Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation.

The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membrane-bound protein with three subunits (PmoB, PmoA, and PmoC) and 12-14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of holo-pMMO from Methylococcus capsulatus (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear CuI at the A site, resolve two CuIs at the B site, and uncover additional CuI clusters at the PmoA/PmoC interface within the membrane (D site) and in the water-exposed C-terminal subdomain of the PmoB (E clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.

Development of the Soft X-ray AGM-AGS RIXS beamline at the Taiwan Photon Source.

We report on the development of a high-resolution and highly efficient beamline for soft X-ray resonant inelastic X-ray scattering (RIXS) located at the Taiwan Photon Source. This beamline adopts an optical design that uses an active grating monochromator (AGM) and an active grating spectrometer (AGS) to implement the energy compensation principle of grating dispersion. Active gratings are utilized to diminish defocus, coma and higher-order aberrations, as well as to decrease the slope errors caused by thermal deformation and optical polishing. The AGS is mounted on a rotatable granite platform to enable momentum-resolved RIXS measurements with scattering angles over a wide range. Several high-precision instruments developed in-house for this beamline are described briefly. The best energy resolution obtained from this AGM-AGS beamline was 12.4 meV at 530 eV, achieving a resolving power of 4.2 × 104, while the bandwidth of the incident soft X-rays was kept at 0.5 eV. To demonstrate the scientific impact of high-resolution RIXS, we present an example of momentum-resolved RIXS measurements on a high-temperature superconducting cuprate, i.e. La2-xSrxCuO4. The measurements reveal the A1g buckling phonons in superconducting cuprates, opening a new opportunity to investigate the coupling between these phonons and charge-density waves.

Highly efficient beamline and spectrometer for inelastic soft X-ray scattering at high resolution.

The design, construction and commissioning of a beamline and spectrometer for inelastic soft X-ray scattering at high resolution in a highly efficient system are presented. Based on the energy-compensation principle of grating dispersion, the design of the monochromator-spectrometer system greatly enhances the efficiency of measurement of inelastic soft X-rays scattering. Comprising two bendable gratings, the set-up effectively diminishes the defocus and coma aberrations. At commissioning, this system showed results of spin-flip, d-d and charge-transfer excitations of NiO. These results are consistent with published results but exhibit improved spectral resolution and increased efficiency of measurement. The best energy resolution of the set-up in terms of full width at half-maximum is 108 meV at an incident photon energy tuned about the Ni L3-edge.

Plasma and saliva levels of nerve growth factor and neuropeptides in chronic migraine patients.

OBJECTIVES: To investigate the level and interrelationship of nerve growth factor (NGF) and sensory neuropeptides [substance P (SP), calcitonin gene-related peptide (CGRP)] in plasma and saliva of chronic migraine patients, and to analyze the association between pain intensity and their concentration. MATERIALS AND METHODS: Plasma and resting whole saliva were collected from 33 chronic migraine patients and 36 control subjects. NGF, SP, and CGRP concentrations were measured by enzyme immunoassay and pain intensity of each subject was measured using the Graded Chronic Pain Scale. RESULTS: Chronic migraine patients showed higher NGF and neuropeptide levels in both plasma and saliva compared to the control subjects. Plasma NGF, and plasma and saliva levels of SP and CGRP were highly associated with pain intensity. There was a significant positive correlation between NGF and both neuropeptide levels in plasma, and between the neuropeptide levels in both plasma and saliva. Plasma levels of SP and CGRP were significantly correlated with their saliva level. CONCLUSIONS: The increased production of NGF and sensory neuropeptides may play an important role in the maintenance of pain in chronic migraine and analysis results of human saliva could act as an index of disease state and therapeutic outcome.

Lung perfusion and chest wall configuration is altered by glossopharyngeal breathing.

