Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate

Di(2‐ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP‐induced BNL CL. 2 cells. [³H]‐thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen‐activated protein kinases [extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK)], activator protein (AP)‐1 (c‐Jun and c‐Fos), proliferating cell nuclear antigen (PCNA) and cell cycle‐related factors (cyclin D1/cyclin‐dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]‐thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP‐induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP‐1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate‐induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

Secreted phosphoprotein‐24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor‐β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W‐20‐17 mesenchymal stem cells with rhBMP‐2, short‐term Smad1/5 phosphorylation was inhibited, intermediate‐term alkaline phosphatase (ALP) induction was blunted, and long‐term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS–PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non‐reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween‐20 at pH 2.7–8.5. LC–MS/MS analysis of affinity‐purified, non‐reducing SDS–PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α₂‐macroglobulins (α₂‐macroglobulin [α₂M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti‐thrombin III (Serpin C1), which may protect Spp24 from proteolysis. J. Cell. Biochem. 114: 378–387, 2013. © 2012 Wiley Periodicals, Inc.     

Photoluminescence properties of LiTi2 − xEux(PO4)3 phosphor

A solid‐state reaction route‐based LiTi_{2 ? x}Eu_x(PO₄)₃ was phosphor synthesized for the first time to evaluate its luminescence performance by excitation, emission and lifetime (τ) measurements. The LiTi_{2 ? x}Eu_x(PO₄)₃ phosphor was excited at λ_exci. = 397 nm to give an intense orange–red (597 nm) emission attributed to the ⁵D₀ → ⁷F₁ magnetic dipole (ΔJ = ±1) transition and red (616 nm) emission (⁵D₀ → ⁷F₂), which is an electric dipole (ΔJ = ±2) transition of the Eu³⁺ ion. Beside this, excitation and emission spectra of host LiTi₂(PO₄)₃ powder were also reported. The effect of Eu³⁺ concentration on luminescence characteristics was explained from emission and lifetime profiles. Concentration quenching in the LiTi_{2 ? x}Eu_x(PO₄)₃ phosphor was studied from the Dexter's model. Dipole–quadrupole interaction is found to be responsible for energy transfer among Eu³⁺ ions in the host lattice. The LiTi_{2 ? x}Eu_x(PO₄)₃ phosphor displayed a reddish‐orange colour realized from a CIE chromaticity diagram. We therefore suggest that this new phosphor could be used as an optical material of technological importance in the field of display devices. Copyright © 2016 John Wiley & Sons, Ltd.     

Luminescent properties of MAl(SO4)2Br:Eu3+ (M = Sr or Mg) red phosphors for near‐UV light‐emitting diodes

Eu³⁺‐activated MAl(SO₄)₂Br phosphors (where M = Mg or Sr) are successfully prepared using a wet chemical reaction technique. The samples are characterized by X‐ray diffraction (XRD) and photoluminescence (PL) spectroscopies. The XRD pattern revealed that both the samples are microcrystalline in nature. PL of Eu³⁺‐doped SrAl(SO₄)₂Br and MgAl(SO₄)₂Br phosphors exhibited characteristic red emission coming from the ⁵D₀ → ⁷F₂ (616 nm) electron transition, when excited by 396 nm wavelength of light. The maximum intensity of luminescence was observed at a concentration of 1 mol% Eu³⁺. The intensity of the electric dipole transition at 616 nm is greater than that of the magnetic dipole transition at 594 nm. The results showed that MAl(SO₄)₂Br:Eu³⁺, (M = Mg, Sr) phosphors have potential application in near‐UV light‐emitting diodes as efficient red‐emitting phosphor. Copyright © 2014 John Wiley & Sons, Ltd.     

Syntheses,Receptor Bindings,in vitro and in vivo Stabilities and Biodistributions of DOTA‐Neurotensin(8–13) Derivatives Containing β‐Amino Acid Residues – A Lesson about the Importance of Animal Experiments

Neurotensin(8–13) (NTS(8–13)) analogs with C‐ and/or N‐terminal β‐amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1 – 6 ). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) derivatives, 6a , into a crystallographically identified receptor NTSR1 (Fig. 1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell‐membrane homogenates, while, with NTSR1‐exhibiting cancer tissues, affinities in the single‐digit nanomolar range can be observed (Table 2). Most of the β‐amino acid‐containing NTS(8–13) analogs (Table 1 and Fig. 2), including the ⁶⁸Ga complexes of the DOTA‐substituted ones ( 6 ; Figs. 2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two ⁶⁸Ga complexes (of 6a and 6b ) in HT29 tumor‐bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET‐imaging experiments with the tumor‐bearing mice (Fig. 6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10–15 min) of the two ⁶⁸Ga complexes shows that they are rapidly cleaved in the animals (Fig. 5).     

