Coadministration of β‐asarone and levodopa increases dopamine in rat brain by accelerating transformation of levodopa: A different mechanism from Madopar

The aim of the present study was to investigate the effect of coadministration of β‐asarone and levodopa (l ‐dopa) on increasing dopamine (DA) in the striatum of healthy rats. Rats were randomly divided into four groups: (i) a normal group, administered normal saline; (ii) a Madopar group, administered 75 mg/kg Madopar (l ‐dopa : benserazide, 4 : 1); (iii) an l ‐dopa group, administered 60 mg/kg l ‐dopa; and (iv) a group coadministered 15 mg/kg β‐asarone and 60 mg/kg l ‐dopa. All drugs (or normal saline) were administered intragastrically twice a day for 7 days. Then, plasma and striatum concentrations of DA, l ‐dopa, 5‐hydroxytryptamine (5‐HT), homovanillic acid (HVA), 3,4‐dihydroxyphenylacetic acid (DOPAC), tyrosine hydroxylase (TH), catechol‐O‐methyltransferase (COMT) and monoamine oxidase B (MAO‐B) were determined. In the group coadministered β‐asarone and l ‐dopa, there was a decline in plasma and striatal concentrations of l ‐dopa; however, DA and DOPAC concentrations increased in the striatum and plasma and plasma HVA concentrations increased, whereas there was no significant change in striatal levels. Concentrations of 5‐HT in the striatum and plasma were similar in the coadministered and Madopar‐treated groups. In addition, plasma and striatal COMT levels decreased after coadministration of β‐asarone and l ‐dopa, whereas there were no significant differences in MAO‐B concentrations among groups. Furthermore, coadministration of β‐asarone and l ‐dopa increased plasma TH concentrations. Altogether, β‐asarone affects the conversion of l ‐dopa to DA by modulating COMT activity and DA metabolism. The mechanism of coadministration is different from that of Madopar in Parkinson's disease (PD) treatment. Thus, the coadministration of β‐asarone and l ‐dopa may be beneficial in the treatment of PD.     

β‐asarone and levodopa co‐administration protects against 6‐hydroxydopamine‐induced damage in parkinsonian rat mesencephalon by regulating autophagy: down‐expression Beclin‐1 and light chain 3B and up‐expression P62

In this study, we investigated Beclin‐1, light chain (LC)3B, and p62 expression in 6‐hydroxydopamine (6‐OHDA)‐induced parkinsonian rats after β‐asarone and levodopa (l ‐dopa) co‐administration. Unilateral 6‐OHDA injection into the medial forebrain bundle was used to create the models, except in sham‐operated rats. Rats were divided into eight groups: sham‐operated group; 6‐OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l ‐dopa group (60 mg/kg, p.o.); β‐asarone group (15 mg/kg, p.o.); β‐asarone + l ‐dopa co‐administered group (15 mg/kg + 60 mg/kg, p.o.); 3‐methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin‐1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin‐1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l ‐dopa, β‐asarone, and co‐administered groups when compared with the 6‐OHDA model. Beclin‐1 and LC3B expression in the β‐asarone and co‐administered groups were less than in the madopar or l ‐dopa groups, whereas p62 expression in the β‐asarone and co‐administered groups was higher than in the madopar or l ‐dopa groups. In addition, a significant decrease in autophagosome was exhibited in the β‐asarone and co‐administered groups when compared with the 6‐OHDA group. Our findings indicate that Beclin‐1 and LC3B expression decreased, whereas p62 expression increased after co‐administration treatment. In sum, all data suggest that the co‐administration of β‐asarone and l ‐dopa may contribute to the treatment of 6‐OHDA‐induced damage in rats by inhibiting autophagy activity.     

