Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH(2) is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH(2), where acyl (R-CO) possesses a series of alkyl groups, R=C(n)H(2n+1) (n=0-5). The isovaleryl derivative with the C(4)H(9) (=(CH(3))(2)CHCH(2)-) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH(2) and producing pure antagonist activity.     

Site-directed affinity-labeling of delta opioid receptors by

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala², Leu⁵]-enkephalyl-Cys(Npys)⁶ and [-Ala²,Leu(CH₂SNpys)⁵]enkephalin, and dynorphin A analogues of [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide and [-Ala²,Cys(Npys)⁸]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide.     

The bifidogenic growth stimulator inhibits the growth and respiration of Helicobacter pylori

Background: Triple therapy with amoxicillin, clarithromycin, and a proton‐pump inhibitor is a common therapeutic strategy for the eradication of Helicobacter pylori (H. pylori). However, frequent appearance of clarithromycin‐resistant strains is a therapeutic challenge. While various quinones are known to specifically inhibit the growth of H. pylori, the quinone 1,4‐dihydroxy‐2‐naphthoic acid (DHNA) produced by Propionibacterium has strong stimulating effect on Bifidobacterium. We were interested to see whether DHNA could inhibit the growth of H. pylori in in vitro or in vivo experimental setting. Materials and Methods: The minimum inhibitory concentration (MIC) of DHNA was determined by the agar dilution method. The inhibitory action of DHNA on the respiratory activity was measured by using an oxygen electrode. Germ‐free mice infected with H. pylori were given DHNA in free drinking water containing 100 μg/mL for 7 days. Results: DHNA inhibited H. pylori growth at low MIC values, 1.6–3.2 μg/mL. Likewise, DHNA inhibited clinical isolates of H. pylori, resistant to clarithromycin. However, DHNA did not inhibit other Gram negative or anaerobic bacteria in the normal flora of the human intestine. Both H. pylori cellular respiration and adenosine 5′‐triphosphate (ATP) generation were dose‐dependently inhibited by DHNA. Similarly, the culture filtrates of propionibacterial strains inhibited the growth of H. pylori, and oral administration of DHNA could eradicate H. pylori in the infected germ‐free mice. Conclusions: The bifidogenic growth stimulator DHNA specifically inhibited the growth of H. pylori including clarithromycin‐resistant strains in vitro and its colonization activity in vivo. The bactericidal activity of DHNA was via inhibition of cellular respiration. These actions of DHNA may have clinical relevance in the eradication of H. pylori.     

Three-dimensional motion of the upper extremity joints during various activities of daily living

Highly reliable information on the range of motion (ROM) required to perform activities of daily living (ADL) is important to allow rehabilitation professionals to make appropriate clinical judgments of patients with limited ROM of the upper extremity joints. There are, however, no data available that take full account of corrections for gimbal-lock and soft tissue artifacts, which affect estimation errors for joint angles. We used an electromagnetic three-dimensional tracking system (FASTRAK) to measure the three-dimensional ROM of the upper extremity joints of healthy adults (N=20, age range 18–34) during 16 ADL movement tasks. The ROM required for the performance of each movement was shown in terms of the joint angle at the completion of the task, using a new definition of joint angle and regression analysis to compensate for estimation errors. The results of this study may be useful in setting goals for the treatment of upper extremity joint function.     

Rotenone, a mitochondrial electron transport inhibitor, ameliorates ischemia-reperfusion-induced intestinal mucosal damage in rats

In ischemia-reperfusion (I/R)-induced tissue injury, oxygen radicals can be generated by several mechanisms. One of the important sources of oxygen radicals is thought to be mitochondrial respiration. The aim of this study was to investigate the antioxidative defense effect of the mitochondrial electron transport inhibitor, rotenone using the I/R-induced rat intestinal mucosal injury model in vivo. Intestinal ischemia was induced for 30 min by applying a small clamp to the superior mesenteric artery in rats. Rotenone at a dose of 100 mg/kg was given to rats orally 2 h before the ischemia. Intraluminal hemoglobin and protein levels, the mucosal content of thiobarbituric acid-reactive substances (TBARS), the mucosal myeloperoxidase activity, and the content of inflammatory cytokines (CINC-1, TNF-alpha) were all significantly increased from mean basal levels after 60 min of reperfusion. These increases after I/R were inhibited by treatment with rotenone at a dose of 100 mg/kg. Co-administration with succinate (100 mg/kg), a substrate of the mitochondrial electron transport system, cancelled significant reduction of intraluminal hemoglobin and mucosal TBARS treated with rotenone alone. The results of the present study indicate that rotenone inhibited lipid peroxidation and reduced development of the intestinal mucosal inflammation induced by I/R in rats. This investigation suggests that rotenone has potential as a new therapeutic agent for reperfusion injury.     

