β‐asarone and levodopa co‐administration increase striatal dopamine level in 6‐hydroxydopamine induced rats by modulating P‐glycoprotein and tight junction proteins at the blood‐brain barrier and promoting levodopa into the brain

Levodopa (L‐dopa) is widely considered as one of the most effective drug constituents in the treatment of Parkinson's disease (PD), but the blood‐brain barrier (BBB) permeability of L‐dopa is <5%, which causes low efficacy. Neuroprotective effects of β‐asarone on 6‐hydroxydopamine (6‐OHDA)‐induced PD rats were demonstrated by our previous studies. Co‐administration of β‐asarone and L‐dopa has not been explored until being investigated on PD rats in this study. PD rats were divided into four groups: untreated, L‐dopa‐treated, β‐asarone‐treated and co‐administered‐treated groups. All of the treatments were administered to the rats twice per day for 30 days. The L‐dopa, dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), S100β and neuron‐specific enolase (NSE) levels were subsequently determined. The P‐glycoprotein (P‐gp), zonula occludens‐1 (ZO‐1), claudin‐5, occludin and actin expression was also assessed in cortex. Changes in BBB ultrastructure were observed using transmission electron microscopy. Our results showed that the co‐administered treatment increased levels of L‐dopa, DA, DOPAC and HVA in striatum, and S100β in plasma, but down‐regulated NSE, P‐gp, ZO‐1, occludin, actin and claudin‐5 in cortex. Crevices were observed between capillary endothelial cells at intercellular tight junction of the striatum in co‐administered‐treated group, while the endothelial cells in untreated group were tightly jointing each other. In addition, the correlations of L‐dopa or DA and P‐gp or tight junction proteins respectively were significantly negative in co‐administered‐ and β‐asarone‐treated groups. These findings suggest that co‐administered treatment may enhance the L‐dopa BBB permeability and attenuate brain injury, which may be beneficial to PD treatment.     

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.     

In‐vitro interaction of l‐dopa with bacterial adhesins of Helicobacter pylori: an explanation for clinicial differences in bioavailability?

Objectives Recent investigations on the pharmacokinetics of levodopa (l ‐dopa) indicated that the presence of Helicobacter pylori in patients with Parkinson's disease, orally treated with l ‐dopa, influences the absorption of this compound, which consequently leads to decreased plasma levels. Therefore this work aims to study a potential in‐vitro interaction of l ‐dopa with H. pylori and its surface adhesins. Methods Solutions containing l ‐dopa of different concentrations were incubated with H. pylori at different bacterial densities and time intervals. Free l ‐dopa was quantified from the incubation supernatants by HPLC. A flow cytometric assay with fluorescence labelled H. pylori was used to investigate the influence of l ‐dopa on the bacterial adhesion of H. pylori: FITC‐labelled bacteria were pre‐incubated with l ‐dopa, followed by incubation with gastric epithelial cells (AGS cells) and FACS quantification of adhering bacteria. Key findings Evaluation of time‐ and concentration‐dependent incubation experiments indicated a significant decrease in l ‐dopa concentrations when coming into contact with H. pylori. The reduction in l ‐dopa concentrations was determined as 47 to 12%, referred to the initial starting concentration, with time‐dependency and dependency of the H. pylori density. FITC‐labelled H. pylori, pre‐incubated with differing l ‐dopa concentrations, were shown to have a significantly reduced bacterial adhesion to AGS cells, with a maximum reduction of 22 ± 9%. These results demonstrate a direct interaction of l ‐dopa with the outer membrane proteins of H. pylori responsible for the adhesion to gastric epithelial cells. By this interaction the unbound l ‐dopa concentration in bacterial suspension was strongly reduced. Conclusions This study suggests a potential in‐vitro interaction of l ‐dopa with H. pylori adhesins, confirming the clinical changes found in pharmacokinetics of l ‐dopa therapy by H. pylori‐positive patients with Parkinson's disease.     

l‐Cysteine enhances nutrient absorption via a cystathionine‐β‐synthase‐derived H2S pathway in rodent jejunum