Transient alterations of the blood–brain barrier tight junction and receptor potential channel gene expression by chlorpyrifos

The blood–brain barrier (BBB) is formed by specialized endothelial cells lining capillaries in the central nervous system (CNS). We previously demonstrated that exposure to very low concentrations of the organophosphorus insecticide chlorpyrifos (CPF) decreased electrical resistance across the BBB in vitro, indicating a loss of BBB integrity. The present study examined the transient effects of CPF on expression of genes contributing to tight junctions of the BBB. Rat brain endothelial cells (RBE4) were co‐cultured with rat astrocytes on membrane inserts to form an in vitro BBB. The RBE4 cells in the BBB were then exposed to CPF for 2, 4 and 12 h. Total RNA was extracted from RBE4 cells and quantitative real‐time PCR (qRT‐PCR) was used to quantify levels of gene expression of tight junction proteins claudin5, scaffold proteins zona occludens (ZO1) and transient receptor potential (canonical) channels (TRPC4). Gene expression decreased 2 h after exposure to CPF, especially TRPC4, but the effects were reversed 12 h later. CPF exposure for only 15 min caused less effect than longer exposures, with TRPC4 gene expression above the control at 4 h. These results suggest that altering gene expression for claudin5, TRPC4 and ZO1 by CPF may directly contribute to BBB disruption, and that the alteration is reversible upon removal of CPF. Copyright © 2012 John Wiley & Sons, Ltd.     

Possible involvement of cationic‐drug sensitive transport systems in the blood‐to‐brain influx and brain‐to‐blood efflux of amantadine across the blood–brain barrier

The purpose of this study was to characterize the brain‐to‐blood efflux transport of amantadine across the blood–brain barrier (BBB). The apparent in vivo efflux rate constant for [³H]amantadine from the rat brain (k_eff) was found to be 1.53 × 10^‐2 min^‐1 after intracerebral microinjection using the brain efflux index method. The efflux of [³H]amantadine was inhibited by 1‐methyl‐4‐phenylpyridinium (MPP⁺), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P‐glycoprotein (P‐gp), did not affect the efflux transport of [³H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [³H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P‐gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [³H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [³H]amantadine was significantly decreased with the co‐perfusion of quinidine or verapamil, which are cationic P‐gp inhibitors, while MPP⁺ did not have a significant effect. It is thus concluded that while P‐gp is not involved, it is possible that rPMAT and the cationic drug‐sensitive transport system participate in the brain‐to‐blood efflux and the blood‐to‐brain influx of amantadine across the BBB, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Decrease of tight junction integrity in the ipsilateral thalamus during the acute stage after focal infarction and ablation of the cerebral cortex in rats

1. Whether damage to the blood–brain barrier (BBB) occurs in remote areas after a focal cortical lesion remains unknown. The present study investigated tight junction‐related proteins and tight junction microstructure in the ipsilateral thalamus during the acute stage after middle cerebral artery occlusion (MCAO) and cortical aspiration lesion (CAL) in rats. 2. Thirty‐six hypertensive and normotensive rats were subjected to MCAO or CAL; another 18 rats in each group were submitted to sham operation. Zonula Occluden (ZO)‐1, occludin and albumin were detected by western blotting 12 and 24 h after surgery. Tight junction microstructure was evaluated using electron microscopy, whereas albumin location in the ipsilateral thalamus was determined using double immunostaining for albumin and occludin or albumin and neuronal nuclei (NeuN) 24 h after surgery. 3. Twenty‐four hours after MCAO or CAL, occludin expression was reduced to 78.4% and 81.3%, respectively, compared with control. A reduction in ZO‐1 expression in the ipsilateral thalamus (to 79%) was seen only after CAL (P < 0.05). Membrane contact at the tight junction was discontinuous in the ipsilateral thalamus in both MCAO and CAL rats. Albumin levels were 23.2% and 82.5% higher in the ipsilateral thalamus after MCAO and CAL, respectively (P < 0.05). The percentage of the albumin‐positive area that coincided with the occludin‐positive area in the MCAO and CAL groups was 76.8% and 64.6%, respectively, indicating that albumin was mainly localized around the microvessels. 4. The results of the present study suggest that tight junction integrity decreases during the acute stage in the ipsilateral thalamus after MCAO and CAL in rats.     

5‐Fluorouracil treatment alters the expression of intestinal transporters in rats