Glossopharyngeal insufflation is used by competitive breath-hold divers to increase lung gas content above baseline total lung capacity (TLC) in order improve performance. Whilst glossopharyngeal insufflation is known to induce hypotension and tachycardia, little is known about the effects on the pulmonary circulation and structural integrity of the thorax. Six male breath-hold divers were studied. Exhaled lung volumes were measured before and after glossopharyngeal insufflation. On two study days, subjects were studied in the supine position at baseline TLC and after maximal glossopharyngeal insufflation above TLC. Tc 99(m) labelled macro-aggregated albumin was injected and a computed tomography (CT) scan of the thorax was performed during breath-hold. Single photon emission CT images determined flow and regional deposition. Registered CT images determined change in the volume of the thorax. CT and perfusion comparisons were possible in four subjects. Lung perfusion was markedly diminished in areas of expanded lung. 69% of the increase in expired lung volume was via thoracic expansion with a caudal displacement of the diaphragm. One subject who was not proficient at glossopharyngeal insufflation had no change in CT appearance or lung perfusion. We have demonstrated areas of hyperexpanded, under perfused lung created by glossopharyngeal insufflation above TLC.

Endoscopic submucosal dissection of gastric lesions by using a Master and Slave Transluminal Endoscopic Robot (MASTER).

BACKGROUND: Performing endoscopic submucosal dissection (ESD) by using standard endoscopy platforms is technically challenging because of the equipment's lack of dexterity. OBJECTIVE: To explore the feasibility of using the Master and Slave Transluminal Endoscopic Robot (MASTER), a novel robotics-enhanced endosurgical system, to perform ESD. DESIGN: ESD was performed on simulated gastric lesions in 5 Erlangen porcine stomach models (ex vivo) and 5 live pigs (in vivo). Performance of ESD by using the MASTER was compared with that using the insulation-tipped (IT) diathermic knife. SETTING: SMART Laboratory, Advance Surgical Training Centre, National University Hospital, Singapore. SUBJECTS: Five Erlangen porcine stomach models and 5 pigs, 5 to 7 months old, each weighing about 35 kg. INTERVENTIONS: ESD. MAIN OUTCOME MEASUREMENTS: Lesion resection time, grasper and hook efficacy grade, completeness of resection, and presence of procedure-related perforation. RESULTS: In the Erlangen stomach models, 15 simulated lesions from the cardia, antrum, and body were removed en bloc (mean dimension, 37.4 x 26.5 mm) by electrocautery excision using the MASTER. The mean ESD time was 23.9 minutes (range 7-48 minutes). There was no difference in the dissection times of lesions at different locations (P = .449). In the live pigs, the MASTER took a mean of 16.2 minutes (range 3-29 minutes) to complete the ESD of 5 gastric lesions, whereas the IT diathermic knife took 18.6 minutes (range 9-34 minutes). There was no significant difference in the times taken (P = .708). All lesions were excised en bloc; the mean dimensions of lesions resected by the MASTER and the IT diathermic knife were 37.2 x 30.1 mm and 32.78 x 25.6 mm, respectively. The MASTER exhibited good grasping and cutting efficiency throughout. Surgical maneuvers were achieved with ease and precision. There was no incidence of excessive bleeding or stomach wall perforation. LIMITATIONS: Exploratory study with limited sample size. CONCLUSIONS: Performing ESD by using the MASTER is feasible.

Natural orifice transgastric endoscopic wedge hepatic resection in an experimental model using an intuitively controlled master and slave transluminal endoscopic robot (MASTER).

