Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A.

When AR42J cells, an amylase-secreting pancreatic exocrine cell line, were treated with activin A, cells extended neuritelike processes, and, concomitantly, amylase-containing vesicles disappeared. Immunofluorescence and immunoelectron microscopy revealed that these processes had neurite-specific cytoskeletal architectures: neurofilaments and microtubule bundles with cross-bridges of microtubule-associated protein 2. In addition to such morphological changes, activin-treated cells exhibited a marked increase in cytoplasmic free calcium concentration in response to depolarizing concentration of potassium. Moreover, activin-treated AR42J cells expressed mRNA for alpha 1 subunit of the neuroendocrine/beta cell-type voltage-dependent calcium channel. In naive AR42J cells, a sulfonylurea compound, tolbutamide, did not affect free calcium concentration, while it induced a marked elevation of free calcium in activin-treated cells. Single channel recording of the membrane patch revealed the existence of ATP-sensitive potassium channel in activin-treated cells. These results indicate that activin A converts amylase-secreting AR42J cells to neuronlike cells. Given that pancreatic endocrine cells possess neuronlike properties and express ATP-sensitive potassium channel as well as neuroendocrine/beta cell-type voltage-dependent calcium channel, activin treatment of AR42J cells may provide an in vitro model system to study the conversion of pancreatic exocrine cells to endocrine cells in islets.

Monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) induces apoptosis in primary renal cell carcinoma cells in vitro and inhibits tumor growth in vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. We investigate the susceptibility of human renal cell carcinoma (RCC) cells to TRM-1 and HGS-ETR2, 2 human monoclonal agonistic antibodies specific for TRAIL-R1 and TRAIL-R2, respectively. HGS-ETR2 effectively induced apoptotic cell death in 10 of 11 cell cultures, including 2 human RCC cell lines and 9 human primary RCC cell cultures, with a more pronounced effect after preincubation with anti-human IgG Fc. In contrast, TRM-1 was effective in only 1 primary RCC cell culture. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell-surface expression of the 2 TRAIL receptors, namely that TRAIL-R2 but not TRAIL-R1 was frequently expressed in most of the RCC cells tested. The activities of caspase-9, -8, -6, and -3 were increased with HGS-ETR2-induced apoptosis, and cell death could be blocked by specific caspase inhibitors for caspase-9, -8, and -3, and the general caspase inhibitor. In vivo administration of HGS-ETR2 with or without cross-linker significantly suppressed tumor growth of subcutaneously inoculated human RCC xenografts in immunodeficient mice. These results suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent in RCC.

Lipid peroxidation and its inhibition by tinoridine, II. Ascorbic acid-induced lipid peroxidation of rat liver mitochondria.

Incubation of rat liver mitochondrial suspension with ascorbic acid and Fe2+ resulted in the formation of malondialdehyde and a decrease in the turbidity of the suspension. The maximum amount of malondialdehyde formed during the peroxidation reaction was estimated to be 1 mol per approximately 6 mol of mitochondrial phospholipids. Tinoridine and alpha-tocopherol at the concentration of 5 micron and 1 mM, respectively, completely inhibited the peroxidative disintegration of mitochondria. From the relationship between the concentration of tinoridine and the amount of malondialdehyde formed, it was demonstrated that 1 mol of tinoridine prevents the formation of about 6 mol of malondialdehyde. These findings suggest that there is a limit in the chain reaction of the lipid peroxidation of mitochondria and that the limit is the membrane sphere which is capable of releasing 6 molecules of malondialdehyde and contains about 36 molecules of the constitutive phospholipids.

Morphological analysis of growth hormone release from rat somatotrophs into blood vessels by immunogold electron microscopy.

The ultrastructure of GH release from the somatotrophs of the rat anterior pituitary was examined in vivo by immunogold electron microscopy. Under pentobarbital anesthesia, injection of GH-releasing factor clearly induced an increase in both plasma GH content and the number of exocytotic GH-immunopositive granules in the cells. The exocytotic events occurred from a part of the plasma membrane facing endocrine cells, including other somatotrophs, and other portions facing folliculo-stellate cells or the walls of blood vessels. When released from the plasma membrane, the GH-immunopositive secretory granules sometimes appeared to aggregate with each other and showed an irregular shape surrounded by a single unit membrane. The exocytotic secretory granules were released into the extracellular space, and then flowed into the sinusoids as irregularly shaped GH-immunopositive electron-dense masses. After reaching the vascular space via the intercellular spaces between the endothelial cells, the contents of each mass became diffusely dispersed into the blood stream, with concomitant disappearance of immunopositivity. The present study thus revealed the morphological aspects of the process of GH secretion from somatotrophs into the blood vessels.

Thymolipoma with striated myoid cells. Histological, immunohistochemical, and ultrastructural study.

