Effect of N-oleoyl dopamine on myofibroblast trans-differentiation of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells contribute to several clinical conditions resulting in retinal fibrotic scars. Myofibroblast trans-differentiation of RPE cells is a critical step in the process of retinal fibrosis. In this study, we investigated the effects of N-oleoyl dopamine (OLDA), a newer endocannabinoid with a structure distinct from classic endocannabinoids, on TGF-ß2-induced myofibroblast trans-differentiation of porcine RPE cells. Using an in vitro collagen matrix contraction assay, OLDA was found to inhibit TGF-ß2 induced contraction of collagen matrices by porcine RPE cells. This effect was concentration-dependent, with significant inhibition of contraction observed at 3 µM and 10 µM. OLDA did not affect the proliferation of porcine RPE cells. Immunocytochemistry showed that at 3 µM, OLDA decreased incorporation of α-SMA in the stress fibers of TGF-ß2-treated RPE cells. In addition, western blot analysis showed that 3 µM OLDA significantly downregulated TGF-ß2-induced α-SMA protein expression. Taken together these results demonstrate that OLDA inhibits TGF-ß induced myofibroblast trans-differentiation of RPE cells. It has been established that classic endocannabinoid such as anandamide, by activating the CB1 cannabinoid receptor, promote fibrosis in multiple organ systems. In contrast, this study demonstrates that OLDA, an endocannabinoid with a chemical structure distinct from classic endocannabinoids, inhibits myofibroblast trans-differentiation, an important step in fibrosis. Unlike classic endocannabinoids, OLDA has weak affinity for the CB1 receptor. Instead, OLDA acts on non-classic cannabinoid receptors such as GPR119, GPR6, and TRPV1. Therefore, our study indicates that the newer endocannabinoid OLDA and its non-classic cannabinoid receptors could potentially be novel therapeutic targets for treating ocular diseases involving retinal fibrosis and fibrotic pathologies in other organ systems.

Opposing roles of ZEB1 in the cytoplasm and nucleus control cytoskeletal assembly and YAP1 activity.

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and is initiated by mesenchyme-driving transcription factors and actin cytoskeletal assembly. We show a cytoplasmic-to-nuclear transport gradient of the EMT transcription factor Zeb1 toward sites of invasion in lung adenocarcinoma (LUAD), driven by the EMT inducer Tgfb, which is expressed in M2 polarized macrophages. We show that Zeb1 binds free actin monomers and RhoA in the cytoplasm to inhibit actin polymerization, blocking cell migration and Yap1 nuclear transport. Tgfb causes turnover of the scaffold protein Rassf1a, which targets RhoA. Release of this RhoA inhibition in response to Tgfb overcomes Zeb1's block of cytoskeleton assembly and frees it for nuclear transport. A ZEB1 nuclear transport signature highlights EMT progression, identifies dedifferentiated invasive/metastatic human LUADs, and predicts survival. Blocking Zeb1 nuclear transport with a small molecule identified in this study inhibits cytoskeleton assembly, cell migration, Yap1 nuclear transport, EMT, and precancerous-to-malignant transition.

Reparative effects of chronic intermittent hypobaric hypoxia pre­treatment on intervertebral disc degeneration in rats.

Previous studies have indicated that chronic intermittent hypobaric hypoxia (CIHH) preconditioning can inhibit TNF­α and other related inflammatory cytokines and exerts protective effect on intervertebral disc degeneration disease (IDD) in rats; however, the mechanism is still unclear. The present study aimed to explore the repair mechanisms of CIHH on IDD in rats. In the experiment, 48 adult Sprague­Dawley rats were selected and randomly divided into an experimental group (CIHH­IDD), a degenerative group (IDD) and a control group (CON). The CIHH­IDD group of rats (n=16) were treated with CIHH (simulated 3000 m altitude, 5 h per day, 28 days; PO2=108.8 mmHg) before disc degeneration surgery. The IDD group of rats (n=16) underwent tail­vertebral intervertebral disc surgery to establish a model of intervertebral disc degeneration. The CON group of rats (n=16) did not receive any treatments. After surgery, the disc height index was calculated using X­ray analysis of rat tail vertebrae, the degeneration process was observed and repair was evaluated by chemically staining degenerative intervertebral disc tissue slices. The expression levels of basic fibroblast growth factor (bFGF), TGFß1, Collagen I and Collagen II were measured in the intervertebral disc tissue using western blotting; while the expression levels of bFGF, TGFß1 and hypoxia­inducible factor 1­α (HIF­1α) were measured in rat serum using ELISA. The results demonstrated that: i) The degree of intervertebral disc height degeneration in CIHH­IDD rats was significantly lower compared with that in IDD rats (P<0.05); ii) the expression levels of bFGF, TGFß1 and HIF­1α were higher in CIHH­IDD rat serum compared with those in IDD rat serum (P<0.05); iii) optical microscopy revealed that the degree of disc degeneration was relatively mild in CIHH­IDD rats; and iv) the protein expression levels of bFGF, TGFß1 and collagen II were increased in CIHH­IDD rat intervertebral disc tissues compared with those of IDD rats, while the overexpression of collagen I protein was inhibited. Overall, after CIHH pre­treatment, the expression levels of bFGF and TGFß1 were up­regulated, which play notable roles in repairing degenerative intervertebral discs in rats.

