Differentially expressed Maf family transcription factors, c-Maf and MafA, activate glucagon and insulin gene expression in pancreatic islet alpha- and beta-cells.

A basic-leucine zipper transcription factor, MafA, was recently identified as one of the most important transactivators of insulin gene expression. This protein controls the glucose-regulated and pancreatic beta-cell-specific expression of the insulin gene through a cis-regulatory element called RIPE3b/MARE (Maf-recognition element). Here, we show that MafA expression is restricted to beta-cells of pancreatic islets in vivo and in insulinoma cell lines. We also demonstrate that c-Maf, another member of the Maf family of transcription factors, is expressed in islet alpha-cells and in a glucagonoma cell line (alphaTC1), but not in gamma- and delta-cells. An insulinoma cell line, betaTC6, also expressed c-Maf, albeit at a low level. Chromatin immunoprecipitation assays demonstrated that Maf proteins associate with insulin and glucagon promoters in beta- and alpha-cell lines, respectively. c-Maf protein stimulated glucagon promoter activity in a transient luciferase assay, and activation of the glucagon promoter by c-Maf was more efficient than by the other alpha-cell-enriched transcription factors, Cdx2, Pax6, and Isl-1. Furthermore, inhibition of c-Maf expression in alphaTC1 cells by specific short hairpin RNA resulted in marked reduction of the glucagon promoter activity. Thus, c-Maf and MafA are differentially expressed in alpha- and beta-cells where they regulate glucagon and insulin gene expression, respectively.

Nitric oxide/cGMP-induced inhibition of calcium and chloride currents in retinal pericytes.

In the CNS, contractile pericytes positioned on endothelium-lined lumens appear to play a role in regulating capillary blood flow. This function may be particularly important in the retina where pericytes are more numerous than in other tissues. Despite the importance of pericytes, knowledge of the effects of vasoactive molecules, such as nitric oxide (NO), on the physiology of these cells is limited. Since it is likely that ion channels play a role in the response of pericytes to signaling molecules from other cells, we used the perforated-patch configuration of the patch-clamp technique to record the whole-cell currents of pericytes located on microvessels freshly isolated from the rat retina. We found that voltage-gated calcium currents and calcium-activated chloride currents were inhibited during exposure to the NO donor, sodium nitroprusside (SNP). 8-Bromo-cyclic guanosine monophosphate (cGMP) mimicked these effects. In contrast, neither SNP nor the cGMP analog significantly affected the potassium or nonspecific cation conductances, which establish the resting membrane potential of retinal pericytes. Consistent with endogenous NO suppressing pericyte channel activity, exposure of isolated microvessels to an inhibitor of NO synthase increased the calcium and chloride currents. Since our experiments indicate that chloride channel activity is dependent, in part, upon the function of voltage-gated calcium channels, we postulate that a NO/cGMP-mediated inhibition of calcium channels reduces calcium influx and, thereby, lessens the opening of the calcium-activated chloride channels. This may be one mechanism by which NO decreases the contractile tone of pericytes.

PDGF-induced coupling of function with metabolism in microvascular pericytes of the retina.

PURPOSE: The aim of this study was to test the hypothesis that platelet-derived growth factor (PDGF)-BB regulates the physiology of retinal pericytes, which are contractile cells located on the abluminal surface of capillaries. The expression of PDGF-BB and its cognate receptor in retinal vessels suggests a vasoactive function. However, although endothelium-derived PDGF-BB appears vital for the development of pericyte-containing microvessels, its role in the mature vasculature remains uncertain. METHODS: Based on the premise that ion channels mediate the responses of pericytes to vasoactive signals, the perforated-patch configuration of the patch-clamp technique was used to determine the effect of PDGF-BB on the ionic currents and membrane potential of pericytes located on microvessels freshly isolated from the adult rat retina. Changes in pericyte calcium levels were monitored with the calcium indicator fluo-4. Differential interference contrast optics and image analysis software aided in assessing the effects of PDGF-BB on the lumens of isolated pericyte-containing microvessels. In some experiments, blockers of adenosine triphosphate (ATP) synthesis created chemical ischemia. RESULTS: Electrophysiological recordings from pericytes showed that PDGF-BB can activate nonspecific cation channels, chloride channels, and ATP-sensitive potassium channels. The metabolic status of an isolated capillary determined which of these ion channels were activated by PDGF-BB and thereby whether the membrane potential decreased or increased, the cell calcium rose or fell, and the vessel lumen constricted or dilated. CONCLUSIONS: The ability of PDGF-BB to be a vasoconstrictor when energy supplies are ample and to be a vasodilator under ischemic conditions may provide an efficient mechanism to link capillary function to local metabolic needs.