A large asymptomatic anterior mediastinal thymolipoma, discovered by chest radiograph during a regular check-up for company employees, was excised from a 33-year-old Japanese man. On immunohistochemical and electron-microscopic examination, clusters of myoglobin-positive cells with cytoplasmic Z band structures were found scattered in the medulla. Myoid cells have been previously seen in the normal thymus as well as in thymic hyperplasia, thymoma, and in the thymus of patients with myasthenia gravis. To our knowledge, this is the first reported instance of myoid cells in a thymolipoma.

Rational design, discovery, and synthesis of a novel series of potent growth hormone secretagogues.

In the joint experimental and computational efforts reported here to obtain novel chemical entities as growth hormone secretagogues (GHSs), a small database of peptides and non-peptides known to have GHS activity was used to generate and assess a 3D pharmacophore for this activity. This pharmacophore was obtained using a systematic and efficient procedure, "DistComp", developed in our laboratory. The 3D pharmacophore identified was then used to search 3D databases to explore chemical structures that could be novel GHSs. A number of these were chosen for synthesis and assessment of their ability to release growth hormone (GH) from rat pituitary cells. Among the compounds tested, those with a benzothiazepin scaffold were discovered with micromolar activity. To facilitate lead optimization, a second program, a site-dependent fragment QSAR procedure was developed. This program calculates a library of chemical and physical properties of "fragments" or chemical components in a known pharmacophore and determines which, if any, of these properties are important for the observed activity. The combined use of the 3D pharmacophore and the results of the site-dependent fragment QSAR analysis led to the discovery and synthesis of a novel series of potent GHSs, a number of which had nanomolar in vitro activity.

Detection of deoxyribonuclease I along the secretory pathway in Paneth cells of human small intestine.

The expression and distribution of deoxyribonuclease I (DNase I) in human duodenum, jejunum and ileum were examined by DNase I activity assay and the reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, in situ hybridization, and immunocytochemical ultrastructural analyses. High levels of DNase I were detected in the cytoplasm of Paneth cells in human small intestine. A tissue homogenate fraction rich in Paneth cells showed strong DNase I-specific enzymatic activity. Immunofluorescence analysis using several specific anti-human DNase I antibodies showed very strong immunoreactivity in the cytoplasm of every Paneth cell. In situ hybridization demonstrated high levels of DNase I mRNA in Paneth cells. Immunogold electron microscopy revealed gold particles localized along the secretory pathway, with the exocrine secretory granules mostly labeled. Our findings strongly suggest that Paneth cells synthesize and secrete DNase I into the intestinal lumen.

Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells.

Incubation of Caco-2 cells, a human intestinal cell line, with 25-hydroxycholesterol (25-HOC) marked enhanced cellular cholesteryl ester formation determined by incorporation of [14C]oleic acid into intracellular cholesteryl [14C]oleate. The stimulation by 25-HOC of cholesteryl ester formation was suppressed by staurosporine, a kinase inhibitor, but not by cycloheximide or actinomycin D. The specific activity of microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT) increased two-fold in cells treated with 10 microM 25-HOC for 5 h. ACAT activity decreased when microsomes were incubated without sodium fluoride, a phosphatase inhibitor, but the decrease in ACAT activity in cells stimulated with 25-HOC was more pronounced. The results suggest that protein phosphorylation may be involved in the stimulation of cholesteryl ester formation by 25-HOC in Caco-2 cells.

[A case of primary intracranial malignant melanoma with characteristic magnetic resonance imaging].

The authors reported a rare case of primary malignant melanoma in the central nervous system and discussed the findings of MRI. A 60-year-old male was admitted for examinations to discover the cause of his generalized tonic convulsions. On admission, he had neither neurological deficits, nor were there any abnormalities revealed during physical examination. Ct scan disclosed a slightly high-density mass with perifocal edema in the right parietal cortex which enhanced markedly after injection of contrast material. This lesion was hypointense on T1 weighted image of MRI (GE: 1.5 Tesla) and a hyperintense band was observed on the surface of the tumor. On T2 weighted image, the tumor showed iso-hypointensity surrounded by an increased signal area compatible with edema. On August 31, 1988, a gross total removal of the tumor was performed. Microscopically, it was identified as a malignant melanoma. No melanoma was found in other parts of the body during careful examinations, especially in dermatologic and ophthalmologic examinations. Characteristic findings of the hyperintense band on T1 weighted image coincided well with the pathological findings of excessive melanin deposition on the surface of the tumor, and which resulted from the paramagnetic free-radicals in the melanin. MR image may be useful for differentiation of intracranial malignant melanomas from other mass lesions.

[A case of primary hypothalamic malignant lymphoma with diabetes insipidus].

