates insulin-like growth factor-1 receptor in human smooth muscle cells and that this is critical for induction of apoptosis. To identify mechanisms, we exposed smooth muscle cells to 60 mug/ml oxidized LDL or native LDL and assessed insulin-like growth factor-1 receptor mRNA levels, protein synthesis rate, and receptor protein stability. Oxidized LDL decreased insulin-like growth factor-1 receptor mRNA levels by 30% at 8 h compared with native LDL, and this decrease was maintained for up to 20 h. However, insulin-like growth factor-1 receptor protein synthesis rate was not altered by oxidized LDL. Pulse-chase labeling experiments revealed that oxidized LDL reduced insulin-like growth factor-1 receptor protein half-life to 12.2+/-1.7 h from 24.4+/-4.7 h with native LDL. This destabilization of insulin-like growth factor-1 receptor protein was accompanied by enhanced receptor ubiquitination. Overexpression of dominant-negative Nedd4 prevented oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor, suggesting that Nedd4 was the ubiquitin ligase that mediated receptor downregulation. However, the proteasome inhibitors lactacystin, MG-132, and proteasome inhibitor-1 failed to block oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor. Thus oxidized LDL downregulates insulin-like growth factor-1 receptor by destabilizing the protein via Nedd4-enhanced ubiquitination, leading to degradation via a proteasome-independent pathway. This finding provides novel insights into oxidized LDL-triggered oxidant signaling and mechanisms of smooth muscle cell depletion that contribute to plaque destabilization and coronary events.     

Complexation between triterpene glycosides of holothurians and cholesterol is the basis of lipid-saponin carriers of subunit protein antigens

The ability of some triterpene glycosides of holothurians: cucumarioside A2-2 from Cucumaria japonica, cucumarioside G1 from Cucumaria fraudatrix, frondoside A from Cucumaria frondosa, and holotoxin A1 from A postichopus japonicus to form lipid-saponin supramolecular complexes was studied. The formation of supramolecular cholesterol-glycosides complexes between cholesterol and these glycosides in water medium was observed by transmission electron microscopy. These complexes were considered as nanoparticles with different structure. Complexes formed by cholesterol with cucumarioside A2-2, holotoxin A1, and frondoside A are tubular nanoparticles. In contrast, complexes between cholesterol and cucumarioside G1 have different structured. The structure of nanoparticles formed in the presence of cucumarioside A2-2, holotoxin A1, and cucumarioside G1 was dependent on the ratio of cholesterol in the lipid-saponin system. On the other hand, frondoside A did not shown this tendency. In lipid-saponin systems with a similar molar ratio cholesterol-glycoside, the ordering of the supramolecular structure decreases in the following order: cucumarioside A2-2, holotoxin A1, frondoside A. A comparative analysis of the morphology of the supramolecular complexes and the peculiarities of the molecular structure of triterpene glycosides studied, demonstrated that the structure of supramolecular complexes formed depends on the branching and length of the glycoside carbohydrate chain. On the other hand, the formation of monomeric cholesterol-glycosides complexes depends on the peculiarities of the structure of aglycone. Thus, the possibility of the formation of a new type of antigen carries on the basis of marine triterpene glycosides was proved.     

A novel aldehyde dextran sulfonate matrix for affinity biosensors

Aldehyde dextran sulfonate (ADS), a modified oligosaccharide polymer, was used to prepare a new matrix structure for affinity biosensors. The principal difference between the ADS matrix and similar structures developed previously results from presence of two active functional groups in the matrix, namely, aldehyde and sulfonate. These groups perform two different functions in the matrix. The aldehyde group is responsible for covalent bonding in the biomaterials, and the negatively charged sulfonate group provides electrostatic attraction of the positively charged biomolecules. By varying the ratio between the aldehyde and sulfonate groups in the matrix, one can control contributions from the two binding modes (covalent and electrostatic). A number of oligosaccharides, such as simple dextran, aldehyde dextran (AD), aldehyde dextran sulfonate (ADS) and aldehyde ethylcellulose (AEC), were used for preparation of matrix structures. The properties of the obtained matrices were analysed and compared. Surface plasmon resonance (SPR) was used as the main technique to characterize the matrix structures.     