BACKGROUND: The lack of triangulation of standard endoscopic devices limits the degree of freedom for surgical maneuvers during natural orifice transluminal endoscopic surgery (NOTES). This study explored the feasibility of adapting an intuitively controlled master and slave transluminal endoscopic robot (MASTER) the authors developed to facilitate wedge hepatic resection in NOTES. METHODS: The MASTER consists of a master controller, a telesurgical workstation, and a slave manipulator that holds two end-effectors: a grasper, and a monopolar electrocautery hook. The master controller is attached to the wrist and fingers of the operator and connected to the manipulator by electrical and wire cables. Movements of the operator are detected and converted into control signals driving the slave manipulator via a tendon-sheath power transmission mechanism allowing nine degrees of freedom. Using this system, wedge hepatic resection was performed through the transgastric route on two female pigs under general anesthesia. Entry into the peritoneal cavity was via a 10-mm incision made on the anterior wall of the stomach by the electrocautery hook. Wedge hepatic resection was performed using the robotic grasper and hook. Hemostasis was achieved with the electrocautery hook. After the procedure, the resected liver tissue was retrieved through the mouth using the grasper. RESULTS: Using the MASTER, transgastric wedge hepatic resection was successfully performed on two pigs with no laparoscopic assistance. The entire procedure took 9.4 min (range, 8.5-10.2 min), with 7.1 min (range, 6-8.2 min) spent on excision of the liver tissue. The robotics-controlled device was able to grasp, retract, and excise the liver specimen successfully in the desired plane. CONCLUSION: This study demonstrated for the first time that the MASTER could effectively mitigate the technical constraints normally encountered in NOTES procedures. With it, the triangulation of surgical tools and the manipulation of tissue became easy, and wedge hepatic resection could be accomplished successfully without the need for assistance using laparoscopic instruments.

Clopidogrel versus aspirin and esomeprazole to prevent recurrent ulcer bleeding.

BACKGROUND: Concurrent therapy with a proton-pump inhibitor is a standard treatment for patients receiving aspirin who are at risk for ulcer. Current U.S. guidelines also recommend clopidrogel for patients who have major gastrointestinal intolerance of aspirin. We compared clopidogrel with aspirin plus esomeprazole for the prevention of recurrent bleeding from ulcers in high-risk patients. METHODS: We studied patients who took aspirin to prevent vascular diseases and who presented with ulcer bleeding. After the ulcers had healed, we randomly assigned patients who were negative for Helicobacter pylori to receive either 75 mg of clopidogrel daily plus esomeprazole placebo twice daily or 80 mg of aspirin daily plus 20 mg of esomeprazole twice daily for 12 months. The end point was recurrent ulcer bleeding. RESULTS: We enrolled 320 patients (161 patients assigned to receive clopidogrel and 159 to receive aspirin plus esomeprazole). Recurrent ulcer bleeding occurred in 13 patients receiving clopidogrel and 1 receiving aspirin plus esomeprazole. The cumulative incidence of recurrent bleeding during the 12-month period was 8.6 percent (95 percent confidence interval, 4.1 to 13.1 percent) among patients who received clopidogrel and 0.7 percent (95 percent confidence interval, 0 to 2.0 percent) among those who received aspirin plus esomeprazole (difference, 7.9 percentage points; 95 percent confidence interval for the difference, 3.4 to 12.4; P=0.001). CONCLUSIONS: Among patients with a history of aspirin-induced ulcer bleeding whose ulcers had healed before they received the study treatment, aspirin plus esomeprazole was superior to clopidogrel in the prevention of recurrent ulcer bleeding. Our finding does not support the current recommendation that patients with major gastrointestinal intolerance of aspirin be given clopidogrel.

Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors.

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.

Celecoxib versus diclofenac and omeprazole in reducing the risk of recurrent ulcer bleeding in patients with arthritis.

BACKGROUND: Current guidelines recommend that patients at risk for ulcer disease who require treatment for arthritis receive nonsteroidal antiinflammatory drugs (NSAIDs) that are selective for cyclooxygenase-2 or the combination of a nonselective NSAID with a proton-pump inhibitor. We assessed whether celecoxib would be similar to diclofenac plus omeprazole in reducing the risk of recurrent ulcer bleeding in patients at high risk for bleeding. METHODS: We studied patients who used NSAIDs for arthritis and who presented with ulcer bleeding. After their ulcers had healed, we randomly assigned patients who were negative for Helicobacter pylori to receive either 200 mg of celecoxib twice daily plus daily placebo or 75 mg of diclofenac twice daily plus 20 mg of omeprazole daily for six months. The end point was recurrent ulcer bleeding. RESULTS: In the intention-to-treat analysis, which included 287 patients (144 receiving celecoxib and 143 receiving diclofenac plus omeprazole), recurrent ulcer bleeding occurred in 7 patients receiving celecoxib and 9 receiving diclofenac plus omeprazole. The probability of recurrent bleeding during the six-month period was 4.9 percent (95 percent confidence interval, 3.1 to 6.7) for patients who received celecoxib and 6.4 percent (95 percent confidence interval, 4.3 to 8.4) for patients who received diclofenac plus omeprazole (difference, -1.5 percentage points; 95 percent confidence interval for the difference, -6.8 to 3.8). Renal adverse events, including hypertension, peripheral edema, and renal failure, occurred in 24.3 percent of the patients receiving celecoxib and 30.8 percent of those receiving diclofenac plus omeprazole. CONCLUSIONS: Among patients with a recent history of ulcer bleeding, treatment with celecoxib was as effective as treatment with diclofenac plus omeprazole, with respect to the prevention of recurrent bleeding. Renal toxic effects are common in high-risk patients receiving celecoxib or diclofenac plus omeprazole.

