Protein-glutaminase improves water-/oil-holding capacity and beany off-flavor profiles of plant-based meat analogs.

An unresolved challenge for plant-based meat analogs (PBMAs) is their lack of juiciness. Saturated fats significantly contribute to the juiciness of PBMAs, but there are concerns about the undesirable health effects related to saturated fats; thus, demand for their replacement with vegetable unsaturated oils has increased. Although many food additives are used to reduce the leakage of unsaturated oils, this solution cannot meet the clean-label requirements that have been trending in recent years. In this study, we aimed to develop better consumer-acceptable methods using protein-glutaminase (PG) to improve the juiciness of PBMA patties to meet clean-label trends. We found no significant difference between the visual surface of control and PG-treated textured vegetable proteins (TVPs). However, the microstructure of PG-treated TVP had a more rounded shape than that of the control TVP as observed under a scanning electron microscope. After grilling process, the PBMA patties composed of PG-treated TVP showed significantly higher liquid-holding capacities (a juiciness indicator) than the control patties. This suggested that PG treatment could potentially produce PBMA patties with increased juiciness. Interestingly, after the PG-treated TVP underwent the wash process, we found that PG treatment of TVP easily reduced the various beany off-flavor compounds by 58-85%. Moreover, the results of the in vitro protein digestion test showed that the amounts of free amino nitrogen released from PBMA patties composed of PG-treated TVP were 1.5- and 1.7-fold higher than those from control patties in the gastric and intestinal phases, respectively. These findings indicate that PG treatment of TVP could enhance the physical, sensory, and nutritional properties of PBMA patties and meet the clean-label requirements.

Decolorization and detoxication of plant-based proteins using hydrogen peroxide and catalase.

The gap between the current supply of meat and its predicted future demand is widening, increasing the need to produce plant-based meat analogs. Despite ongoing technical developments, one of the unresolved challenges of plant-based meat analogs is to safely and effectively decolor plant proteins that originally exhibit yellow-brown or strong brown color. This study aimed to develop an effective and safe decoloring system for soy-based protein products using food-grade hydrogen peroxide and catalase. First, soy-based protein isolate (PI) and textured vegetable protein (TVP) were treated with hydrogen peroxide, and then the residual hydrogen peroxide was degraded using catalase. This process caused notable decolorization of PI and TVP, and residual hydrogen peroxide was not detected in these products. These findings indicate that this process could safely and effectively decolorize soy-based proteins. Interestingly, this decoloring process enhanced the solubility, water- and oil-holding capacities, foaming capacity, and emulsifying stability of decolored soy-based PI. Additionally, cooking loss and juiciness of decolored TVP-based foods were improved compared to those of non-treated foods. These findings indicate that the decoloring process also enhances the physical properties of soy-based protein products.

Cyclodextrins produced by cyclodextrin glucanotransferase mask beany off-flavors in plant-based meat analogs.

The widening gap between the supply and demand for meat products has increased the need to produce plant-based meat analogs as protein sources. Meat analogs are principally composed of soy-based textured vegetable proteins. Despite ongoing technical developments, one of the unresolved challenges for plant-based meat analogs is the off-flavor from soy, which limits their consumer acceptability. Among the various methods developed for overcoming this challenge, masking the beany flavors with cyclodextrins (CDs) is an attractive, cost-effective, and safe strategy. However, the current established CD treatment method does not meet the requirement for a clean-label. This study aimed to develop more acceptable off-flavor-masking technologies for plant-based patties for modern clean-label preferences using enzymatic methods. We used the cyclodextrin glucanotransferase (CGT), "Amano," as a commercially available food-grade CGT. The CGT-catalyzed reaction in plant-based patties yielded 17.1 g/L CD. As CGT could yield sufficient CD in the patties, we investigated whether CDs produced by CGT could mask the off-flavors released from the plant-based patties. The CGT-treated patties had significantly lower volatilization amounts of the known beany off-flavor-generating compounds compared to the non-treated patties. Moreover, CGT treatment improved the texture of the patties and increased their water- and oil-holding capacity. As CGT is rendered inactive after cooking, it would not be considered an additive. These findings indicated that CDs produced by the CGT reaction could effectively mask off-flavors of meat analogs and improve their physical properties while meeting clean-label requirements.