Treatment of posterolateral tibial plateau fractures with a rotational support plate and special pressurizer: technical note and retrospective case series.

BACKGROUND: In tibial plateau fractures, the posterolateral segment of the tibia plateau is frequently affected and challenging to treat. Although there are many surgical approaches and fixation methods for the treatment of these fractures, all of these methods have limitations. We designed a new rotational support plate (RSP) and a special pressurizer that can fix the fracture directly via the anterolateral approach. This method is advantageous because it leads to little trauma, involves a simple operation, and has a reliable fixation effect. This study details the technique of treating these fractures with the RSP and special pressurizer and provides the outcomes. METHODS: From May 2016 to January 2019, the data of 12 patients with posterolateral tibial plateau fractures treated with the RSP and special pressurizer in our hospital were retrospectively analyzed. Postoperative rehabilitation was advised, knee X-rays were taken at follow-ups, and fracture healing, complications, and knee range of motion were assessed. The Hospital for Special Surgery (HSS) knee score and Knee Injury and Osteoarthritis Outcome Score (KOOS) were used to evaluate knee function at the last follow-up. RESULTS: The average follow-up time of all patients was 16.5 months (range, 12-25 months). The average bony union time was 3.2 months (range, 3-4.5 months). At the last follow-up, the average knee range of motion was 138° (range, 107-145°). The average HSS score was 91 (range, 64-98). The average KOOS Symptoms score was 90 (range, 75-96). The average KOOS Pain score was 91 (range, 72-97). The average KOOS ADL score was 91 (range, 74-97). The average KOOS sport/recreation score was 83 (range, 70-90). The average KOOS QOL score was 88 (range, 69-93). Skin necrosis, incision infections, and fixation failure did not occur during the follow-up period. CONCLUSIONS: With our newly designed RSP and special pressurizer, posterolateral tibial plateau fractures can be easily and effectively reduced and fixed through the anterolateral approach, which serves as a novel treatment for posterolateral tibial plateau fractures.

Discovery of endogenous inverse agonists for G protein-coupled receptor 6.

The orphan G protein-coupled receptor 6 (GPR6) is highly expressed in the striatum and has been linked to multiple striatal pathologies. The identification of endogenous ligands and their mechanisms of action at GPR6 will help to elucidate the physiological and pathological roles of the receptor. In the current study, we tested the concentration-dependent effects of a variety of endocannabinoid-like N-acylamides on GPR6 signaling. Here, we demonstrate for the first time that N-arachidonoyl dopamine, N-docosahexaenoyl dopamine, N-oleoyl dopamine and N-palmitoyl dopamine exert inverse agonism at GPR6. This effect was concentration-dependent, with potencies in the micromolar range, and functionally selective for ß-arrestin2 recruitment. Structure-activity relationship studies demonstrate that both the N-acyl side chain and the dopamine head group are important for these ligands to act on GPR6. Our discovery of these N-acyl dopamines as endogenous inverse agonists for GPR6 moves us one step further in understanding the roles GPR6 play in neurodegenerative and neuropsychiatric disorders related to striatal dysfunction.

GPR3, GPR6, and GPR12 as novel molecular targets: their biological functions and interaction with cannabidiol.

