Automated measurement of spontaneous pain-associated limb movement and drug efficacy evaluation in a rat model of neuropathic pain.

BACKGROUND: The withdrawal response elicited by a nociceptive stimulus, i.e., evoked pain measure, is commonly used as an efficacy endpoint in neuropathic pain animal models. It, however, has several limitations, which highlight the importance of examining spontaneous pain. The present study describes an automated method for measuring spontaneous pain behaviour in a rat model of neuropathic pain caused by chronic constriction injury (CCI) of sciatic nerve. METHODS: After CCI surgery, a small magnet was implanted into the operated limb. The rat was placed in a test chamber that was surrounded by wire coil. Limb movements, including lifting/guarding, flinching/shaking, licking and walking in the operated limb, caused changes in the electromagnetic field, including a change in voltage and transformed into a signal via an amplifier. RESULTS: CCI rats consistently showed more frequent limb movement than sham rats. There was no significant correlation between the frequency of spontaneous pain behaviour and the evoked pain symptoms. Treatment with duloxetine (30 mg/kg p.o.) and amitriptyline (30 and 100 mg/kg p.o.) significantly reduced this frequency. Pregabalin at 30 mg/kg p.o. tended to reduce the frequency, and diclofenac up to 10 mg/kg p.o. had no effect. CONCLUSION: A non-subjective automated method for measuring spontaneous pain behaviour in an animal model of neuropathic pain was established. It is expected that the current system will greatly enhance the analysis of spontaneous pain-related behaviour, which is a predominant symptom in patients with neuropathic pain. The current system may also be valuable in the screening of potential analgesic treatments.

Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt ("Caspian Sea yogurt") in Japan.

Lactobionic acid was first found in a Caucasian fermented milk product popularly known as "Caspian Sea yogurt" in Japan. The presence of lactobionic acid in the fermented milk was indicated by the results of both high-performance anion-exchange chromatographic analysis with pulsed amperometric detection and mass spectrometric analysis. Thereafter, the acid was purified from the yogurt and analyzed by nuclear magnetic resonance. A substantial amount of lactobionic acid was found to be accumulated in the upper layer of the yogurt, especially within 10 mm from the surface. A total of 45 mg of lactobionic acid per 100 g of the upper yogurt layer was collected after 4 d of fermentation. The annual intake of lactobionic acid in individuals consuming 100 g of the yogurt every day would be 0.5 to 1.0 g. A lactose-oxidizing bacterium was isolated from the fermented milk and was identified as Acetobacter orientalis. Washed A. orientalis cells oxidized monosaccharides such as d-glucose at considerable rates, although their activities for substrates such as lactose, maltose, and cellobiose were much lower. When A. orientalis cells were cultivated in cow's milk, they exhibited lactose-oxidizing activity, suggesting that this bacterium was the main organism involved in the production of lactobionic acid in the yogurt.

Pharmacological characterization and gene expression profiling of an L5/L6 spinal nerve ligation model for neuropathic pain in mice.

L5/L6 spinal nerve ligation (SNL) in rodents induces behavioral signs similar to the symptoms of neuropathic pain in humans. L5/L6 SNL in rats has been well characterized so far, but there have been few studies using mice. In this study, we established an L5/L6 SNL model in mice and examined the effects of known antinociceptive drugs in the model. We also analyzed the changes in gene expression in dorsal root ganglions with special reference to those which are known to change in a neuropathic pain state to validate the model. Mechanical allodynia in the ipsilateral side paw was observed beginning on day 1 and lasted for at least 2 months following surgery. Diclofenac showed no significant effect on the mechanical allodynia. Gabapentin and pregabalin completely reversed allodynia, but they also caused a decrease in locomotor activity. Duloxetine caused a partial recovery of the threshold. Mexiletine completely reversed allodynia, but it also caused sedation or motor impairment. Morphine caused a partial recovery of the threshold and hyper-locomotion. This mouse L5/L6 SNL model represents a robust mechanical allodynia, which shows a similar pharmacological response to that reported in rats and human patients with neuropathic pain. The pattern changes in gene expression also resembled those reported in rats. This model will therefore be useful for investigation of the effects of novel antinociceptive compounds and the mechanisms of neuropathic pain.