Intervention Strategies to Reduce Surgical Site Infection Rates in Patients Undergoing Rectal Cancer Surgery.

BACKGROUND/AIM: This study aimed to determine the effectiveness of surgical site infection (SSI) prevention approaches in rectal cancer surgery. PATIENTS AND METHODS: A total of 1,408 patients who underwent elective rectal cancer surgery between 1995 and 2017 were reviewed. Patients were divided into three groups: control group (group A, n=245), SSI prevention intervention group (group B, n=516), and laparoscopic or robotic surgery group (group C, n=647). The groups were compared in terms of SSI and anastomotic leakage (AL) incidences, and risk factors for SSI were investigated. RESULTS: The overall SSI and AL rates were 19.4% and 3.6%, respectively. These rates were significantly lower in Group C (9.3%, 1.7%), compared to Groups A (40.0%, 6.1%) and B (22.5%, 3.5%). Abdominoperineal resection, open surgery, operation time, intraoperative bleeding, lack of absorbable sutures, lack of mechanical bowel preparation, and lack of oral antibiotics were independently associated with SSI. CONCLUSION: SSI reduction after rectal cancer surgery was achieved through various intervention strategies.

Synergistic effects of laccase and pectin on the color changes and functional properties of meat analogs containing beet red pigment.

The widening gap between current supply of meat and its future demand has increased the need to produce plant-based meat analogs. Despite ongoing technical developments, one of the unresolved challenges of plant-based meat analogs is to safely and effectively imitate the appearance of raw and cooked animal-based meat, especially the color. This study aimed to develop a more effective and safe browning system for beet red (BR) in plant-based meat analog patties using laccase (LC) and sugar beet pectin (SBP). First, we investigated the synergistic effects of SBP and LC on BR decolorization of meat analog patties. We discovered that the red tones of LC-treated patties containing BR and SBP were remarkably browned after grilling, compared to patties that did not contain SBP. Notably, this color change by LC + SBP was similar to that of beef patties. Additionally, the hardness of LC-treated meat analog patties containing BR was higher than those that did not contain BR. Interestingly, the presence of SBP and LC enhanced the browning reaction and functional properties of meat analogs containing BR. This is the first report on a browning system for meat analogs containing BR using enzymatic methods to the best of our knowledge.

Improved functional properties of meat analogs by laccase catalyzed protein and pectin crosslinks.

The gap between the current supply and future demand of meat has increased the need to produce plant-based meat analogs. Methylcellulose (MC) is used in most commercial products. Consumers and manufacturers require the development of other novel binding systems, as MC is not chemical-free. We aimed to develop a novel chemical-free binding system for meat analogs. First, we found that laccase (LC) synergistically crosslinks proteins and sugar beet pectin (SBP). To investigate the ability of these SBP-protein crosslinks, textured vegetable protein (TVP) was used. The presence of LC and SBP improved the moldability and binding ability of patties, regardless of the type, shape, and size of TVPs. The hardness of LC-treated patties with SBP reached 32.2 N, which was 1.7- and 7.9-fold higher than that of patties with MC and transglutaminase-treated patties. Additionally, the cooking loss and water/oil-holding capacity of LC-treated patties with SBP improved by up to 8.9-9.4% and 5.8-11.3%, compared with patties with MC. Moreover, after gastrointestinal digestion, free amino nitrogen released from LC-treated patties with SBP was 2.3-fold higher than that released from patties with MC. This is the first study to report protein-SBP crosslinks by LC as chemical-free novel binding systems for meat analogs.

Epithelial-mesenchymal transition and metastatic ability of CD133+ colorectal cancer stem-like cells under hypoxia.