Hydrogen sulphide (H₂S) is generated endogenously from l ‐cysteine (l ‐Cys) by the enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE). In addition, l ‐Cys is commonly used as a precursor in the food and pharmaceutical industries. The aim of the present study is to determine whether l ‐Cys regulates intestinal nutrient transport. To that end, the presence of CBS and CSE in the jejunum epithelium was assessed by immunohistochemistry, Western blotting and the methylene blue assay. In addition, in vivo l ‐Cys (100 mg/kg, administered immediately after the glucose load) significantly increased blood glucose levels 30 min after the oral administration of glucose to mice. This effect of l ‐Cys was completely blocked by amino‐oxyacetic acid (AOA; 50 mg/kg; administered at the same time as l ‐Cys) an inhibitor of CBS. Measurements of the short‐circuit current (I_sc) in the rat jejunum epithelium revealed that l ‐Cys (1 mmol/L; 6 min before the administration of l ‐alanine) enhances Na⁺‐coupled l ‐alanine or glucose transport, and that this effect is inhibited by AOA (1 mmol/L;10 min before the administration of l ‐Cys), but not d ,l ‐propargylglycine (PAG;1 mmol/L; 10 min before the administration of l ‐Cys), a CSE inhibitor. Notably, l ‐Cys‐evoked enhancement of nutrient transport was alleviated by glibenclamide (Gli;0.1 mmol/L; 10 min before the administration of l ‐Cys), a K⁺ channel blocker. Together, the data indicate that l ‐Cys enhances jejunal nutrient transport, suggesting a new approach to future treatment of nutrition‐related maladies, including a range of serious health consequences linked to undernutrition.     

Physiological Effects of a Novel Immune Stimulator Drug, (1,4)‐α‐d‐Glucan,in Rats

Abstract: The (1,4)‐α‐d ‐glucan (α‐d ‐glucan), derived from medicinal plant, Tinospora cordifolia, activates human lymphocytes with downstream synthesis of the pro‐ and anti‐inflammatory cytokines, in vitro. We investigated physiological and immunological effects of a low and a high dose of α‐d ‐glucan (0.5 and 10 mg/kg), in vivo, testing the hypothesis that intravenous administration of α‐d ‐glucan does not affect haemodynamic, respiratory, haematological, and immune responses in normal rats. Male rats (300–400 g) were anaesthetized, tracheostomized, and catheterized in one femoral artery and vein. The mean arterial blood pressure and heart rate were continuously recorded. The baselines for gas exchange, differential blood cell count, and plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ were determined. Rats were then randomly assigned to controls (n = 7), a low dose (0.5 mg/kg; n = 10), and a high dose (10 mg/kg; n = 7) of α‐d ‐glucan for a six 6 hr study period. Gas exchange, differential cell count, plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ, and mean arterial blood pressure values remained within physiological range. Intravenous administration of 10 mg/kg α‐d ‐glucan created tachycardia, associated with hyperventilation, and significant reductions in the blood haemoglobin and haematocrit concentrations. We suggest that these in vivo effects of α‐d ‐glucan should be considered for future clinical and/or experimental trials.     

4‐Aminoquinoline Derivatives as Potential Antileishmanial Agents

The leishmanicidal activity of a series of 4‐aminoquinoline ( AMQ ) derivatives was assayed against Leishmania amazonensis. This activity against the intracellular parasite was found stronger than for L. amazonensis promastigotes. Neither compound was cytotoxic against macrophages. The compound AMQ ‐j , which exhibited a strong activity against promastigotes and amastigotes of L. amazonensis (IC₅₀ values of 5.9 and 2.4 μg/mL, respectively) and similar leishmanicidal activity to reference drugs, was chosen for studies regarding its possible mechanism of action toward parasite death. The results showed that the compound AMQ ‐j induced depolarization of the mitochondrial membrane potential in promastigotes and in L. amazonensis‐infected macrophages, but not in uninfected macrophages. Furthermore, the depolarization of the mitochondrial membrane potential was dose dependent in infected macrophages. We have established that promastigotes and L. amazonensis‐infected macrophages treated with AMQ ‐j were submitted to oxidative stress. This is in line with the increase in the level of reactive oxygen species (ROS). Leishmania amazonensis‐infected macrophages treated with AMQ ‐j did not show a significant increase in the production of nitric oxide. Our results indicate the effective and selective action of AMQ ‐j against L. amazonensis, and its mechanism of action appears to be mediated by mitochondrial dysfunction associated with ROS production.     