Platelet-derived growth factor-BB: a survival factor for the retinal microvasculature during periods of metabolic compromise.

PURPOSE: The aim of this study was to test the hypothesis that platelet-derived growth factor (PDGF) reduces ischemia-induced damage to cells in the retinal microvasculature. METHODS: As a model of ischemia, pericyte-containing microvessels freshly isolated from the adult rat retina were exposed to the inhibitors of ATP synthesis, iodoacetate and antimycin A. Cell viability was assayed by trypan blue exclusion. RESULTS: PDGF-BB significantly reduced cell death induced by chemical ischemia. The half-maximally effective concentration was approximately 15 pM. In contrast to PDGF-BB, which is the specific ligand for PDGF-beta receptors, ischemic death was not reduced by PDGF-AA, which does not activate the beta-receptors. The protective effect of PDGF-BB was blocked by tolbutamide, which is an inhibitor of ATP-sensitive potassium (K(ATP)) channels and mimicked by the K(ATP) channel opener, pinacidil. Nifedipine, which blocks voltage-gated calcium channels (VGCC's), also mimicked the protective effect of PDGF-BB. Consistent with PDGF-BB and nifedipine preventing cell death via a common mechanism, i.e., reducing VGCC activity, the maximal effects of this growth factor and the calcium channel blocker were not additive. CONCLUSIONS: Our results indicate that PDGF-BB significantly reduces the vulnerability of retinal microvessels to damage caused by profound ischemia. During episodes of metabolic compromise, it appears likely that the opening of K(ATP) channels via activation of PDGF-beta receptors initiates an adaptive mechanism to enhance the survival of the retinal microvasculature.

Diffuse submucosal cysts of the stomach associated with gastric cancer: contribution of Helicobacter pylori infections.

Diffuse submucosal cysts (DSCs) in the stomach are often associated with gastric cancer and a high occurrence of multiple gastric cancers. We studied the clinicopathological features of four early gastric cancer patients with DSCs in the submucosal layer of the stomach. All patients had early gastric cancers with gastritis and erosion in the gastric mucosa, and were positive for Helicobacter pylori (H. pylori). Based on a review of the reported cases, we found that a very high proportion (>94%) of DSCs are associated with infection by H. pylori. Although DSCs have previously been considered to be paracancerous lesions of gastric cancer, we speculate that DSCs might be post-inflammatory changes following infection by H. pylori, which may result in the high incidence of gastric cancer development.

Synthesis, SARs, and pharmacological characterization of 2-amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists.

