Side‐effects of thiamethoxam on the brain andmidgut of the africanized honeybee Apis mellifera (Hymenopptera: Apidae)

The development of agricultural activities coincides with the increased use of pesticides to control pests, which can also be harmful to nontarget insects such as bees. Thus, the goal of this work was assess the toxic effects of thiamethoxam on newly emerged worker bees of Apis mellifera (africanized honeybee—AHB). Initially, we determined that the lethal concentration 50 (LC₅₀) of thiamethoxam was 4.28 ng a.i./μL of diet. To determine the lethal time 50 (LT₅₀), a survival assay was conducted using diets containing sublethal doses of thiamethoxam equal to 1/10 and 1/100 of the LC_50. The group of bees exposed to 1/10 of the LC₅₀ had a 41.2% reduction of lifespan. When AHB samples were analyzed by morphological technique we found the presence of condensed cells in the mushroom bodies and optical lobes in exposed honeybees. Through Xylidine Ponceau technique, we found cells which stained more intensely in groups exposed to thiamethoxam. The digestive and regenerative cells of the midgut from exposed bees also showed morphological and histochemical alterations, like cytoplasm vacuolization, increased apocrine secretion and increased cell elimination. Thus, intoxication with a sublethal doses of thiamethoxam can cause impairment in the brain and midgut of AHB and contribute to the honeybee lifespan reduction. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1122–1133, 2014.     

New morpholine‐liganded palladium(II) N‐heterocyclic carbene complexes: Synthesis,characterization, crystal structure,and DNA‐binding studies

A series of the morpholine‐liganded palladium(II) complexes ( 1a–e ) bearing N‐heterocyclic carbene (NHC) functionalized by benzonitrile were synthesized. These complexes were synthesized from (NHC)Pd(II)(pyridine) complexes (PEPPSI) and morpholine. The new complexes were fully characterized by using ¹H NMR, ¹³C NMR, Fourier‐transform infrared spectroscopy, and elemental analysis techniques. Single‐crystal X‐ray diffraction was used to determine the structure of a derivative. The DNA‐binding studies of the new (NHC)Pd(II)morpholine complexes were examined using the pBR322 plasmid. The 2,4,6‐trimethylbenzyl derivative compound has the most DNA binding activity. In addition, for the 3‐methylbenzyl derivative compound, oxidation effects were observed at concentrations higher than 100 µg/ml. Also, the molecular and crystal structures of the complex 3‐methylbenzyl derivative compound were recorded by using a single‐crystal X‐ray diffraction method.     

Antibacterial activities and conformations of synthetic α‐defensin HNP‐1 and analogs with one,two and three disulfide bridges

Abstract: Structure and biological activities of synthetic peptides corresponding to human α‐defensin HNP‐1, AC¹YC²RIPAC³IAGERRYGTC⁴IYQGRLWAFC⁵C⁶ with the S–S connectivities: C¹–C⁶, C²–C⁴, C³–C⁵, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP‐1 yielded a peptide with S–S connectivities C¹–C³, C²–C⁴ and C⁵–C⁶, and not with the S–S linkages as in naturally occurring HNP‐1. Selective protection of cysteine sulfhydryls was necessary for the formation of S–S bridges as in native HNP‐1. Likewise, oxidation of peptide encompassing the segment from C² to C⁵, resulted in the S–S linkages C²–C³ and C⁴–C⁵ instead of the expected linkage C²–C⁴ and C³–C⁵. Antibacterial activities were observed for all peptides, irrespective of how the S–S bridges were linked. Linear peptides without S–S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S–S bridges do not form rigid β‐sheet structures in an aqueous environment. The spectrum of HNP‐1 in an aqueous environment suggests the presence of a β‐hairpin conformation. In the presence of lipid vesicles, the S–S constrained peptides tend to adopt a β‐structure. Although the S–S connectivities observed in HNP‐1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Assessment of in vitro metabolic stability,plasma protein binding,and pharmacokinetics of E‐ and Z‐guggulsterone in rat