Although CD133 is a representative cancer stem cell marker, its function in tumor aggressiveness under hypoxia remains unclear. Therefore, the present study aimed to investigate the associations between CD133, the epithelial-mesenchymal transition and distant metastasis in colorectal cancer. CD133+ and CD133- cells were isolated from a single colorectal cancer cell line LoVo, and their adhesive and migratory properties were compared under hypoxic conditions. Immunostaining analysis was performed to determine CD133 expression in clinical samples of primary tumors, as well as liver and peritoneal metastases. Under hypoxia, the expression levels of hypoxia-inducible factor (HIF)-1α and the epithelial-mesenchymal transition markers N-cadherin and vimentin were significantly higher in the CD133+ compared with those in the CD133- cells. Furthermore, the migratory ability of the CD133+ cells was higher compared with that of the CD133- cells under hypoxia. By contrast, the expression levels of ß1 integrin were significantly lower in the CD133+ cells under hypoxia compared with those in the CD133- cells. Immunohistochemical analysis of clinical samples revealed that the levels of CD133 expression in metastatic tissues from the liver were significantly higher compared with those in the corresponding primary tumors, whereas CD133 expression levels in peritoneal metastatic tissues were significantly lower compared with those in the corresponding primary tumors. In conclusion, compared with the CD133- cells, the CD133+ colorectal cancer cells exhibited enhanced levels of HIF-1α expression and tumor cell migration during hypoxia. This was associated with an increased ability of epithelial-mesenchymal transition, possibly leading to the acquisition of an increased hematogenous metastatic potential and eventually resulting in liver metastasis. High ß1 integrin expression levels in the CD133- cells under hypoxia may serve a key role in cell adhesion to the peritoneum, resulting in peritoneal metastasis.

Metastatic role of mammalian target of rapamycin signaling activation by chemoradiotherapy in advanced rectal cancer.

Postoperative distant metastasis dramatically affects rectal cancer patients who have undergone neoadjuvant chemoradiotherapy (NACRT). Here, we clarified the association between NACRT-mediated mammalian target of rapamycin (mTOR) signaling pathway activation and rectal cancer metastatic potential. We performed immunohistochemistry for phosphorylated mTOR (p-mTOR) and phosphorylated S6 (p-S6) on surgical specimen blocks from 98 rectal cancer patients after NACRT (cohort 1) and 80 colorectal cancer patients without NACRT (cohort 2). In addition, we investigated the association between mTOR pathway activity, affected by irradiation, and the migration ability of colorectal cancer cells in vitro. Based on the results of the clinical study, p-mTOR was significantly overexpressed in cohort 1 (with NACRT) as compared to levels in cohort 2 (without NACRT) (P < .001). High p-mTOR and p-S6 levels correlated with the development of distant metastasis only in cohort 1. Specifically, high p-S6 expression (HR 4.51, P = .002) and high pathological T-stage (HR 3.73, P = .020) after NACRT were independent predictors of the development of distant metastasis. In vitro, p-S6 levels and migration ability increased after irradiation in SW480 cells (TP53 mutation-type) but decreased in LoVo cells (TP53 wild-type), suggesting that irradiation modulates mTOR signaling and migration through cell type-dependent mechanisms. We next assessed the expression level of p53 by immunostaining in cohort 1 and demonstrated that p-S6 was overexpressed in samples with high p53 expression as compared to levels in samples with low p53 expression (P = .008). In conclusion, p-S6 levels after NACRT correlate with postoperative distant metastasis in rectal cancer patients, suggesting that chemoradiotherapy might modulate the mTOR signaling pathway, promoting metastasis.

[Rectal cancer with local re-recurrence successfully treated by carbon ion radiotherapy].