The G protein-coupled receptors 3, 6, and 12 (GPR3, GPR6, and GPR12) comprise a family of closely related orphan receptors with no confirmed endogenous ligands. These receptors are constitutively active and capable of signaling through G protein-mediated and non-G protein-mediated mechanisms. These orphan receptors have previously been reported to play important roles in many normal physiological functions and to be involved in a variety of pathological conditions. Although they are orphans, GPR3, GPR6, and GPR12 are phylogenetically most closely related to the cannabinoid receptors. Using ß-arrestin2 recruitment and cAMP accumulation assays, we recently found that the nonpsychoactive phytocannabinoid cannabidiol (CBD) is an inverse agonist for GPR3, GPR6, and GPR12. This discovery highlights these orphan receptors as potential new molecular targets for CBD, provides novel mechanisms of action, and suggests new therapeutic uses of CBD for illnesses such as Alzheimer's disease, Parkinson's disease, cancer, and infertility. Furthermore, identification of CBD as a new inverse agonist for GPR3, GPR6, and GPR12 provides the initial chemical scaffolds upon which potent and efficacious agents acting on these receptors can be developed, with the goal of developing chemical tools for studying these orphan receptors and ultimately new therapeutic agents.

Cannabidiol, a novel inverse agonist for GPR12.

GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119.

The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

CB2 cannabinoid receptor is a novel target for third-generation selective estrogen receptor modulators bazedoxifene and lasofoxifene.

The purpose of the current study was to investigate the ability of the third-generation selective estrogen receptor modulators (SERMs) bazedoxifene and lasofoxifene to bind and act on CB2 cannabinoid receptor. We have identified, for the first time, that CB2 is a novel target for bazedoxifene and lasofoxifene. Our results showed that bazedoxifene and lasofoxifene were able to compete for specific [(3)H]CP-55,940 binding to CB2 in a concentration-dependent manner. Our data also demonstrated that by acting on CB2, bazedoxifene and lasofoxifene concentration-dependently enhanced forskolin-stimulated cAMP accumulation. Furthermore, bazedoxifene and lasofoxifene caused parallel, rightward shifts of the CP-55,940, HU-210, and WIN55,212-2 concentration-response curves without altering the efficacy of these cannabinoid agonists on CB2, which indicates that bazedoxifene- and lasofoxifene-induced CB2 antagonism is most likely competitive in nature. Our discovery that CB2 is a novel target for bazedoxifene and lasofoxifene suggests that these third-generation SERMs can potentially be repurposed for novel therapeutic indications for which CB2 is a target. In addition, identifying bazedoxifene and lasofoxifene as CB2 inverse agonists also provides important novel mechanisms of actions to explain the known therapeutic effects of these SERMs.

Identification of raloxifene as a novel CB2 inverse agonist.

The purpose of the current study was to apply a high throughput assay to systematically screen a library of food and drug administration (FDA)-approved drugs as potential ligands for the cannabinoid receptor 2 (CB2). A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring changes in intracellular cAMP levels was validated and found to be suitable for testing ligands that may act on CB2. Among the 640 FDA-approved drugs screened, raloxifene, a drug used to treat/prevent post-menopausal osteoporosis, was identified for the first time to be a novel CB2 inverse agonist. Our results demonstrated that by acting on CB2, raloxifene enhances forskolin-stimulated cAMP accumulation in a concentration-dependant manner. Furthermore, our data showed that raloxifene competes concentration-dependently for specific [(3)H]CP-55,940 binding to CB2. In addition, raloxifene pretreatment caused a rightward shift of the concentration-response curves of the cannabinoid agonists CP-55,940, HU-210, and WIN55,212-2. Raloxifene antagonism is most likely competitive in nature, as these rightward shifts were parallel and were not associated with any changes in the efficacy of cannabinoid agonists on CB2. Our discovery that raloxfiene is an inverse agonist for CB2 suggests that it might be possible to repurpose this FDA-approved drug for novel therapeutic indications for which CB2 is a target. Furthermore, identifying raloxifene as a CB2 inverse agonist also provides important novel mechanisms of actions to explain the known therapeutic effects of raloxifene.

Effects of palmitoylethanolamide on aqueous humor outflow.

