Light-dependent phosphorylation of the gamma subunit of cGMP-phophodiesterase (PDE6gamma) at residue threonine 22 in intact photoreceptor neurons.

The gamma subunit of rod-specific cGMP phosphodiesterase 6 (PDE6gamma), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6gamma on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6gamma may increase or decrease the ability of PDE6gamma to deactivate phototransduction. To resolve role of phosphorylation of PDE6gamma in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6gamma (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6gamma, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6gamma is essential for the regulation of G-protein signaling.

Cellular and molecular origin of circumpapillary dysgenesis of the pigment epithelium.

PURPOSE: We studied clinical phenotyping and TEAD1 expression in mice and humans to gain a better understanding of the primary origin in the pathogenesis of circumpapillary dysgenesis of the pigment epithelium. DESIGN: Observational case series and experimental study. PARTICIPANTS: Three female patients from an affected family were included for phenotypic study. Mice and human tissues were used for biochemistry and immunohistochemistry studies. METHODS: We performed genetic analyses and longitudinal clinical, imaging, and electrophysiologic studies in a 3-generation family. Western blotting and immunohistochemistry were used to detect TEAD1 expression in mice and human retinal tissues. MAIN OUTCOME MEASURES: Autofluorescence and optical coherence tomography (OCT) imaging were compared and reviewed from 3 patients. TEAD1 expression was compared in different tissues from mice and human samples. RESULTS: A point mutation at T1261 in TEAD1 was detected in the mother. Autofluorescence and OCT imaging studies revealed choroid is involved earlier than retinal pigment epithelium (RPE). From immunoblot analysis, we discovered that TEAD1 and its cofactors YAP65 and FOXA2 are expressed in the choroid. Immunohistochemical analysis on frozen sections of mouse retina supports immunoblot results. CONCLUSIONS: The primary cellular origin of circumpapillary dysgenesis of the pigment epithelium is within the choroid instead of the pigment epithelium. The loss of the RPE and photoreceptors in later stages of the disease is a secondary consequence of choroidal degeneration. Studies of the downstream targets of TEAD1 in choroidal cells will provide promising new research opportunities for the development of treatments for choroidal diseases. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Indications for the use of endoscopic mucosal resection for early gastric cancer in Japan: a comparative study with endoscopic submucosal dissection.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) has been reported to produce excellent treatment results for early gastric cancer. In terms of lesions that previously met the criteria for endoscopic mucosal resection (EMR), there is now controversy about which of the two methods is superior, and whether the two methods are comparable. PATIENTS AND METHODS: A total of 177 patients (202 lesions) with early gastric cancer who met the guidelines for EMR and who underwent either EMR or ESD were studied. The rates of en bloc resection, complete resection, local recurrence, and complications were compared between EMR and ESD. RESULTS: The overall en bloc and complete resection rates were lower in patients undergoing EMR than in those undergoing ESD (en bloc: 53.8 % vs. 94.3 %, P < 0.001; complete: 37.5 % vs. 92.6 %, P < 0.001). The overall 5-year recurrence-free rate was lower in the EMR group than in the ESD group (82.5 % vs. 100 %; P < 0.001). However, with regard to the tumor size, the two groups did not differ in en bloc ( P = 1.0) or complete resection rate ( P = 0.8) for tumors < or = 5 mm and in 5-year recurrence-free rate ( P = 0.19) for tumors < or = 10 mm. The mean time required for resection was longer for ESD than for EMR ( P < 0.001). Perforation and bleeding requiring blood transfusion occurred in a small percentage in the ESD group, but in none in the EMR group. CONCLUSION: In this study, EMR was comparable to ESD for the millimeter-sized lesions. We suggest that such small lesions might be well suited to treatment with EMR.

Is somatostatin a true local inhibitory regulator of insulin secretion?

Pancreatic somatostatin was depleted after oral administration of cysteamine to rats, yet the B-cells in the isolated islets were morphologically and functionally intact. Compared with islets from the rats not given cysteamine, the somatostatin-depleted islets released larger amounts of insulin during 1 h of incubation by glucose or 3-isobutyl-1-methylxanthine stimulation. Therefore, the possibility that pancreatic somatostatin may locally regulate the inhibitory effects of insulin secretion has to be considered.

Effect of glucose on somatostatin secretion from isolated pancreatic islets of normal and streptozotocin-diabetic rats.

