It is now well established that several G protein- coupled receptors can signal without agonist stimulation (constitutive receptors). Inverse agonists have been shown to inhibit the activity of such constitutive G protein-coupled receptor signaling. Agonist activation of the Gi/o-coupled peripheral cannabinoid receptor CB2 normally inhibits adenylyl cyclase type V and stimulates adenylyl cyclase type II. Using transfected COS cells, we show here that application of SR144528, an inverse agonist of CB2, leads to a reverse action (stimulation of adenylyl cyclase V and inhibition of adenylyl cyclase II). This inverse agonism of SR144528 is dependent on the temperature, as well as on the concentration of the cDNA of CB2 transfected. Pertussis toxin blocked the regulation of adenylyl cyclase activity by SR 144528.
Adenylate Cyclase/antagonists&inhibitors/genetics/metabolism ; Animals ; Binding, Competitive ; Bornanes/metabolism/*pharmacology ; COS Cells ; Cannabinoids/metabolism ; Cercopithecus aethiops ; Isoenzymes/antagonists&inhibitors/genetics/metabolism ; Pyrazoles/metabolism/*pharmacology ; Rats ; *Receptor, Cannabinoid, CB2 ; Receptors, Cannabinoid ; Receptors, Drug/agonists/*antagonists&inhibitors/genetics/metabolism ; Signal Transduction/drug effects/physiology ; Transfection

Using site-directed mutagenesis technique, I have replaced serine 285 and serine 292 with the alanine, and assessed the binding of agonist and signaling such as the inhibition of adenylyl cyclase activity.I have found that serine 292 has an important role in the signal transduction of cannabinoid agonists, HU-210 and CP55940, but not in that of aminoalkylindoles derivatives WIN55,212-2. All mutants express well in protein level determined by western blot using monoclonal antibody HA 11 as compared with the wild type receptor.Interestingly, binding affinity of S285A and S292A mutants with classical cannabinoid agonist HU-243 was somewhat decreased. In signaling assay, the inhibition of adenylyl cyclase by HU-210, CP55940 and WIN55, 212-2 is the same order in both wild type receptor and S285A mutant receptor. However, S292A have been shown that the inhibition curves of adenylyl cyclase activity moved to the right by HU-210 and CP55940, but those of adenylyl cyclase activity did not by aminoalkylindole WIN55,212-2, which is indicating that this residue is closely related to the binding site with HU-210 and CP55940. In addition, serine 292 might take more important role in CB2 receptor and G-protein signaling than serine 285.
Adenylate Cyclase/*metabolism ; Animals ; Binding, Competitive ; Blotting, Western ; COS Cells ; Cannabinoids/metabolism ; Cercopithecus aethiops ; Cyclohexanols/metabolism ; Excitatory Amino Acid Antagonists/metabolism ; Mutagenesis, Site-Directed ; Protein Conformation ; Protein Structure, Tertiary ; Receptors, Cannabinoid ; Receptors, Drug/genetics/metabolism/*physiology ; Serine/metabolism/*physiology ; Signal Transduction/physiology ; Tetrahydrocannabinol/*analogs&derivatives/metabolism ; Transfection

Cudrania tricuspidata (C. tricuspidata) is a deciduous tree found in Japan, China and Korea. The root, stems, bark and fruit of C. tricuspidata has been used as traditional herbal remedies such as eczema, mumps, acute arthritis and tuberculosis. In this study, we investigated the potential efficacies of this natural compound by focusing on the inhibitory effect of cudraxanthone L (CXL) isolated from the roots of C.tricuspidata on human platelet aggregation. Our study focused on the action of CXL on collagen-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane A 2 secretion. In addition, we investigated the inhibitory effect of CXL on thrombin-induced clot retraction. Our results showed that CXL inhibited collagen-induced human platelet aggregation, intracellular calcium mobilization, fibrinogen binding, fibronectin adhesion and clot retraction without cytotoxicity. Therefore, we confirmed that CXL has inhibitory effects on human platelet activities and has potential value as a natural substance for preventing thrombosis.

Cudrania tricuspidata (C. tricuspidata) is a deciduous tree found in Japan, China and Korea. The root, stems, bark and fruit of C. tricuspidata has been used as traditional herbal remedies such as eczema, mumps, acute arthritis and tuberculosis. In this study, we investigated the potential efficacies of this natural compound by focusing on the inhibitory effect of cudraxanthone L (CXL) isolated from the roots of C.tricuspidata on human platelet aggregation. Our study focused on the action of CXL on collagen-stimulated human platelet aggregation, inhibition of platelet signaling molecules such as fibrinogen binding, intracellular calcium mobilization, fibronectin adhesion, dense granule secretion, and thromboxane A 2 secretion. In addition, we investigated the inhibitory effect of CXL on thrombin-induced clot retraction. Our results showed that CXL inhibited collagen-induced human platelet aggregation, intracellular calcium mobilization, fibrinogen binding, fibronectin adhesion and clot retraction without cytotoxicity. Therefore, we confirmed that CXL has inhibitory effects on human platelet activities and has potential value as a natural substance for preventing thrombosis.