PURPOSE: To study the effects of palmitoylethanolamide (PEA), a fatty acid ethanolamide, on aqueous humor outflow facility. METHODS: The effects of PEA on outflow facility were measured using a porcine anterior segment-perfused organ culture model. The involvements of different receptors in PEA-induced changes were investigated using receptor antagonists and adenovirus delivered small hairpin RNAs (shRNAs). PEA-induced activation of p42/44 mitogen-activated protein kinase (MAPK) was determined by Western blot analysis using an antiphospho p42/44 MAPK antibody. RESULTS: PEA caused a concentration-dependent enhancement of outflow facility, with the maximum effect (151.08 ± 11.12% of basal outflow facility) achieved at 30 nM of PEA. Pretreatment of anterior segments with 1 µM cannabinoid receptor 2 antagonist SR144528 and 1 µM PPARα antagonist GW6471, but not 1 µM cannabinoid receptor 1 antagonist SR141716A, produced a partial antagonism on the PEA-induced increase of outflow facility. Treatment of TM cells with PEA for 10 minutes activated phosphorylation of p42/44 MAPK, which was blocked by pretreatment with SR1444528 and GW6471, but not SR141716A. Knocking down the expression of either GPR55 or PPARα receptors with specific shRNAs for these receptors partially blocked PEA-induced increase in outflow facility and abolished PEA-induced phosphorylation of p42/44 MAPK. PD98059, an inhibitor of the p42/44 MAPK pathway, blocked both PEA-induced enhancement of aqueous humor outflow facility and PEA-induced phosphorylation of p42/44 MAPK. CONCLUSIONS: Our results demonstrate that PEA increases aqueous humor outflow through the TM pathway and these effects are mediated by GPR55 and PPARα receptors through activation of p42/44 MAPK.

Functional consequences of nonsynonymous single nucleotide polymorphisms in the CB2 cannabinoid receptor.

OBJECTIVE: To test the hypothesis that the two nonsynonymous single nucleotide polymorphisms at the CB2 cannabinoid receptor gene may have functional consequences on human CB2. METHODS: Q63R, H316Y, and Q63R/H316 mutations were made in recombinant human CB2 by the method of site-directed mutagenesis. After these mutant CB2 receptors were stably transfected into HEK293 cells, ligand binding, ligand-induced activity, and constitutive activity assays were performed to test the functional significance of these mutations. RESULTS: In general, our results showed that the CB2 polymorphic receptors are able to bind cannabinoid ligands and mediate signal transduction. However, in ligand-induced cyclic AMP accumulation assays, the cannabinoid agonists WIN55212-2 and 2-arachidonoylglycerol had reduced efficacy in cells expressing the polymorphic receptors as compared with the CB2 wild-type receptor. Furthermore, in constitutive activity assays, the H316Y and Q63R/H316Y polymorphic receptors exhibited higher constitutive activity than the CB2 wild-type receptor. CONCLUSION: Our data shows that the presence of the polymorphisms at both positions 63 and 316 produce alterations in the CB2 receptor functions. Moreover, these findings strengthen the idea that the CB2 polymorphic receptors may contribute to the etiology of certain diseases.

The role of the calcar femorale in stress distribution in the proximal femur.

OBJECTIVE: To investigate the role of the calcar femorale in stress distribution in the proximal femur. METHODS: Twenty-five specimens of proximal femurs were fixed to simulate single-limb stance. Strain gauges were applied to record the strain under different loads. Strain values of 27 selected sites in the proximal femur were recorded and analyzed at the level of 100 N, 200 N, 300 N, 400 N, 500 N, 600 N and 700 N, respectively before and after disruption of the calcar femorale. RESULTS: When a normal load was being borne, strain values measured in the posterior and medial aspects of the proximal femur were greater than those measured in the anterior and lateral aspects, no matter whether the calcar femorale was disrupted or not. However after disruption of the calcar femorale, strain values in the posterior and medial aspects of the proximal femur increased significantly, whereas those of the anterior and lateral aspects decreased significantly. CONCLUSION: The calcar femorale redistributes stress in the proximal femur by decreasing the load in the posterior and medial aspects and increasing the load in the anterior and lateral aspects.

Residues accessible in the binding-site crevice of transmembrane helix 6 of the CB2 cannabinoid receptor.

We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [(3)H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore, the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a alpha-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors.

[Treatment of complex calcaneal fractures under computer navigation: report of 130 feet].

OBJECTIVE: To investigate the effects of computer navigation in the treatment of intra-articular calcaneal fractures. METHODS: 130 feet in 110 patients with intra-articular calcaneal fractures, 57 calcanei with fracture of Sander's type II, 45 of type III, and 28 cases of type IV, were treated with internal fixation under computer navigation, and were followed up for 16.3 months (6 - 24 months). RESULTS: According to the Maryland Foot Score system, excellent result was noted in 63 feet, good result in 57 feet, and fair result in 10 feet, with the excellent and good rates being 92.31% together. CONCLUSION: Using computer navigation to treat intra-articular calcaneal fractures is one of the best ways for treatment of calcaneal fractures.