Pancreatic somatostatin (SRIF) secretion was examined using the RIA described in earlier paper. Ten isolated rat pancreatic islets were incubated for 30 min in 1 ml Krebs-Ringer bicarbonate buffer. Glucose (5.6 mM) caused a small but significant increase of SRIF secretion. The maximal secretion rate was observed at 16.7 mM glucose, and the half-maximal rate was seen at about 9.7 mM. Islets preincubated with 16.7 mM glucose released higher levels of SRIF and insulin during the subsequent incubation with 16.7 mM glucose than did islets preincubated with 2.8 mM glucose. Glucose-induced SRIF secretion was suppressed by epinephrine, but beta-adrenergic stimulation (epinephrine and phentolamine) produced an increase in SRIF secretion. Islets taken from rats 2 days after streptozotocin administration released minimal amounts of insulin. Basal and glucose-induced SRIF secretion from these islets, which had relatively unchanged SRIF contents and D cell numbers, equaled SRIF secretion from control rat islets. Islets taken from rats 6 weeks after streptozotocin administration, however, had increased SRIF content and D cell numbers, and they oversecreted SRIF. We conclude that pancreatic SRIF secretion can be induced by glucose and modulated by catecholamines and preexposure to high glucose, and the duration and severity of diabetes may be an important determinant of the changes in pancreatic D cell structure and function.

Effect of calcium on the secretion of somatostatin and insulin from pancreatic islets.

Calcium ionophore A23187 (20 micrometer) evoked the secretion of somatostatin (SRIF) as well as insulin from isolated rat pancreatic islets in a medium containing a relatively low concentration of calcium (0.9 mM) and a low concentration of glucose (5.5 mM). A high level of extracellular calcium (7.5 mM) also had a stimulatory effect on SRIF and insulin release. On the other hand, in the presence of high glucose (16.7 mM), A23187 had different effects on D and B cells; insulin release was markedly suppressed by A23187, but SRIF secretion was significantly enhanced. A high concentration of glucose (16.7 mM) did not stimulate SRIF secretion at low extracellular calcium concentration (0.25 mM), at which level insulin release is significantly enhanced. These findings indicate that calcium may play an important role in the regulation of the secretion of SRIF as well as insulin and suggest that the B and D cells differ in their sensitivity to the calcium ion.

Somatostatin and insulin secretion from pancreatic islets: studies on the effect of high K+, 9-aminoacridine and valinomycin.

We attempted to determine whether a decrease in the potassium permeability of the D cell membrane plays a role in the stimulus-secretion coupling, as it does in the pancreatic B cell. Elevation in the extracellular potassium concentration from 5.5 to 16.5 mM, or 0.2 mM 9-aminoacridine, which decreases potassium permeability in plasma membrane, stimulated the release of somatostatin as well as insulin from the isolated rat pancreatic islets. Valinomycin (1 microM), a potassium ionophore inhibited the secretion in response to high glucose, high extracellular potassium or 9-aminoacridine. These findings indicate that a reduction in potassium permeability in the D cell membrane, as induced by glucose or other stimulants, may be a major step in secretion of somatostatin.

Significant increase of interleukin 6 production in blood mononuclear leukocytes obtained from patients with active inflammatory bowel disease.

In the present study, we compared the potency of interleukin 6 production in peripheral blood mononuclear leukocytes between paired patients with active stage and inactive stage of inflammatory bowel disease. Subjects included nine patients with ulcerative colitis, ten patients with Crohn's disease and sex-matched nine healthy volunteers. Mononuclear leukocytes were stimulated with concanavalin A for 24 h to induce interleukin 6 production. Interleukin 6 content in the culture medium was assayed by using specific ELISA and interleukin 6 dependent cell line MH-60. Interleukin 6 production was found to be significantly increased in mononuclear leukocytes from both active ulcerative colitis and Crohn's disease as compared to that from control subjects. There was no significant difference in interleukin 6 production between ulcerative colitis and Crohn's disease. The potency of interleukin 6 production was returned to the control level when the diseases became inactive. The present results, therefore, may indicate some important role of interleukin 6 in the pathogenesis of inflammatory bowel disease and also the potency of interleukin 6 production in mononuclear leukocytes can be an indicator of the activity of inflammatory bowel disease.

[Characteristic features of the atrophic border of gastric ulcers in elderly].