BACKGROUND: The development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with beta-lactams against MRSA. METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and beta-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production. RESULTS: The MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 microg/mL, respectively. The PBEAE significantly reduced MICs of all beta-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-beta-lactams. Time-killing assays showed that the synergistic effects of two beta-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Deltalog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the beta-lactams and the PBEAE. CONCLUSIONS: PBEAE can enhance the efficacy of beta-lactams for combined therapy in patients infected with MRSA.
Acetates/chemistry ; Agaricales/*chemistry/metabolism ; Anti-Infective Agents/chemistry/*pharmacology ; Drug Synergism ; Enzyme-Linked Immunosorbent Assay ; Methicillin-Resistant Staphylococcus aureus/*drug effects/metabolism ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins/analysis/metabolism ; Plant Extracts/chemistry/*pharmacology ; beta-Lactams/*pharmacology

BACKGROUND: Postoperative urinary retention is defined as the inability to void with a full bladder during the postoperative period. It affects both sexes in all ages following all types of operation, including patients who previously had no micturition problems. We investigated the incidence and risk factors of urinary retention following long spinal anesthesia for total knee replacement. METHODS: We retrospectively studied a number of factors that may be associated with urinary retention in 98 women. The outcome variable of logistic regression models are urinary retention and severe urinary retention. The potential explanatory variables are age, height, weight, history of hypertension, DM and abnormal urology, heavy bupivacaine dose, types of patient-controlled analgesia, time to regression of spinal block to sacral segments (Tregression), amount of fluid and duration of surgery. We constructed a multiple linear regression model of the time from subarachnoid injection to spontaneous voiding (Tvoiding) in relation to above variables. RESULTS: The overall rate of urinary retention and severe retention were 57.1% and 30.6%. Tregression was identified as significant explanator of an increased probability for urinary retention (P = 0.002), Tregression and DM for severe urinary retention (P <0.001, P = 0.054). In the multiple linear regression model, three variables - Tregression, age, abnormal urological history were identified to have significant t-values (3.902, 3.107, 2.284) with Tvoiding (P <0.001, P = 0.003, P = 0.025). CONCLUSION: Old age, DM, abnormal urological history, delayed recovery of spinal anesthesia are risk factors to urinary retention or delayed spontaneous voiding.
Analgesia, Patient-Controlled ; Anesthesia, Spinal ; Arthroplasty, Replacement, Knee* ; Bupivacaine ; Female* ; Humans ; Hypertension ; Incidence ; Linear Models ; Logistic Models ; Postoperative Period ; Retrospective Studies ; Risk Factors ; Urinary Bladder ; Urinary Retention* ; Urination ; Urology

The present study was carried out to investigate the effects of biomedicinal agents on Ca2+, P and alkaline phosphatase (ALP) levels in ovariectomized rats. Rats were ovariectomized bilaterally and were fed up with Ca2+ and P-free diet during 8(9,10) weeks to induce osteoporosis. Osteoporosis was determined by the extent of bone density and by lowering the concentrations of serum Ca2+, P and ALP activity every week. Rats in antler, safflower, ipriflavon, or coadminisrated with estrogen groups were administrated with feed supplement for 5 weeks to elucidate the protective and therapeutic effects against osteoporosis. The bone tissue was examined with electron microscope to determine the effects of each treatment on osteoporosis. 1. The levels of serum Ca2+ and P in osteoporosisinduced rats, administrated with antler, ipriflavon and estrogen groups, were little higher than those of control rats. However, the levels of serum Ca and P in ovariectomized rats were significantly higher than those of control group (p<0.05). 2. The activities of serum ALP in osteoporosisinduced rats, administrated with antler extract, safflower, ipriflavon, or co-admistrated with estrogen, were little increased in comparing with those of control group, but were significantly decreased in with combination of estrogen for 5 weeks. However, The connections were interrupted and the bone matrix was destroyed in the osteoporosis-induced rats. 3. The inter-trabecular connections were examined under electron microscope. The connections were well maintained and bone loss was without in the administration with antler, safflower, and ipriflavon with combination of estrogen for 5 weeks. However, The connections were interrupted and the bone matrix was destroyed in the osteoporosis-induced rats.
Alkaline Phosphatase/*blood ; Animals ; Antlers ; Bone Density/drug effects/*physiology ; Bone Remodeling/drug effects ; Calcium/*blood ; Dietary Supplements ; Disease Models, Animal ; Female ; Isoflavones/administration & dosage/pharmacology ; Osteoporosis/*blood/enzymology/prevention & control ; Ovariectomy ; Phosphates/*blood ; Phytotherapy ; Rats ; Safflower Oil/administration & dosage/therapeutic use ; Tissue Extracts/administration & dosage/therapeutic use