(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (4, LY354740), a highly selective and orally active group II metabotropic glutamate receptor (mGluR) agonist, has increased interest in the study of group II mGluRs. Our interest focused on a conformationally constrained form of compound 4, because it appeared that the rigid form resulted in not only selectivity for group II mGluR but was orally active. Therefore, we introduced a fluorine atom to compound 4, based on the molecular size (close resemblance to hydrogen atom) and electronegativity (effects on the electron distribution in the molecule) of this atom and carbon-fluorine bond energy. Compound (+)-7 (MGS0008), the best compound among 3-fluoro derivatives 7-10, retained the agonist activity of compound 4 for mGluR2 and mGluR3 ((+)-7: EC(50) = 29.4 +/- 3.3 nM and 45.4 +/- 8.4 nM for mGluR2 and mGluR3, respectively; 4: EC(50) = 18.3 +/- 1.6 nM and 62.8 +/- 12 nM for mGluR2 and mGluR3, respectively) and increased the oral activity of compound 4 ((+)-7: ED(50) = 5.1 mg/kg and 0.26 mg/kg for phencyclidine (PCP)-induced hyperactivity and PCP-induced head-weaving behavior, respectively; 4: ED(50) = >100 mg/kg and 3.0 mg/kg for PCP-induced hyperactivity and PCP-induced head-weaving behavior, respectively). In addition, a compound [(3)H]-(+)-7 binding study using mGluR2 or 3 expressed in CHO cells was successful ((+)-7: K(i) = 47.7 +/- 17 nM and 65.9 +/- 7.1 nM for mGluR2 and mGluR3, respectively; 4: K(i) = 23.4 +/- 7.1 nM and 53.5 +/- 13 nM for mGluR2 and mGluR3, respectively). On the basis of a successful result of compound 7, we focused on the introduction of a fluorine atom on the C6 position of compound 4. (1R,2S,5R, 6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid ((-)-11) exhibited a high degree of agonist activity for group II mGluRs equal to that of compound 4 or 7 ((-)-11: K(i) = 16.6 +/- 5.6 and 80.9 +/- 31 nM for mGluR2 and mGluR3, respectively). Our interest shifted to modification on CH(2) at C4 position of compound 11, since replacement of the CH(2) group with either an oxygen atom or sulfur atom yielded compound 5 or 6, resulting in increased agonist activity. We selected a carbonyl group instead of CH(2) at the C4 position of compound 11. The carbonyl group might slightly change the relative conformation of three functional groups, the amino group and two carboxylic acids, which have important roles in mediating the interaction between group II mGluRs and their ligand, compared with the CH(2) group of 4, oxygen atom of 5, and sulfur atom of 6. (1R,2S,5S,6S)-2-Amino-6-fluoro-4-oxobicyclo[3.1. 0]hexane-2,6-dicarboxylic acid monohydrate ((+)-14, MGS0028) exhibited a remarkably high degree of agonist activity for mGluR2 (K(i) = 0.570 +/- 0.10 nM) and mGluR3 (K(i) = 2.07 +/- 0.40 nM) expressed in CHO cells but not mGluR4, 6, 7, 1a, or 5 expressed in CHO cells (K(i) = >100 000 nM). Furthermore, compound (+)-14 strongly inhibited phencyclidine (PCP)-induced head-weaving behavior (ED(50) = 0.090 microg/kg) and hyperactivity (ED(50) = 0.30 mg/kg) in rats. Thus, (+)-7 and (+)-14 are potent, selective, and orally active group II mGluR agonists and might be useful not only for exploring the functions of mGluRs but in the treatment of schizophrenia.

Multiple hepatic adenomas caused by long-term administration of androgenic steroids for aplastic anemia in association with familial adenomatous polyposis.

We report a rare case of hepatic adenomas (HA), in a 20-year-old Japanese girl treated for 6 years with anabolic androgens for aplastic anemia. In a review of the world literature using computer MEDLINE search, we found only 17 cases of androgen-induced HA published between 1975 and 1998 in the English-language literature. The patient was referred to us because of liver lesions detected during a follow-up examination for familial adenomatous polyposis. After being diagnosed with aplastic anemia at 14 years of age, she had been treated with oxymetholone (30 mg/day) for 6 years. Laboratory evaluation revealed normal liver function. Ultrasonography (US) and computed tomography (CT) demonstrated multiple liver lesions. Histopathological examinations of biopsied specimens from the liver tumor showed HA. After the patient was diagnosed with HA, oxymetholone was tapered off. Patients taking androgenic-anabolic steroids should be carefully monitored with US and CT and tumor markers should be measured. This report may be helpful in identifying the population who is at risk of developing hepatic sex hormone-related tumors.

[Use of the multifocal electroretinogram to evaluate retinal function after pars plana vitrectomy for diabetic macular edema].

PURPOSE: Multifocal electroretinograms (multifocal ERGs) were performed to evaluate retinal function in patients with diabetic macular edema. MATERIALS & METHODS: Eight eyes of 5 patients underwent pars plana vitrectomy for diabetic macular edema. Multifocal ERGs, fundus examinations, and fluorescent angiographies were done both before and after the operation. RESULTS: Post-operative visual acuity was improved by two or more lines in 3 eyes. In multifocal ERGs, the amplitude of the positive wave (P1) from the macular area decreased transiently after surgery (p < 0.05), but it improved with the passage of time. The P1 amplitude from the peripheral area was decreased throughout the observation period of six months (p < 0.05). For both areas, the P1 latency was increased significantly (p < 0.05) at one month post-operatively, and then decreased. CONCLUSION: These findings are consistent with the possibility that the surgical method, especially removal of the posterior cortical vitreous, can cause retinal damage.

Organophosphate metabolic changes in the rat lens during the development of galactose-induced cataract.

