Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Correlation Between the Molecular Structure and the Biological Activity of Co-ARIS, a Cofactor for Acrosome Reaction-Inducing Substance

Two components in the egg jelly are required for inducing the acrosome reaction in starfish; a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and its cofactor called Co-ARIS. Three distinct molecules were isolated as the major Co-ARIS' and designated as Co-ARIS' I, II and III. Structural analysis of Co-ARIS' revealed that they are steroidal saponins comprising a sulfated steroid and a pentasaccharide chain. Co-ARIS' I and II differ only in the steroidal side chain. In the presence of ARIS, each Co-ARIS induced the acrosome reaction with a maximal effect at 100–200 μM (Co-ARIS I) or 25–50 μM (Co-ARIS' II and III). Mixtures of Co-ARIS' I, II and III were more effective than the individuals. The activity of Co-ARIS was considerably reduced by solvolytic desulfation but was not affected at all by periodate oxidation. Reduction by NaBH₄ decreased the activity of Co-ARIS I and enhanced that of Co-ARIS II. Treatment of Co-ARIS III with NaBH₄ did not affect the activity as anticipated from its structure. These results suggest that the sulfate moiety and the side chain of steroid are important for the activity of Co-ARIS. The saccharide chain, however, seems not necessarily to be strictly specified for the activity.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Ecological distribution and phenology of an invasive species, Cardamine hirsuta L., and its native counterpart, Cardamine flexuosa With., in central Japan

Cardamine hirsuta is a European species that was recently introduced into Japan and its wide distribution has been confirmed in the Kanto district. To understand mechanisms of the recent spread of C. hirsuta in Japan, a comparative study of the alien species and its native congeneric species, C. flexuosa, was conducted. Habitat preferences, phenology and seed germination were examined. Cardamine hirsuta and C. flexuosa showed distinctive habitat-preferences; the former was most common in open habitats created by recent man-made constructions, and the latter was common in rice paddy fields and surrounding areas. The results indicate that C. flexuosa is a year-long annual, with a mixed phenology of summer and winter germination and growth. Seed dormancy during summer was relatively weak for C. flexuosa, and some plants that germinated early in summer reproduced during the same summer–autumn period. Plants that germinated in late summer and autumn behaved as winter annuals. In rice paddy fields, C. flexuosa is a winter annual because germination is prevented by submergence during summer. Plants flower during the following spring and complete their life cycle before the fields are flooded for rice cultivation. Cardamine hirsuta showed strong seed dormancy during summer and behaved as a typical winter annual. Seeds of C. hirsuta were intolerant to submergence in water, a condition that breaks seed dormancy of C. flexuosa. The results explain the absence of C. hirsuta from rice paddy fields. It was concluded that the spread of C. hirsuta is attributable to the recent expansion of urban habitats created by human activity and has occurred without direct competition with C. flexuosa. Considering recent urbanization in many areas, it is suggested that C. hirsuta has been spreading rapidly in Japan.     

Determination and Regulation Capacities of the Wing Disc of the Fleshfly, Sarcophaga peregrina

Unilateral extirpation of the wing discs was performed on mid- and late-third instar larvae of Sarcophaga peregrina to investigate the regulation capacities of the thoracic discs. Out of 636 operated larvae, 175 became adult and all of them exhibited hemithoracic structures, lacking any adult structures in the operated side, which were normally produced from a wing disc. These findings suggest that the wing disc does not possess the capacity of bicentric regulation to produce the thoracic structures of the opposite side.     

Methane-flux-dependent lateral faunal changes in a Late Cretaceous chemosymbiotic assemblage from the Nakagawa area of Hokkaido, Japan