Catechol‐O‐methyltransferase Inhibition in Erythrocytes and Liver by BIA 3‐202 (1‐[3,4‐dibydroxy‐5‐nitrophenyl]‐2‐phenyl‐ethanone)

Abstract: The present study evaluated the relationship between the degree of catechol‐O‐methyltransferase (COMT) inhibition in erythrocytes and liver by BIA 3‐202 (1‐[3,4‐dihydroxy‐5‐nitrophenyl]‐2‐phenyl‐ethanone) and determined its effects upon the O‐methylation of L‐DOPA in rats orally treated with L‐DOPA plus benserazide. The soluble form of COMT (S‐COMT) in erythrocytes was endowed with the same affinity as liver S‐COMT for the substrate adrenaline. BIA 3‐202 inhibited erythrocytes and liver S‐COMT with ED50's of 1.9 (0.7, 3.1) and 1.9 (0.5, 3.2) (95% confidence limits) mg kg^?1, respectively. BIA 3‐202 reduced the L‐DOPA‐induced rise of 3‐O‐methyl‐L‐DOPA in the peripheral circulation, striatal dialysate levels and striatum, and increased dopamine striatal levels. In BIA 3‐202‐treated rats the increase in L‐DOPA in peripheral blood and striatal dialysates was significantly greater than in vehicle‐treated rats. It is concluded that S‐COMT activity in erythrocytes may provide important information on the pharmacodynamic profile of COMT inhibitors. The novel COMT inhibitor BIA 3‐202 is a potent COMT inhibitor that enhances the availability of L‐DOPA to the brain by reducing its O‐methylation, which may prove beneficial in patients with Parkinson's disease treated with L‐DOPA.     

Effect of Chain Elongation on Biological Properties of the Toxin Paralysin β‐Alanyl‐tyrosine

In hemolymph of insect species, compounds with remarkable properties for pharmaceutical industry are present. At the first line, there were found compounds of low molecular mass, less than 1 kDa. One of such compounds, β‐alanyl‐tyrosine (252 Da), was isolated from larval hemolymph of some species of holometabolous insects (e.g. Neobellieria bullata). Its paralytic activity and antimicrobial properties were described until now. In this study, we present the effect of elongation of β‐alanyl‐tyrosine by repeating of this motive on the biological and physical properties of prepared analogues. For assessment of antimicrobial properties of these new compounds strains of Gram‐positive, Gram‐negative bacteria and fungi were used, we also followed the haemolytic activity and toxic effect on human cell culture HepG2. On the base of ECD spectroscopy measurement, subsequent molecular modelling and known secondary structure of original β‐alanyl‐tyrosine dipeptide, the secondary structures of repeating sequences of β‐AY were specified. The repeating structures of β‐alanyl‐tyrosine show increase in antimicrobial activity; for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, minimal inhibitory concentration was decreased from 30 to 15 mm for 2xβ‐AY, 0.4 mm for 4xβ‐AY and 0.25 mm for 6xβ‐AY.     

4‐Methylthioamphetamine Increases Dopamine in the Rat Striatum and has Rewarding Effects In Vivo

4‐Methylthioamphetamine (MTA) is a phenylisopropylamine derivative whose use has been associated with severe intoxications. MTA is usually regarded as a selective serotonin‐releasing agent. Nevertheless, previous data have suggested that its mechanism of action probably involves a catecholaminergic component. As little is known about dopaminergic effects of this drug, in this work the actions of MTA upon the dopamine (DA) transporter (DAT) were studied in vitro, in vivo and in silico. Also, the possible abuse liability of MTA was behaviourally assessed. MTA exhibited an in vitro affinity for the rat DAT in the low micromolar range (6.01 μM) and induced a significant, dose‐dependent increase in striatal DA. MTA significantly increased c‐Fos‐positive cells in striatum and nucleus accumbens, induced conditioned place preference and increased locomotor activity. Docking experiments were performed in a homology model of the DAT. In conclusion, our results show that MTA is able to increase extracellular striatal DA levels and that its administration has rewarding properties. These effects were observed at concentrations or doses that can be relevant to its use in human beings.     