Using phosphorous-31 nuclear magnetic resonance (31P-NMR) spectroscopy, we observed the metabolic kinetics of organophosphate compounds in the rat lens during cataract development induced by different doses of galactose (5%, 15%, 25%, and 40%) added to rat chow. The metabolic and histologic changes in the lenses were compared among the rats fed with different doses of galactose. alpha-Glycerophosphate significantly increased in response to the galactose doses, followed by a decrease to steady values of approximately 120% of the base line value, except in the 40% galactose group, which had a marked decrease to 68% of the base line value. Choline phosphate decreased rapidly during dosing in all but the 5% group, but no changes in those levels were observed after 2 weeks of galactose dosing. ATP decreased significantly when the histologic destruction progressed in the entire lens. Although inorganic phosphate increased significantly in a dose-dependent manner, it did not exceed the peak level. The histologic changes were closely related to the dynamic changes in the phosphorous compounds in the rat lens during formation of the galactose-induced cataract. Our results indicated that the development of a galactose-induced cataract was associated with the metabolic changes of the phosphate compounds.

Benign fibrous mesothelioma successfully treated by thoracoscope-assisted surgery: report of a case.

We report herein the case of an 84-year-old man in whom a benign fibrous mesothelioma (BFM) was successfully treated by thoracoscope-assisted surgery. In September 1996, the patient underwent a sigmoidectomy for colon cancer, soon after which a followup examination disclosed a coin lesion on his chest X-ray film. The patient was readmitted to our hospital in November 1996 for further investigation of this tumor. A chest computed tomography scan revealed a 3.5 x 3.0 cm homogeneous tumor arising from the pleura. A needle biopsy was performed under ultrasonographic guidance, and the specimen was histopathologically diagnosed as a BFM. In view of his poor respiratory function, the tumor was excised by thoracoscope-assisted surgery. This case report serves to demonstrate the benefits of thoracoscope-assisted surgery for such patients.

[A case of retroperitoneal malignant lymphoma successfully treated by chemotherapy].

A 71-year-old woman was admitted to our hospital with the complaint of lower abdominal pain. CT scan revealed a huge retroperitoneal mass. On laparotomy the tumor was found to have extensively invaded the retroperitoneum, and an incisional biopsy was performed. Histopathological diagnosis of the biopsied specimen demonstrated diffuse malignant lymphoma of the pleomorphic type. Six cycles of CHOP chemotherapy were very effective for complete remission. Our review of 18 cases in the Japanese literature showed that chemotherapy and radiotherapy were effective for retroperitoneal malignant lymphoma, so due care must be taken in opting for surgery.

Carcinosarcoma of the colon: report of a case and review of the literature.

We report a case of carcinosarcoma in the transverse colon in a 60-year-old woman. She was admitted to our hospital for further examination of occult blood in October, 1995. Colonoscopy disclosed an elevated lesion with ulceration in the transverse colon, and she underwent right hemicolectomy. Histopathological examination revealed the tumor to consist of both carcinomatous and sarcomatous elements, the latter being more predominant. Immunohistochemistry revealed vimentin immunoreactivity in most of the sarcomatous cells, and S-100 and myoglobin in a few carcinomatous cells. Distinct carcinomatous features were noted in one superficial portion of the tumor, and these carcinomatous cells showed immunoreactivity for epithelial membrane antigen. The patient is alive 14 months after surgery without evidence of recurrence. To our knowledge, this is the fourth reported case of carcinosarcoma of the colon. Our review of the literature disclosed poor prognosis in colonic carcinosarcoma.

[Emulsification experiments with dimethylsiloxane/phenylmethylsiloxane copolymer].

We compared the emulsification tendency of dimethylsiloxane/phenylmethylsiloxane copolymer (DPC; 5%-phenylated, specific gravity 0.984) with that of silicone oil (dimethylsiloxane; SO, specific gravity 0.966) and fluorosilicone oil (methyl-3,3,3-trifluoropropylsiloxane; FSO, specific gravity 1.256), which are clinically used as an intraocular tamponade. After SO, DPC and FSO were placed in a glass container with an equal amount of albumin solution (1 mg/ml) or gamma-globulin solution (1 mg/ml), and shaken SO and DPC, with their specific gravities being closer to water, showed less emulsification than FSO. Following vitrectomy, DPC was injected into the rabbit vitreous cavity, but did not seem to show any severe histological damage. Highly-phenylated DPC, which is slightly heavier than water, may be useful to treat inferior retinal detachment in place of FSO.

Double cancers of the gallbladder and bile duct associated with anomalous choledochopancreatic duct junction.

