The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

Induction of structural and numerical changes of chromosome, centrosome abnormality, multipolar spindles and multipolar division in cultured Chinese hamster V79 cells by exposure to a trivalent dimethylarsenic compound

Dimethylarsine iodide (DMI) was used as a model compound of trivalent dimethylarsenicals [DMA(III)], and the biological effects were extensively investigated in cultured Chinese hamster V79 cells. When the cytotoxic effects of DMA(III) were compared with those of inorganic arsenite and dimethylarsinic acid [DMA(V)], DMA(III) was about 10,000 times more potent than DMA(V), and it was even 10 times more toxic than arsenite. Depletion of cell glutathione (GSH) did not influence the cytotoxic effects of DMA(III), whereas it enhanced the cytotoxicity of arsenite. Chromosome structural aberrations, such as gaps, breaks and pulverizations, and numerical changes, such as aneuploidy, hyper- and hypo-tetraploidy, were induced by DMA(III) in a concentration-dependent manner. Mitotic index increased 9-12h after the addition of DMA(III), and then declined. By contrast, the incidence of multinucleated cells increased conversely with the decrease in mitotic index at and after 24h of exposure. The mitotic cell-specific abnormality of centrosome integrity and multipolar spindles were induced by DMA(III) in a time- and concentration-dependent manner. Moreover, DMA(III) caused abnormal cytokinesis (multipolar division) at concentrations that were effective in causing centrosome abnormality, multipolar spindles and aneuploidy. These results showed that DMA(III) was genotoxic on cultured mammalian cells. Results also suggest that DMA(III)-induced multipolar spindles and multipolar division may be associated with the induction of aneuploidy. In addition, the centrosome may be a primary target for cell death via multinucleated cells.     

Genistein represses the induction of prostatic buds by testosterone]

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.     

Secretion of endothelin-1 in human endothelial cell line but not in B cell line by transfection of preproendothelin-1 cDNA.

Stable transformants with preproendothelin-1 (preproET-1) cDNA were established for the study of the regulation of endothelin-1 (ET-1) biosynthesis in human cells. ET-1, a potent vasoconstrictor peptide, is produced by endothelial cells and is secreted into the blood at a low level. Human preproET-1 cDNA was introduced into two immortal human cell lines, t-HUE2, an endothelial cell line, and Raji, a B cell line, with Ecogpt selection. Several stable transformants of t-HUE2 expressed extraordinarily high levels of preproET-1 specific mRNA and secreted ET-1 into serum-free culture medium, while the transformants of Raji cells expressed high levels of ET-1 mRNA, but secreted a negligible amount of ET-1. Immunocytochemical studies of intracellular ET-1 content revealed that there were some defects in the translation or processing of preproET-1 in the B cell line transformants. In addition, the ratio of ET-1 to ET-1 precursor (big ET-1) was much higher in the t-HUE2 transformants than in normal endothelial cells, suggesting that t-HUE2 transformants (for example t-HUE2-1) possess high levels of endothelin converting enzyme (ECE). The establishment of stable transformants producing high levels of ET-1 in serum-free medium will be useful for the study of cell-type-specific translation and processing to mature ET-1, and of the regulatory factors of ECE.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

A case of renin producing leiomyosarcoma originating in the lung.

A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.     

Langerhans cells exhibit low responsiveness to double-stranded RNA

Double-stranded RNA (dsRNA) is a viral product recognized by Toll-like receptor 3 (TLR3), and it is a potent activator of dendritic cells (DC). We compared Langerhans cells (LC) and splenic CD11c(+) DC and investigated the responsiveness to dsRNA. We prepared highly purified LC (> 95%) using the panning method. TLR3 mRNA was expressed in LC, splenic DC, and keratinocytes (KC). The expression of IFN-beta mRNA was enhanced in LC and splenic DC by Poly(I:C) stimulation. However, cytokine/chemokine production in response to Poly(I:C) by LC was much lower than that by splenic DC. In addition, Poly(I:C) induced further maturation in splenic DC, but not in LC. Finally, we found that the mouse KC cell line, PAM212, produced a great amount of IL-1alpha by Poly(I:C) stimulation, and that IL-1alpha promoted the maturation of LC. These data altogether indicate that LC exhibit low responsiveness to dsRNA. It is possible that KC may primarily trigger anti-viral immune responses in the skin via cytokine production such as IL-1alpha.     

Docking study and binding free energy calculation of poly (ADP-ribose) polymerase inhibitors

Recently, the massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed. The present study aimed to determine whether the MP-CAFEE method is useful for drug discovery research. In the drug discovery process, it is important for computational chemists to predict the binding affinity accurately without detailed structural information for protein / ligand complex. We investigated the absolute binding free energies for Poly (ADP-ribose) polymerase-1 (PARP-1) / inhibitor complexes, using the MP-CAFEE method. Although each docking model was used as an input structure, it was found that the absolute binding free energies calculated by MP-CAFEE are well consistent with the experimental ones. The accuracy of this method is much higher than that using molecular mechanics Poisson-Boltzmann / surface area (MM / PBSA). Although the simulation time is quite extensive, the reliable predictor of binding free energies would be a useful tool for drug discovery projects.     

Effect of compressive force on the expression of MMPs, PAs, and their inhibitors in osteoblastic Saos-2 cells

Bone matrix turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor type-1 (PAI-1). We previously demonstrated that 1.0g/cm(2) of compressive force was an optimal condition for inducing bone formation by osteoblastic Saos-2 cells. Here, we examined the effect of mechanical stress on the expression of MMPs, TIMPs, tPA, uPA, and PAI-1 in Saos-2 cells. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and with or without continuously compressive force (0.5-3.0g/cm(2)) for up to 24h. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 gene expression were estimated by determining the mRNA levels using real-time PCR, and the protein levels were determined using ELISA. The expression levels of MMP-1, MMP-2, MMP-14, and TIMP-1 markedly exceeded the control levels at 1.0g/cm(2) of compressive force, whereas the expression levels of MMP-3, MMP-13, TIMP-2, TIMP-3, TIMP-4, tPA, uPA, and PAI-1 markedly exceeded the control levels at 3.0g/cm(2). These results suggest that mechanical stress stimulates bone matrix turnover by increasing these proteinases and inhibitors, and that the mechanism for the proteolytic degradation of bone matrix proteins differs with the strength of the mechanical stress.