Surgery‐induced changes in rat IL‐1β and acetylcholine metabolism: role of physostigmine

Pharmacological enhancement of cholinergic activity following administration of physostigmine is known to induce protective effects generally. However, it is unclear whether the effect of physostigmine on inflammation and acetylcholine (ACh) metabolism is related to different types of surgical intervention or anaesthesia alone. To investigate this, rats were subjected to partial liver resection (PLR) or sham surgery, with a control group receiving anaesthesia alone. Half of each treatment group received a single intra‐operative dose of physostigmine (0.04 mg/kg); the others received placebo. Acetylcholinesterase (AChE) activity and plasma and brain concentrations of interleukin (IL)‐1β and ACh were determined. Both PLR and sham operation induced a time‐dependent increase in plasma concentrations of IL‐1β compared with rats receiving anaesthesia alone (3.9‐ and 4.8‐fold increases, respectively). In the brain, IL‐1β concentrations had increased approximately twofold after surgery compared with the control group. Blood AChE was transiently decreased after surgery. Brain AChE activity increased 1.3‐fold (P = 0.014) only after PLR; consequently, cerebral ACh concentrations were significantly reduced. Physostigmine administration significantly reduced IL‐1β and AChE levels. Cerebral ACh concentrations were markedly increased from 544 ± 122 ng/mg protein following placebo administration to 654 ± 93 ng/mg protein after physostigmine administrations (P < 0.001). We conclude that a single dose of physostigmine intra‐operatively has a sustained anti‐inflammatory effect (up to 120 min after injection) that is especially pronounced under the conditions of PLR surgery. In addition to its protective peripheral action, physostigmine exerts a neuroprotective action by increasing levels of the neurotransmitter ACh.     

Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract: Gramicidin S (GS) is a 10‐residue cyclic β‐sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure–activity studies of de novo‐designed 14‐residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single l ‐amino acid with its corresponding d ‐enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274 , 13181]. The diastereomer with a d ‐Lys substituted at position 4 caused the greatest improvement in specificity vs. other l to d substitutions within the cyclic 14‐residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted β‐sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275 , 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a d ‐ or l ‐amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the d ‐ and l ‐substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic d ‐amino acids d ‐Lys and d ‐Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the l ‐diastereomers, which demonstrated strong hemolysis. All of the l ‐substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the d ‐substituted peptides tended to improve based on the hydrophilicity of the residue. d ‐Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic d ‐amino acid substitutions had superior antimicrobial activity vs. the l ‐enantiomers although substitution of a hydrophobic d ‐amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted β‐sheet structure on antimicrobial activity.     

Catechol-O-methyltransferase inhibition by U-0521 increases striatal utilization of levodopa

Summary The effects of U-0521, a catechol-O-methyltransferase (COMT) inhibitor, were studied on this enzyme activity and on Dopa metabolism in rat striatum. In vivo maximal inhibition (95%) of COMT activity was obtained at 5 min with enzyme recovery to 64% of basal activity at 120 min. When injected in increasing doses U-0521 (200 mg·kg^–1) inhibited, at 10 min, COMT activity by 85% with an IC₅₀=80 mg·kg^–1. In rats pretreated with U-0521 and then with DOPA the accumulation of 3-O-methyldopa (OMD) in the plasma was essentially blocked while Dopa, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) accumulation in the striatum was significantly higher than in DOPA treated controls. U-0521, a potent COMT inhibitor, enhances the availability and utilization of levodopa in the brain and may thus be helpful in future treatment of parkinsonian patients.     

