Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.     

Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.     

Purification and Characterization of a Fibrinogen-Degrading Protease in Bacteroides fragilis Strain YCH46

A novel fibrinogenolytic protease was purified from Bacteroides fragilis strain YCH46. The protease was extracted from cells by ultrasonic treatment and was purified 425-fold with a recovery of 2.1% by sequential procedures using azocasein as a substrate. The purified protease showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 100 kDa, which was consistent with the value obtained by gel filtration, indicating a monomeric native structure. Its optimal pH, K_m, and V_max for azocasein were 7.5, 0.2%, and 286 U/min/mg, respectively. The protease activity was completely inhibited by addition of 1 mM Hg²⁺, Cu²⁺, Zn²⁺, diisopropyl fluorophosphate, N-ethylmaleimide or p-chloromercuribenzoate but not by the inhibitors of metalloprotease or aspartic protease, suggesting that the enzyme is a serine-thiol-like protease. The protease hydrolyzed azocasein, casein, fibrinogen, gelatin, and azocoll, but not bovine serum albumin, ovalbumin, fibrin, fibronectin, immunoglobulins, transferrin, hemoglobin or types I, III, and IV collagen. The enzyme also hydrolyzed the chromogenic substrates alanyl-alanine p-nitroanilide, L -valyl-alanine p-nitroanilide, alanyl-alanyl-valyl-alanine p-nitroanilide, and glycyl-proline p-nitroanilide, but was inert toward L -alanine p-nitroanilide, alanyl-alanyl-alanine p-nitroanilide, and N-α-benzoyl-DL -arginine p-nitroanilide. The protease completely hydrolyzed the α-chain of fibrinogen at 37 C within 10 hr and at the same time the time required for clotting of protease-treated fibrinogen by thrombin was prolonged. The fibrinogenolytic activity of a crude extract of B. fragilis was stronger than that of other species of the Bacteroides fragilis group tested: B. ovatus, B. distasonis, B. eggerthii, B. uniformis, and B. thetaiotaomicron. These results suggest that the fibrinogenolytic protease is an important biological factor in Bacteroides infection.     

Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Binding and labeling of omega-conotoxin GVIA in crude membranes from subfractionated fractions and various areas of chick brain

Specific binding and specific labeling of¹²⁵I-ω-CgTX were investigated in crude membranes from both subfractionated fractions and various brain areas in chick whole brain. The specific activities of the marker enzymes 2′,3′-cyclic nucleotide 3′-phosphorylase, Na/K ATPase and succinic dehydrogenase in the subfractionated fractions were three- to five-fold higher than those in the P₂ fraction. However, the amount of specific [¹²⁵I]ω-CgTX binding in the fractions of synaptosomes and synaptic plasma membranes was only about 1.2-times higher than that in the P₂ fraction. The characteristics of specific¹²⁵I-ω-CgTX labeling with disccinimidyl suberate to the 135-kDa band were generally comparable to those of specific [¹²⁵I]ω-CgTX binding sites. These results suggest that the specific binding sites of [¹²⁵I]ω-CgTX were not localized the synaptosomes and synaptic plasma membranes fractions, although each fraction was well isolated from the others from which were decided by the strength of specific activity for marker enzymes.     

First evidence of Wolbachia infection in populations of grasshopper Podisma sapporensis (Orthoptera: Acrididae)

The brachypterous grasshopper Podisma sapporensis (Orthoptera: Acrididae) is distributed throughout the Sakhalin, Kunashir and Hokkaido Islands. Karyotypes of this species consist of two major chromosomal races with different sex chromosome systems, XO/XX and XY/XX. Molecular phylogeographic analysis of the chromosome races and subraces confirms the genetic divergence of the races and subraces in P. sapporensis. Here we first report that P. sapporensis is infected with Wolbachia consisting of three variants on wsp locus, while gatB locus was monomorphic. Furthermore, observation of cell tissue of P. sapporensis using electron microscopy confirmed the infection of Wolbachia that was inferred from polymerase chain reaction and revealed the distribution of the bacteria in the head, thorax and abdomen of P. sapporensis embryos. Our finding may shed new light on Wolbachia as a possible agent causing hybrid dysfunction resulting from experimental crosses between chromosome races or subraces of P. sapporensis.     

Androgen receptor, testosterone uptake and karyotype in androgen-dependent mouse tumor (SC 115) and its androgen-independent subline (CS 2)

Content of androgen receptor, retention of injected testosterone and karyotype of SC 115, androgen-dependent tumor, were compared with those of CS 2, an androgen-independent subline derived from SC 115. Although Bmax was less than that of SC 115, androgen receptor was present in the cytosol and the nuclear extract from CS 2. To examine the ability for androgen retention, a large amount of testosterone was injected into tumor-bearing mice, and the amount of androgen in the crude nuclear and postnuclear fractions of tumors was compared. In both fractions, retention of injected androgen was higher in the SC 115 than in the CS 2. Since most of the injected testosterone was not metabolized in the tissues and the injection of testosterone 5 alpha-reductase inhibitor showed no significant influence on the growth rate of the SC 115, intracellular active androgen was assumed to be testosterone in these tumor cells. As the CS 2 was tetraploid, the androgen independency of the CS 2 seems to be related to chromosomal changes.     

Properties of progestin-binding protein in benign hypertrophic human prostate

RU 27987 is a new ligand for progesterone receptor and binds in high affinity to nuclei of target tissues of progesterone. Using this compound, progestin-binding components in the benign hypertrophic human prostate were studied, and compared with those examined with R 5020, a conventional ligand, in the study of progesterone receptor. In cytosols, the binding affinity of RU 27987 was higher than that of R 5020, and the number of maximum binding sites for RU 27987 seemed to be large but correlated well with those of R 5020. The binder for RU 27987 sedimented at 8.6 S, and the binding was specific to progestational steroids, indicating that binding properties of this binder in the cytosols are identical to those for R 5020. Although there was no binding with R 5020 in the nuclear extract, a small amount of specific binding with RU 27987 was detected. However, the cytosol bound with RU 27987 was not retained in DNA Sepharose and no specific binder for RU 27987 in the nuclear extract was observed in a sucrose density gradient centrifugation. From these observations, it was assumed that the nuclear binding observed was attributable to contamination of the cytosolic binder. The results obtained in the present study suggest that the progestin-binding component in the benign prostatic hypertrophy is not the progesterone receptor but a high affinity binder for progestins whose physiological role is not clear at present.