The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein

We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Reformulation of a new vancomycin analog: An example of the importance of buffer species and strength

The purpose of this research was to use our previously validated dynamic injection apparatus as a rapid method for screening pH-adjusted formulations of a new vancomycin analog, Van-An, for their potential to precipitate upon dilution. In 1 vial, Van-An was reconstituted according to the manufacturer’s instructions. In a separate vial, the Van-An formulation’s existing phosphate buffer species was supplemented with acetate buffer, which has a pKa in the desired range: between the pH values of the formulation (pH 3.9) and blood (pH 7.4). The formulations were injected using the dynamic injection apparatus into a flowing stream of isotonic Sorensen’s phosphate buffer at rates of 0.25, 0.5, 1, and 2 mL/min. The peaks obtained with the spectrophotometer were reproducible for each injection rate/formulation combination. For the phosphate-buffered formulation, the least amount of precipitation was obtained at the 0.25 mL/min injection rate. Acetate buffer was able to substantially reduce such precipitation, even at the highest injection rate. The opacity peaks for the formulation with the acetate addition were significantly smaller (P<.05) than those obtained for the unaltered formulation at all 4 injection rates. The results suggest that acetate is a better buffer species than phosphate for the pH range defined. Furthermore, we present evidence to support a generally applicable approach to screening new formulations of drug products that may be clinically useful for reducing the incidence of phlebitis in humans. Published: January 13, 2006     

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.     

Solubility of soy lipophilic proteins: comparison with other soy protein fractions

Solubility of soy lipophilic proteins (LP) was studied as compared with that of other soy protein fractions. LP, β-conglycinin, glycinin, and soy protein isolate (N-SPI) were prepared under the condition to avoid heat denaturation. Solubility of LP was lower than that of other soy protein fractions under all the tested conditions varying in pH values and ionic strength. The solubility of LP was increased constantly by elevating temperature until 90 °C, whereas that of β-conglycinin and glycinin dropped at high temperature. Temperature-dependent change in solubility of N-SPI might reflect the balance among that of glycinin, β-conglycinin and LP. Based on the results of SDS-PAGE, determination of phospholipid content and Fourier Transform Infrared spectroscopy, we discussed the solubilization behavior of LP relating to its origin and composition.     

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.     

Cloning of acryIIIA endotoxin gene ofBacillus thuringiensis var.tenebrionis and its transient expression inindica rice

Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was considered desirable. CryIIIA endotoxin ofBacillus thuringiensis var.tenebrionis, a 65 kDa protein toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was isolated from a local isolate ofBacillus thuringiensis var.tenebrionis. The gene was tailored at the N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon next to ATG of threonine to proline. This modification did not affect the functional property of the gene product. A chimeric construct of the modifiedcryIIIA gene was developed containing CaMV35S promoter andnos terminator for plant expression. The expressibility of thecryIIIA gene inindica rice was judged through test for transient expression in indica rice protoplasts.