Guggulsterone is a racemic mixture of two stereoisomers (E‐ and Z‐), obtained from the gum resin of Commiphora mukul and it is marketed as an antihyperlipidemic drug. The aim of our study was to assess the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties namely solubility, in vitro metabolism, plasma protein binding and oral pharmacokinetic studies of E‐ and Z‐guggulsterone. In vitro metabolism experiments were performed by using rat liver and intestinal microsomes. In vitro intrinsic clearance (CL_int) was found to be 33.34 ± 0.51 and 39.23 ± 8.12 μL/min/mg protein in rat liver microsomes for E‐ and Z‐isomers, respectively. Plasma protein binding was determined by equilibrium dialysis method and in vivo pharmacokinetic studies were performed in male Sprague Dawley (SD) rats. Both isomers were highly bound to rat plasma proteins (>95% bound). Plasma concentration of E‐ and Z‐isomers decreased rapidly following oral administration and were eliminated from systemic circulation with a terminal half‐life of 0.63 ± 0.25 and 0.74 ± 0.35 h, respectively. The clearance (CL) for E‐isomer was 2.79 ± 0.73 compared to 3.01 ± 0.61 L/h/kg for Z‐isomer, indicating no significant difference (student t test; p <0.05) in their elimination.The pharmacokinetics of both isomers was characterized by extensive hepatic metabolism as seen with rat liver microsomes with high clearance and low systemic availability in rats. In brief, first‐pass metabolism seems to be responsible factor for low bioavailability of guggulsterone. Copyright © 2015 John Wiley & Sons, Ltd.     

In‐situ absorption,protein binding and pharmacokinetic studies of S002‐853, a novel antidiabetic and antidyslipidaemic flavone derivative in rats

Objectives The aim of the study was to investigate the in‐situ absorption kinetics, plasma protein binding and pharmacokinetic characteristics of a novel synthetic flavone derivative, S002‐853, which shows pronounced antidiabetic and antidyslipidaemic activity. Methods Quantification of S002‐853 in plasma was performed by the LC‐MS/MS method and in‐situ sample analysis was carried out by the HPLC‐UV method. Key findings The absorption rate constant was 0.274/h in a mild alkaline environment, which S002‐853 experiences in the intestine following oral dose administration. Plasma protein binding was found to be 26.37 ± 2.58% at a concentration of 1 μg/ml. The pharmacokinetic parameters were determined in male rats after administration of a single 40 mg/kg oral dose and 10 mg/kg intravenous dose. The peak plasma concentration (C_max) was found to be 60.93 ng/ml at 8 h after oral administration. Irregular concentration–time profiles with secondary peaks were observed after oral dose administration. The elimination half‐life of the compound was 19.56 h and 16.30 h after oral and intravenous doses, respectively. Comparison of the AUC after oral and intravenous dosing of S002‐853 indicates that only about 29.48% (bioavailability) of the oral dose reaches the systemic circulation. Conclusions In‐situ study of S002‐853 shows slow absorption from the gastrointestinal tract. S002‐853 also shows low plasma protein binding. The pharmacokinetic parameters after oral and intravenous dose reveal low oral bioavailability and high mean residence time.     

Growth receptor binding protein 10 inhibits glucose‐stimulated insulin release from pancreatic β‐cells associated with suppression of the insulin/insulin‐like growth factor‐1 signalling pathway

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Design,Synthesis, and Antitumor Activity of (E,Z)‐1‐(dihydrobenzofuran‐5‐yl)‐3‐phenyl‐2‐(1,2,4‐triazol‐1‐yl)‐2‐propen‐1‐ones

A series of (E,Z)‐1‐(dihydrobenzofuran‐5‐yl)‐3‐phenyl‐2‐(1,2,4‐triazol‐1‐yl)‐2‐propen‐1‐ones ( C1 – C35 ) were designed and synthesized, and the structures of compounds (Z)‐ C27 and (Z)‐ C29 were confirmed by single‐crystal X‐ray diffraction. The antitumor activities of these novel compounds against cervical cancer (HeLa), lung cancer (A549), and breast cancer (MCF‐7) cell lines were evaluated in vitro. Majority of the title compounds exhibited strong antitumor activities and were much more promising than the positive control Taxol, which were also accompanied by lower cytotoxicity to normal cells. In particular, compounds (E,Z)‐ C24 exhibited the most consistent potent activities against three neoplastic cells with IC₅₀ values ranging from 3.2 to 7.1 μm . Further researches demonstrated that compounds (E,Z)‐ C24 could induce cell apoptosis and arrest cell cycle at the G2/M and S phases. Meanwhile, the structure–activity relationship between the configurations and cytotoxicity of the compounds was also investigated.     