Prolonged dual antiplatelet therapy in stable coronary disease: comparative observational study of benefits and harms in unselected versus trial populations.

OBJECTIVE:  To estimate the potential magnitude in unselected patients of the benefits and harms of prolonged dual antiplatelet therapy after acute myocardial infarction seen in selected patients with high risk characteristics in trials. DESIGN:  Observational population based cohort study. SETTING:  PEGASUS-TIMI-54 trial population and CALIBER (ClinicAl research using LInked Bespoke studies and Electronic health Records). PARTICIPANTS:  7238 patients who survived a year or more after acute myocardial infarction. INTERVENTIONS:  Prolonged dual antiplatelet therapy after acute myocardial infarction. MAIN OUTCOME MEASURES:  Recurrent acute myocardial infarction, stroke, or fatal cardiovascular disease. Fatal, severe, or intracranial bleeding. RESULTS:  1676/7238 (23.1%) patients met trial inclusion and exclusion criteria ("target" population). Compared with the placebo arm in the trial population, in the target population the median age was 12 years higher, there were more women (48.6% v 24.3%), and there was a substantially higher cumulative three year risk of both the primary (benefit) trial endpoint of recurrent acute myocardial infarction, stroke, or fatal cardiovascular disease (18.8% (95% confidence interval 16.3% to 21.8%) v 9.04%) and the primary (harm) endpoint of fatal, severe, or intracranial bleeding (3.0% (2.0% to 4.4%) v 1.26% (TIMI major bleeding)). Application of intention to treat relative risks from the trial (ticagrelor 60 mg daily arm) to CALIBER's target population showed an estimated 101 (95% confidence interval 87 to 117) ischaemic events prevented per 10 000 treated per year and an estimated 75 (50 to 110) excess fatal, severe, or intracranial bleeds caused per 10 000 patients treated per year. Generalisation from CALIBER's target subgroup to all 7238 real world patients who were stable at least one year after acute myocardial infarction showed similar three year risks of ischaemic events (17.2%, 16.0% to 18.5%), with an estimated 92 (86 to 99) events prevented per 10 000 patients treated per year, and similar three year risks of bleeding events (2.3%, 1.8% to 2.9%), with an estimated 58 (45 to 73) events caused per 10 000 patients treated per year. CONCLUSIONS:  This novel use of primary-secondary care linked electronic health records allows characterisation of "healthy trial participant" effects and confirms the potential absolute benefits and harms of dual antiplatelet therapy in representative patients a year or more after acute myocardial infarction.

Upregulation of heme oxygenase-1 and p21 confers resistance to apoptosis in human gastric cancer cells.

