A mouse model of a human multiple GIST family with KIT-Asp820Tyr mutation generated by a knock-in strategy.

Several families exhibiting multiple gastrointestinal stromal tumours (GISTs) and germline c-kit gene mutations at exons 8, 11, 13, or 17 have been reported. These patients also exhibit diffuse hyperplasia of the interstitial cells of Cajal (ICCs) as a pre-existing lesion of multiple GISTs. We generated a mouse model of a family with germline c-kit gene mutation at exon 17, and compared the phenotypes between the mice and humans. The mouse counterpart (KIT-Asp818Tyr) of the human KIT-Asp820Tyr mutation was transmitted into germline by a knock-in strategy. Mating of male and female heterozygotes (KIT-Asp818Tyr/+) resulted in the generation of homozygotes (KIT-Asp818Tyr/KIT-Asp818Tyr). Histological examination revealed that all heterozygotes had both a small KIT-positive mesenchymal tumour at the caecum, consistent with GIST, and KIT-positive diffuse spindle-shaped cell proliferation in the distal oesophagus, stomach, proximal duodenum, and colon consistent with ICC hyperplasia. All homozygotes exhibited a larger caecal tumour and more prominent spindle-shaped cell proliferation compared with the heterozygous mice, and they usually died within 10 weeks after birth, likely due to ileus. The small intestine of both genotypes showed no apparent morphological abnormality, and autonomous contraction of the ileal segments appeared normal. Western blotting demonstrated that the caecal tumours expressed phosphorylated KIT, MAPK, Stat1, and Stat5. These mutant mice are considered to be useful for further investigation of the mechanism of GIST development as a result of ICC hyperplasia and for assessment of the in vivo effects of drugs against molecular targets.

A versatile dual-channel carbon dioxide (CO2) insufflator for various CO2)applications. The prototype.

BACKGROUND: Carbon dioxide (CO2), with its rapid absorptive nature, has been proven superior to atmospheric air as an insufflating agent in various clinical settings. However, CO2 insufflation has not gained wide clinical acceptance, mainly because there has been no suitable feeding system. The authors therefore have developed a versatile "dual-channel" CO2 insufflator that facilitates wider use of CO2. The objectives of this study were to introduce the authors' prototype insufflator, to evaluate its safety and performance, and to validate CO2 application using the prototype. METHODS: The prototype insufflator provides one CO2 inlet connected to a regular CO2 gas cylinder and two CO2 outlets positioned on the front and back of the device, respectively. The CO2 gas fed from the cylinder is pressure-regulated and divided into two independent conduits inside the device. The front outlet feeds CO2 gas for pneumoperitoneum at an electronically controlled pressure and flow rate. The back channel supplies CO2 gas at a fixed flow rate, allowing manual control of insufflation for various purposes. The device was evaluated with canine models. RESULTS: The prototype was safe and performed well. The CO2 application (colonoscopy in this series) using the back channel was feasible while intact CO2 pneumoperitoneum was simultaneously maintained via the front channel. There were no device malfunctions. The serial abdominal x-rays indicated that intraluminal CO2 insufflation such as that used for CO2 colonoscopy caused less residual intestinal gas than conventional air insufflation. CONCLUSIONS: The dual-channel CO2 insufflator enabled two different modes of CO2 insufflation at the same time from a single CO2 cylinder. The authors are now improving the prototype to allow safer and wider usage of CO2 in the operating room.

Large and asymptomatic pancreatic islet cell tumor in a patient with multiple endocrine neoplasia type 1.

The major phenotypes of multiple endocrine neoplasia type 1 (MEN 1) consist of three lesions characterized by hyperparathyroidism, pituitary tumors, and endocrine pancreatic tumors. The endocrine pancreatic tumors are a significant cause of disease-related mortality in MEN 1. Although symptomatic pancreatic tumors such as insulinoma and gastrinoma should be resected, the management of asymptomatic pancreatic tumors is not established. In asymptomatic pancreatic tumors, the most important factor is the propensity for malignant transformation of the tumors. Although there are no means to foresee it, the size of the pancreatic tumors might be predictive of malignant development in MEN 1. We report here a patient with MEN 1 who had a large asymptomatic pancreatic tumor. The patient (72-yr-old man) was diagnosed with primary hyperparathyroidism and underwent a total parathyroidectomy. Genetic examination showed a germline mutation of the MEN1 gene (E45G). Abdominal magnetic resonance imaging revealed a large (>6 cm) tumor with a heterogeneous pattern in the tail of the pancreas. No metastases of the tumor were evident. Serum levels of insulin, gastrin, and glucagon were normal, and the patient had no symptoms. Operative resection was performed, and microscopic examination revealed that the tumor was an islet cell tumor stained with multiple hormones. This is a case indicating that asymptomatic pancreatic tumors associated with MEN 1 might be indolent independent of their size.

Successful autotransplantation of an adrenal gland using a new method of omental wrapping: report of a case.

