Amphotericin B covalent dimers bearing a tartarate linkage

Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 microM) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB.     

Germitorosone and methylgermitorosone,two hydroanthracene derivatives from seedlings of Cassia torosa

Two new yellow pigments, germitosone and methylgermitorosone, were isolated from the seedling of Cassia torosa. The structures of these substances were established as 3,7 dimethyl - 6 - methoxy - 1 - oxo - 2,3,8,9 - tetrahydroxy - 1,2,3,4 - tetrahydroanthracene and 6,9 dimethoxy - 3,7 - dimethyl - 1 - oxo - 2,3,8 - trihydroxy - 1,2,3,4 - tetrahydroanthracene respectively.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Augmentation of Interferon Production after Cell-Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2′,5′-Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.     

A human case of gnathostomiasis nipponica confirmed indirectly by finding infective larvae in leftover largemouth bass meat

A human case of creeping eruption due to Gnathostoma nipponicum was confirmed indirectly by finding infective advanced third-stage larvae in leftover largemouth bass meat. This is the first report indicating that the largemouth bass (Micropterus salmoides) serves as a source of G. nipponicum infection in humans.     

Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with multiple eyespots

We have isolated a new Chlamydomonas reinhardtii Dangeard (Chlamydomonadales, Chlorophyceae) mutant with from one up to more than four eyespots cell^?1. It was designated mes (multiple eyespots)‐10 A wild‐type cell has a single eyespot, located under the chloroplast envelope, at a certain position near the cell's equator where the chloroplast envelope is in contact with the cell membrane. The eyespot(s) in mes‐10, however, are located at various positions on its chloroplast. The mes‐10 cells displayed negative phototaxis to 480–500 nm light. This behavior differed from that of a similar mutant, ptx4, which has been shown to have multiple eyespots and display no phototaxis (Pazour et al., J. Cell Biol. 1995; 131 : 427–40). Mes‐10 may retain a functional photoreceptor and a photosignal transduction system independently of its multiple eyespots. This mutant should be useful for studying how C. reinhardtii responds to light signals, as well as how eyespots are formed in the cell.     

Formation of Ascorbate Oxidase in Cultured Pumpkin Cells

Ascorbate oxidase activity rapidly increased during callus formationfrom pumpkin fruit tissue. The activity reached a maximum at5 days after transfer and then declined. In callus which hadbeen subcultured at about 4-week intervals for more than oneyear, the activity also increased after transfer to fresh mediumand reached a maximum in the early logarithmic phase of growth.Light had little effect on the appearance of ascorbate oxidaseactivity in pumpkin callus. In the callus grown in the presenceof 10µM CuSO₄, the activity was about 10 times that inthe presence of 0.1 µM CuSO₄, suggesting that the formatonof ascorbate oxidase in pumpkin callus is stimulated by copper,a prosthetic metal of the enzyme. From 45 to 75% of the totalascorbate oxidase activity in pumpkin cell suspension cultureswas found in the medium. Ascorbate oxidase activity in the medium,as well as that in the cells, increased soon after transferto fresh medium, and reached a maximum at about 5 days. (Received July 2, 1987; Accepted November 21, 1987)