Both heme oxygenase-1 (HO-1) and p21(WAF1/Cip1) (p21) are involved in the pathogenesis of human cancer and their functions are closely associated with apoptosis. However, how these two molecules regulate apoptosis in human gastric cancer is unknown. In this study, we studied how HO-1 and p21 were regulated in two gastric cancer cell lines, MKN-45 with wild p53 and MKN-28 with mutant p53. The cells were treated with hemin and cadmium to induce HO-1. The result showed that HO-1 protein was significantly induced by hemin and cadmium in both cells tested. Following the HO-1 expression, p21 level was also markedly induced. The cells with increased HO-1 and p21 showed obviously resistantance to apoptotic stimuli. The levels of HO-1 and p21 induced were significantly inhibited by p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) and extracellular-regulated kinase (ERK) inhibitor (PD098059). Parallel to decreased HO-1 and p21 expression, the kinase inhibitors also significantly attenuated the resistance of the cells to apoptosis. The elevated HO-1 and p21 was further found to be associated with increase activity of the nuclear NF-kappaB and the inhibition of NF-kappaB led to the block of their induction. The elevated HO-1 and p21 were also demonstrated to be related to increased cellular inhibitor of caspase inbitory protein-2 (c-IAP2) and decreased caspapse-3 activity. It was noted that the above changes observed were not different between MKN-45 and MKN-28 cells, suggesting the functions of HO-1 and p21 were irrespective of the status of p53. In conclusion, we demonstrate that the resistance to apoptosis in gastric cancer cells with elevated HO-1 and p21 is independent of p53 status in a p38 MAPK- and ERK-mediated pathway with elevated c-IAP2 and decreased caspase-3 activity and that this pathway is sensitive to the inhibition of NF-kappaB.

Circulating Epstein-Barr virus DNA in the serum of patients with gastric carcinoma.

PURPOSE: We investigated the detectability of EBV DNA in the serum of gastric carcinoma patients in Hong Kong. Previous data have shown that approximately 10% of gastric carcinomas in Hong Kong are associated with EBV. EXPERIMENTAL DESIGN: We recruited 51 patients with gastric carcinoma, 30 patients with gastritis, and 197 apparently healthy controls. For gastric carcinoma patients, blood samples were obtained before surgery. After surgery, the resected tumor samples from the cancer cases were subjected to in situ hybridization for small EBV-encoded RNA (EBER). Serum EBV DNA in all cases was measured by real-time quantitative PCR. RESULTS: Serum EBV DNA was detectable in 5 of 5 (100%) EBER-positive gastric carcinoma cases (median concentration, 1063 copies/ml), in 13 of 14 (93%) EBER-negative gastric carcinoma cases with EBER-positive infiltrating lymphocytes (median concentration, 50 copies/ml), and in 0 of 32 (0%) EBER-negative cases. In the nontumor controls, serum EBV DNA was detectable in 7 of 30 (23%) gastritis cases (median concentration, 0 copies/ml) and in 7 of 197 (3.6%) apparently healthy individuals (median concentration, 0 copy/ml). CONCLUSIONS: Our data indicate that serum EBV DNA reflects tumoral EBER status and opens up the possibility that circulating EBV DNA may be used as a tumor marker for the EBER-positive gastric carcinomas. The biological and clinical significance of the presence of low levels of circulating EBV DNA in the minority of gastritis patients and healthy individuals remains to be elucidated.

Detection of gene promoter hypermethylation in the tumor and serum of patients with gastric carcinoma.

PURPOSE: Aberrant promoter methylation, an alternative mechanism for gene silencing, is frequently detected in gastric cancer. We studied the feasibility of detecting aberrant methylation in serum of gastric cancer patients. EXPERIMENTAL DESIGN: Patients (54) with gastric adenocarcinoma were studied. The tumor and the paired serum were examined for aberrant methylation in DAP-kinase, E-cadherin, GSTP1, p15, and p16 by methylation-specific PCR. Serum from 30 age-matched noncancer patients was used as control. RESULTS: Promoter methylation in DAP-kinase, E-cadherin, GSTP1, p15, and p16 were detected in 70.3, 75.9, 18.5, 68.5, and 66.7% of primary tumor. In serum of gastric cancer patients, methylation in DAP-kinase, E-cadherin, GSTP1, p15, and p16 were detected in 48.1, 57.4, 14.8, 55.6, and 51.9%, respectively. None of the control serum showed aberrant methylation. Aberrant methylation in serum DNA was all accompanied with methylation in the corresponding tumor samples. In general, >60% of serum from cancers with aberrant methylation demonstrated these epigenetic alterations. CONCLUSION: Our findings suggest that aberrant promoter methylation in serum can be detected in a substantial proportion of gastric cancer patients, and this strategy should be evaluated in the screening and surveillance of gastric cancer.