Morphologic and histopathologic changes in the bowel after super-selective transcatheter embolization for focal lower gastrointestinal hemorrhage.

PURPOSE: To evaluate the safety of superselective arterial embolization therapy in the lower gastrointestinal tract. The sequelae on normal enteric tissue in lower gastrointestinal arterial embolization were retrospectively reviewed. MATERIAL AND METHODS: To control hemorrhage and tumor blood supply, 14 patients were treated by superselective transcatheter embolization at different levels of the colonic and small intestine vessels via the superior and inferior mesenteric arteries using microcoils and/or gelatin sponge. Normal enteric tissues in the embolized areas were analyzed for the occurrence of ischemic changes by clinical follow-up, colonoscopy, and surgery. RESULTS: Normal bowel function was preserved in 13 patients. In 1 patient treated with numerous gelatin sponge particles delivered from the proximal arcade of the superior mesenteric artery, significant muscular fibrosis occurred. CONCLUSION: Superselective arterial embolization for lower gastrointestinal hemorrhage can be safely performed by minimizing the amount of embolic materials and delivering them as distally as possible.

Enhanced expression of decay-accelerating factor and CD59/homologous restriction factor 20 in intestinal metaplasia, gastric adenomas and intestinal-type gastric carcinomas but not in diffuse-type carcinomas.

AIMS: Variable expression of the complement regulatory proteins, decay-accelerating factor, CD59/homologous restriction factor 20 (HRF20) and membrane cofactor protein has been shown in human gastrointestinal malignancies, but their expression in gastric cancer has not been fully described. Thus, we immunohistochemically defined the distribution of these proteins in human normal gastric mucosa, intestinal metaplasia, adenomas and gastric cancers. METHODS AND RESULTS: Gastric tissues were obtained by endoscopic biopsy or surgical resection and stained with mouse monoclonal antibodies to decay-accelerating factor, CD59/HRF20, and membrane cofactor protein. In the normal gastric mucosa, membrane cofactor protein was diffusely stained on the basolateral surface of epithelial cells, whereas the expression of decay-accelerating factor and CD59/HRF20 was inconspicuous. In intestinal metaplasia, adenoma and intestinal-type gastric carcinoma cells, decay-accelerating factor and HRF20 were intensely stained on the apical surface; membrane cofactor protein retained its location on the basolateral surface. In diffuse-type gastric carcinomas, the expression of decay-accelerating factor, CD59/HRF20 was lost, but membrane cofactor protein was present on the tumour cell surface. CONCLUSIONS: These findings suggest that membrane cofactor protein plays a primary role in the regulation of complement activation in normal and neoplastic gastric cells and that the expression pattern of the complement regulatory proteins is closely related to gastric carcinoma development.

A novel 5-HT3 receptor agonist, YM-31636, increases gastrointestinal motility without increasing abdominal pain.

We examined the effects of YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate), a novel 5-HT3 receptor agonist, on gastrointestinal functions including visceral pain reflex in rats. Injection of YM-31636 increased the number of fecal pellets. This effect was completely inhibited by ramosetron, a 5-HT3 receptor antagonist. YM-31636 also increased the intracolonic pressure measured in both conscious and anesthetized rats. In isolated distal colon, YM-31636 increased the short-circuit current response. This effect was abolished by ramosetron. Both the maximal response and the potency of YM-31636 were weaker than those of other 5-HT3 receptor agonists. In two visceral pain reflex models, YM-31636 neither changed the magnitude of pressor response to colonic distension in anesthetized rats nor affected the visceromotor threshold to colorectal distension in conscious rats. In conclusion, YM-31636 facilitated defecation without increasing visceral pain. Consequently, 5-HT3 receptor agonists like YM-31636 would be promising in the treatment of chronic constipation.

Investigation of the effects of YM-31636, a novel 5-HT3 receptor agonist, on defecation in normal and constipated ferrets.

