Effects of three peptidase inhibitors, amastatin, captopril and phosphoramidon, on the hydrolysis of [Met5]-enkephalin-Arg6-Phe7 and other opioid peptides

The contents of [Met⁵]-enkephalin-Arg⁶-Phe⁷ (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37° C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enk-hydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, estimations of [Leu⁵]-enkephalin (leu-enk), α- and β-neoendorphins (α- and β-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: α-neoend, β-neoend, and dyn B. Received: 15 July 1997 / Accepted: 20 November 1997     

TrkA expression is associated with an elevated level of apoptosis in classic medulloblastomas

Medulloblastomas represent the most common central nervous system malignancies in children. Despite intensive modality treatment with craniospinal irradiation and multiple drug chemotherapy, their prognosis remains dismal. In the present study, we examined the potential roles of cellular differentiation, proliferation and apoptosis in 21 pediatric patients with newly diagnosed classic medulloblastomas treated by conventional radiation therapy and adjuvant chemotherapy. The expression of glial fibrillary acidic protein, S‐100, synaptophysin, TrkA and TrkC, and the proliferation index of MIB‐1 were evaluated by immunohistochemistry and the apoptotic index was determined using terminal deoxytransferase‐mediated deoxyuridine‐5′‐triphosphate nick‐end labeling assay. The prognostic value of these biological markers was also assessed. Immunoreactive glial fibrillary acidic protein, S‐100, synaptophysin, TrkA and TrkC were observed in seven (33%), four (19%), 12 (57%), 14 (67%) and 11 (52%) of the 21 cases, respectively. TrkA expression was positively correlated with the MIB‐1 staining index (P = 0.0228) and the apoptotic index (P = 0.0058). None of the immunohistochemical markers was found to be of value in predicting the prognosis. Although the present small sample size does not provide sufficient power to discount biological variables as prognostic markers, it was the well‐established clinical prognostic factors, i.e. tumor stage and extent of surgery, that stood out as the most important predictors of survival. The close association between apoptosis and TrkA expression is consistent with in vitro data demonstrating the capacity of the NGF/TrkA signaling pathway to increase medulloblastoma apoptotic cell death, suggesting that this pathway may yield alternative therapeutic targets for novel therapies.     

Urolithiasis--changes in its treatment in the traditional surgical management

Surgical treatments for urolithiasis in the upper urinary tract are reviewed on the basis of operation statistics at the Department of Urology, Osaka University Hospital for the past 30 years from 1957 to 1986. Open surgery was applied for 1,624 patients with urolithiasis in the kidney and ureter during this period. These operations accounted for 14.4% of the 11,300 cases of urological surgery at our department. Types and frequency of operations for urolithiasis were as follows: ureterolithotomies 697 cases (6.2%), pyelolithotomies including extended pyelotomy 376 cases (3.3%), nephrolithotomies 294 cases (2.6%), partial nephrectomies 132 cases (1.2%) and nephrectomies 125 cases (1.1%).     

Perifusion of isolated rat pancreatic acini: carbamylcholine-induced biphasic amylase release

To solve many problems in other in-vitro methods such as perfusion and static incubation, perifusion of isolated rat pancreatic acini was established. Continuous stimulation by carbamylcholine induced a biphasic secretory response, a sharp initial phase and a slow-varying second phase. The junction of these two phases occurred at almost the same time (in about 7 min of stimulation) at any concentration of carbamylcholine. The slow-rising former part of a second phase indicated that a biphasic pattern is produced by overlapping of two kinds of real phases and the beginning of a real second phase occurs at the time of the junction. The concentration dependence of amylase release showed a bell-shaped pattern and the maximal amylase release at an optimal concentration (10(-6)M) was 5.43 +/- 0.24% total/30 min. The sustained latter part of a second phase showed a slow decline at optimal or less concentrations but a plateau at supraoptimal concentrations.     

Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets.

Human epidermal growth factor (hEGF) has previously been isolated from urine and probably is identical to human beta-urogastrone (hUG). Immunoreactive hEGF/UG has been found in the plasma of normal subjects. In this study, using immunoaffinity chromatography to extract hEGF/UG from plasma, we found that immunoreactive hEGF/UG in blood was associated with blood platelets. It was present in platelet-rich, but not platelet-poor plasma and serum, and was found predominantly in the platelet fraction of whole blood. Sephadex G-50 Fine gel-exclusion chromatography of an extract of outdated blood bank platelets revealed two hEGF/UG components, one of which eluted in the void volume, and the other of which coeluted with purified standard hEGF/UG. The former hEGF/UG component was a high-molecular weight form that was cleaved into hEGF/UG by incubation with either mouse EGF/UG-associated arginine esterase or trypsin. It appeared to be identical to the high-molecular weight hEGF/UG previously reported in human urine, except for its apparently equal activities in radioimmunoassay and radioreceptor assay. The latter hEGF/UG component was immunologically, biologically, and physiochemically indistinguishable from highly purified hEGF/UG from human urine and was immunologically different from purified human platelet-derived growth factor. Platelet-associated hEGF/UG may account for the mitogenic activity of serum in cell lines in which platelet-derived growth factor is not active. Since hEGF/UG appears to be liberated from platelets during coagulation, platelet-associated EGF/UG may be involved in normal vascular and tissue repair and in the pathogenesis of atherosclerotic lesions. The discovery that the EGF/UG in plasma is associated with blood platelets raises important new possibilities for its role in human health and disease.     

Intracytoplasmic localization of CD3 antigen in NKH1+ azurophilic granular T-lymphoblastic lymphoma cells

The intracytoplasmic localization of CD3 antigen was examined in azurophilic granular T-lymphoblastic lymphoma cells obtained from a 27-year-old female. Examination by flow cytometry and an immunohistological method revealed that the surface phenotype of the lymphoma cells were NKH1+, CD5+, CD7+, CD1-, CD2-, CD4-, CD8- and CD16-. CD3 was not detected on the cell-surface by flow cytometry but was found in the cytoplasm using the immunohistological method. CD3 was detected in the perinuclear space and rough endoplasmic reticulum (rough ER) by an immunoelectron microscopic study. An electron microscopic observation of the granules revealed lysosomal structures with low electron density. The lymphoma cells proliferated when cultured in the presence of interleukin-2 but showed no NK (K562) activity before and after culture. The molecular size of metabolically labeled intracytoplasmic CD3 precipitated by Leu 4 monoclonal antibody was identical to that of normal CD3. These results indicate that the lymphoma cells are neoplastic counterparts of immature thymocyte at a very early stage of differentiation and may be related to the lineage of CD3+ NKH1+ T-lymphocyte.