Physical gills in Elmidae (Coleoptera: Byrrhoidea): Structure and evolutionary pattern of plastron in Stenelmis and related genera

Many species within Elmidae (Coleoptera: Byrrhoidea) have plastrons composed of flattened setae. However, some genera display fine plastrons on the epicuticle, called plastron hairs. In Japanese elmids, members of the genera Stenelmis, Ordobrevia, Nomuraelmis and Leptelmis bear ventral plastron hairs. Based on a maximum likelihood tree including most Japanese genera within Elmidae, we found that these genera are monophyletic and that plastron hairs are a derived character in Elmidae. We also found that the genus Graphelmis bears jigsaw puzzle‐like plastron scales with plastron hair‐like projections, and is sister to the group with plastron hairs.     

A novel site-specific recombination system derived from bacteriophage phiMR11

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.     

Poxviruses and the Origin of the Eukaryotic Nucleus

A number of molecular forms of DNA polymerases have been reported to be involved in eukaryotic nuclear DNA replication, with contributions from α-, δ-, and ε-polymerases. It has been reported that δ-polymerase possessed a central role in DNA replication in archaea, whose ancestry are thought to be closely related to the ancestor of eukaryotes. Indeed, in vitro experiment shown here suggests that δ-polymerase has the potential ability to start DNA synthesis immediately after RNA primer synthesis. Therefore, the question arises, where did the α-polymerase come from? Phylogenetic analysis based on the nucleotide sequence of several conserved regions reveals that two poxviruses, vaccinia and variola viruses, have polymerases similar to eukaryotic α-polymerase rather than δ-polymerase, while adenovirus, herpes family viruses, and archaeotes have eukaryotic δ-like polymerases, suggesting that the eukaryotic α-polymerase gene is derived from a poxvirus-like organism, which had some eukaryote-like characteristics. Furthermore, the poxvirus's proliferation independent from the host-cell nucleus suggests the possibility that this virus could infect non-nucleated cells, such as ancestral eukaryotes. I wish to propose here a new hypothesis for the origin of the eukaryotic nucleus, posing symbiotic contact of an orthopoxvirus ancestor with an archaebacterium, whose genome already had a δ-like polymerase gene. Received: 26 October 2000 / Accepted: 16 January 2001     

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity. Ad4-1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4-1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1xanti-CD3). Cocultivation of Ad4-1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4-1BBL and T-LAK cells were administered when tumor size exceeded 5 mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4-1BBL for genetic immunotherapy of cancer.     

Method of localized removal of cells using a bolt‐clamped Langevin transducer with an ultrasonic horn

Cell isolation by eliminating undesirable cell aggregations or colonies with low activity is essential to improve cell culture efficiency. Moreover, when creating tissues from induced pluripotent stem cells, residual undifferentiated cells must be removed to prevent tumor formation in vivo. Here, we evaluated the use of ultrasonic irradiation, which can apply energy locally without contact, and proposed a method to eliminate cells in a small area of culture by ultrasonic irradiation from a Langevin transducer. We constructed a device that incorporated a bolt‐clamped 19.84 kHz Langevin transducer with an ultrasonic horn and determined the optimal conditions for stable elimination of cells in small areas of a 35‐mm culture dish. The optimal conditions were as follows: number of cycles = 400, clearance distance = 1 mm, volume of medium = 4 mL, and distance from the center of culture surface = 0 mm. The mean cell elimination area under these conditions was 0.097 mm². We also evaluated the viability of neighboring cells after ultrasonic irradiation by fluorescent staining and found that most cells around the elimination area survived. These findings suggest that the proposed method has potential for localized elimination of cells without the need for contact with the cell surface.     