A Late Cretaceous carbonate body (2 m in maximum diameter) surrounded by clastic rocks, recently discovered in the Nakagawa area (Hokkaido, Japan), is interpreted as a methane‐seep deposit, on the basis of negative carbon isotopic composition (as low as ?43.5‰), variable sulphide sulphur isotopic composition, high carbonate content, and in situ fractures. It most likely formed owing to methane‐bearing pore‐water diffusion. We estimate that the concentration of methane decreased toward the margin of the carbonate body, and that only small carbonate concretions were precipitated at a certain distance from the methane‐seep centre. These spatial characteristics coincide well with the observed pattern of faunal distribution. The gastropod‐dominated association (indeterminate abyssochrysids and ataphrids and the acmaeid limpet Serradonta sp. are most common) co‐occurs with lucinid and thyasirid bivalves (Thyasira sp., Myrtea sp., and Miltha sp.), and was found within and just above the methane‐derived carbonate body. Acharax and Nucinella (solemyoid bivalves) are more typical of the peripheral part of the methane‐influenced sediments. We suggest that this pattern of faunal distribution reflects the decreasing concentration of methane and apparently also hydrogen sulphide when moving from the centre of discharge toward the periphery of the methane seep.     

Histopathological Analysis of Acinetobacter baumannii Lung Infection in a Mouse Model

Acinetobacter baumannii is the main causative pathogen of nosocomial infections that causes severe infections in the lungs. In this study, we analyzed the histopathological characteristics of lung infection with two strains of A. baumannii (ATCC 19606 and the clinical isolate TK1090) and Pseudomonas aeruginosa PAO-1 in C3H/HeN mice to evaluate the virulence of A. baumannii. Survival was evaluated over 14 days. At 1, 2, 5, or 14 days postinfection, mice of C3H/HeN were sacrificed, and histopathological analysis of lung specimens was also performed. Histopathological changes and accumulation of neutrophils and macrophages in the lungs after infection with A. baumannii and P. aeruginosa were analyzed. Following intratracheal inoculation, the lethality of ATCC 19606- and TK1090-infected mice was lower than that of PAO-1-infected mice. However, when mice were inoculated with a sub-lethal dose of A. baumannii, the lung bacterial burden remained in the mice until 14 days post-infection. Additionally, histopathological analysis revealed that macrophages infiltrated the lung foci of ATCC 19606-, TK1090-, and PAO-1-infected mice. Although neutrophils infiltrated the lung foci of ATCC 19606- and TK1090-infected mice, they poorly infiltrated the lung foci of PAO-1-infected mice. Accumulation of these cells in the lung foci of ATCC 19606- and TK1090-infected mice, but not PAO-1-infected mice, was observed for 14 days post-infection. These results suggest that A. baumannii is not completely eliminated despite the infiltration of immune cells in the lungs and that inflammation lasts for prolonged periods in the lungs. Further studies are required to understand the mechanism of A. baumannii infection, and novel drugs and vaccines should be developed to prevent A. baumannii infection.     

TRANSCELLULAR BIOSYNTHESIS OF CYSTEINYL LEUKOTRIENES BY KUPFFER CELL—HEPATOCYTE COOPERATION IN RAT LIVER

We previously proposed that an enzymatic cooperation between Kupffer cells and hepatocytes may play an important role in cysteinyl leukotriene (LT) production in rat liver. Anin vitrotranscellular synthesis cysteinyl LTs by a Kupffer cell—hepatocyte coculture system was characterized here. Kupffer cells alone, with A23187 stimulation, did not generate cysteinyl LTs until supplemented either with isolated hepatocytes or with LTC₄synthase and glutathione, indicating that Kupffer cells can synthesize LTA₄but not convert it into LTC₄. In contrast, hepatocytes converted the LTA₄into cysteinyl LTs and further degraded the cysteinyl LTs. Cysteinyl LT production by the Kupffer cell—hapatocyte coculture system was optimized by addition of 1–3% serum albumin to the culture and by bringing the cell—cell distance closer to less than 3μ. Tumour necrosis factor also stimulated cysteinyl LT production by the coculture system. From these results, it is expected that the Kupffer cell—hepatocyte transcellular system for cysteinyl LT production actually functionsin vivo.     

A DETAILED FATE MAP OF THE WING DISC OF SARCOPHAGA PEREGRINA

In order to construct an anlageplan of the imaginal disc, various fragments of the wing disc from mature larvae of Sarcophaga peregrina were implanted into host larvae of the same age. The implants were metamorphosed with the host and the differentiated adult structures were analyzed.
On the basis of the results obtained from the transplanted disc fragments, a fate map of the wing disc was satisfactorily constructed.
The fate map of S. peregrina is similar to an earlier study of Drosophila , but the PAA, PWP, and AP are located in different positions from those of Drosophila .