A rare case of double cancers of the gallbladder and bile duct associated with anomalous choledochopancreatic duct junction (ACPDJ) is reported. The patient was a 61-year-old Japanese woman who with presented right upper quadrant abdominal pain. Liver function tests results were normal. Computed tomography showed a polypoid lesion in the gallbladder, and endoscopic retrograde cholangiopancreatography (ERCP) demonstrated ACPDJ and irregular wall of the inferior bile duct. A diagnosis of double cancers of the gallbladder and bile duct was made and a pancreaticoduodenectomy and liver bed resection was performed. His topathological examination showed papillary adenocarcinoma of the gallbladder and mucosal adenocarcinoma of the bile duct. The patient is in good health 15 months after the operation and shows no signs of recurrence. A review of the literature is presented.

Synthesis of cyclodeca-1,5-diyne derivatives having o-nitrophenylseleno group.

7-Benzoyloxy-3-(2-nitrophenylseleno)-1,5-cyclodecadiyne (13) was prepared from 5-hexyn-1-ol and propargyl bromide via 10-iodo-7-(t-butyldiphenylsiloxy)-5,9-decadiynal (10). The facile cyclization of the acyclic precursor 10 would be rationalized in terms of "bulky group (TBDPSO) assisted conformational control". Oxidation of 13 by NaIO4, followed by thermal treatment gave tetrahydronaphthalene derivative 15, indicating the generation of biradical.

Estimation of the optimal cut off point in a new immunological faecal occult blood test in a corporate colorectal cancer screening programme.

OBJECTIVE: To estimate the optimal cut off point in a new immunological method (OC-Hemodia) for faecal occult blood testing (FOBT). SETTING: A corporate colorectal cancer screening programme in Japan. METHOD - The screening programme targeted colorectal cancer and adenomatous polyps > or = 10 mm, and was conducted on 27 860 participants (age > or = 40 ) during 1991-92. The follow up consisted of diagnostic management by total colonoscopy on positive screened subjects exceeding the manufacturer recommended cut off level of 50 ng/ml faecal haemoglobin, and the identification of false negative cases by health insurance claims. The optimal cut off point was estimated by the positive predictive value, receiver operating characteristic (ROC) curve, and a cost effectiveness analysis. In this study evaluation was carried out only for cancer as the target disease. RESULTS: - At the current cut off level of 50 ng/ml the sensitivity and specificity were 86.5% and 94.9%. When the optimal cut off point was estimated the highest positive predictive value was obtained at 250-350 ng/ml. The ROC curve showed that the sum of sensitivity and specificity is maximised at 50 ng/ml, but evaluation of the ratio, change in sensitivity/change in false positive rate, pointed to higher optimal cut off points, showing marked changes occurring at about 200 ng/ml. The average cost per case was lowest at 250-300 ng/ml. Overall, the optimal cut off point was estimated to be about 200 ng/ml, at which the sensitivity and specificity of the test would be 77.5% and 98.9%, respectively. CONCLUSION: The optimal cut off point of the new immunological method of FOBT was estimated to be about 200 ng/ml, a value which, more than the current cut off value, favours specificity over sensitivity.

[Efficacy of treatment with frequent and low-dose epirubicin in two cases of pulmonary metastases after surgery of liver cancer].

Case 1 was a 55-year-old male with multiple pulmonary metastases after two operations to excise hepatocellular carcinoma. Treatment consisted of initial administration of 5'-DFUR (po), followed by frequent administration of low-dose epirubicin (20 mg/body once every 2 weeks iv). This resulted in disappearance of the pulmonary metastases and a striking decrease in the AFP level. Case 2 was a 68-year-old male with multiple pulmonary metastases after surgery for hepatocellular carcinoma. Treatment with 5-FU (iv; once a week) and epirubicin (20 mg/body once every 2 weeks iv) resulted in disappearance of the pulmonary metastases and a marked decrease in the AFP level. It was concluded that administration of epirubicin in frequent, low doses is a useful method for the treatment of pulmonary metastasis after surgery for liver cancer, and it can be safely performed even on an outpatient basis.

Gene targeting with a replication-defective adenovirus vector.

Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gene inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants examined had experienced a precise correction of the mutant neo gene. Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy.

A rapid method for isolation of retinal pigment epithelial cells from rat eyeballs.

A new, simple method for isolating retinal pigment epithelial (RPE) cells from rat eyeballs was developed: incubation of the eyeballs in 0.1% proteinase K solution. This method yielded an average of 4 x 10(4) viable RPE cells from each Sprague-Dawley rat eyeball in 1 h. These RPE cells were hexagonal and had cytokeratin in their cytoplasm and numerous microvilli on their surface, similar to RPE cells evaluated in vivo. The results of this study thus show that our method for incubating eyeballs in proteinase K can provide good-quality RPE cells in sufficient quantities for study in a short time.