Simulation of Cell Patterning Triggered by Cell Death and Differential Adhesion in Drosophila Wing

The Drosophila wing exhibits a well-ordered cell pattern, especially along the posterior margin, where hair cells are arranged in a zigzag pattern in the lateral view. Based on an experimental result observed during metamorphosis of Drosophila, we considered that a pattern of initial cells autonomously develops to the zigzag pattern through cell differentiation, intercellular communication, and cell death (apoptosis) and performed computer simulations of a cell-based model of vertex dynamics for tissues. The model describes the epithelial tissue as a monolayer cell sheet of polyhedral cells. Their vertices move according to equations of motion, minimizing the sum total of the interfacial and elastic energies of cells. The interfacial energy densities between cells are introduced consistently with an ideal zigzag cell pattern, extracted from the experimental result. The apoptosis of cells is modeled by gradually reducing their equilibrium volume to zero and by assuming that the hair cells prohibit neighboring cells from undergoing apoptosis. Based on experimental observations, we also assumed wing elongation along the proximal-distal axis. Starting with an initial cell pattern similar to the micrograph experimentally obtained just before apoptosis, we carried out the simulations according to the model mentioned above and successfully reproduced the ideal zigzag cell pattern. This elucidates a physical mechanism of patterning triggered by cell apoptosis theoretically and exemplifies, to our knowledge, a new framework to study apoptosis-induced patterning. We conclude that the zigzag cell pattern is formed by an autonomous communicative process among the participant cells.     

Production and Secretion of Adrenomedullin by Cultured Choroid Plexus Carcinoma Cells

Abstract: Adrenomedullin is a potent vasodilator peptide that was originally isolated from pheochromocytoma. The production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells were studied by radioimmunoassay and northern blot hybridization. Choroid plexus carcinoma is a rare malignant tumor derived from the epithelium of the choroid plexus. Immunoreactive adrenomedullin was detected in the conditioned medium of choroid plexus carcinoma cells (40.8 ± 7.5 fmol/10⁵ cells/24 h; mean ± SEM, n = 5). Reverse-phase HPLC of the conditioned medium showed one major peak of the immunoreactive peptide eluting in the position of synthetic human adrenomedullin and two smaller peaks eluting earlier. Addition of interleukin-1β (10 ng/ml) alone or in combination with three cytokines, interferon-γ (100 U/ml), tumor necrosis factor-α (20 ng/ml), and interleukin-1β (10 ng/ml), caused significant increases in the immunoreactive adrenomedullin concentrations in the medium (∼175 and 293% of the control level, respectively). Northern blot analysis showed the expression of 1.6-kb adrenomedullin mRNA in the total RNA sample prepared from cultured choroid plexus carcinoma cells. Treatment with either interleukin-1β or the combination of three cytokines caused significant increases in levels of adrenomedullin mRNA in parallel with those in immunoreactive adrenomedullin concentrations in the conditioned medium. These findings raise a possibility that adrenomedullin is secreted from the choroid plexus and has physiological roles in the CNS via the CSF. In addition, adrenomedullin secreted from choroid plexus carcinoma may be related to the pathophysiology of the tumor.     

Effect of certain growth factors on proliferation in serum-free collagen gel culture of vaginal epithelial cells from prepuberal mice exposed neonatally to diethylstilbestrol

Neonatal treatment with diethylstilbestrol (DES) induces ovary-independent vaginal epithelial changes in mice. The response of vaginal epithelial cells from intact prepuberal BALB/cCrgl mice treated neonatally with 2 micrograms of DES for 5 days to growth-stimulatory and -inhibitory factors was studied using a serum-free collagen gel culture system that sustains the growth of normal vaginal epithelial cells. Cells from control and DES-exposed mice at 21 days of age showed about a 5-fold increase in number during 10 days in a serum-free medium supplemented with transferrin, bovine serum albumin fraction V, insulin, and epidermal growth factor. Epidermal growth factor and insulin stimulated dose-related proliferation of vaginal epithelial cells from both control and DES-exposed mice; however, cells from DES-exposed mice showed a reduced growth response to epidermal growth factor and an increased growth response to insulin, compared with control cells. Insulin-like growth factor I (1-100 ng/ml) tested in the absence of insulin failed to stimulate cell growth. Transforming growth factor-beta (0.05-5 ng/ml) consistently inhibited cell growth in a dose-dependent manner.