Elevated vascular γ‐butyrobetaine levels attenuate the development of high glucose‐induced endothelial dysfunction

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Asymmetric dimethylarginine does not inhibit arginase activity and is pro‐proliferative in pulmonary endothelial cells

Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. l ‐Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater l ‐arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (l ‐arginine, ADMA, N^G‐monomethyl‐l ‐arginine (L‐NMMA) and N^G‐nitro‐l ‐arginine methyl ester (l ‐NAME)). Only l ‐arginine resulted in measurable urea production (K_m = 6.9 ± 0.8 mmol/L; V_max = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, l ‐NMMA and l ‐NAME in the presence of 1 mmol/L l ‐arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.     

Design of high affinity cyclic pentapeptide ligands for κ‐opioid receptors

Abstract: Using results from our previously reported cyclic opioid peptide series and reliable models for μ‐, δ‐, and κ‐opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr‐c[d ‐Cys‐Phe‐Phe‐X]‐NH₂, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = d ‐ or l ‐Cys; or d ‐ or l ‐penicillamine (Pen; β,β‐dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = d ‐ or l ‐Cys display KOR affinities in the low nanomolar range, demonstrating that a ‘DPDPE‐like’ tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8 , Tyr‐c[d ‐Cys‐Phe‐Phe‐Cys]‐NH₂‐cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm ) with more than 100‐fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Synthesis and Biological Evaluation of a Series of 2‐((1‐substituted‐1H‐1,2,3‐triazol‐4‐yl)methylthio)‐6‐(naphthalen‐1‐ylmethyl)pyrimidin‐4(3H)‐one As Potential HIV‐1 Inhibitors

A series of novel S‐DABO derivatives with the substituted 1,2,3‐triazole moiety on the C‐2 side chain were synthesized using the simple and efficient CuAAC reaction, and biologically evaluated as inhibitors of HIV‐1. Among them, the most active HIV‐1 inhibitor was compound 4‐((4‐((4‐(2,6‐dichlorobenzyl)‐5‐methyl‐6‐oxo‐1,6‐dihydropyrimidin‐2‐ylthio)methyl)‐1H‐1,2,3‐triazol‐1‐yl)methyl)benzenesulfonamide ( B5b7) , which exhibited similar HIV‐1 inhibitory potency (EC₅₀ = 3.22 μm ) compared with 3TC (EC₅₀ = 2.24 μm ). None of these compounds demonstrated inhibition against HIV‐2 replication. The preliminary structure–activity relationship (SAR) of these new derivatives was discussed briefly.     

Pyocyanin inhibits both nitric oxide‐dependent and ‐independent relaxation in porcine coronary arteries

The effects of the Pseudomonas aeruginosa virulence factor pyocyanin (PCN) on the contractile function of porcine coronary arteries was investigated in vitro. Artery rings (5 mm) were suspended in organ baths containing Krebs' solution for the measurement of isometric tension. The effect of PCN on resting and precontracted coronary arteries was initially investigated with various agents. Arteries were precontracted with prostaglandin (PG) F_2α or potassium chloride and endothelium‐dependent relaxations were induced by various agents in the presence of PCN. Pyocyanin (0.1–10 μmol/L) evoked small‐amplitude, dose‐dependent contractions in resting porcine coronary arteries. In addition, PCN amplified the contractile response to PGF_2α, but did not alter responses to carbachol. Pyocyanin (0.1–10 μmol/L) significantly inhibited endothelium‐dependent relaxations evoked by neurokinin A. Pyocyanin also inhibited relaxations evoked by diethylamine nitric oxide (a nitric oxide donor), forskolin (an adenylate cyclase activator), dibuytyryl‐cAMP (a cAMP analogue), 8‐bromo‐cGMP (a cGMP analogue) and P1075 (a K_ATP channel activator), but not isoprenaline (β‐adrenoceceptor agonist). These results indicate that physiological concentrations of PCN interfere with multiple intracellular processes involved in vascular smooth muscle relaxation, in particular pathways downstream of nitric oxide release. Thus, PCN may alter normal vascular function in patients infected with P. aeruginosa.     