5‐Fluorouracil (5‐FU), an anticancer drug, causes severe gastrointestinal damage, which may affect the absorption of orally administered drugs including the substrates of intestinal uptake and efflux transporters. This study aimed to investigate quantitatively the effect of 5‐FU‐induced intestinal damage on the expression of intestinal transporters: P‐glycoprotein (P‐gp), breast cancer resistance protein (BCRP) and peptide transporter 1 (PEPT1) in rats. The rats were treated with 5‐FU (30 mg/kg/day, p.o. ) for 5 days to induce intestinal damage, and then the upper, middle and lower intestinal segments were removed. The mRNA and protein expression levels of these transporters in each segment were determined using quantitative real‐time PCR and Western blotting, respectively. In the 5‐FU‐treated rats, the protein levels of P‐gp and Bcrp in the upper segment were significantly increased to 15‐ and 2.6‐fold of the control, respectively, while those in other segments were unaffected. Pept1 expression was increased by 5‐FU in almost all segments. A remarkable increase in P‐gp expression was shown, the uptake of digoxin, a P‐gp substrate, in each intestinal segment was measured using a rat everted sac. As a result, the uptake of digoxin in the upper segments of 5‐FU‐treated rats was decreased compared with that of the control. In conclusion, 5‐FU‐induced intestinal damage was shown to alter the expression of these transporters, especially in the upper intestinal segment, while the characteristics of the influence varied among the transporters. The 5‐FU‐induced intestinal damage may affect transporter‐mediated drug absorption of orally administered drugs in the clinical setting.     

Enhanced oral bioavailability and anti‐tumour effect of paclitaxel by 20(s)‐ginsenoside Rg3 in vivo

The purpose of this study was to investigate the effect of paclitaxel in combination with 20(s)‐ginsenoside Rg3 on its anti‐tumour effect in nude mice. In the Caco‐2 transport assay, the apparent permeability from the apical side to the basal side (P_app) (A‐B) and P_app (B‐A) of paclitaxel were measured when co‐incubated with different concentrations of 20(s)‐ginsenoside Rg3. The results indicated that the penetration of paclitaxel through the Caco‐2 monolayer from the apical side to the basal side was facilitated by 20(s)‐ginsenoside Rg3 in a concentration‐dependent manner. Meanwhile, 20(s)‐ginsenoside Rg3 inhibited P‐glycoprotein (P‐gp), and the maximum inhibition was achieved at 80 µ m (p < 0.05). The pharmacokinetic parameters of paclitaxel after oral co‐administration of paclitaxel (40 mg/kg) with various doses of 20(s)‐ginsenoside Rg3 in rats were investigated by an in vivo pharmacokinetic experiment. The results showed that the AUC of paclitaxel co‐administered with 20(s)‐ginsenoside Rg3 was significantly higher (p < 0.001 at 10 mg/kg) compared with the control. The relative bioavailability (RB) % of paclitaxel with 20(s)‐ginsenoside Rg3 was 3.4‐fold (10 mg/kg) higher than that of the control. The effect of paclitaxel orally co‐administered with 20(s)‐ginsenoside Rg3 against human tumour MCF‐7 xenografts in nude mice was also evaluated. Paclitaxel (20 mg/kg) co‐administered with 20(s)‐ginsenoside Rg3 (10 mg/kg) exhibited an effective anti‐tumour activity with the relative tumor growth rate (T/C) values of 39.36% (p <0.05). The results showed that 20(s)‐ginsenoside Rg3 enhanced the oral bioavailability of paclitaxel in rats and improved the anti‐tumour activity in nude mice, indicating that oral co‐administration of paclitaxel with 20(s)‐ginsenoside Rg3 could provide an effective strategy in addition to the established i.v. route. Copyright © 2012 John Wiley & Sons, Ltd.     

Unpredictable Rotational Responses to L‐dopa in the Rat Model of Parkinson’s Disease: the Role of L‐dopa Pharmacokinetics and Striatal Dopamine Depletion

Abstract: L‐dopa is still the gold standard in the symptomatic treatment of Parkinson’s disease (PD), and thus, it is the most commonly used drug in the non‐clinical assessment of new drug therapies to PD, including those intended to improve the effect of L‐dopa. In unilaterally 6‐hydroxydopamine (6‐OHDA)‐lesioned rat model of PD, the results from L‐dopa‐induced rotation tests are often unpredictable. While repeated administration of L‐dopa improves the rotation, the exact mechanisms underlying the extensive variability in rotation responses between rats and testing times are unclear. In the present study, we aimed to assess whether the route of administration (oral or intraperitoneal) or the form of L‐dopa (base or methyl ester) is associated with the extensive variation in rotation responses to L‐dopa in 6‐OHDA rats. We also wanted to examine the dependence between L‐dopa (base or methyl ester)‐induced rotational behaviour and the extent of dopamine and dopa decarboxylase enzyme loss in the lesioned striatum. It was found that variation in plasma levels of L‐dopa as well as the administration route explains a part of the variability in rotation. There were small but significant differences in striatal dopamine depletion (indicative of degree of lesion) between the groups, which may partially account for the various patterns in L‐dopa‐induced rotational behaviour. While apomorphine‐induced rotation test is a useful tool for primary screening of the success of 6‐OHDA lesion, it is not useful at predicting the rotational performance of 6‐OHDA rats to L‐dopa. The exact mechanisms and causes of the variability in the rotation responses to L‐dopa in 6‐OHDA rats still remain to be clarified.     