Although the effects of the rice bran have recently been investigated, there is no information regarding platelet physiology available. However, it is well known that fermented natural plants have a beneficial effect on cardiovascular diseases. Therefore, this study was conducted to investigate whether fermented rice bran extract (FRBE) with several plants (Artemisia princeps, Angelica Gigantis Radix, Cnidium officinale, and Camellia sinensis) affected agonist-induced platelet aggregation, and if so, what the underlying mechanism of its activity was. We performed several experiments, including in vitro platelet aggregation, intracellular calcium concentration and adenosine triphosphate release. In addition, the activation of integrin alphaIIbbeta3 was determined using fibrinogen binding. Thrombus formation was also evaluated in vivo using an arterio-venous shunt model. The FRBE inhibited collagen-induced platelet aggregation in a concentration-dependent manner. FRBE significantly and dose dependently attenuated thrombus formation using rat arterio-venous shunt. FRBE suppressed the intracellular calcium mobilization in collagen-stimulated platelets. We also found that FRBE inhibited extracellular stimuli-responsive kinase 1/2, p38-mitogen-activated protein kinases and c-Jun N-terminal kinase phosphorylation. These results suggested that FRBE inhibited collagen-induced platelet aggregation, which was mediated by modulation of downstream signaling molecules. In conclusion, FRBE could be developed as a functional food against aberrant platelet activation-related cardiovascular diseases.
Adenosine Triphosphate ; Angelica ; Animals ; Blood Platelets ; Calcium ; Camellia ; Cardiovascular Diseases ; Cnidium ; Collagen ; Fibrinogen ; Functional Food ; JNK Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Physiology ; Platelet Aggregation ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Kinases ; Rats ; Thrombosis

BACKGROUND: Surgery may have a role when medical treatment alone is not successful in patients with multi-drug resistant (MDR) pulmonary tuberculosis (PTB). To document the role of resection in MDR PTB, we analyzed 4 years of our experience. METHODS: A retrospective review was performed on thirteen patients that underwent pulmonary resection for MDR PTB between May 1996 and February 2000. All patients had organisms resistant to many of the first-line drugs including isoniazid (INH) and rifampicin (RFP). RESULTS: The thirteen patients were 37.5±12.4 years old (mean±S.D.)(M:F=5:8), and their sputum was culture positive even with adequate medication for prolonged periods (109.7±132.0 months), resistant to 2-8 drugs including isoniazid and rifampin. All patients had localized lesion(s) and most (92.3%) had cavities. At least 3 sensitive anti-TB medications were started before surgery in all patients according to the drug sensitivity test. The preoperative FE1 was 2.37±0.83 L. Lobectomy was performed in 11 patients and pleuropneumonectomy in two. Postoperative mortality did not occur, but pneumonia occurred as a complication in one (7.7%). After 41.5±58.9 days (range 1~150 days) follow up, negative conversion of sputum culture was achieved in all patients within 5 months. Only one patient (7.7%) recurred 32 months after lung resection. CONCLUSION: When medical treatment alone is not successful, surgical resection can be a good treatment option in patients with localized MDR PTB.
Follow-Up Studies ; Humans ; Isoniazid ; Lung ; Mortality ; Pneumonia ; Retrospective Studies ; Rifampin ; Sputum ; Tuberculosis, Pulmonary*

Suaeda asparagoides (Miq.) has long been used as a Korean folk herbal medicine for the treatment of functional gastrointestinal disorders. However, reports on its pharmacological activity on gastrointestinal motility are scarce. The present study investigated the effects of Suaeda asparagoides water fraction of the extract (SAWF) on antral motility in vitro. Muscle strips from rat gastric antrum were set up in an organ bath in a circular orientation. SAWF (100 microg/mL) inhibited the spontaneous contraction of antral circular muscle strips. These inhibitory effects were not significantly affected by tetrodotoxin (1 microM), N omega-Nitro-L-arginine methyl ester hydrochloride (100 microM), 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 microM), ryanodine (10 microM) and phentolamine (10 microM). SAWF-induced inhibition was mostly restored by cyclopiazonic acid (10 microM). Furthermore, the beta-adrenergic receptor antagonist, propranolol (10 microM), abolished SAWF-induced inhibition. These results suggest that SAWF may exert its activity on gastrointestinal smooth muscle via a-adrenergic receptors and sarcoplasmic reticulum Ca2+ ATPase.
Animals ; Baths ; Calcium-Transporting ATPases ; Carbamates ; Chenopodiaceae ; Contracts ; Gastrointestinal Diseases ; Gastrointestinal Motility ; Herbal Medicine ; Indoles ; Muscle, Smooth ; Muscles ; Organometallic Compounds ; Orientation ; Oxadiazoles ; Phentolamine ; Propranolol ; Pyloric Antrum ; Quinoxalines ; Rats ; Ryanodine ; Sarcoplasmic Reticulum ; Tetrodotoxin ; Water