Millimeter‐wave emissivity as a metric for the non‐contact diagnosis of human skin conditions

A half‐space electromagnetic model of human skin over the band 30–300 GHz was constructed and used to model radiometric emissivity. The model showed that the radiometric emissivity rose from 0.4 to 0.8 over this band, with emission being localized to a layer approximately one millimeter deep in the skin. Simulations of skin with differing water contents associated with psoriasis, eczema, malignancy, and thermal burn wounds indicated radiometry could be used as a non‐contact technique to detect and monitor these conditions. The skin emissivity of a sample of 30 healthy volunteers, measured using a 95 GHz radiometer, was found to range from 0.2 to 0.7, and the experimental measurement uncertainty was ±0.002. Men on average were found to have an emissivity 0.046 higher than those of women, a measurement consistent with men having thicker skin than women. The regions of outer wrist and dorsal forearm, where skin is thicker, had emissivities 0.06–0.08 higher than the inner wrist and volar forearms where skin is generally thinner. Recommendations are made to develop a more sophisticated model of the skin and to collect larger data sets to obtain a deeper understanding of the signatures of human skin in the millimeter wave band. Bioelectromagnetics. 38:559–569, 2017. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.     

Temperature effects on alpha-crystallin structure probed by 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive two-wavelength fluorescent dye covalently attached to the single Cys residue

The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.     

Capacitative cation entry in human myometrial cells and augmentation by hTrpC3 overexpression

Transient receptor potential (Trp) channels have been implicated in mediating store- and receptor-activated Ca2+ influx. Different properties of this influx in various cell types may stem from the assembly of these Trp proteins into homo- or heterotetramers or association with other regulatory proteins. We examined the properties of endogenous capacitative Ca2+ entry in PHM1 immortalized human myometrial cells that express endogenous hTrpCs 1, 3, 4, 6, and 7 mRNA and in primary human myocytes. In PHM1 cells, activation of the oxytocin receptor or depletion of intracellular Ca2+ stores with the endoplasmic reticulum calcium pump-inhibitor thapsigargin induced capacitative Ca2+ entry, which was inhibited both by SKF 96365 and gadolinium (Gd3+). Whereas unstimulated cells did not exhibit Sr2+ entry, oxytocin and thapsigargin enhanced Sr2+ entry that was also inhibited by SKF 96365 and Gd3+. In contrast, Ba2+, a poor substrate for Ca2+ pumps, accumulated in these cells in the absence of the capacitative entry stimulus and also after oxytocin and thapsigargin treatment. Both types of entry were markedly decreased by SKF 96365 and Gd3+. The membrane-permeant derivative of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), elicited oscillatory increases in PHM1 intracellular Ca2+ that were dependent on extracellular Ca2+. These properties were also observed in primary human myocytes. Overexpression of hTrpC3 in PHM1 cells enhanced thapsigargin-, oxytocin-, and OAG-induced Ca2+ entry. These data are consistent with the expression of endogenous hTrpC activity in myometrium. Capacitative Ca2+ entry can potentially contribute to Ca2+ dynamics controlling uterine smooth muscle contractile activity.     

Cyanine dyes as intercalating agents: kinetic and thermodynamic studies on the DNA/Cyan40 and DNA/CCyan2 systems

The interaction of cyanines with nucleic acids is accompanied by intense changes of their optical properties. Consequently these molecules find numerous applications in biology and medicine. Since no detailed information on the binding mechanism of DNA/cyanine systems is available, a T-jump investigation of the kinetics and equilibria of binding of the cyanines Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with CT-DNA is performed at 25 degrees C, pH 7 and various ionic strengths. Bathochromic shifts of the dye absorption band upon DNA addition, polymer melting point displacement (DeltaT = 8-10 degrees C), site size determination (n = 2), and stepwise kinetics concur in suggesting that the investigated cyanines bind to CT-DNA primary by intercalation. Measurements with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) reveal fair selectivity of CCyan2 toward G-C basepairs. T-jump experiments show two kinetic effects for both systems. The binding process is discussed in terms of the sequence D + S left arrow over right arrow D,S left arrow over right arrow DS(I) left arrow over right arrow DS(II), which leads first to fast formation of an external complex D,S and then to a partially intercalated complex DS(I) which, in turn, converts to DS(II), a more stable intercalate. Absorption spectra reveal that both dyes tend to self-aggregate; the kinetics of CCyan2 self-aggregation is studied by T-jump relaxation and the results are interpreted in terms of dimer formation.