RNA‐Interference Approach to Study Functions of NADPH : Cytochrome P450 Oxidoreductase in Human Hepatocytes

Human NADPH : cytochrome P450 oxidoreductase (POR) is encoded by a single gene on chromosome 7q11.2. This flavoprotein donates electrons derived from NADPH to a variety of acceptor proteins, including squalene monooxygenase, heme oxygenase, cytochrome b₅, and many microsomal cytochromes P450 (CYPs), which are involved in oxidative drug metabolism, steroidogenesis, and other functions. Numerous aspects related to cellular POR expression have not been systematically investigated. Interestingly, POR expression is lower compared to CYPs and may thus be limiting for monooxygenase activities, but conversely, POR knock‐out in mice resulted in compensatory upregulation of CYPs. POR may also influence intracellular cholesterol and lipid homeostasis. To systematically investigate such effects, we developed specific POR gene silencing in cell lines and primary human hepatocytes by RNA interference using small interfering RNAs (siRNAs). In HepG2 cells, POR mRNA could be reduced by 95% over 4 days accompanied by reduced protein content and activity. In primary human hepatocytes, POR mRNA knock‐down was less effective and more variable. Analysis of CYPs indicated induction of CYP3A4 but not CYP1A2 or CYP2D6. These results demonstrate that POR can be efficiently and almost completely silenced in HepG2 cells and, at least partially, in primary human hepatocytes. This will allow systematic studies of various consequences of POR variability in human cells.     

Seizure‐induced impairment in neuronal ketogenesis: Role of zinc‐α2‐glycoprotein in mitochondria

Ketone bodies (KBs) were known to suppress seizure. Untraditionally, neurons were recently reported to utilize fatty acids and produce KBs, but the effect of seizure on neuronal ketogenesis has not been researched. Zinc‐α2‐glycoprotein (ZAG) was reported to suppress seizure via unclear mechanism. Interestingly, ZAG was involved in fatty acid β‐oxidation and thus may exert anti‐epileptic effect by promoting ketogenesis. However, this promotive effect of ZAG on neuronal ketogenesis has not been clarified. In this study, we performed immunoprecipitation and mass spectrometry to identify potential interaction partners with ZAG. The mechanisms of how ZAG translocated into mitochondria were determined by quantitative coimmunoprecipitation after treatment with apoptozole, a heat shock cognate protein 70 (HSC70) inhibitor. ZAG level was modulated by lentivirus in neurons or adeno‐associated virus in rat brains. Seizure models were induced by magnesium (Mg²⁺)‐free artificial cerebrospinal fluid in neurons or intraperitoneal injection of pentylenetetrazole kindling in rats. Ketogenesis was determined by cyclic thio‐NADH method in supernatant of neurons or brain homogenate. The effect of peroxisome proliferator–activated receptor γ (PPARγ) on ZAG expression was examined by Western blot, quantitative real‐time polymerase chain reaction (qRT‐PCR) and chromatin immunoprecipitation qRT‐PCR. We found that seizure induced ketogenesis deficiency via a ZAG‐dependent mechanism. ZAG entered mitochondria through a HSC70‐dependent mechanism, promoted ketogenesis by binding to four β‐subunits of long‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (HADHB) and alleviated ketogenesis impairment in a neuronal seizure model and pentylenetetrazole‐kindled epileptic rats. Additionally, PPARγ activation up‐regulated ZAG expression by binding to promoter region of AZGP1 gene and promoted ketogenesis through a ZAG‐dependent mechanism.     

Dual effects of [Tyr6]‐γ2‐MSH(6–12) on pain perception and in vivo hyperalgesic activity of its analogues

[Tyr⁶]‐γ2‐MSH(6–12) with a short effecting time of about 20 min is one of the most potent rMrgC receptor agonists. To possibly increase its potency and metabolic stability, a series of analogues were prepared by replacing the Tyr⁶ residue with the non‐canonical amino acids 3‐(1‐naphtyl)‐L ‐alanine, 4‐fluoro‐L ‐phenylalanine, 4‐methoxy‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine. Dose‐dependent nociceptive assays performed in conscious rats by intrathecal injection of the MSH peptides showed [Tyr⁶]‐γ2‐MSH(6–12) hyperalgesic effects at low doses (5–20 nmol) and analgesia at high doses (100–200 nmol). This analgesic activity is fully reversed by the kyotorphin receptor‐specific antagonist Leu–Arg. For the two analogues containing in position 6, 4‐fluoro‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine, a hyperalgesic activity was not observed, while the 3‐(1‐naphtyl)‐L ‐alanine analogue at 10 nmol dose was found to induce hyperalgesia at a potency very similar to γ2‐MSH(6–12), but with longer duration of the effect. Finally, the 4‐methoxy‐L ‐phenylalanine analogue (0.5 nmol) showed greatly improved hyperalgesic activity and prolonged effects compared to the parent [Tyr⁶]‐γ2‐MSH(6–12) compound. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Drug‐binding energetics of human α‐1‐acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