The effects of charge-neutralizing mutation D6.30N on the functions of CB1 and CB2 cannabinoid receptors.

Charge-neutralizing mutation D6.30N of the human cannabinoid receptor subtype 1 (CB1) and cannabinoid receptor subtype 2 (CB2) cannabinoid receptors was made to test two hypotheses: (1) D6.30 may be crucial for the functions of CB1 and CB2 receptors. (2) D6.30 may participate in an ionic lock with R3.50 that keeps the receptors in an inactive conformation. Specific ligand binding and ligand-induced inhibition of forskolin-stimulated cAMP accumulation were observed with human embryonic kidney epithelial cell line (HEK293) cells expressing wild-type CB1 and CB2, as well as CB1D6.30N and CB2D6.30N mutant receptors. There was however a decrease in maximum response of the mutant receptors compared to their wild-type counterparts, suggesting that D6.30 is essential for full activation of both CB1 and CB2 receptors. Both CB1D6.30N and CB2D6.30N demonstrated a level of constitutive activity no greater than that of their wild-type counterparts, indicating that either D6.30 does not participate in a salt bridge with R3.50, or the salt bridge is not critical for keeping cannabinoid receptors in the inactive conformation.

[Intra-operative stereotactic accuracy of computer-assisted robot orthopaedic trauma surgery planning system].

OBJECTIVE: To investigate the feasibility and accuracy of the software computer-assisted robot orthopaedic trauma surgery planning system (CAOTS). METHODS: The software CAOTS was developed and used on 85 cases of stereotactic operation, 24 model bones, 21 cadaveric extremity bones, and 40 patients. 307 groups of directional targets in these cases were randomly sampled. The distribution of error sources for evaluating system performance was embodied using Monte-Carlo method in order to derive the theoretic guarantees for further optimizing and enhancing the system performance, then the software SPSS 2.0 was used to analyze the errors. RESULTS: The statistical area of deviation number was 0.0408 +/- 0.4578 mm, corresponding to the result by Monte-Carlo method. Punching succeeded at the first run for all 307 cases without wrong locking and other clinical complications. CONCLUSION: Accurate and reliable, CAOTS improves the intra-operative navigation techniques and facilitates the orthopedists to perform operation.

Noladin ether acts on trabecular meshwork cannabinoid (CB1) receptors to enhance aqueous humor outflow facility.

PURPOSE: To study the effects of 2-arachidonyl glyceryl ether (noladin ether), an endocannabinoid ligand selective for cannabinoid (CB)1 receptor, on aqueous humor outflow facility, to investigate the involvement of trabecular meshwork CB1 receptors and the p42/44 MAP kinase signaling pathway and to explore the cellular mechanisms of noladin ether-induced changes of outflow facility. METHODS: The effects of noladin ether on aqueous humor outflow facility were measured in a porcine anterior-segment-perfused organ culture model. The expression of CB1 receptors on cultured porcine trabecular meshwork cells and the coupling of these receptors to p42/44 MAP kinase was determined by immunofluorescence microscopy and Western blot analysis. Both Western blot and zymography were used to monitor the effects of noladin ether on matrix metalloproteinase (MMP)-2. In morphologic studies, AlexaFluor 488-labeled phalloidin staining was used to examine actin filament, and immunohistochemistry with anti-paxillin antibodies was used to detect focal adhesions. RESULTS: Within 1 hour after adding 3, 30, or 300 nM of noladin ether, the aqueous humor outflow facility increased concentration dependently. The effect of 30 nM of noladin ether was completely blocked by SR141716A, a selective CB1 antagonist. Positive signals were detected on cultured porcine trabecular meshwork cells with an anti-CB1 antibody in immunofluorescence microscopy and Western blot studies. Treatment of trabecular meshwork cells with 30 nM of noladin ether activated p42/44 MAP kinase, whereas pretreatment with SR141716A blocked the p42/44 MAP kinase-activating effects of noladin ether. In addition, the enhancement of outflow facility induced by noladin ether was blocked by pretreatment of porcine anterior segments with PD98059, an inhibitor of p42/44 MAP kinase pathway. Furthermore, noladin ether treatment caused rounding of trabecular meshwork cells, and there was a decrease of actin stress fibers, as well as a decrease in focal adhesions. These noladin ether-induced morphologic changes were also blocked by SR141716A and PD98059. CONCLUSIONS: The results demonstrate for the first time that administration of noladin ether, an endocannabinoid agonist selective for the CB1 receptor, increases aqueous humor outflow facility. The data also show that noladin ether-induced enhancement of outflow facility is mediated through the trabecular meshwork CB1 receptor, with an involvement of p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons.