We examined the effects of YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate), a newly synthesized 5-HT(3) receptor agonist, on defecation in normal and constipated ferrets, and evaluated it as an agent against constipation. YM-31636 facilitated defecation without inducing diarrhea or emetic episodes. This effect occurred within 1 h after oral administration, mostly within 30 min, whereas sodium picosulfate, a widely used laxative, tended to increase the frequency of defecation for several hours with much lower peak incidence than that of YM-31636, and induced diarrhea. UK14304 (brimonidine), an alpha2 receptor agonist, and morphine reduced the frequency of defecation and YM-31636 restored it. These effects of YM-31636 were antagonized by ramosetron, a 5-HT(3) receptor antagonist. These results suggest that YM-31636 could be promising in the treatment of constipation. Because of an early and reliable onset of action compared with sodium picosulfate, YM-31636 could make it easier to control the time of defecation.

Neurometer measurement of current stimulus threshold in rats.

We examined the current stimulus threshold in rats with the Neurometer, a device used clinically for measuring perception and pain thresholds. Although many studies have indicated the usefulness of this device in the quantification of nerve dysfunction in patients, we have found no published reports on the use of the Neurometer in animals. Transcutaneous nerve stimuli of the three sine-wave pulses produced by the Neurometer (at 2000, 250, and 5 Hz) were applied to plantar surface of rats. The intensity of each stimulation at which rats vocalized or were hardly startled was defined as the current stimulus threshold. With repeated stimulation, the thresholds were almost constant. Repeated topical application to the area around the stimulating electrode of a high concentration of capsaicin, which acts on small-diameter fibers, increased the thresholds at 250 and 5 Hz, but did not affect the 2000-Hz threshold. Intravenous morphine (2-5 mg/kg) increased all three thresholds, whereas intrathecal morphine (20 or 80 microg) increased only the 5-Hz threshold. Intravenous injection of a minor tranquilizer, diazepam, at 1 mg/kg raised the thresholds at 2000 and 250 Hz, but did not affect the 5-Hz threshold. Higher dose of diazepam increased all three thresholds. These results suggest that the Neurometer makes possible selective examination of subsets of nerve fibers that differ in diameter not only in humans but also in animals. The present study in rats, in which we established a method of measurement, may provide helpful suggestions for the interpretation of data in humans.

L-2,5-dihydrophenylalanine, an inducer of cathepsin-dependent apoptosis in human promyelocytic leukemia cells (HL-60).

L-2,5-Dihydrophenylalanine (DHPA), a phenylalanine analogue, induced apoptosis in human promyelocytic leukemia cells (HL-60). This apoptosis was demonstrated by morphological changes of the cells, such as fragmentation of nuclei and chromatin condensation, and by some evidence found in biochemical analysis, such as DNA ladder and activation of caspase 3. The DHPA-induced apoptosis was prevented by a pan-caspase inhibitor, Z-VAD-fmk, and a cysteine protease inhibitor, E-64d, which inhibits calpains and cathepsin B and L. A calpain inhibitor, Z-LL-H, did not affect this apoptosis. A cathepsin B specific inhibitor, CA074-Me, prevented only chromatin condensation. However, E-64d and a cathepsin L specific inhibitor, Z-FY(t-Bu)-dmk, protected the cells from both chromatin condensation and oligonucleosomal DNA fragmentation. As proceeding to the apoptotic process, the activities of both cathepsin B and L increased gradually. These results indicated that DHPA was an inducer of cathepsin-dependent apoptosis in HL-60 cells.

Pharmacological profile of YM-31636, a novel 5-HT3 receptor agonist, in vitro.