Icariside II attenuates cerebral ischemia/reperfusion-induced blood–brain barrier dysfunction in rats via regulating the balance of MMP9/TIMP1

Cerebral ischemia/reperfusion (I/R) results in harmful consequences during ischemic stroke, especially the disruption of the blood–brain barrier (BBB), which leads to severe hemorrhagic transformation through aggravation of edema and brain hemorrhage. Our previous study demonstrated that icariside II (ICS II), which is derived from Herba Epimedii, attenuates cerebral I/R injury by inhibiting the GSK-3β-mediated activation of autophagy both in vitro and in vivo. However, the effect of ICS II on the BBB remains unclear. Thus, in this study, we investigated the regulation of BBB integrity by ICS II after cerebral I/R injury and further explored the underlying mechanism in rats. Cerebral I/R injury was induced by middle cerebral artery occlusion (MCAO), and the treatment groups were administered ICS II at a dose of 16 mg/kg by gavage twice a day for 3 days. The results showed that ICS II effectively prevented BBB disruption, as evidenced by Evans Blue staining. Moreover, ICS II not only significantly reduced the expression of MMP2/9 but also increased TIMP1 and tight junction protein (occludin, claudin 5, and ZO 1) expression. Intriguingly, ICS II may directly bind to both MMP2 and MMP9, as evidenced by molecular docking. In addition, ICS II also inhibited cerebral I/R-induced apoptosis and ameliorated the Bax/Bcl-2 ratio and cleaved-caspase 3 level. Collectively, our findings reveal that ICS II significantly ameliorates I/R-induced BBB disruption and neuronal apoptosis in MCAO rats by regulating the MMP9/TIMP1 balance and inhibiting the caspase 3-dependent apoptosis pathway.     

2‐Hydroxypropyl‐β‐cyclodextrin‐modified SLN of paclitaxel for overcoming p‐glycoprotein function in multidrug‐resistant breast cancer cells

Objectives This study aimed to evaluate the potential of solid lipid nanoparticles (SLNs) of paclitaxel (PTX) modified with a 2‐hydroxypropyl‐β‐cyclodextrin system to enhance cellular accumulation of PTX into p‐glycoprotein (p‐gp)‐expressing cells. Methods The PTX‐loaded‐SLNs consisted of lipid (stearic acid) and surfactants (lecithin and poloxamer 188) and were then modified with 2‐hydroxypropyl‐β‐cyclodextrin by a sonication method. Key findings In terms of cytotoxicity, PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin showed higher cytotoxicity than other formulations. In particular, the cellular uptake of PTX from PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin was about 5.8‐ and 1.5‐fold higher than that from PTX solution and unmodified PTX‐loaded SLNs in MCF‐7/ADR cells, respectively. After a 4‐h incubation, clear fluorescence images inside cells were observed over time. When PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin were incubated with MCF‐7/ADR cells for 4 h, cellular uptake of PTX increased 1.7‐fold versus that of PTX in the presence of verapamil. Conclusions These results suggest that optimized SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin may have potential as an oral drug delivery system for PTX.     

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine‐inspired P‐glycoprotein inhibitors

P‐glycoprotein (P‐gp)/MDR‐1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P‐gp inhibitors were reported to reverse the MDR when co‐administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P‐gp inhibitors tested in the clinics are reported to have cross‐reactivity with CYP450 drug‐metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co‐administered chemotherapeutic drugs. In this study, two piperine analogs ( 3 and 4 ) having lower cross‐reactivity with CYP3A4 drug‐metabolizing enzyme are identified as P‐glycoprotein (P‐gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over‐expressing P‐gp (KB Ch^R 8–5, SW480‐VCR, and HCT‐15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB Ch^R 8–5 cells and found to act as competitive inhibitor of P‐gp as well as potentiated the vincristine‐induced NF‐KB‐mediated apoptosis. Therefore, 3 ((2E,4E)‐1‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)‐5‐(4‐hydroxy‐3‐methoxyphenyl)penta‐2,4‐dien‐1‐one) can serve as a potential P‐gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.     

