Blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathways by statins reduces the expression of bFGF, HGF, and TGF-β as angiogenic factors in mouse osteosarcoma

The tumor microenvironment plays a critical role in modulating malignant behavior and can dramatically influence cancer treatment strategies. We investigated whether statins inhibit the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGF-β) mRNA in the mouse osteosarcoma cell line LM8. We found that statins significantly inhibited mRNA expressions of bFGF, HGF, and TGF-β, and bFGF, HGF, and TGF-β secretions at concentrations that did not have antiproliferative effects on LM8 cells, but had no effect on the mRNA expression and secretion of VEGF. The inhibition of bFGF, HGF, and TGF-β mRNA expression, and bFGF, HGF, TGF-β secretions was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination with statins. Furthermore, statins reduced the membrane localization of K-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our research indicates that statins inhibit GGPP biosynthesis in the mevalonate pathway, and then inhibit signal transduction in the Ras/ERK and Ras/Akt pathways, thereby inhibiting bFGF, HGF, TGF-β expression in LM8 cells. These results suggest that statins are potentially useful as anti-angiogenic agents for the treatment of osteosarcoma.     

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

To test the feasibility of CO₂ remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO₂-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO₂ concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO₂ fixation rate of 16.7 g CO₂ l⁻¹ 24 h⁻¹ (or 200.4 g CO₂ m⁻² 24 h⁻¹) could readily be sustained at a light intensity of 2000 μmol m⁻² s⁻¹ at 25 °C, and an ultrahigh cell density of well over 80 g l⁻¹ could be maintained by daily replacing the culture medium. Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998     

Identification of vitamin A-free cells in a stellate cell-enriched fraction of normal rat liver as myofibroblasts

Myofibroblasts (MFs) as well as hepatic stellate cells (HSCs) are known to be involved in liver fibrogenesis. Quiescent HSCs (qHSCs) in culture have been thought to differentiate to replicative activated HSCs (aHSCs). In this study a qHSC-enriched fraction isolated by Nycodenz-isodensity centrifugation was separated with a fluorescence-activated cell sorter, which revealed the presence of a small fraction (occupancy rate = 0.4%) of cells that did not show vitamin A-autofluorescence under ultraviolet (UV)-irradiation (UV⁻ cells). The remaining vitamin A-containing cells were autofluorescent (UV⁺) and originally expressed markers of qHSCs, and, in culture, did not grow, lost vitamin A, and expressed markers of aHSCs. UV⁻ cells showed morphology of MFs, and, in culture, grew to form colonies and expressed markers of MFs. These results indicated that UV⁺ and UV⁻ cells represent qHSCs and MFs, respectively, and that aHSCs have no growth potential and are a cell-type distinct from proliferative MFs. Gene expression profiles of UV⁻ cells (MFs) newly identified gremlin as one of MF-preferential genes and its proteins were localized around fibrotic septa in rat and human livers. In addition, we suggested that the qHSC-enriched fraction included approximately 6% of liver MFs.     

Mitochondrial damage in galactosamine-induced liver intoxication in rats

Mitichondria isolated from livers of rats which received D-galactosamine (375 mg/kg body wt., four times) demonstrated a marked decrease in respiratory control ratios, the ADP/O ratios, and state 3 respiration rates and an increase in state 4 respiration rates. The aberration was profound with site I being altered prior to sites II and III. Quantitation of phospholipids revealed a reduction of total phospholipids per mg protein with decreases in phosphatidylcholine and phosphatidylethanolamine contents. Caldiolipin was the only phospholipid which remained unaltered. Fatty acid composition was altered in these phospholipids; caldiolipin was altered most severely, showing reductions in linoleic and arachidonic acids, and an elevation in saturated fatty acids and in some other small components of fatty acids. In phosphatidylethanolamine, palmitic acid decreased, whereas stearic and docosahexonoic acids increased. These changes were smaller in phosphatidylcholine fatty acids. These mitochondria were also characterized by an altered composition in high molecular weight polypeptide components. By experiments with normal mitochondria in vitro, galactosamine, but not other aminohexoses, was proved to be an uncoupling agent of the oxidative phosphorylation system. Electron microscopic observation demonstrated that both in vivo and in vitro treatments with galactosamine induced marked disorganization of mitochondral structures. These results suggest that mitochondrial damage is also included in galactosamine-induced hepatic lesion.     