Receptor binding characteristics of the endocrine disruptor bisphenol A for the human nuclear estrogen-related receptor gamma. Chief and corroborative hydrogen bonds of the bisphenol A phenol-hydroxyl group with Arg316 and Glu275 residues

Bisphenol A, 2,2-bis(4-hydroxyphenyl)propane, is an estrogenic endocrine disruptor that influences various physiological functions at very low doses, even though bisphenol A itself is ineffectual as a ligand for the estrogen receptor. We recently demonstrated that bisphenol A binds strongly to human estrogen-related receptor gamma, one of 48 human nuclear receptors. Bisphenol A functions as an inverse antagonist of estrogen-related receptor gamma to sustain the high basal constitutive activity of the latter and to reverse the deactivating inverse agonist activity of 4-hydroxytamoxifen. However, the intrinsic binding mode of bisphenol A remains to be clarified. In the present study, we report the binding potentials between the phenol-hydroxyl group of bisphenol A and estrogen-related receptor gamma residues Glu275 and Arg316 in the ligand-binding domain. By inducing mutations in other amino acids, we evaluated the change in receptor binding capability of bisphenol A. Wild-type estrogen-related receptor gamma-ligand-binding domain showed a strong binding ability (K(D) = 5.70 nm) for tritium-labeled [(3)H]bisphenol A. Simultaneous mutation to Ala at positions 275 and 316 resulted in an absolute inability to capture bisphenol A. However, individual substitutions revealed different degrees in activity reduction, indicating the chief importance of phenol-hydroxyl<-->Arg316 hydrogen bonding and the corroborative role of phenol-hydroxyl<-->Glu275 hydrogen bonding. The data obtained with other characteristic mutations suggested that these hydrogen bonds are conducive to the recruitment of phenol compounds by estrogen-related receptor gamma. These results clearly indicate that estrogen-related receptor gamma forms an appropriate structure presumably to adopt an unidentified endogenous ligand.     

The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio)

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.     

Treatment of Near-Infrared Photodynamic Therapy Using a Liposomally Formulated Indocyanine Green Derivative for Squamous Cell Carcinoma

Introduction

Photodynamic therapy (PDT) is a less invasive option for cancer treatment that has evolved through recent developments in nanotechnology. We have designed and synthesized a novel liposome system that includes an indocyanine green (ICG) derivative, ICG-C18, in its bilayer. In addition to its use as an optical imager to visualize blood, lymphatic, and bile flow, ICG has also been used as an optical sensitizer. In the present report, we evaluate the use of our novel liposome system, LP-ICG-C18, in PDT for squamous cell carcinoma in an autologous murine model.

Materials and Methods

An excitation pulse beam (300 μJ/pulse) of a single band (800 nm) was used for sensitization. The cytotoxicity of the photodynamic therapy was evaluated in terms of cellular morphology changes, methyl thiazolyl tetrazolium (MTT) assay results, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. We tested the enhanced permeability and retention effect of LP-ICG-C18 in tumor-bearing C3H/He mice using a near-infrared fluorescence imaging system and fluorescence microscopy. We also examined the antitumor effect of PDT by measuring tumor volume in tumor-bearing mice.

Results

Cell death and apoptosis were only observed in the PDT group receiving LP-ICG-C18. LP-ICG-C18 itself had no cytotoxic activity and showed good biocompatibility. LP-ICG-C18 accumulated on the tumor 24 hours after injection and was retained for approximately 3 weeks. Tumor cell apoptosis following PDT with LP-ICG-C18 was also observed under optical microscopy, MTT assay, and TUNEL staining.

Conclusion

These findings suggest that LP-ICG-C18 may be an effective intervening material in PDT for malignant disease.