Enalapril attenuates ischaemic brain oedema and protects the blood–brain barrier in rats via an anti‐oxidant action

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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 ).     

Irinotecan‐induced gastrointestinal damage impairs the absorption of dabigatran etexilate

Irinotecan causes serious gastrointestinal damage. Dabigatran etexilate (DABE), an oral anticoagulant and substrate of P‐glycoprotein (P‐gp), is poorly absorbed and exhibits low bioavailability in humans. The aim of this study was to evaluate the effects of irinotecan‐induced gastrointestinal damage on the pharmacokinetics/pharmacodynamics (PK/PD) of DABE. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. To investigate the PK profile of dabigatran (DAB), an active moiety of DABE, DABE was administered orally on day 5, and then DAB was administered intravenously on day 6. To evaluate the PD profile of DAB, the activated partial thromboplastin time (APTT) was measured. The protein expression level of intestinal P‐gp was evaluated. In the irinotecan‐treated rats, the area under the concentration–time curve of DAB after the oral administration of DABE and the bioavailability of DABE were decreased significantly. The APTT ratio also decreased, suggesting that the impaired efficacy of DABE was attributable to a reduction in its bioavailability. The expression of intestinal P‐gp was higher in the irinotecan‐treated rats. Taking into consideration the histological damage caused to the intestinal epithelium, both the increased P‐gp expression and the reduced passive diffusion were considered to be responsible for the reduction in the bioavailability of DABE.     

Simplified synthesis of N‐(3‐[18F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane ([18F]β‐CFT‐FP) using [18F]fluoropropyl tosylate as the labelling reagent

A synthesis method has been developed for the labelling of N‐(3‐[¹⁸F]fluoropropyl)‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane ([¹⁸F]β‐CFT‐FP), a potential radioligand for visualization of the dopamine transporters by positron emission tomography. The two‐step synthesis includes preparation of [¹⁸F]fluoropropyl tosylate and its use without purification in the fluoroalkylation of 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)nortropane (nor‐β‐CFT). The final product is purified by HPLC. Optimization of the two synthesis steps resulted in a greater than 30% radiochemical yield of [¹⁸F]β‐CFT‐FP (decay corrected to end of bombardment). The synthesis time including HPLC‐purification was approximately 90 min. The radiochemical purity of the final product was higher than 99% and the specific radioactivity at the end of synthesis was typically 20 GBq/µmol. In comparison to alkylation by [¹⁸F]fluoropropyl bromide, the procedure described here results in an improved overall radiochemical yield of [¹⁸F]β‐CFT‐FP in a shorter time. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of (R)‐ and (S)‐[O‐methyl‐11C]N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐methoxy‐phenyl)‐urea as candidate high affinity β1‐adrenoceptor PET radioligands

Molecular imaging and quantification of myocardial β₁‐adrenoceptor (AR) rather than total β‐AR density is of great clinical interest since cardiac biopsy studies suggest that myocardial β₁‐AR density is reduced in patients with chronic heart failure whereas cardiac β₂‐AR density may vary. Positron emission tomography (PET), with appropriate radioligands, offers the possibility to assess β‐AR density non‐invasively in humans. However, no PET radioligand for the selective imaging of cardiac β₁‐ARs is clinically available. Here some derivatives of the well characterized β₁‐AR selective antagonist, ICI 89,406, namely the enantiomers of N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐hydroxy‐phenyl)‐urea ( 5a and 5b ) were synthesized and evaluated in vitro. The (R)‐isomer 5a was more β₁‐selective but has lower affinity than its (S)‐enantiomer 5b (β₁‐AR selectivity: 6100 vs 1240; β₁‐affinity: K₁ = 0.288 nM vs K₁ = 0.067 nM). Etherification of the analogous desmethyl precursors, 5e and 5f , respectively, with [¹¹C]iodomethane gave ¹¹C‐labelled versions of 5a and 5b , namely 5g and 5h , in 44 ± 5% radiochemical yield (decay‐corrected) and 97.4 ± 1.3% radiochemical purity with specific radioactivities of 26.4 ± 9.4 GBq/µmol within 41.2 ± 3.4 min from the end of bombardment (n = 14). 5g and 5h are now being evaluated as candidate radioligands for myocardial β₁‐ARs. Copyright © 2005 John Wiley & Sons, Ltd.     