This study utilizes sensitive, modern isothermal titration calorimetric methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α‐1‐acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug–AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine, all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug‐binding thermodynamics was characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed that an enthalpy–entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (?Hº) and favorable (positive) entropic (?Sº) contributions to the Gibbs free energy (?Gº). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side‐chain sub‐domains within the multi‐lobed AGP ligand binding cavity.Copyright © 2012 John Wiley & Sons, Ltd.     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

Differential effects of 24‐hydroxycholesterol and 27‐hydroxycholesterol on tyrosine hydroxylase and α‐synuclein in human neuroblastoma SH‐SY5Y cells

Evidence suggests that environmental and dietary factors may contribute to the pathogenesis of Parkinson’s disease (PD). High dietary intake of cholesterol is such a factor that has been shown to increase or decrease the risk of PD. However, because circulating cholesterol does not cross the blood–brain barrier, the mechanisms linking dietary cholesterol to the pathogenesis of PD remain to be understood. In contrast to cholesterol, the oxidized cholesterol metabolites (oxysterols), 24S‐hydroxycholesterol (24‐OHC) and 27‐hydroxycholesterol (27‐OHC), can cross the blood–brain barrier and may place the brain at risk of degeneration. In this study, we incubated the human neuroblastoma SH‐SY5Y cells for 24 h with 24‐OHC, 27‐OHC, or a mixture of 24‐OHC plus 27‐OHC, and have determined effects on tyrosine hydroxylase (the rate‐limiting enzyme in dopamine synthesis) levels, α‐synuclein levels, and apoptosis. We demonstrate that while 24‐OHC increases the levels of tyrosine hydroxylase, 27‐OHC increases levels of α‐synuclein, and induces apoptosis. Our findings show for the first time that oxysterols trigger changes in levels of proteins that are associated with the pathogenesis of PD. As steady state levels of 24‐OHC and 27‐OHC are tightly regulated in the brain, disturbances in these levels may contribute to the pathogenesis of PD.     

Anti‐feeding and insecticidal efficacy of a topical administration of dinotefuran–pyriproxyfen–permethrin spot‐on (Vectra® 3D) on mice against Stegomyia albopicta (= Aedes albopictus)

An ectoparasiticide combining three active ingredients [dinotefuran, permethrin and pyriproxyfen (DPP)] was used in mice in an experiment designed to evaluate its anti‐feeding and insecticidal efficacy against Stegomyia albopicta (= Aedes albopictus) (Diptera: Culicidae) mosquitoes. Twenty‐two adult mice were randomly allocated into two groups consisting of an untreated control group and a DPP‐treated group. Mice were exposed individually for 1 h to a mean ± standard deviation of 27 ± 2 starved female mosquitoes on days 1, 7, 14, 21 and 28 post‐treatment. At the end of the exposure (1 h), mosquitoes were assessed for immediate survival and engorgement status. Additionally, live mosquitoes in both groups were incubated separately and observed for mortality at 24 h after the end of the exposure. The anti‐feeding efficacy of DPP after the 1‐h exposure period was 99.2, 100, 98.0, 89.3 and 87.4% at 1, 7, 14, 21 and 28 days, respectively. Levels of insecticidal efficacy evaluated at 1 h and 24 h after exposure on days 1, 7, 14, 21 and 28 were 36.7, 28.9, 30.8, 23.1 and 11.9%, and 68.4, 45.0, 43.3, 37.9 and 19.9%, respectively. Based on the mouse model, the present study demonstrates that the DPP combination has significant anti‐feeding and insecticidal efficacy against S. albopicta for at least 4 weeks.     

Effects of solvent fractions of Allomyrina dichotoma larvae through the inhibition of in vitro BACE1 and β‐amyloid(25–35)‐induced toxicity in rat pheochromocytoma PC12 cells

Amyloid‐β peptide (Aβ) generation initiated by β‐site amyloid precursor protein cleaving enzyme 1 BACE1 is a critical cause of Alzheimer's disease. In the course of our ongoing investigation of natural anti‐dementia resources, the ethyl acetate (EtOAc) fraction exerted strong BACE1‐specific inhibition with the half maximal inhibitory concentration (IC₅₀) value of 9.2 × 10^?5 μg/mL. Furthermore, Aβ(25–35)‐induced cell death was predominantly prevented by the EtOAc fraction of Allomyrina dichotoma larvae through diminishing of cellular oxidative stress and attenuating apoptosis by inhibiting caspase‐3 activity. Taken together, the present study demonstrated that A. dichotoma larvae possess novel neuroprotective properties not only via the selective and specific inhibition of BACE1 activity but also through the alleviation of Aβ(25–35)‐induced toxicity, which may raise the possibility of therapeutic application of A. dichotoma larvae for preventing and/or treating dementia.