Potentiation of μ-opioid receptor-mediated signaling by ketamine

Ketamine, a clinically relevant drug, has been shown to enhance opioid-induced analgesia and prevent hyperalgesia. However, the molecular mechanisms involved are not clearly understood. As previous studies found that activation of opioid receptors leads to the phosphorylation of mitogen-activated protein kinases, we investigated whether ketamine could modulate μ-opioid receptor (μOR)-mediated ERK1/2 phosphorylation. We find that acute treatment with ketamine enhances (～2- to 3-fold) the levels of opioid-induced ERK1/2 phosphorylation in recombinant as well as cells endogenously expressing μOR. Interestingly, we find that in the absence of ketamine ERK1/2 signaling is desensitized 10 min after opioid exposure whereas in its presence significant levels (～3-fold over basal) are detected. In addition, ketamine increases the rate of resensitization of opioid-mediated ERK1/2 signaling (15 min in its presence vs. 30 min in its absence). These results suggest that ketamine increases the effectiveness of opiate-induced signaling by affecting multiple mechanisms. In addition, these effects are observed in heterologous cells expressing μOR suggesting a non-NMDA receptor-mediated action of ketamine. Together this could, in part, account for the observed effects of ketamine on the enhancement of the analgesic effects of opiates as well as in the duration of opiate-induced analgesia.     

Masticatory muscle and temporomandibular joint pain in Croatian war veterans with posttraumatic stress disorder

The aim of this study was to investigate the prevalence and intensity of masticatory muscle and temporomandibular joint (TMJ) pain in Croatian war veterans with posttraumatic stress disorder (PTSD). The examined group consisted of 100 Croatian war veterans, in whom PTSD had previously been diagnosed. Patients were compared with 92 subjects who had not taken part in the war and in whom PTSD was excluded by psychiatric examination. The clinical examination consisted of palpation of the masticatory muscles, the prominent neck musculature, and TMJ. The examination technique used and the definition of items were previously tested for reliability and validity. 93% of the subjects with PTSD had masticatory muscle tenderness compared to 45.65% of the subjects in the control group (chi2 = 51.46, p < 0.0001). The most frequent painful location in the subjects with PTSD was the left lateral pterygoid site in 88%, and in subjects of the control group the right lateral pterygoid site in 28.26% of cases. The most painful location in the PTSD group was the left lateral pterygoid site in 72%, and in the control group the left posterior digastric in 4.35% of cases. 58% of the subjects with PTSD had TMJ tenderness compared to 3.26% of subjects in the control group (chi2 = 66.23, p < 0.0001). The most frequent painful location of TMJ in both groups was the left posterior capsule; in the PTSD group 38% and in subjects in the control group 2.17% of cases. The most painful location was the left posterior capsule in 28% of subjects with PTSD, while not one subject in the control group reported severe painful sensitivity. The very high frequency and intensity of pain in subjects with PTSD confirms the effect of stress on muscle and joint sensitivity, i.e. perception of pain.     

Targeted mutagenesis of zebrafish: use of zinc finger nucleases

The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.     

Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N(3)-Gal (commercial), 6-N(3)-Gal, 3-N(3)-Glc and 3-N(3)-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N(3)-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzitrans-sialidase (TcTS) inhibitory activity at 1.0mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC(50) 260μM). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease.     

Exploratory Studies on the in Vitro Anti‐inflammatory Potential of Two Herbal Teas (Annona muricata L. and Jasminum grandiflorum L.), and Relation with Their Phenolic Composition