Effects of N‐Methylated Amyloid‐β30–40 Peptides on the Fibrillation of Amyloid‐β1–40

Alzheimer's disease is a neurodegenerative disorder associated with amyloid‐β (Aβ) fibrillation. N‐Methylated amyloid‐β peptides are potent inhibitors of amyloid‐β fibrillation. We investigated the inhibitory effect of N‐Methylated Aβ_30–40 peptides on Aβ_1–40 fibrillation. N‐Methylated Aβ_30–40 peptides affected the fibrillation, and this effect was dependent on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. N‐Methylated Aβ_30–40 peptides were co‐aggregated with Aβ_1–40. Spectroscopic technique was adopted to investigate an origin of the observed dependence. Suppression of thioflavin T (ThT) fluorescence count was correlated with the dissociation constant K_d of monomer–dimer equilibrium of each N‐Methylated Aβ_30–40 peptide. Monomeric N‐Methylated peptides decreased ThT fluorescence count during Aβ_1–40 fibrillation. Secondary structure content was not largely different between Aβ_1–40 fibrils and co‐aggregates. These results suggested that N‐Methylated Aβ_30–40 peptides disrupted the regular β‐sheet structure of Aβ_1–40 fibrils and affected the ThT fluorescence count. The monomer–dimer equilibrium of N‐Methylated peptides was (partly) responsible for the observed dependence of their inhibitory effect on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. Our study provides a hint to design new N‐Methylated inhibitor peptides of fibrillation.     

Nasal epithelial barrier disruption by particulate matter ≤2.5 μm via tight junction protein degradation

Upper airway diseases including sinonasal disorders may be caused by exposure to fine particulate matter (≤2.5 μm; PM2.5), as proven by epidemiological studies. PM2.5 is a complex entity whose chemical constituents and physicochemical properties are not confined to a single, independent “particle” but which in this study means a distinctive environmental “toxin.” The mechanism whereby PM2.5 induces nasal epithelial barrier dysfunction leading to sinonasal pathology remains unknown. In the present study, human nasal epithelial cells were exposed to non‐cytotoxic doses of PM2.5 to examine how PM2.5 affects the nasal epithelial barrier. Tight junction (TJ) integrity and function were assessed by transepithelial electric resistance and paracellular permeability. The expression levels of TJ proteins such as zona occludens‐1, occludin and claudin‐1 were assessed by immunofluorescence staining and western blot. PM2.5 exposure induced epithelial barrier dysfunction as reflected by increased paracellular permeability and decreased transepithelial electric resistance. TJ proteins zona occludens‐1, occludin and claudin‐1 were found to be downregulated. Pretreatment with N‐acetyl‐l ‐cysteine alleviated PM2.5‐mediated reactive oxygen species generation in RPMI 2650 cells, further preventing barrier dysfunction and attenuating the degradation of TJ proteins. These results suggest that PM2.5 induces nasal epithelial barrier disruption via oxidative stress, and N‐acetyl‐l ‐cysteine counteracts this PM2.5‐mediated effect. Thus, nasal epithelial barrier disruption caused by PM2.5, which leads to sinonasal disease, may be prevented or treated through the inhibition of reactive oxygen species.     

Synthesis of all‐trans‐[10′‐3H]‐8′‐apo‐β‐carotenoic acid

The enzyme, 15,15′‐β‐carotene dioxygenase (BCDOX), facilitates the oxidation of β‐carotene to yield retinal. This is a remarkable process in which one of 11 double bonds in β‐carotene is selectively oxidized. To further probe the mechanistic aspects of BCDOX, the synthesis of all‐trans‐[10′‐³H]‐8′‐apo‐β‐carotenoic acid is reported. This compound will be used as a photoaffinity labeling reagent to probe the β‐carotene binding pocket within BCDOX. The synthesis outlines a simple and efficient route for the incorporation of tritium at the 10′ olefinic carbon of 8′‐apo‐β‐carotenoic acid. Copyright © 2002 John Wiley & Sons, Ltd.