Cysteine 2.59(89) in the second transmembrane domain of human CB2 receptor is accessible within the ligand binding crevice: evidence for possible CB2 deviation from a rhodopsin template.

In this study, the sensitivity of the CB2 receptor to methanethiosulfonate (MTS) derivatives was tested, and a native cysteine residue conferring the sensitivity was identified. By incubating human embryonic kidney 293 cells stably transfected with CB2 receptors and MTS derivatives such as MTS ethylammonium (MTSEA), [(3)H]HU-243 binding was inhibited. Pretreatment of the CB2 receptor with cannabinoid ligands prevented this inhibition, suggesting that MTSEA modification occurred within the binding crevice. To identify the cysteine(s) responsible for the MTSEA sensitivity, 10 CB2 mutants were prepared in which the eight cysteines in transmembrane domains or extracellular loop 2 were mutated to serine or alanine, one at a time or in combination. Five mutants exhibited specific [(3)H]HU-243 binding, with K(d) and B(max) values similar to those of wild-type CB2. However, five other mutants had no detectable ligand binding and were not detected on cell membranes by Western blot analysis. Among the five mutants with normal binding, only the sensitivity to MTSEA of the C2.59(89)S mutant was reduced significantly. These data demonstrate that C2.59(89) is the residue that mainly confers the inhibitory effect of MTSEA on ligand binding. Furthermore, the magnitude of the second-order rate constant (1.14 +/- 0.28 M(-1)s(-1)) for the MTSEA reaction with wild-type CB2 suggests that C2.59(89) resides at the margin of the CB2 binding site crevice. The accessibility of C2.59(89) to MTSEA provides experimental evidence for a possible conformational difference between TMH2 of CB2 versus Rho. Modeling studies undertaken to explore the origin of such differences suggest it is possibly caused by the conformational influence of S2.54(84).

[Three-dimensional spiral CT in advanced gastric cancer].

OBJECTIVE: To study the usefulness of three-dimensional spiral CT (3DCT) in the diagnosis of advanced gastric cancer (AGC). METHODS: Between June 1999 and December 2000, 54 patients with AGC were consecutively examined. On the 3D Virtuoso workstation, source images were uploaded to create a 3DCT volume block that was then processed with volume rendering technology (VA30C) to achieve virtual-reality endoscopy (VE), clipped volume block (CVB), and ray sum (RS). After the above scanning, all the patients were examined by a two-phase enhanced spiral CT (2DCT). The visualization, manifestation, and Borrman's classification of lesions in VE, CVB, RS, and 2DCT were evaluated and correlated with gastroscopic, surgical, and pathological findings. Respiratory artifact and gastric residue were also observed. RESULTS: (1) CVB showed the excellent visualization in 88.9% of cases, in contrast to VE and RS (50.0% and 38.9%) (P < 0.01). (2) The accuracy in evaluating mucous membrane, ulceration, lumen, wall, cardia, pylorus, and extension of the tumor were more than 90.0% except mucosa by RS (84.4%) and ulceration by VE (87.5%) or RS (81.6%) which was significantly different from CVB (96.0%) and 2DCT (96.1%) (P < 0.05). VE demonstrated an accuracy of 95.8% in diagnosis of mucosal abnormality. (3) The correct Borrman's classification was obtained in 83.3% cases by VE, 79.6% by CVB, 72.2% by RS, 88.9% by 2DCT and 85.2% by 3DCT with significant difference between 2DCT and RS (P < 0.05), but not between 3DCT and 2DCT (P > 0.05). (4) In addition to 2DCT which had no step-like artifacts, they were invisible in 53.7% of VE, 40.7% of CVB, and 81.5% of RS, with RS showing the least artifacts among 3DCT (P < 0.01). A few of gastric residues caused by pre-scanning intake of water to swallow effervescent agent could be found on 3DCT images which caused no evident influence on diagnosis. CONCLUSION: Additional information on the diagnosis of AGC can be obtained by use of 3DCT, especially the visualization of a lesion in clipped volume block and the observation of mucosa in virtual-reality endoscopy.