Bilateral total adrenalectomy results in the need for patients to take lifelong supplements of adrenal steroids, with the risk of possible Addisonian crisis. Few reports of the successful autotransplantation of adrenal tissue in muscular pockets have been documented; however, we describe herein the case of a 22-year-old woman in whom autotransplantation of an adrenal gland was successfully performed employing a new method of omental wrapping. The patient underwent bilateral total adrenalectomy for bilateral pheochromocytoma at which time adrenal tissue was sliced into 1-2-mm thick pieces, half of which were placed in muscular pockets in the abdominal rectus muscles, and the remaining half put onto the omentum and wrapped with it. Laboratory examinations done 6 months after surgery showed recovery of her adrenal function, and 4 months later steroid supplements were able to be discontinued. Scintigraphic studies using [131I]-iodomethyl-norcholesterol ([131]I-Adosterol) demonstrated clear activity in the omentum, weak activity in the rectus muscles, and no activity in the adrenal beds. These findings suggest that the omentum may be more suitable as an implantation site for adrenal tissue than muscular pockets.

A family of MEN1 with a novel germline missense mutation and benign polymorphisms.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1) has recently been cloned, and its germline mutations were identified in patients with this syndrome. The majority of the mutations, frameshift or nonsense mutations, are expected to result in a loss of function of the gene product menin. Since the consequence of less common missense or in-frame deletion mutations is not clear, careful judgment is necessary regarding the role(s) of such mutations in MEN1 disease. Here we describe a large multigenerational MEN1 family with a novel germline missense mutation and three benign polymorphisms. The proband was a man with hyperparathyroidism and thymic carcinoid. We performed biochemical studies and DNA analyses of the MEN1 gene simultaneously and independently as family screening studies. Seven patients including the proband were identified, and all of them carried a heterozygous germline missense mutation E45G, but 5 members with normal biochemical results did not. This mutation was not observed in 50 normal volunteers. This novel missense mutation is therefore almost conclusively responsible for the disease. Although all of the mutant gene carriers in the present study already had clinical diseases, an MEN1 gene analysis in younger individuals at risk would be very useful in identifying carriers before the onset of the symptoms.

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.

A sensitive determination method for mexiletine derivatized with dansyl chloride in rat plasma utilizing a HPLC peroxyoxalate chemiluminescence detection system.

A sensitive determination method for a non-fluorescent anti-arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO-CL) detection system. After an internal standard (4-methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 microL rat plasma, the solution was poured onto an Extrelut 1 column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH 8.5) and mixed with dansyl chloride (75 nmol) in acetronitrile. After standing of 90 min at room temperature, 0.5 N HCl was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO-CL detection system using bis(4-nitro-2(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20-100 ng/mL plasma (20.6-103 fmol/injection). The detection limit (S/N = 2) was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug [25 mg (115.9 mumol)/kg] to rats.

[EEG data filing system with personal computer and magneto-optical disc].

We established a new computerized EEG filing system, in which an EEG machine was connected to an EEG filing unit which consisted of a personal computer with a 32 bit CPU, 18 channel analog -to-digital and digital-to-analog converters, and a magneto-optical disc driver. This system has the following merits. The system was space-saving, the space required to store the EEG data, being only 1/500 of that required for storing EEG record. Though the system had two independent EEG filing units, our original file configuration enabled us to have access to all of the recorded EEG data anytime for more than 10 years. The system enabled us to display EEG data continuously on the high resolution CRT, just as if turning the page of an EEG record paper, and through the quick scanning, enabled us to view the entire EEG pattern. Furthermore, the system enabled us to conduct reliable digital analysis for EEG data by entering the EEG data without artifacts into a data processor after confirmation on the display. In the system, we used data files with the MS-DOS operating system. This enabled us to analyze the data obtained by a personal computer operating on other systems, by transferring the data to a floppy disc operating on our system. Our EEG filing system allows quick access to data as well as mass data storage.

Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography.

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO-CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO-CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4-dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5-7 and pH 3-4, respectively. Dipyridamole and benzydamine hydrochloride (anti-inflammatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.

Application of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)-phenyl]oxalate to post-column chemiluminescence detection in high-performance liquid chromatography.

Bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) was used to examine the peroxyoxalate chemiluminescence (CL) reaction for the detection of fluorescent compounds. Some fluorescent compounds (perylene, eosine, rhodamine, Rose Bengal, fluorescein and umbelliferone) gave higher CL intensities as the proportion of water in the reaction medium increased to ca. 40%, whereas dansylalanine, 8-anilinonaphthalene-1-sulphonic acid, 7-nitrobenzo-2-oxa-1,3-diazol-4-ylproline and dihydronicotinamide adenine dinucleotide gave opposite results. The effects of temperature and time on the post-column reaction in reversed-phase high-performance liquid chromatography (HPLC) were investigated. Under the optimal conditions, the detection limit for dansylamino acids was at the sub-femtomole level. The advantage of using TDPO in HPLC is its stability in the presence of hydrogen peroxide [ca. 10% loss of activity per 8 h vs. 60% per 8 h for bis(2,4,6-trichlorophenyl) oxalate].