We investigated the in vitro pharmacological profile of YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate). In cloned human 5-HT3A receptors, YM-31636 had a pKi value of 9.67 vs. ramosetron and pKi values for other 5-HT3 receptor agonists were less than 7. YM-31636 showed very low affinities for other receptors. YM-31636 induced contraction of isolated guinea pig distal colon. The intrinsic activity was approximately 0.90 compared with 5-hydroxytryptamine's (5-HT) 1.0, and the potency was 26 times greater than that of 5-HT. YM-31636 increased short-circuit current (Isc) in the isolated guinea pig distal colon. In this case, the relative intrinsic activity was approximately 0.19. In isolated guinea pig right atrium, YM-31636 induced tachycardia with the relative intrinsic activity of approximately 0.23. All these effects of YM-31636 were antagonized by ramosetron, a selective 5-HT3 receptor antagonist. These results suggest that YM-31636 is a potent and selective 5-HT3 receptor agonist, preferentially acting on the contraction of the colon.

Hydrolysis of beta-galactosyl ester linkage by beta-galactosidases.

p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.

Transfructosylation of thiol group by beta-fructofuranosidases.

Beta-fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers' yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-fructofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the beta-fructofranosidase.

Galactosylation of thiol group by beta-galactosidase.

beta-Galactosidase catalyzed beta-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-beta-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzae beta-galactosidase hydrolyzed p-nitrophenyl S-beta-D-galactoside most rapidly among several beta-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolor enzyme was as effective as the A. oryzae enzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulans galactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8-2.8 M did not affect the synthetic yield of the thiogalactoside so greatly.

The role of 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in the regulation of gut motility in the ferret.

The role of 5-hydroxytryptamine (5-HT)3 and 5-HT4 receptors in the regulation of gut motility in the ferret was investigated. The selective 5-HT3 receptor antagonist ramosetron (1 - 10 microg/kg s.c.) prolonged the interval of gastric antral migrating motor complex, but had only slight effect on small intestinal and colonic motility in unfed animals. The selective 5-HT4 receptor antagonist SB 204070 did not affect motility throughout gut in unfed animals. Neither ramosetron nor SB 204070 affected the motility throughout gut in fed animals. In conclusion, neither 5-HT3 nor 5-HT4 receptors tonically regulate ferret gut motility except that 5-HT3 receptors have a key role in the occurrence of migrating motor complex specifically in the stomach. The role of 5-HT3 and 5-HT4 receptor system in the regulation of gut motility in ferrets is similar to that in other mammalian species studied, including humans. This similarity suggests that the ferret is a suitable model animal to study gut motor functions in humans.

Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae.

Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.

Hydrolysis of beta-glucosyl ester linkage of p-hydroxybenzoyl beta-D-glucose, a chemically synthesized glucoside, by beta-glucosidases.

To investigate the hydrolysis of glucosyl esters by beta-glucosidase, p-hydroxybenzoyl beta-D-glucose (pHBG) was chemically synthesized. The hydrolytic activity of some beta-glucosidases for pHBG was compared to that for p-nitrophenyl beta-glucoside (pNPG). The Clavibacter michiganense and Flavobacterium johnsonae enzymes could hydrolyze pHBG and steviol glycosides which are natural glucosyl esters. The commercial beta-glucosidase originating from Caldocellum saccharolyticum also hydrolyzes pHBG despite having no activity for steviol glycosides. The beta-glucosidase from Aspergillus niger cleaved the glucosyl ester linkage much more weakly than the glucosidic linkage. The pH-activity profile for the hydrolysis of pHBG was similar to that of pNPG by the C. saccharolyticum beta-glucosidase. The similar profiles for these substrates suggested that the active site for the glucosyl ester chemically resembles that for glucoside with respect to catalysis. Kinetic analysis of the C. saccharolyticum beta-glucosidase for mixed substrates of pHBG and pNPG showed that the hydrolysis of pHBG competed with that of pNPG. This result indicated that there is only one active site for both the glucosyl ester and glucoside. Mass spectroscopic analysis of the hydrolysates of pHBG in H218O suggested that beta-glucosidase hydrolyzes glucosyl esters between the anomeric carbon of glucose and the carbonyl oxygen, not between the carbonyl carbon and the carbonyl oxygen.