Combined sinus tract endoscopy and endoscopic retrograde cholangiopancreatography in management of pancreatic necrosis and abscess.

BACKGROUND: We report our experience of sinus tract endoscopy (STE) and endoscopic retrograde cholangiopancreatography (ERCP) in the treatment of pancreatic necrosis and abscess. METHODS: Thirteen patients with extensive pancreatic necrosis were firstly managed with either percutaneous drainage (PD group; n = 9) or open necrosectomy (ON group; n = 4). Debridement of necrotic tissue was subsequently performed via the drain tract by STE. ERCP was performed only when there was a suspicious of persistent pancreatic duct disruption or choledocholithiasis. RESULTS: In the PD group, the median number of STE sessions required was 3 (range 2-8). The median hospital and ICU stay were 84 days (range 29-163 days) and 0 day (range 0-64 days), respectively, with an overall success rate of 67%. In the ON group, the median number of STE sessions required was 6.5 (range 1-18). The median hospital and ICU stay were 82 days (range 58-194 days) and 19 days (range 4-24 days), respectively. No mortality or failure was noted in the latter group. ERCP was required in nine of 13 patients. CONCLUSION: Combined ERCP and STE is a useful adjunct in treating pancreatic necrosis or abscess.

The effect of endoscopic therapy in patients receiving omeprazole for bleeding ulcers with nonbleeding visible vessels or adherent clots: a randomized comparison.

BACKGROUND: The optimal treatment of ulcers with nonbleeding visible vessels and adherent clots is unclear. OBJECTIVE: To compare intravenous omeprazole infusion plus endoscopic therapy with intravenous omeprazole infusion alone for prevention of recurrent bleeding from ulcers with nonbleeding visible vessels or adherent clots. DESIGN: Single-blind randomized study with blinded evaluation of study end points. SETTING: An endoscopy center in a university hospital in Hong Kong. PATIENTS: 156 persons with upper gastrointestinal bleeding and ulcers showing nonbleeding visible vessels or adherent clots. INTERVENTION: Combination of endoscopic therapy and omeprazole infusion versus sham endoscopic therapy and omeprazole infusion. MEASUREMENTS: Recurrent ulcer bleeding before discharge and within 30 days. RESULTS: 78 patients were recruited in each group. Ulcer bleeding recurred before discharge in seven patients who received intravenous omeprazole alone (9%) and no patients who received combined therapy (difference, 9 percentage points [95% CI, 1.7 to 17.6 percentage points]; P = 0.01). The probability of recurrent bleeding within 30 days was 11.6% (9 patients) in the omeprazole-alone group and 1.1% (1 patient) in the combined therapy group (difference, 10.5 percentage points [CI, 1.7 to 19.8 percentage points]; P = 0.009). Patients in the combined therapy group required less transfusion (difference in median units of blood transfused, 1 unit [CI, 0 to 2 units]; P = 0.02). One patient in the combined therapy group had surgery for ulcer perforation. Four patients receiving omeprazole alone (5.1%) and two patients receiving combined therapy (2.6%) died within 30 days. CONCLUSION: The combination of endoscopic therapy and omeprazole infusion is superior to omeprazole infusion alone for preventing recurrent bleeding from ulcers with nonbleeding visible vessels and adherent clots.

Inhibition by SK&F 96365 of Ca2+ current, IL-2 production and activation in T lymphocytes.

