Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

The first synthesis and antifungal activities of 9-methoxystrobilurin-type beta-substituted beta-methoxyacrylate

The first synthesis of 9-methoxystrobilurin-type beta-substituted MOAs was successfully achieved. A chiral oudemansin-type beta-substituted MOA was also synthesized utilizing Mukaiyama's asymmetric aldol reaction. Antifungal activities of the synthesized compounds against several representative fungi were examined by disk-diffusion assay. As a result, unique and superior antifungal properties of 9-methoxystrobilurin-type beta-substituted MOAs compared with those of oudemansin-type analogue were clearly revealed.     

The relationship and its change with aging between ADL and daily life satisfaction characteristics in independent Japanese elderly living at home

The purposes of this study were to examine the characteristics of the relationship between ADL ability and daily life satisfaction and the pattern change with aging in independent Japanese elderly, and to compare these tendencies between males and females. The characteristics of ADL ability and daily life satisfaction of 482 subjects (213 males, 269 females) were investigated in a self-response survey. Seventy-four ADL items, considered from previous studies, were selected from nine ADL domains of 1) movement, 2) going up and down stairs, 3) changing and holding posture, 4) bathing, 5) toileting, 6) dressing, 7) grooming, 8) eating, and 9) manual activities, and nine items of daily life satisfaction were selected from physical, psychological and sociological factors. Both ADL ability and life satisfaction of independent elderly tended to decline with aging. From correlation analysis, since life satisfaction of the elderly was higher with high ADL ability level, it was considered that ADL ability level is one of the important factors in providing for life satisfaction of independent elderly. The subjective symptoms of inconvenience in the lower extremity and lumbar region increased from the 70s in both genders, and the use of assisting devices for movement remarkably increased in the 80s in both genders. The use of assisting devices closely related to the activity area in daily life and influenced the characteristics of life satisfaction and its age-related change in the relationship between ADL ability and life satisfaction. The relationship between ADL ability and satisfaction with physical function was similar in both genders, while the relationship between ADL ability and satisfaction with sociological and psychological factors was different between males and females. Since the relationship between ADL ability and life satisfaction of independent elderly is influenced by a combination of personal, cultural, and environmental factors, additional study must investigate in detail the influence of these factors.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Chemokine receptor inhibitor,Antileukinate, suppressed ovalbumin-induced eosinophilic inflammation in the airway

Accumulating evidence suggests that eosinophils play an important role in the pathogenesis of allergic diseases. An eosinophil-active chemokine, eotaxin, and its receptor, C-C chemokine receptor 3, are particularly attractive as novel targets of immunological intervention for the disease. In this study, we examine the effects of a hexa-peptide (Ac-RRWWCR-NH(2)), Antileukinate, which we have previously defined as a potent inhibitor of CXC chemokine receptor 1 and 2, on eotaxin in vitro and in vivo. Antileukinate inhibited the binding of 125I-labeled human eotaxin to human eosinophils with an IC(50) of approximately 10 microM and eosinophil chemotaxis to human eotaxin was significantly inhibited by 10 microM of Antileukinate. We examined the effects of Antileukinate on eosinophil accumulation induced by intraperitoneal administration of murine eotaxin, and confirmed that Antileukinate is also active in the murine system. When Antileukinate was tested in ovalbumin-induced airway inflammation model in vivo, Antileukinate significantly inhibited eosinophil accumulation and allergen-induced increase in total protein in bronchoalveolar lavage fluids. Furthermore, Antileukinate suppressed fibrous thickening of submucosal tissue induced by chronic antigen challenge. These results suggest that eotaxin is involved in the pathogenesis of eosinophilic airway inflammation, and that Antileukinate may be a promising tool to control allergic diseases.     

Studies on Japanese Botryllid Ascidians. III. A new species of the genus Botryllus with a vivid colony color from the vicinity of Shimoda

The morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae are described in detail. This ascidian was collected from the stony shore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). The ascidian colony was easily distinguished from colonies of other botryllids because it was very thin and bright pink in color. The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus. This ascidian was prolific, with 1-5 embryos on each side of a zooid, and the embryos of this ascidian developed in the peribranchial cavity without any brooding organs as in Botryllus scalaris. We observed the processes and features of the allorecognition reaction in colony specificity and found that allorejection occurred after fusion of the vascular system between two incompatible colonies. This manner of allorejection is also shown in B. scalaris and Botryllus delicatus; however, the reaction speed of allorejection is faster than that of B. delicatus and similar to that of B. scalaris. These results indicate that this ascidian might be closely related to B. scalaris.     

Effects of Ovariectomy and Prolactin on Mammary Gland Expressions of Transforming Growth Factor alpha and Epidermal Growth Factor Receptor mRNAs in Mice

Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice at 3 months of age received subcutaneous injections of danazol (0.5 mug / 0.1 ml olive oil, once a day), perphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice a day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased by danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression of both transforming growth factor alpha (TGFalpha) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary gland was increased by danazol. However, TGFalpha mRNA expression was decreased by perphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings suggest that the manifestation of the effect of TGFalpha on mammary gland is rather suppressed by PRL, while mammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGFalpha on the mammary gland growth. OVX resulted in a significant decrease of TGFalpha mRNA expression in the mammary gland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was observed in ICR mice; however, the response to hormonal modulation is generally less susceptible than SHN mice.     

Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47(phox), p67(phox), and Rac with the membrane-associated flavocytochrome b(558). Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529-7538). To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed. In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K(m) for NADPH. O(2) generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b(558) relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b(558) in the active complex.     

Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae

The name Enterobacter kobei sp. nov. is proposed for a group of organisms referred to as NIH Group 21 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of 23 strains of NIH Group 21 to the representative proposed as the type strain of this species averaged 82% at 70°C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 42%. Because the phenotypic resemblance to Enterobacter cloacae is very close and the DNA relatedness (12–42%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 21 were placed in the genus Enterobacter. Close phenotypic and genetic relationships were also found between NIH Group 21 and a member of a group of organisms referred to as Enteric Group 69 at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. It is suggested that the latter could be regarded as a subspecific rank of E. kobei, though this is subject to study of further strains. The majority of strains of E. kobei were isolated from clinical specimens. A culture of the type strain (NIH 1485-79) has been deposited in the Japan Collection of Microorganisms as JCM 8580. Received: 22 March 1996 / Accepted: 19 April 1996