A case of primary malignant lymphoma involving the hypothalamus is reported. A 31-year-old man was admitted with progressive disorientation. A computed tomography (CT) scan demonstrated a normodense, homogenously enhanced, and nodular shaped mass lesion involving the suprasellar region and extending to the third ventricle. In view of marked cerebral edema, dexamethasone was intravenously administered, at a dose of 4 mg/4 hr, and gradually tapered off. CT scans obtained 10 days after the admission revealed dramatic regression of the lesion, which corresponded with the clinical remission. However, on the 4th day after the discontinuance of corticosteroid, a CT scan demonstrated the recurrence of the mass lesion. Because of the rapid deterioration of consciousness and the progressive enlargement of the mass and dilatation of the ventricles, partial removal of tumor and ventricular drainage were carried out on the 24th day after admission. Dexamethasone was intravenously administered again at a dose of 4 mg/4 hr. A CT scan obtained five days after the operation showed complete disappearance of the lesion, and the patient had became almost alert. CT scans obtained on the 18th day and the 35th day after the operation showed complete disappearance of the mass. However, Magnetic Resonance Imaging revealed a localized area of abnormal signal intensity in the hypothalamus. Diabetes insipidus was continuously observed from the time of admission. A concomitant change of beta-2-microglobulin (beta-2-MG) and immunoglobulin (IgA, IgG, IgM) concentrations in serum and cerebrospinal fluid (CSF) with clinical profiles and CT findings were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Quality assurance for histodiagnosis and cytodiagnosis with a model of proficiency testing].

A quality assurance (QA) program for histopathology and cytology has not yet been completed although an inhouse quality control for technical standardization is being tested. The fact that a strict Performance Improvement Program of CAP (College of American Pathologists) for cytology is required separately from other laboratory tests emphasizes the importance of cytology, since erroneous cytology reports would directly cause incorrect clinical diagnosis. The results of questionnaires gathered by the QA Committee of the Japan Association of Registered Clinical Laboratories on preparation techniques, workload limits of pathologists and technologists, double diagnostic systems, error detection programs, and specialization of personnel are presented. In addition to pathology and cytology proficiency testing provided by CAP survey, a model of blind QC (quality control) in our laboratory is discussed; for diagnostic standardization of histopathology, reexamination of randomly selected cases by another pathologist and reexamination of previously diagnosed cases by the same doctor are periodically performed. For error detection of cytoscreening, cytologists are obligated to reexamine random samples of 1 to 10% of negative gynecological slides. Evaluation of sufficiency of slides as required by the Bethesda System is referred to diagnostic interpretation.

[Histogenesis of the clear cytoplasm in the tumor cells of smooth muscle origin].

Perinuclear clear cytoplasm observed in the light microscopic specimens of the tumor cells of smooth muscle origin is in general, understood as the artefact caused by the formalin fixation. However, the precise mechanisms of the histogenesis of clear cytoplasm are still not clear. We observed the clear cytoplasm directly by mean of the electron microscopy of materials detached from light microscopic specimen. Furthermore, we observed the light microscopic specimens made by varying types of methods, examining whether the clear cytoplasm was present or not. The electron microscopy of materials detached from light microscopic specimens revealed the band-like defects of cytoplasm along the long axes of tumor cells. These defects were thought to result from the falling off of cytoplasm. The 1 mu section of the epon embedded block derived from the paraffin embedded block for light microscopic specimen presented no clear cytoplasm, suggesting that the cytoplasm falls off at the procedure of deparaffinization and staining. Although the specimen of conventional frozen section showed no clear cytoplasm, the specimen made by the frozen sectioning after formalin fixation revealed clear cytoplasm. Consequently, it is thought that the fixation of the tissue before the sectioning makes the cytoplasm fragile, thereafter, the cytoplasm falls off at the procedure of deparaffinization and staining.

[Morphological methods for analysing hormone secretory mechanism in the pituitary cells].

We introduce here two morphological methods of the rat anterior pituitary cells. In the first experiment, we showed immunofluorescence combined with a confocal laser scanning microscope. Distribution of actin was examined in growth-hormone (GH) secreting cells and folliculo-stellate cells and compared between in vivo and in vitro cells. In tissue, actin immunoreactivity was mainly limited near the plasma membrane. In cultured cells localization of actin clearly changed and the cells sometimes showed stress fiber-like structures in the cytoplasm. The cells also showed a small amount of alpha-actinin- and myosin-like immunoreactivities along stress fiber-like structures, which cannot be detected in the tissue cells. These data clearly showed differences in the localization of cytoskeletal proteins between in vivo and in vitro. In the second experiment, we showed electron microscopic analysis combined with monitoring hormone secretion in vivo. A catheter was inserted through the left external jugular vein into the right atrium of the heart. Through the catheter GH-releasing factor (GRF) and/or somatostatin was administered to freely-moving rats. Only about 30% of rats showed an increase in blood GH after GRF injection. Such responded rats were selected for our morphological analysis. From 5 min after GRF injection, the rats were infused with somatostatin or saline for 10 min. The treatment of somatostatin clearly diminished exocytotic figures from the plasma membrane.