We studied the characteristic features of gastric ulcers in 50 elderly cases (over 60 years old) concerning 2 points. Initially, we compared the clinical features of the elderly patients with those of the non-elderly (under 60 years old) patients. Secondly, we compared the endoscopic appearance of the atrophic border of elderly patients with that of elderly persons without gastric ulcer, using the Takemoto and Kimura's classification of endoscopic atrophic borders. The results were that elderly patients with gastric ulcer had few symptoms. The symptoms associated with bleeding, however, were recognized. Many of them had some complications. There was no difference in the healing rate at 8 weeks from the beginning of the treatment by histamine H2-receptor blocker (H2 blocker) between the elderly and non-elderly with gastric ulcer. Concerning the endoscopic atrophic border of the elderly persons without ulcers, the number of the open type cases was significantly greater than that of the closed type. On the contrary, in elderly persons with gastric ulcer, the number of the closed type was significantly greater than that of the open type, especially the number of C-2 type was the greatest. The healing rate of the elderly with closed type border was higher than that of elderly cases with open type border in 4 and 8 weeks from the beginning of the treatment.

Rapid and noninvasive imaging of retinal ganglion cells in live mouse models of glaucoma.

PURPOSE: We report a noninvasive method for the monitoring of retinal ganglion cell (RGC) survival in live mice utilizing standard fluorescence microscopy. PROCEDURES: Transgenic mice expressing cyan fluorescent protein (CFP) under the regulation of an RGC-specific promoter Thy1 were used in this study. RESULTS: We established that Thy1-CFP expression is a quantitative reflection of the number of surviving RGCs, the fluorescence emission is stable for at least a year and that the loss of fluorescence correlates directly to glaucomatous damage. In high pressure glaucoma model, the peripheral retina is preferentially affected. CONCLUSIONS: Our live-imaging technique allows for the longitudinal assessment of RGC survival from the same animal. Noninvasive monitoring of neuronal cell death and survival is a powerful technique that would allow investigators to validate new potential glaucoma therapy based on neuroprotection.

Functional rescue of degenerating photoreceptors in mice homozygous for a hypomorphic cGMP phosphodiesterase 6 b allele (Pde6bH620Q).

PURPOSE: Approximately 8% of autosomal recessive retinitis pigmentosa (RP) cases worldwide are due to defects in rod-specific phosphodiesterase PDE6, a tetramer consisting of catalytic (PDE6alpha and PDE6beta) and two regulatory (PDE6gamma) subunits. In mice homozygous for a nonsense Pde6b(rd1) allele, absence of PDE6 activity is associated with retinal disease similar to humans. Although studied for 80 years, the rapid degeneration Pde6b(rd1) phenotype has limited analyses and therapeutic modeling. Moreover, this model does not represent human RP involving PDE6B missense mutations. In the current study the mouse missense allele, Pde6b(H620Q) was characterized further. METHODS: Photoreceptor degeneration in Pde6b(H620Q) homozygotes was documented by histochemistry, whereas PDE6beta expression and activity were monitored by immunoblotting and cGMP assays. To measure changes in rod physiology, electroretinograms and intracellular Ca(2+) recording were performed. To test the effectiveness of gene therapy, Opsin::Pde6b lentivirus was subretinally injected into Pde6b(H620Q) homozygotes. RESULTS: Within 3 weeks of birth, the Pde6b(H620Q) homozygotes displayed relatively normal photoreceptors, but by 7 weeks degeneration was largely complete. Before degeneration, PDE6beta expression and PDE6 activity were reduced. Although light-/dark-adapted total cGMP levels appeared normal, Pde6b(H620Q) homozygotes exhibited depressed rod function and elevated outer segment Ca(2+). Transduction with Opsin::Pde6b lentivirus resulted in histologic and functional rescue of photoreceptors. CONCLUSIONS: Pde6b(H620Q) homozygous mice exhibit a hypomorphic phenotype with partial PDE6 activity that may result in an increased Ca(2+) to promote photoreceptor death. As degeneration in Pde6b(H620Q) mutants is slower than in Pde6b(rd1) mice and can be suppressed by Pde6b transduction, this Pde6b(H620Q) model may provide an alternate means to explore new treatments of RP.

Effect of famotidine on gastric mucosal lesions induced by gold thioglucose in rats.

Rats that were given an intraperitoneal injection of gold thioglucose (GTG, 0.3 mg/g) developed hemorrhagic lesions in the gastric mucosa 24 hours after the injection. The gastric juice volume and the generation of acid and pepsin were significantly increased 24 hours after the GTG injection (p < 0.01). Simultaneous administration of famotidine (3 mg/kg) with GTG significantly reduced gastric juice secretion and the generation of acid and pepsin (all p < 0.01), as well as suppressing the development of hemorrhagic lesions. These results indicate that there is a relationship between the hemorrhagic lesions induced by GTG and increased acid and pepsin generation. Rats treated with GTG seem to be a useful new animal model for the investigation of pepsin secretion.