Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.     

Elevated immunoreactive-somatostatin levels in the brain of ataxic mutant mice

Immunoreactive-somatostatin (IR-SRIF) levels were investigated in the brain of 4 types of ataxic mice (Rolling Mouse Nagoya, Weaver, PCD, Staggerer) with different cerebellar pathologies. IR-SRIF concentrations (ng/mg) were found to be significantly elevated in both cerebellum and cerebrum of all ataxic mutant mice, IR-SRIF (ng/organ) was found to be increased in the cerebellum and cerebrum in Rolling Mouse Nagoya and PCD compared with control mice. The gel-filtration profile (Sephadex G-50) in the cerebellar extracts of Rolling Mouse Nagoya proved to be identical to that of control mice. Three peaks of IR-SRIF were found to be uniformly elevated in Rolling Mouse Nagoya, with the highest peak coinciding with authentic somatostatin-14. The present results suggest that elevated levels of IR-SRIF in the brain may play a role in the mechanism underlying the manifestation of ataxia in ataxic mutant mice, especially in Rolling Mouse Nagoya and PCD.     

Down-regulation of macrophage Ia mRNA expression by interferon (IFN)-alpha and IFN-beta mediated by de novo synthesized protein

We investigated the regulation of class I and class II major histocompatibility complex (MHC) antigen expression of murine peritoneal macrophages (M phi) by interferons (IFNs) at the mRNA level. Enhancement of class I antigen expression by IFNs (IFN-alpha, beta, and gamma), induction of class II antigen expression by IFN-gamma, and inhibition of class II antigen expression by IFN-alpha or IFN-beta all corresponded to steady-state levels of these MHC-specific mRNAs. Cycloheximide (CHX), a protein synthesis inhibitor, was used to elucidate whether IFN regulation of MHC mRNA expression depends on the newly synthesized proteins. CHX concentration was carefully chosen so that M phi viability was not decreased, total protein synthesis was considerably but not completely inhibited, and suppression of surface class II expression was virtually perfect. Under these conditions CHX did not affect the levels of either class I or class II mRNA, but it prevented IFN-beta from interfering with class II mRNA induction by IFN-gamma. These results indicate that the augmentation of induction and/or accumulation of MHC mRNA by IFNs is not dependent on the de novo synthesis of protein, but the down-regulation of class II mRNA level by IFN-beta is mediated by some newly synthesized protein(s).     

A case of glucocorticoid-suppressible hyperaldosteronism with aldosterone producing adenoma

A 34-yr-old woman with hypertension (142/102 mmHg), hypokalemia, high plasma and urinary aldosterone and low plasma renin activity was studied. A left adrenal tumor and enlarged right adrenal gland were demonstrated by adrenal venography. During administration of dexamethasone (2 mg daily, for 3 weeks), urinary aldosterone excretion decreased abruptly from 22.5 to 9-11 micrograms/day, serum potassium increased and blood pressure fell to 120-130/80-90 mmHg. After left adrenalectomy, all manifestations improved with no medication. The resected adrenal gland revealed clear cell adenoma and micronodular adrenocortical hyperplasia. The patient was considered to be a rare case of glucocorticoid-suppressible hyperaldosteronism with an aldosterone-producing adenoma.     

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.     

Purification and identification of [hydroxyprolyl3]bradykinin in ascitic fluid from a patient with gastric cancer

Kinins in the ascitic fluid from a patient with gastric cancer were purified by gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two fractions (fractions I and II) showed kinin activity. Fraction I did not correspond to either bradykinin or other known kinins, whereas fraction II corresponded to bradykinin. Fraction I contained 8 amino acid residues from bradykinin minus 1 proline plus 1 additional hydroxyproline. Sequence analysis of fraction I showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of fraction I on reversed-phase HPLC was exactly the same as that of synthetic [hydroxyprolyl3]bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of [hydroxyprolyl3]bradykinin in vivo. This is also the first report of the presence of bradykinin in human tumor ascites.     

Tyrosinase-catalyzed binding of 3,4-dihydroxyphenylalanine with proteins through the sulfhydryl group

The cytotoxicity of catechols has been ascribed to covalent binding of the omicron-quinone oxidation products to proteins through sulfhydryl groups. The nature of the covalent binding was studied with dopaquinone formed on tyrosinase oxidation of 3,4-dihydroxyphenylalanine (DOPA). After acid hydrolysis of the reaction products, cysteinyldopas liberated (protein-bound cysteinyldopas) were determined by HPLC with electrochemical detection. When 0.1 mM DOPA was oxidized in the presence of 0.2 mM bovine serum albumin, alcohol dehydrogenase or isocitrate dehydrogenase, protein-bound cysteinyldopas were formed in yields of 5.4, 44, or 33%, respectively. The covalent binding was almost completely inhibited by 1 mM cysteine or 1 mM ascorbic acid, but 10 mM lysine had no effect. These results unambiguously demonstrate that dopaquinone can bind with proteins mostly through sulfhydryl groups.     

Growth experiment of Hantaan virus in A549 cells: an attempt to improve the immunofluorescent antibody technique for hemorrhagic fever with renal syndrome

An assay method for the infectivity of Hantaan virus, a causative agent of HFRS (hemorrhagic fever with renal syndrome), was developed by the use of IFA (immunofluorescent antibody technique). With the aid of this method, the growth characteristics of Hantaan virus, 76-118 strain, were followed in A549 cells. At a maximal MOI (multiplicity of infection) of 1.6 VAIU (viral antigen-inducing units) per cell, the conventionally available value, plateau level potencies of the viral antigen and virus infectivity were attained at eight and ten days postinfection, respectively, and most of the infective virus produced accumulated in the culture fluids of infected cells. When infections were defined with MOI values in terms of VAIU per cell, development of the viral antigen was highly consistent and followed a given pattern of kinetics. Based on these findings, a protocol for preparation of the viral antigen in IFA was presented, wherein spot culture and FBS treatment were emphasized as effective procedures to minimize non-specific staining.     

Diurnal rhythms of proopiomelanocortin-derived N-terminal peptide, beta-lipotropin, beta-endorphin and adrenocorticotropin in normal subjects and in patients with Addison's disease and Cushing's disease

In order to clarify the diurnal pattern of secretion of plasma immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides, IR-N-terminal peptide (Nt), IR-beta-endorphin (Ep), IR-beta-lipotropin (LPH), and IR-ACTH (ACTH) in normal subjects and in patients with Addison's disease and Cushing's disease, we measured these 4 peptides in the same plasma obtained at 0900 h and then every three hours until 0600 h at the next day. All four peptides showed diurnal rhythms with the peaks at 0600 h, and the nadirs of ACTH, LPH, Ep and Nt were at 0000 h, 0000 h, 1800 h and 0300, respectively in normal subjects. In patients with Addison's disease, these four peptides also showed diurnal rhythms with the peaks at 0600 h for ACTH and Ep and at 0900 h for LPH and Nt, and the nadirs at 2100 h for ACTH and Ep and at 0000 h for LPH and Nt. The molar ratios of Ep/ACTH, LPH/ACTH and Nt/ACTH in plasma also presented diurnal variations in normal subjects and in patients with Addison's disease. On the other hand, in patients with Cushing's disease, ACTH, LPH and Nt showed no rhythmicity or change in molar ratios of Ep/ACTH, LPH/ACTH or Nt/ACTH. Only Ep showed diurnal variation. The molar ratios of Ep/ACTH, LPH/ACTH and Nt/ACTH in patients with Cushing's disease were significantly higher than those in normal subjects and in patients with Addison's disease at 0000 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.