Preparation and a simple one‐step purification of [His1‐mono‐125I‐Tyr10,Nle27]‐hGHRH(1‐32)‐NH2

A one‐step purification of [His¹‐mono‐¹²⁵I‐Tyr¹⁰,Nle²⁷]‐hGHRH(1‐32)‐NH₂, prepared using chloramine‐T, by HPLC with isocratic elution is described. The labeled GHRH analog was suitable for GHRH receptor binding assays. Copyright © 2002 John Wiley & Sons, Ltd.     

Evaluation of oral pharmacokinetics,in vitro metabolism,blood partitioning and plasma protein binding of novel antidiabetic agent,S009‐0629 in rats

S009‐0629 [methyl‐8‐(methylthio)‐2‐phenyl‐6‐p‐tolyl‐4,5‐dihydro‐2H‐benzo[e]indazole‐9‐carboxylate] is a novel antidiabetic agent with PTP1B inhibitory activity. In this study, we have investigated the in vitro metabolic stability, plasma protein binding, blood partitioning, and oral pharmacokinetic study of S009‐0629 in rats. The plasma protein binding, blood partitioning, and metabolic stability were determined by HPLC method. The oral pharmacokinetic study was analyzed by liquid chromatography coupled mass spectrometry (LC‐MS/MS) method. The plasma protein binding of S009‐0629 using modified charcoal adsorption method at 5 and 10 µg/mL was 80.58 ± 1.04% and 81.95 ± 1.15%, respectively. The K_RBC/PL of S009‐0629 was independent of concentration and time. The in‐vitro half‐life of S009‐0629 at 5 and 10 µM using rat liver microsomes was determined as 273 ± 24.46 and 281.67 ± 26.53 min, respectively. After oral administration, S009‐0629 exhibited C_max 55.51 ± 1.18 ng/mL was observed at 18 hr (t_max). S009‐0629 was found to have the large apparent volume of distribution (1,894.93 ± 363.67 L/kg). Oral in‐vivo t_1/2 of S009‐0629 was found to be 41.23 ± 5.96 hr. A rapid and highly sensitive LC‐MS/MS method was validated for S009‐0629 in rat plasma. S009‐0629 has high plasma protein binding and low hepatic extraction. S009‐0629 has no affinity with human P‐gp and BCRP in ATPase assay. After oral dosing, S009‐0629 has slow absorption and elimination in rats.     

Concentration‐dependent plasma protein binding of the novel dipeptidyl peptidase 4 inhibitor BI 1356 due to saturable binding to its target in plasma of mice,rats and humans

Objectives The purpose of this study was to characterise the plasma protein binding of BI 1356. Methods BI 1356 (proposed trade name ONDERO) is a novel dipeptidyl peptidase 4 (DPP‐4) inhibitor, which is under clinical development for the treatment of type 2 diabetes. DPP‐4 is expressed in various tissues but soluble DPP‐4 is also present in plasma. Therefore, binding to soluble DPP‐4 may influence the pharmacokinetics of BI 1356. Plasma protein binding of BI 1356 was determined in vitro for wild type mice and rats and the results compared with those for DPP‐4 knockout mice and DPP‐4 deficient Fischer rats. In addition, protein binding of BI 1356 was examined in plasma from healthy human volunteers and renal excretion of the compound in the DPP‐4 knockout mice was compared with that occurring in wild type mice. Key findings The results showed that BI 1356 exhibited a prominent concentration‐dependent plasma protein binding due to a saturable high affinity binding to the DPP‐4 target in plasma. Differences in renal excretion of BI 1356 between DPP‐4 knockout mice and wild type mice suggested that saturable binding of BI 1356 to DPP‐4 in the body also influenced elimination. Conclusions High affinity, but readily saturable binding of BI 1356 to its target DPP‐4 accounted primarily for the concentration‐dependent plasma protein binding at therapeutic plasma concentrations of BI 1356.     