1. By use of whole cell patch-clamp and Indo-1 fluorescence studies of the Jurkat T leukaemic cell line, we show that the new organic antagonist of receptor-mediated Ca2+ entry, SK&F 96365, inhibits the T cell Ca2+ current in a dose-dependent fashion, with an IC50 of 12 microM. 2. SK&F 96365 also inhibits [3H]-thymidine incorporation and interleukin-2 (IL-2) synthesis in peripheral blood lymphocytes. 3. SK&F 96365 has no effect on Ca2+ stores release or K+ channels. 4. This is the first account of an organic inhibitor of the T cell Ca2+ current. The ability of SK&F 96365 to inhibit IL-2 synthesis and cell proliferation suggests that a new class of related Ca2+ channel blockers can be developed as immunosuppressive agents.

Inverse association between cyclooxygenase-2 overexpression and microsatellite instability in gastric cancer.

This study examined the association between cyclooxygenase-2 (COX-2) overexpression and microsatellite instability (MSI) in gastric cancer. COX-2 expression was assessed by immunohistochemistry and scored in a semi-quantitative manner whereas MSI status was characterized by nine microsatellite markers. The clinicopathological features of cancers including survival data were analyzed. Of the 109 gastric cancers studied, COX-2 overexpression and high level of MSI (MSI-H) was detected in 64.2 and 22.0% cases respectively. Gastric tumors with MSI-H phenotypes had significantly lower level of COX-2 expression levels when compared to MSI-L and MSS tumors (P=0.002). Moreover, COX-2 overexpression was associated with tumor invasion beyond submucosa (P=0.045) and there was a trend favoring better survival in gastric cancers without COX-2 overexpression (P=0.07). The results from this study suggest that gastric cancer with microsatellite instability or COX-2 overexpression present with diverse clinicopathological features.

Somatic beta-catenin mutation in gastric carcinoma--an infrequent event that is not specific for microsatellite instability.

We screened 90 cases of gastric carcinoma (GCA) samples for beta-catenin exon 3 mutation and assessed its possible relationship with microsatellite instability (MSI). Three mutations were detected in two samples, including a single mutation in an intestinal type and double mutations in a diffuse type GCA. One of the mutations found in the diffuse type GCA sample was a non-sense mutation at codon 68 (CAG-->TAG). This novel mutation was predicted to disrupt the binding of beta-catenin to alpha-catenin and may be related to the diffuse type morphology. The other two mutations were missense mutations involved or related to the GSK-3beta phosphorylation site, which have been reported previously. No MSI can be demonstrated in the two cases with beta-catenin mutation. Our results suggested that beta-catenin mutation was infrequent in GCA and appeared not specific for MSI.

Cyclooxygenase-2 upregulates vascular endothelial growth factor expression and angiogenesis in human gastric carcinoma.

Although gastric cancer with cyclooxygenase (COX)-2 overexpression is associated with poor prognosis, the mechanistic pathway remains unknown. We examined the associations between expressions of COX-2 and vascular endothelial growth factor (VEGF) in both gastric cancer cells and in human gastric cancer. The gastric cell line, Kato III, was transiently transfected with cox-2 expressing vector. The levels of COX-2, prostaglandin (PG) E2 and VEGF expression were measured post-transfection. Additionally, expressions of COX-2 and VEGF in human gastric cancer were determined by immunohistochemistry in archive gastrectomy specimens. Tumor angiogenesis was assessed by the microvessel density (MVD), which was determined by anti-CD34 immunostaining. Transient transfection of Kato III with cox-2 was associated with increased COX-2 expression, higher PGE2 production and upregulated VEGF expressions. Treatment with NS398, a specific COX-2 inhibitor, reduced VEGF expression in COX-2 expressing Kato III cells by 25%. Among the 67 gastric cancers examined, COX-2 overexpression was found in 45 (67%) cases whereas increased VEGF expression was detected in 46 (69%) cases. There was a significant association between COX-2 and VEGF expressions in gastric cancer (r=0.25, p=0.041). Additionally, tumor MVD was associated with both COX-2 (r=0.32, p=0.008) and VEGF (r=0.39, p=0.001) expressions. Our results showed that overexpression of COX-2 in both gastric cells and primary gastric cancer is associated with upregulation of VEGF and angiogenesis. Future studies should evaluate the potential anti-angiogenic effect of COX-2 inhibitors on human gastric cancer.