A 61 year-old male with rectal cancer underwent anterior resection with D2 lymph node dissection in August 2007. Carcinoembryonic antigen (CEA) level was 5.6 before the operation. Pathological findings were Rs, tub2¼>tub1, type 3, pSE, ly1, v2, pN1 (1/23), H0, P0, M0 , pStage IIIA. Adjuvant chemotherapy with tegafur-uracil (UFT) 600 mg/Leucovorin (LV) 75 mg was administered for 1 year. A recurrence at a site of anastomosis developed and lower anterior resection was required in September 2010. CEA level was 5.4 before the operation. After 7 courses of capecitabine plus oxaliplatin (XELOX) treatment, the right #283 lymph node increased to 8 mm in October 2011 and the patient was diagnosed with a re-recurrence of the original tumor (CEA level, 4.6). Carbon ion radiotherapy (73.6 Gy/16 Fr/4 weeks) was performed between November 28 and December 22, 2011. Although the right #283 lymph node had shrunk by January 2012, a single node in the S3 domain of the right lung was observed and became progressively larger, indicating a lung metastasis (CEA level, 5.4). The patient received carbon ion radiotherapy (60.0 Gy/4 Fr) for the lung metastasis between July 30 and August 2, 2012. No additional recurrences have been seen through February 2014.

ASP6537, a novel highly selective cyclooxygenase-1 inhibitor, exerts potent antithrombotic effect without "aspirin dilemma".

INTRODUCTION: Aspirin inhibits both the cyclooxygenase (COX)-1-dependent production of thromboxane A2 (TXA2) in platelets and COX-2-dependent production of anti-aggregatory prostaglandin I2 (PGI2) in vessel walls, resulting in "aspirin dilemma." Our objective is to investigate whether ASP6537 can overcome aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect. METHODS: We evaluated the inhibitory effects of ASP6537 on recombinant human COX-1 (rhCOX-1) and rhCOX-2 activities using a COX-1/2 selectivity test. To determine whether ASP6537 induces aspirin dilemma, we examined the effects of ASP6537 on in vitro TXA2 and PGI2 metabolite production from platelets and isolated aorta of guinea pigs, and on plasma concentrations of TXA2 and PGI2 metabolites in aged rats. Finally, we evaluated the antithrombotic effects and ulcerogenic activity of ASP6537 using an electrically induced carotid arterial thrombosis model and a gastric ulcer model in guinea pigs. RESULTS: The IC50 ratios of rhCOX-2 to rhCOX-1 for ASP6537 and aspirin were >142,000 and 1.63 fold, respectively. ASP6537 inhibited TXA2 production more selectively than aspirin in in vitro and in vivo TXA2/PGI2 production studies. ASP6537 exerted a significant antithrombotic effect at ≥3 mg/kg, while aspirin tended to inhibit thrombosis at 300 mg/kg but it was not statistically significant. Further, ASP6537 did not induce ulcer formation at 100 mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of ≥100 mg/kg. CONCLUSIONS: ASP6537 functions as a highly selective COX-1 inhibitor with a superior ability to aspirin for normalizing TXA2/PGI2 balance, and exerts antithrombotic effect without ulcerogenic effect.

Renoprotective properties of pirfenidone in subtotally nephrectomized rats.

Renal fibrosis is the final common pathway of chronic kidney disease, and its progression predicts the degree of renal dysfunction. We investigated the renoprotective properties of pirfenidone in a remnant kidney model of chronic renal failure to determine its pharmacological potency compared to enalapril. Five-sixths nephrectomized rats were fed diet containing pirfenidone (approximately 700mg/kg/day) for 8weeks. Pirfenidone steadily inhibited the progression of proteinuria, but not to a significant degree. Pirfenidone prevented the elevation of plasma creatinine and blood urea nitrogen. At the end of the experiment, pirfenidone had reduced systolic blood pressure by means of its renoprotective effect. In a histological study, pirfenidone improved interstitial fibrosis in the renal cortex. These effects were supported by the suppression of the expression of TGF-beta and fibronectin in the mRNA of the kidney. In contrast, pirfenidone had little effect on the expression of alpha-smooth muscle actin, which is one of the proteins responsible for epithelial-mesenchymal transition. This property was confirmed by the TGF-beta-induced transdifferentiation observed in cultured normal rat kidney tubular epithelial NRK52E cells. These results suggest that pirfenidone improves the progression of chronic renal failure via its antifibrotic action, although pirfenidone has less effective TGF-beta-induced epithelial to mesenchymal transdifferentiation.