Anti-obesity and anti-diabetic activities of a new beta3 adrenergic receptor agonist, (S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino]ethyl]-1-propenyl] phenoxy] acetic acid ethanedioic acid (SWR-0342SA), in KK-Ay mice.

We investigated the beta-adrenergic receptor (AR) agonistic activities in rats and humans, and the anti-obesity and anti-diabetic activities in KK-Ay mice, of a new beta3-AR agonist, SWR-0342SA ((S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino]ethyl]-1-pro penyl]phenoxy] acetic acid ethanedioic acid). With regards to its beta-AR agonistic activity in rats, SWR-0342SA stimulated the atrial beating rate (beta1-AR activity) and white adipocyte lipolysis (beta3-AR activity), but did not induce uterine muscle relaxation (beta2-AR activity). The beta3-AR agonistic activity of SWR-0342SA was about 20 times stronger than its beta1-AR agonistic activity. Similarly, SWR-0342SA enhanced the accumulation of cAMP in Chinese hamster ovary (CHO) cells expressing human beta1- and beta3-ARs, while having no effect in CHO cells expressing beta2-ARs. Adenylyl cyclase stimulation by SWR-0342SA in CHO cells expressing beta3-ARs was about 35 times higher than that in CHO cells expressing beta1-ARs. With regards to anti-obesity and anti-diabetic activities, SWR-0342SA had no effect on body weight or food intake, but slightly decreased the fat pads weight in KK-Ay mice, an animal model of obesity and non-insulin-dependent diabetes mellitus (NIDDM). On the other hand, SWR-0342SA significantly decreased both blood glucose (to about 46% of control) and serum insulin levels (to about 40% of control) in KK-Ay mice. These results indicated that SWR-0342SA is a selective beta3-AR agonist, and possesses potent anti-diabetic activity, and that the anti-obesity activity is inferior to the anti-diabetic activity.

Structural analyses of a precursory substance of bitterness: new polyisoprenepolyols isolated from an edible mushroom (Hypsizigusmarmoreus) by fast atom bombardment mass spectrometry.

New polyisoprenepolyols (hypsiziprenol AA and BA) were isolated from an edible mushroom (Hypsizigus marmoreus). These polyols occur as a mixture of homologous polyisoprene derivatives with 40-70 carbon atoms. Analyses by FAB/MS in the positive and negative ion modes are complementary with each other in that the former provides information on the number of hydroxy groups present while the latter specifies the isoprenoid sequence, and thus become a powerful tool for analyzing the structures of polyisoprenepolyols. No polyisoprenepolyols obtained here were found to have antitumor activity on NCI-H292 and EL-4 cell lines.

cAMP-independent chloride secretion activated by a vasoactive intestinal peptide in a monolayer culture of human bronchial epithelial cells.

To investigate the effects of vasoactive intestinal peptide (VIP) on Cl- transport across normal human bronchial epithelial (NHBE) cells grown in a monolayer, changes in short-circuit current (Isc) were measured in Ussing chamber systems. In the presence of 10(-4) M amiloride, the addition of VIP to the serosal solution led to an increase in the Isc in a concentration-dependent manner, the 50% effective concentration (EC50) being 2.6 x 10(-11) M. However, the addition of 10(-5) M forskolin had little effect on the increase in Isc. On the other hand, in the intracellular cAMP measurement, 10(-5) M forskolin remarkably increased the cAMP levels, but 10(-7) M VIP did not. This result suggests that Cl- secretion by VIP is not related to the raised intracellular cAMP levels in NHBE cells.

Isolation and characterization of new limonoid glycosides from Citrus unshiu peels.

Three limonoid glycosides were isolated from Citrus unshiu peels, and their structures were determined based on MS and NMR spectroscopic data as nomilinic acid 17-O-beta-D-glucopyranoside (1), methyl nomilinate 17-O-beta-D-glucopyranoside (2), and obacunone 17-O-beta-D-glucopyranoside (3). In particular, the location of the sugar moiety was clearly determined by the B/E constant linked scan FABMS method. No limonoid glycosides obtained here were found to have antitumor activity in NCI-H292 and EL-4 cell lines.