The role of actin in changes of cell shape and steroidogenesis in mouse Y-1 cells stimulated by ACTH--immunocytochemical studies

We investigated the immunocytochemical localization of two kinds of representative cytoskeletal proteins (tubulin and actin) and the morphology of Y-1 mouse adrenal tumor cells under ACTH stimulation at the light and electron microscopic levels. After ACTH stimulation, Y-1 cells showed the rounding-up phenomenon and increased steroidogenesis. Meanwhile, actin was accumulated near the cell membrane and formed filaments. Electron microscopically, stress fibers disappeared with ACTH administration. However, changes in the tubulin localization (main element of the microtubules) were not conspicuous. Cytochalasin B which blocks formation of actin filaments induced rounding-up of Y-1 cells and inhibited increases of steroid synthesis induced by ACTH. Colchicine caused disappearance of the microtubule-organizing center, but the shape of Y-1 cells was not changed. These results suggested that actin-filaments may be the main filament involved in the changes of cell shape and the increased steroidogenesis induced by ACTH.     

Ubiquitinated cytokeratin inclusions in lichen amyloidosus: An immunohistochemical analysis

Eosinophilic hyaline inclusions were consistently seen in the perinuclear cytoplasm of suprabasal keratinocytes in lichen amyiddosus. The inclusions, negative with amylold staining, were immunoreective for ubiquitin and cytokeratin, and uitrastructuraliy showed aggregations of fine filaments of two size (central thin and peripheral thick). The thin filaments were the main component in the upper epidermal layer. Four monoclonal antibodies (AE1, AE3, KL1 and CAM5.2) and om antiserum (WSS) were used for characterizing cytokeratin expression. The AE1 antibody normally stained the basal cells, but in lichen amyloidosus basal staining mostly disappeared. Instead, groups of suprabasal keratrnocytss were labeled, with AE1 -reactive inclusions disfributed therein. In contrast, the KL1 antibody, showing suprabasal staining, failed to react with the inclusions. The includms were weakly reactive with the AE3 and WSS antibodies, which stained all keratinocytes. The CAM5.2 antibody was unreactive. The subepidermal amyloid deposits were negative with all the antibodies. The inclusions were ubiquitinated especialty in the granular layer. Immuno-electron microscopy disclosed that ubiquitin was more denseiy localized in the thin filaments than in the thick ones. This indicated that cytokeratin expression and metabolism are altered in the affected epidermis, and that ubiquitin functions in the process of degradation of abnormal cytokeratin filaments.     

Expression of carbohydrate antigens (SSEA-1, sialyl-Lewis X, DU-PAN-2 and CA19-9) and E-selectin in urothelial carcinoma of the renal pelvis, ureter, and urinary bladder

Carbohydrate antigens and E-selectin play important roles in the invasion and metastasis of cancers. We examined the expression of these antigens and their ligand protein, E-selectin, in urothelial carcinomas to evaluate whether their staining is correlated with the grade and stage of cancer. We studied the expression of carbohydrate antigens (type 1 and type 2 blood-group antigens) and E-selectin in urothelial carcinomas of the renal pelvis, ureter, and urinary bladder in 52 patients by staining SSEA-1 (LeX), sialyl LeX (sLeX), DU-PAN-2, CA19-9, and E-selectin with 5 different monoclonal antibodies (MAbs) to evaluate whether their staining correlated with cancer grade and stage. The differences between organs with regard to the degree of expression of these antigens were not evident. Type 2 antigens (SSEA-1 and sialyl LeX) are frequently expressed in the tumor cells regardless of atypical grade. The expression level of type 1 antigens (DU-PAN-2 and CA19-9) is lower than that of type 2 antigens. However, the presence of DU-PAN-2 tends to correlate with the grade of atypia; however, that of CA19-9 is inversely proportional to the grade of atypia. The lack of CA19-9 and appearance of DU-PAN-2 in urothelial carcinoma implies a high malignant potential. The expression of E-selectin can be correlated with stage and grade of tumor atypia. Type 2 antigen and E-selectin may be involved in tumor invasion and metastasis.     