Epigallocatechin gallate attenuates fibroblast proliferation and excessive collagen production by effectively intervening TGF‐β1 signalling

Pulmonary fibrosis (PF) poses a huge burden to the patients and society due to lack of an effective treatment drug. Activation of fibrocyte, fibroblast and myofibroblasts are important steps in the development of PF. Targeting this common pathway with natural chemicals may lead to the development of new drug regimens for PF treatment. In this study, PF was induced in male Wistar rats by intratracheal administration of Bleomycin (BLM). Epigallocatechin gallate (EGCG) was administered to one of the groups of rats to test its efficacy against the development of PF. Bleomycin‐induction resulted in significant elevation of matrix metalloproteinase (MMP)‐2 and ‐9 expression, increased RNA and protein expression of transforming growth factor (TGF)‐β1, Smads and alpha‐smooth muscle actin (α‐SMA). EGCG treatment normalized the BLM induced aberrations in these rats. The protective role of EGCG was also validated in vitro using the WI‐38 fibroblast cell line. TGF‐β1 incubated cells exhibited increased fibroblast proliferation and hydroxyproline levels with a concomitant decrease in the expression of MMPs 2 and 9. An increase in protein expression levels of p‐Smad, α‐SMA and type I collagen (COL1A) was also exhibited by fibroblasts upon TGF‐β1 incubation. Simultaneous treatment of EGCG to WI‐38 cells significantly decreased these protein expressions alongside normalizing the MMPs expression. The study revealed that EGCG inhibited fibroblast activation and collagen accumulation by inhibiting TGF‐β1 signalling and thus can be considered as an effective drug against PF.     

Radiosynthesis of 1′‐[18F]fluoroethyl‐β‐D‐lactose ([18F]‐FEL) for early detection of pancreatic carcinomas with PET

Introduction: The hepatocellular carcinoma–intestine–pancreas and pancreatitis‐associated proteins, also known as lactose‐binding protein, is upregulated in peritumoral pancreatic tissue. Previously, we reported ethyl‐ β ‐D ‐galactopyranosyl‐(1,4′)‐2′‐deoxy‐2′‐[¹⁸F]fluoro‐ β ‐D ‐glucopyranoside (Et‐[¹⁸F]‐FDL), a radiofluorinated lactose analog for positron emission tomography (PET) of small pancreatic carcinomas in mice. However, synthesis of the precursor for Et‐[¹⁸F]‐FDL involves 11 steps, which is quite lengthy, and produces overall low yields. Here, we report on synthesis and radiolabeling of another analog of lactose, the 1′‐[¹⁸F]fluoroethyl‐ β ‐D ‐lactose for PET imaging of pancreatic carcinomas. Methods: Two precursor compounds, 1′‐bromoethyl‐2′,3′,6′,2,3,4,6‐hepta‐O‐acetyl‐ β ‐D ‐lactose 4, and 1′‐p‐toluenesulfonylethyl‐2′,3′,6′,2,3,4,6‐hepta‐O‐acetyl‐ β ‐D ‐lactose 5, were synthesized in two and three steps, respectively; then, cold fluorination and radiofluorination of these precursors were performed. The reaction mixture was passed through a silica gel Sep‐pack cartridge, eluted with EtOAc, and the 1′‐[¹⁸F]fluoroethyl‐2′,3′,6′,2,3,4,6‐hepta‐O‐acetyl‐ β ‐D ‐lactose ([¹⁸F]‐6) purified by HPLC. After hydrolysis of the protecting groups, the 1′‐[¹⁸F]fluoroethyl‐ β ‐D ‐lactose [¹⁸F]‐7 was neutralized, diluted with saline, filtered through a sterile Millipore filter, and analyzed by radio‐TLC. Results: The average decay‐corrected radiochemical yield was 9% (n = 7) with>99% radiochemical purity and specific activity of 55.5 GBq/ µ mol. Conclusion : A new analog of lactose, 1′‐[¹⁸F]fluoroethyl‐ β ‐D ‐lactose, has been synthesized in good yields, with high purity and high specific activity suitable for PET imaging of early pancreatic carcinomas. Copyright © 2011 John Wiley & Sons, Ltd.