Pharmacology and anti-addiction effects of the novel κ opioid receptor agonist Mesyl Sal B, a potent and long-acting analogue of salvinorin A.

BACKGROUND AND PURPOSE: Acute activation of κ opioid (KOP) receptors results in anticocaine-like effects, but adverse effects, such as dysphoria, aversion, sedation and depression, limit their clinical development. Salvinorin A, isolated from the plant Salvia divinorum, and its semi-synthetic analogues have been shown to have potent KOP receptor agonist activity and may induce a unique response with similar anticocaine addiction effects as the classic KOP receptor agonists, but with a different side effect profile. EXPERIMENTAL APPROACH: We evaluated the duration of effects of Mesyl Sal B in vivo utilizing antinociception assays and screened for cocaine-prime induced cocaine-seeking behaviour in self-administering rats to predict anti-addiction effects. Cellular transporter uptake assays and in vitro voltammetry were used to assess modulation of dopamine transporter (DAT) function and to investigate transporter trafficking and kinase signalling pathways modulated by KOP receptor agonists. KEY RESULTS: Mesyl Sal B had a longer duration of action than SalA, had anti-addiction properties and increased DAT function in vitro in a KOP receptor-dependent and Pertussis toxin-sensitive manner. These effects on DAT function required ERK1/2 activation. We identified differences between Mesyl Sal B and SalA, with Mesyl Sal B increasing the Vmax of dopamine uptake without altering cell-surface expression of DAT. CONCLUSIONS AND IMPLICATIONS: SalA analogues, such as Mesyl Sal B, have potential for development as anticocaine agents. Further tests are warranted to elucidate the mechanisms by which the novel salvinorin-based neoclerodane diterpene KOP receptor ligands produce both anti-addiction and adverse side effects. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

Epidermal growth factor and human growth hormone accelerate adaptation after massive enterectomy in an additive, nutrient-dependent, and site-specific fashion.

BACKGROUND: After massive enterectomy (ME), remnant intestine undergoes compensatory adaptation. Epidermal growth factor (EGF) and human growth hormone (hGH) have each been shown to enhance total length small intestine nutrient transport after ME. This study aims to determine the differential effects of EGF and hGH on proximal and distal small intestinal remnants after ME. METHODS: New Zealand white rabbits underwent 70% mid-jejunoileal resection. After 1 week, animals received hGH (0.2 mg/kg/day), EGF (1.5 micrograms/kg/hr), hGH + EGF, or vehicle (equal volume) for 7 days. Sodium-dependent uptake of glucose, glutamine, alanine, leucine, and arginine into brush border membrane vesicles was quantitated. Serum insulin-like growth factor-I concentrations as well as proximal and distal villus and microvillus heights were measured. IGF binding protein-3 and -4 mRNA expression was determined in full-thickness proximal and distal gut remnants. RESULTS: Concomitant hGH and EGF treatment up-regulates glucose (100%), glutamine (80%), and leucine (60%) transport in the proximal remnant; alanine (150%) and arginine (400%) transport in the distal remnant; and microvillus height (25% to 35%) both proximally and distally. Serum IGF-I levels and gross villus heights were not different among groups. CONCLUSIONS: Co-infusion of hGH and EGF accelerates intestinal adaptation after ME in an additive, nutrient-dependent, and site-specific fashion via enhanced nutrient transport as well as microvillus hypertrophy.

Function of the zinc finger in Escherichia coli Fpg protein.

Fpg protein of Escherichia coli cleaves duplex DNA containing the oxidatively damaged base 8-oxo-7,8-dihydroguanine (Tchou, J., Kasai, H., Shibutani, S., Chung, M.-H., Laval, J., Grollman, A. P., and Nishimura, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4690-4694). This DNA repair enzyme contains one zinc atom/protein molecule (Boiteux, S., O'Connor, T. R., Lederer, F., Gougette, A., and Laval, J. (1990) J. Biol. Chem. 265, 3916-3922); its N-glycosylase and apurinic/apyrimidinic lyase activities are physically associated. Amino acid sequence analysis reveals a putative single zinc finger motif of the CC/CC type located near the carboxyl terminus. A gel mobility shift assay was used to assay binding of Fpg protein to a noncleavable substrate analog, namely an oligodeoxynucleotide duplex containing a single tetrahydrofuran residue. High resolution hydroxyl radical DNA footprinting showed protection centered around the tetrahydrofuran residue. No footprint was observed on the complementary strand. To establish the role of COOH-terminal zinc finger in DNA binding and/or DNA cleavage, amino acid substitutions and an amber mutation were introduced at Cys-244 (C244S, C244H, C244A, and C244amber). In addition, a double amino acid substitution was generated at Cys-244 and Cys-247 (C244S/C247S). These mutant Fpg proteins lack DNA binding or cleavage activity, as tested in crude lysates of Escherichia coli. Wild type Fpg protein contains one zinc/protein molecule, whereas the mutant Fpg protein (C244S/C247S) lacks zinc, as measured by atomic absorption spectroscopy. This mutation did not significantly alter secondary structure, as assessed by circular dichroism spectroscopy. Our results suggest that Fpg protein utilizes its single COOH-terminal zinc finger motif in DNA binding.

High-flux hemodiafiltration: under six hours/week treatment.

Better utilization of existing high blood flow in mature vascular accesses with the described new technique of simultaneous high diffusion and convection results in a marked increase of treatment efficiency. Coupled with the better tolerance to high solute and weight removal rates, this approach permits drastic reduction of treatment time over conventional hemodialysis without sacrificing treatment adequacy.

Combined computed tomographic and radionuclide imaging in the long-term follow-up of children with primary intra-axial intracranial neoplasms.

Radionuclide and computed tomographic (CT) scanning was performed for the long-term follow-up of 63 patients who had been treated for primary intracranial central nervous system tumors. This group included 23 children with supratentorial lesions and 40 with infratentorial lesions. The results of imaging were correlated with clinical assessment and the results of cytologic evaluation of the cerebrospinal fluid and, when available, surgical or autopsy findings. The sensitivity, specificity, and positive predictive value of both CT and radionuclide scanning were evaluated for each type of tumor. These two modalities play a complementary role in the long-term follow-up of children with primary intra-axial neoplasms.

Radiological investigation of sellar region masses in children.

The treatment and prognosis of sellar region masses in children differ according to the type of tumor, and thus accurate determination of the extent and characteristics of the lesion is important. Evaluation by conventional skull radiography, complex-motion tomography, radionuclide imaging, computed tomography, cerebral air studies, and cerebral angiography has demonstrated definable characteristics of the various types of tumors and often allows distinction from benign processes.

Hyperbaric system for oxygen toxicity studies in the toad urinary bladder.

A hyperbaric research system to study the effects on membrane function of oxygen at pressures as high as 20 ATA has been designed. The system includes a pressure chamber capable of holding eight isolated toad bladder short-circuit current apparatuses and unique experimental gassing, diluent addition, and sampling systems which are monitored by a control panel mounted on the side of the pressure chamber. These externally controlled gassing, diluent, and sampling systems permit experimental manipulations at any ambient pressure and are a significant addition to the methodology of hyperbaric research.