Minodronic acid, a third-generation bisphosphonate, antagonizes purinergic P2X(2/3) receptor function and exerts an analgesic effect in pain models.

The P2X(2/3) receptor has an important role in the nociceptive transmission. Minodronic acid is a third third-generation bisphosphonate and a potent inhibitor of bone resorption. We found that minodronic acid inhibited alpha,beta-methylene ATP-induced cation uptake with the potency higher than that of suramin in the P2X(2/3) receptor receptor-expressing cells. Other bisphosphonates did not show such activity. Subcutaneously administered (10-50 mg/kg) minodronic acid significantly inhibited the alpha,beta-methylene ATP-, acetic acid- and formalin-induced nociceptive behaviors in mice. These unique effects of minodronic acid would be beneficial for the treatment of accelerated bone turnover diseases accompanied by bone pain, including bone metastases.

Comparison of chronic renal failure rats and modification of the preparation protocol as a hyperphosphataemia model.

BACKGROUND: Several animal models with chronic renal failure have been established and used for demonstrating complications including hyperphosphataemia. Although long-time feeding is required to cause hyperphosphataemia in animals, a few modifications have been reported to provide more useful models for research. METHODS: Three separate experiments were carried out in the present study. First, characteristics of commonly used subnephrectomized (5/6Nx) rats and rats fed an adenine diet (0.75% adenine in normal diet) were compared as hyperphosphataemia models. Next, using adenine-diet rats, the inhibitory effect of sevelamer hydrochloride (Sev) on serum phosphorus elevation was examined. Third, oral adenine dosing for induction of hyperphosphataemia and validation as a model using Sev were examined. RESULTS: Serum phosphorus in 5/6Nx rats became elevated in 8-17 weeks, but the levels and time points of elevation differed among animals. In adenine-fed rats, the elevation was more clearly demonstrated with less diversity at 4 weeks. The data revealed a potential shorter model preparation period and the importance of controlling feeding amounts. Oral adenine dosing induced hyperphosphataemia by 12 days, and Sev treatment was inhibitory. After a maintenance period of over a month (no treatments), Sev-treated rats showed hyperphosphataemia as did oral adenine-dosed control rats. The serum phosphorus levels significantly decreased on further Sev treatment. CONCLUSION: Oral dosing with adenine made the model preparation period definitely shorter, and its usefulness as a hyperphosphataemia model was revealed using Sev.

Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models.

We describe in vitro properties and in vivo neuroprotective effects of a newly synthesized, high-affinity, selective allosteric metabotropic glutamate receptor type 1 (mGluR(1)) antagonist, N-cyclohexyl-6-{[(2-methoxyethyl)(methyl)amino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074). YM-202074 bound an allosteric site of rat mGluR(1) with a K(i) value of 4.8+/-0.37 nM. YM-202074 also inhibited the mGluR(1)-mediated inositol phosphates production in rat cerebellar granule cells with an IC(50) value of 8.6+/-0.9 nM, while showing selectivity over mGluR(2-7). When YM-202074 was infused intravenously at an initial dose of 20 mg/kg/h for 0.5 h followed by a dose of 5 mg/kg/h for 7.5 h, the free concentration of YM-202074 in the brain rapidly (<12 min) reached approximately 0.3 microM, reaching a steady-state phase within 1.5 h. We first treated rats such that they developed transient middle cerebral artery (MCA) occlusion. Results clearly demonstrate a dose-dependent improvement of neurological deficit and reduction of the infarct volume in both the hemisphere and cortex when YM-202074 was infused intravenously immediately after occlusion at a dose of 10 or 20 mg/kg/h for 0.5 h followed by a dose of 2.5 or 5 mg/kg/h for 23.5 h, respectively. Significant neuroprotection was maintained even when the administration of drugs was delayed by up to 2 h following the onset of ischemia. Furthermore, the improvement of neurological deficit and the reduction of infarct volume were sustained for 1 week following the onset of ischemia. These results suggest that YM-202074 exhibits great potential as a novel neuroprotective agent for the treatment of stroke.