The Structural and Functional Role of the B‐chain C‐terminal Arginine in the Relaxin‐3 Peptide Antagonist,R3(BΔ23‐27)R/I5

Relaxin‐3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin‐3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin‐3/RXFP3 pair. The analog R3(BΔ23‐27)R/I5, in which a C‐terminally truncated human relaxin‐3 (H3) B‐chain is combined with the INSL5 A‐chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B‐chain C‐terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BΔ23‐27)R/I5 and R3(BΔ23‐27)R containing the B‐chain C‐terminal Arg as well as R3(BΔ23‐27)/I5 and R3(BΔ23‐27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BΔ23‐27)R and R3(BΔ23‐27)R/I5, the peptide R3(BΔ23‐27) is a weak agonist. This suggests that the C‐terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BΔ23‐27)R/I5 its potent antagonistic activity.     

Effects of poloxamer 407‐induced hyperlipidemia on hepatic multidrug resistance protein 2 (Mrp2/Abcc2) and the pharmacokinetics of mycophenolic acid in rats

Hepatic multidrug resistance‐associated protein 2 (Mrp2) is responsible for the majority of the biliary elimination of endogenous and exogenous substances, therefore it is important to evaluate possible functional changes in Mrp2 activity under conditions of hyperlipidemia (HL). Thus, the present study assessed the protein expression and transporting activity of hepatic Mrp2 based on the in vivo biliary excretion of phenolsulfonphthalein (PSP) as a model anionic substrate for Mrp2 in poloxamer 407‐induced hyperlipidemic rats (HL rats) and compared these values with those for control rats. The pharmacokinetics of mycophenolic acid (MPA) and mycophenolic acid‐7‐O‐glucuronide (MPAG) were evaluated after the intravenous (5 mg/kg) and oral (10 mg/kg) administration of MPA to control and HL rats. In HL rats, the protein expression of hepatic Mrp2 and its biliary transporting activity exhibited significant reductions (by 24.3% and 24.6%, respectively) in the absence of a change in bile flow rate. Unexpectedly, HL and control rats showed comparable biliary excretion rates of MPAG due to the counter effects of the reduced expression and activity of Mrp2 and a 484% increase in the free fraction of MPAG in HL rats. The estimated biliary clearance value of free MPAG in HL rats was considerably slower (by 77.1%) than that in control rats. Although significant pharmacokinetic changes in total MPA and MPAG levels were not observed in HL rats, there was a marked increase in free MPA and MPAG levels. Clinically relevant pharmacokinetic changes in subjects with HL that are related to MRP2 could not be ruled out. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of an herbal protein,CI‐1, purified from Cajanus indicus,in models of liver failure in mice

Acute liver damage models in Swiss albino mice (male; 25–30 g) induced by paracetamol (300–500 mg/kg); β‐galactosamine (500 mg/kg); and 40% ethanol (2 ml / 100 g) were studied. Clinical indications of chronic hepatocellular necrosis were manifested within 24 h of toxic doses. Initially, serum transaminases, alkaline phosphatase, lactate dehydrogenase, bilirubin, and triglycerides were markedly increased; in addition, the plasma prothrombin time was prolonged. The total serum protein and serum albumin were significantly decreased in comparison to controls. Treatment of severely liver‐injured mice with a protein, CI‐1, purified from the leaves of Cajanus indicus at a dose of 100 μg/ml i.p. regularly for 14 days significantly lowered the respective serum enzymes such as transaminases (SGOT, SGPT), alkaline phosphatases (ALK), and lactate dehydrogenase (LDH), prevented the loss of liver protein content, and improved the altered plasma coagulability. Histological studies of liver of treated (hepatoxins) mice reveal massive centrilobular necrosis, Kupffer's cells hyperplasia, and periportal fatty changes in contrast to control animals. Treatment with CI‐1 in liver‐damaged animals showed near normal histology. CI‐1 may be a useful approach in the treatment of liver disorders. Drug Dev. Res. 48:76–83, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Synthesis of [3H] and [2H6]AZD6642, an inhibitor of 5‐lipoxygenase activating protein (FLAP)