Japanese guidelines for the management of intussusception in children, 2011

Background: The Japanese Society of Emergency Pediatrics has formulated evidence‐based guidelines for the management of intussusception in children in order to diagnose intussusceptions promptly, to initiate appropriate treatment as early as possible, and to protect intussuscepted children from death. Methods: Literature was collected systematically via the Internet using the key words “intussusception” and “children.” The evidence level of each paper was rated in accordance with the levels of evidence of the Oxford Center for Evidence‐based Medicine. The guidelines consisted of 50 clinical questions and the answers. Grades of recommendation were added to the procedures recommended on the basis of the strength of evidence levels. Results: Three criteria of “diagnostic criteria,”“severity assessment criteria,” and “criteria for patient transfer” were proposed aiming at an early diagnosis, selection of appropriate treatment, and patient transfer for referral to a tertiary hospital in severe cases. Barium is no longer recommended for enema reduction (recommendation D) because the patient becomes severely ill once perforation occurs. Use of other contrast media, such as water‐soluble iodinated contrast, normal saline, or air, is recommended under either fluoroscopic or sonographic guidance. Delayed repeat enema improves reduction success rate, and is recommended if the initial enema partially reduced the intussusception and if the patient condition is stable. Conclusions: The guidelines offer standards of management, but it is not necessarily the purpose of the guidelines to regulate clinical practices. One should judge each individual clinical situation in accordance with experiences, available devices, and the patient's condition.     

Sevoflurane stimulates MAP kinase signal transduction through the activation of PKC alpha and betaII in fetal rat cerebral cortex cultured neuron

Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral -cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (alpha, betaI, betaII, gamma, delta, varepsilon and zeta) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC alpha and PKC betaII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC alpha and betaII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5-10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10-15 min after sevoflurane treatments. That of ERK proteins was induced at 15-60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the -localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC alpha and PKC betaII species.     

Growth hormone-releasing hormone expression in pituitary somatotroph adenomas, studied by immunohistochemistry and in situ hybridization using catalyzed signal amplification system

Growth hormone-releasing hormone (GHRH) is a well-known hypothalamic hormone that stimulates the synthesis and release of growth hormone (GH) as well as the proliferation of GH-producing cells in the anterior pituitary gland. Recent reports have shown GHRH synthesis in pituitary somatotroph adenomas, but GHRH immunoreactivity has not been shown in previous studies. To confirm the role of locally generated GHRH for the progression of somatotroph adenomas, we investigated the expression of GHRH in 25 pituitary somatotroph adenomas immunohistochemically, through the use of both conventional avidin-biotin-complex (ABC) method and novel catalyzed signal amplified (CSA) system. In addition, we investigated the expression of GHRH mRNA and GHRH receptor mRNA with in situ hybridization (ISH) using the CSA system. The weak immunopositivity of GHRH was observed in only 2 adenomas (8.0%) of 25 somatotroph adenomas using the ABC method. In contrast, 15 adenomas (60.0%) of 25 somatotroph adenomas were immunopositive for GHRH, as shown by CSA system. Very few of nonsomatotroph adenomas were immunopositive for GHRH using the CSA system. The expression of GHRH mRNA was confirmed, using the CSA-ISH system in 13 adenomas (72.2%) of 18 somatotroph adenomas. In 11 adenomas (61.1%) of 18 somatotrophic adenomas, the expression of GHRH receptor mRNA was demonstrated using the CSA-ISH system. This is a first report that clarified histopathologically GHRH production in pituitary somatotrophic adenomas. The demonstration of GHRH and its receptor expression is meaningful in clarifying the autocrine or paracrine regulation of GHRH in GH production and progression of pituitary somatotroph adenomas.