Experimental model of lower limb ischemia in rats and the effect of YM466, an oral direct factor Xa inhibitor.

A simple, quantitative, and reproducible model of lower limb ischemia was developed. Vascular injury was induced by ferric chloride (FeCl(3)) solution to the rat iliac artery, after which blood flow in all of the lower limbs were continuously monitored using a scanning laser Doppler blood flowmeter. After FeCl(3) injury, a distinct decrease in blood flow in the ischemic lower limb was observed and blood flow did not recover during the 30 min after vascular injury. YM466, an oral direct factor Xa inhibitor, dose-dependently inhibited the reduction of peripheral blood flow. The area under the blood flow-time curve during 30 min after vascular injury improved dose-dependently, with significance at doses of 3 and 10 mg/kg. These results suggest that factor Xa inhibitors are effective in patients with peripheral arterial disease, and that this vascular injury model is a useful tool for the screening and evaluation of the efficacy of new antithrombotic agents.

Antinociceptive profile of a selective metabotropic glutamate receptor 1 antagonist YM-230888 in chronic pain rodent models.

Metabotropic glutamate receptor 1 (mGlu(1) receptor) has been suggested to play an important role in pain transmission. In this study, the effects of a newly-synthesized mGlu(1) receptor antagonist, (R)-N-cycloheptyl-6-({[(tetrahydro-2-furyl)methyl]amino}methyl)thieno[2,3-d]pyrimidin-4-ylamine (YM-230888), were examined in a variety of rodent chronic pain models in order to characterize the potential analgesic profile of mGlu(1) receptor blockade. YM-230888 bound an allosteric site of mGlu(1) receptor with a K(i) value of 13+/-2.5 nM and inhibited mGlu(1)-mediated inositol phosphate production in rat cerebellar granule cells with an IC(50) value of 13+/-2.4 nM. It showed selectivity for mGlu(1) versus mGlu(2)-mGlu(7) subtypes and ionotropic glutamate receptors. YM-230888 recovered mechanical allodynia with an ED(50) value of 8.4 mg/kg p.o. in L5/L6 spinal nerve ligation models. It also showed antinociceptive response at doses of 10 and 30 mg/kg p.o. in streptozotocin-induced hyperalgesia models. In addition, it significantly reduced pain parameters at a dose of 30 mg/kg p.o. in complete Freund's adjuvant-induced arthritic pain models. Although YM-230888 showed no significant effect on rotarod performance time at doses of 10 or 30 mg/kg p.o., it significantly decreased it at a dose of 100 mg/kg p.o. On the other hand, YM-230888 showed no significant sedative effect in locomotor activity measurement up to 100 mg/kg p.o. These results suggest that the blockade of mGlu(1) receptors is an attractive target for analgesics. YM-230888 has potential as a new analgesic agent for the treatment of various chronic pain conditions. In addition, YM-230888 may be a useful tool for the investigation of mGlu(1) receptors.

Reverse reaction of Aspergillus niger APC-9319 alpha-galactosidase in a supersaturated substrate solution: production of alpha-linked galactooligosaccharide (alpha-GOS).

The alpha-galactosidase that effectively catalyzes a reverse reaction of galactose, Aspergillus niger APC-9319 alpha-galactosidase, was screened from industrial enzyme preparations for food processing containing alpha-galactosidase activity. Reverse reaction of A. niger APC-9319 alpha-galactosidase was performed using a supersaturated solution (90% galactose [w/v]). A. niger APC-9319 alpha-galactosidase was not inhibited even in high substrate concentration, and effectively catalyzed the reverse reaction. The yield of the reaction product, alpha-linked galactooligosaccharide (alpha-GOS), increased greatly as the initial concentration of galactose increased to 90% (w/v), and was more than 50%. Furthermore, the half life of enzyme activity was about three times as long as that using 60% galactose (w/v). alpha-GOS (1.4 g) was prepared from galactose (3.0 g) by reverse reaction of A. niger APC-9319 alpha-galactosidase. The alpha-GOS contained 58% alpha-galactobiose (alpha-Gal2), 28% alpha-galactotriose, and 14% oligosaccharides larger than alpha-galactotriose. The main component of positional isomers in alpha-Gal2 was alpha-1,6Gal2.