An AstraZeneca effort to identify a 5‐lipoxygenase activating protein inhibitor with good drug‐like properties resulted in the identification of AZD6642. To further understand its drug metabolism and pharmacokinetic properties, it was required labeled with tritium. The tritiation of AZD6642 was effected by Ir‐catalyzed exchange chemistry to give an average of one tritium per molecule. Additionally, a stable isotope labeled version of AZD6642 was required to support bioanalytical studies. The synthesis originated from [²H₆]acetone which was converted to the trimethylsilyl cyanide adduct and subsequently reduced to give 2‐(aminomethyl)‐[1,1,1,3,3,3‐²H₆]propan‐2‐ol in good yield. Carbonylation to give an amide adduct resulted in an intermediate that was converted to the final compound in four steps.     

Synthesis and immunological studies of α‐conotoxin chimera containing an immunodominant epitope from the 268–284 region of HSV gD protein

Abstract: We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as ‘guest’ sequence in the ‘host’ structure of α‐conotoxin GI, a 13‐residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276–284 region of HSV gD‐1 selected for these studies is highly hydrophilic and adopts a β‐turn. The α‐conotoxin GI also contains a β‐turn in the 8–12 region, stabilized by two disulfide bridges at positions 2–7 and 3–13. Thus, the tetramer sequence of α‐conotoxin, ⁸Arg‐His‐Tyr‐Ser¹² has been replaced by Asp‐Pro‐Val‐Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3–13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys²‐Cys⁷ three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 m HCl or Tl(tfa)₃ in trifluoroacetic acid (TFE)] were compared. The high‐performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimeric peptide and native α‐conotoxin GI showed similar circular dichroism spectra in phosphate‐buffered saline (PBS) and in a PBS‐TFE 1 : 1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M‐ and IgG‐type antibody responses showed that the bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV‐α‐[Tyr¹]‐conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV‐α‐[Tyr¹]‐conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG‐specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.     

Influence of EGCG on α‐synuclein (αS) aggregation and identification of their possible binding mode: A computational study using molecular dynamics simulation

The accumulation of intrinsically disordered α‐synuclein (αS) protein that can form β‐sheet‐rich fibrils is linked to Parkinson's disease. (−)‐Epigallocatechin‐3‐gallate (EGCG) is the most abundant active component in green tea and can inhibit the fibrillation of αS. The elucidation of this molecular mechanism will be helpful to understand the inhibition mechanism of EGCG to the fibrillation of αS and also to find more potential small molecules that can inhibit the aggregation of αS. In this work, to study the influence of EGCG on the structure of β‐sheet‐rich fibrils of αS and identification of their possible binding mode, molecular dynamics simulations of pentamer and decamer aggregates of αS in complex with EGCG were performed. The obtained results indicate that EGCG can remodel the αS fibrils and break the initial ordered pattern by reducing the β‐sheet content. EGCG can also break the Greek conformation of αS by the disappeared H‐bond in the secondary structure of turn. The results from our study can not only reveal the specific interaction between EGCG and β‐sheet‐rich fibrils of αS, but also provide the useful guidance for the discovery of other potential inhibitors.     

Primary structure,spectroscopic and inhibitory properties of a two-chain trypsin inhibitor from the seeds of charlock (Sinapis arvensis L), a member of the napin protein family

A protein with inhibitory activity toward trypsin has been isolated from Sinapis arvensis L (charlock). It has a molecular weight of 15500 and consists of two chains connected by disulfide bonds. The amino acid sequence was determined and showed that it belongs to the napin family of storage proteins. CD studies showed an α-helix content of 12% and a β-structure of about 50%.