The need of new anti‐inflammatory drugs has led to the search for safer and more potent molecules in distinct sources, such as natural products. This work aimed to explore the anti‐inflammatory potential of aqueous extracts from two herbal teas (Annona muricata L. and Jasminum grandiflorum L.) in RAW 264.7 macrophages cells and in cell‐free assays. Furthermore, the phenolic composition of both extracts and of their hydrolysates was characterized by HPLC‐DAD, in order to establish possible relationships with the biological activity. In a general way, A. muricata displayed a stronger capacity to inhibit nitric oxide (NO) production and the activity of phospholipase A₂ (PLA₂), displaying an IC₅₀ value of 142 μg/ml against this enzyme. A deeper look at phenolic compounds revealed that aglycones had more capacity to inhibit NO and PLA₂ than their corresponding glycosides, quercetin being clearly the most potent one (IC₅₀ = 7.47 and 1.36 μm , respectively). In addition, 5‐O‐caffeoylquinic acid, at 1.56 μm , could also inhibit PLA₂ (ca. 35%). Our findings suggest that the consumption of both herbal teas may be a preventive approach to inflammatory disorders.     

Re-expression of estrogen receptor alpha using a tetracycline-regulated gene expression system induced estrogen-mediated growth inhibition of the MDA-MB-231 breast cancer cell line.

Estrogen receptor (ER)-negative breast carcinomas are often difficult to treat with antiestrogens. This work was performed to determine if the re-expression of the human ER alpha could restore the hormone response of these cells. We have transfected the human wild-type ER alpha to an ER-negative breast cancer cell line (MDA-MB-231) using a tetracycline-regulated gene expression system. We obtained a new cell line, MDA-A4-5/2. Cell count and flow cytometry "S" phase cell fraction showed that 17-beta-estradiol induced an inhibition on the proliferation of these cells; on the contrary, the antiestrogens ICI 182 780, and tamoxifen blocked this effect. Finally, we demonstrated an induction of the endogenous progesterone receptor gene when ER alpha was present. These results suggest that the re-expression of ER alpha in ER-negative breast cancer cells recreate, at least partially, a hormone-responsive phenotype and may be useful as a therapeutic approach to control this pathology.     

Highly potent and selective aryl-1,2,3-triazolyl benzylpiperidine inhibitors toward butyrylcholinesterase in Alzheimer's disease

Acetylcholinesterase (AChE) is the key enzyme targeted in Alzheimer's disease (AD) therapy, nevertheless butyrylcholinesterase (BuChE) has been drawing attention due to its role in the disease progression. Thus, we aimed to synthesize novel cholinesterases inhibitors considering structural differences in their peripheral site, exploiting a moiety replacement approach based on the potent and selective hAChE drug donepezil. Hence, two small series of N-benzylpiperidine based compounds have successfully been synthesized as novel potent and selective hBuChE inhibitors. The most promising compounds (9 and 11) were not cytotoxic and their kinetic study accounted for dual binding site mode of interaction, which is in agreement with further docking and molecular dynamics studies. Therefore, this study demonstrates how our strategy enabled the discovery of novel promising and privileged structures. Remarkably, compound 11 proved to be one of the most potent (0.17?nM) and selective (>58,000-fold) hBuChE inhibitor ever reported.     

The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1(+) shut-off and bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-d-glucan and primary septum formation. bgs1(+) shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die.     

Aquaporin molecular characterization in the sea-bass (Dicentrarchus labrax): the effect of salinity on AQP1 and AQP3 expression

Euryhaline fish possess the ability to compensate for environmental salinity changes through hydro-mineral regulation. A number of proteins have been studied in order to understand water and ion exchanges, known as fish osmoregulation. Sea-bass (Dicentrarchus labrax) cDNA sequences encoding a homologue of mammalian aquaporin (termed AQP1) and a homologue of mammalian aquaglyceroporin (termed AQP3) have been isolated and sequenced. The aquaporin amino acid sequences share respectively more than 60% and 65% identity with other known aquaporins. We have shown that salinity influences aquaporin expression levels in the gill, kidney and digestive tract, the main osmoregulatory organs. AQP1 may have a major osmoregulatory role in water transport in kidney and gut in SW-acclimated fish, whereas AQP3 could be implicated in gill water transport in FW-acclimated fish.