Radioligand binding properties and pharmacological characterization of 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198), a high-affinity, selective, and noncompetitive antagonist of metabotropic glutamate receptor type 1.

Metabotropic glutamate receptor type 1 (mGluR1) is thought to play important roles in the neurotransmission and pathogenesis of several neurological disorders. Here, we describe the radioligand binding properties and pharmacological effects of a newly synthesized, high-affinity, selective, and noncompetitive mGluR1 antagonist, 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198). YM-298198 inhibited glutamate-induced inositol phosphate production in mGluR1-NIH3T3 cells with an IC50 of 16 +/- 5.8 nM in a noncompetitive manner. Its radiolabeled form, [3H]YM-298198, bound to mGluR1-NIH3T3 cell membranes with a KD of 32 +/- 8.5 nM and a Bmax of 2297 +/- 291 fmol/mg protein. In ligand displacement experiments using rat cerebellum membrane, an existing noncompetitive mGluR1 antagonist 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) competitively displaced [3H]YM-298198 binding, although glutamate and other mGluR1 ligands acting on a glutamate site failed to inhibit [3H]YM-298198 binding, suggesting that YM-298198 binds to CPCCOEt (allosteric) binding sites but not to glutamate (agonist) binding sites. Specificity was demonstrated for mGluR1 over mGluR subtypes 2 to 7, ionotropic glutamate receptors, and other receptor, transporter, and ion channel targets. In in vivo experiments, orally administered YM-298198 showed a significant analgesic effect in streptozotocin-induced hyperalgesic mice at doses (30 mg/kg) that did not cause Rotarod performance impairment, indicating that it is also useful even for in vivo experiments. In conclusion, YM-298198 is a newly synthesized, high-affinity, selective, and noncompetitive antagonist of mGluR1 that will be a useful pharmacological tool due to its highly active properties in vitro and in vivo. Its radiolabeled form [3H]YM-298198 will also be a valuable tool for future investigation of the mGluR1.

Allergic airway eosinophilia is suppressed in ovalbumin-sensitized Brown Norway rats fed raffinose and alpha-linked galactooligosaccharide.

We recently found that dietary raffinose suppressed allergic airway eosinophilia in ovalbumin-sensitized Brown Norway rats. Using this model in the present study, we compared the efficacy of other oligosaccharides with that of raffinose. Brown Norway rats were immunized s.c. with ovalbumin on d 0 and exposed to aerosolized ovalbumin on d 20; broncho-alveolar lavage fluid was obtained on d 21. In Expt. 1, rats were fed a control diet or diets supplemented with different oligosaccharides (50 g/kg diet, raffinose, alpha-linked galactooligosaccharide, fructooligosaccharide, and xylooligosaccharide). The number of eosinophils in the fluid was significantly lower in rats fed raffinose and alpha-linked galactooligosaccharide diets than in those fed the control diet. Dietary fructooligosaccharide and xylooligosaccharide did not affect airway eosinophilia. In Expt. 2, i.p. administration of raffinose and alpha-linked galactooligosaccharide, but not fructooligosaccharide and xylooligosaccharide, suppressed airway eosinophilia in rats fed the control diet. In Expt. 3, suppression of airway eosinophilia by dietary alpha-linked galactooligosaccharide occurred in cecectomized rats administered neomycin. Reduced levels of interleukin (IL)-4 and IL-5 mRNA in lung tissue were associated with the suppression of airway eosinophilia. We propose that indigestible oligosaccharides differ in their suppressive effect on allergic airway eosinophilia in ovalbumin-sensitized Brown Norway rats and that the effect appears not to be mediated by intestinal microflora.