Mode of Subacute Sclerosing Panencephalitis (SSPE) Virus Infection in Tissue Culture Cells: III. Neurovirulence of Cell-Free SSPE Viruses of Niigata-1, Kitaken-1, and Biken Strains

Cell-free viruses recovered from virus-carrying cultures of the Niigata-1, Kitaken-1, and Biken strains of SSPE virus were examined for neurovirulence. The cell-free viruses were prepared by freezing and thawing or by EDTA treatment of the virus-carrying cultures and inoculated into adult mice intracerebrally. A considerable number of the inoculated mice showed clinical signs about 1 to 5 weeks after the inoculation. The first symptom was hyperreactivity, which was followed by paresis and myoclonus. All of the affected mice fell in paralysis and finally died. The virus could be recovered from the moribund mice by cocultivation of the brain cells with Vero cells. Immunofluorescence staining of the brain tissue revealed that infected cells containing viral antigens were distributed sparsely. No inflammatory feature, however, was observed in the brain as far as examined and neutralizing antibody against SSPE virus was not detected in sera from the mice inoculated with the cell-free SSPE viruses.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

Intergeneric hybrids ofAster ageratoides subsp.ovatus (2n=36)×Kalimeris pinnatifida (2n=18) were produced artificially. The chromosomes of the hybrid were found to be 2n=27, and to consist of 9 large chromosomes and 18 small chromosomes. In meiosis of the PMCs of the hybrid, a chromosome configuration of 9_II+9_I was regularly observed. While all the univalents were large, and all the bivalents were comparatively small. The large and small chromosomes ofA. ageratoides subsp.ovatus were found to be rather distant in homology, and the small chromosomes of the subspecies and the chromosomes ofK. pinnatifida were found to have a high degree of homology. The tetraploidovatus was concluded to be an amphidiploid, composed of the large chromosomes ofAster and the small chromosomes ofKalimeris.     

Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy.

We describe here the use of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca2+]i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca2+]i in optically sectioned unit volumes of the cell. Images of the calcium wave obtained during propagation between paired cells revealed that the wavefront is constant in shape and propagates at constant velocity without any delay at the cell-to-cell junction. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living cells.     

Reduction of patient dose in medical radiography by utilizing scattered X-rays: Relation between permissible limit of scatter fraction,viewer brightness,and perceptibility of vision

This paper proposes a new technique for reducing the patient dose when employing medical radiographs prepared by using screen-film systems. In this technique the patient dose can be reduced by employing scattered X-rays in order to obtain the same film density as that realized without the use of scattered X-rays. The minimum perceptible thickness difference ΔX_min, which can be recognized by liminal vision, was psychophysically calculated by considering the energy spectrum of incident X-ray, sensitivity spectrum of the screen layer, and the perception capability of human vision. From the calculated ΔX_mins in various conditions, the permissible upper limit of scatter fraction for obtaining the same ΔX_min for three kinds of luminances, and the fraction of reduction in the primary X-rays were determined.As an example of the results, when the object size required for perception is 1.3 mm, a scatter fraction up to 42% can be permitted at a density D of 1.0 for a luminance of 2548 cd m^?2. When we increase the luminance of the viewer from 478 cd m^?2 to 2548 cd m^?2, the upper limit of the permitted scatter fraction varies from 30% to 42% at a D of 1.0, i.e., the patient dose can be reduced by 17% under the same perceptibility of ΔX_min by utilizing scattered X-rays. This reduction can be successfully achieved by changing the lead content of the grid from 0.45 to 0.38 g cm^?2.