Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.     

Quantitative examination of a perfusion microscope for the study of osmotic response of cells

The perfusion microscope was developed for the study of the osmotic response of cells. In this microscope, the cells are immobilized in a transparent chamber mounted on the stage and exposed to a variety of milieus by perfusing the chamber with solutions of different concentrations. The concentration of the supplied solution is controlled using two variable-speed syringe pumps, which supply an isotonic solution and a hypertonic solution. Before using this system to characterize the osmotic response of cells, the change in the concentration of NaCl solution flowing through the chamber is examined quantitatively using a laser interferometer and an image processing technique. The NaCl concentration is increased from an isotonic condition to a hypertonic condition abruptly or gradually at a given constant rate, and decreased from a hypertonic condition to an isotonic condition. It is confirmed that the concentration is nearly uniform in the cross direction at the middle of the chamber, and the change in the NaCl concentration is reproducible. The average rate of increase or decrease in the measured concentration agrees fairly well with the given rate when the concentration is changed gradually at a constant rate. The rate of the abrupt change is also determined to be the highest limit achieved by the present method. As the first application of using the perfusion microscope for biological studies, the volume change of cells after exposure to a hypertonic solution is measured. Then, the hydraulic conductivity of the cell membrane is determinedfrom the comparison of the volume change between the experiment and the theoretical estimation for the measured change in the NaCl concentration of the perfused solution.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.     

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.     

Intramuscular gene transfer of FGF-2 attenuates endothelial dysfunction and inhibits intimal hyperplasia of vein grafts in poor-runoff limbs of rabbit

We previously demonstrated that sustained disturbance of endothelium-dependent vasorelaxation and poor distal runoff in ischemic limbs were critical factors affecting the neointimal development of autologous vein grafts (VGs). Also, we recently showed the superior therapeutic potential of basic fibroblast growth factor (bFGF/FGF-2) boosted by the recombinant Sendai virus (SeV) for severe limb ischemia compared with that of vascular endothelial growth factor. Here, the effect of FGF-2 on neointimal hyperplasia of VGs was examined in a rabbit model of poor-runoff limbs. Two weeks after initial surgery for the induction of poor-runoff, SeV-expressing human FGF-2 (SeV-hFGF2) or that encoding firefly luciferase (109 plaque-forming units/head) was injected into the thigh and calf muscle. At that time, the femoral vein was implanted in the femoral artery in an end-to-end manner in some groups. FGF-2 gene-transferred limbs demonstrated significantly increased blood flow assessed not only by laser Doppler flow image but also by ultrasonic transit-time flowmeter (USTF). USTF also showed a significant increase in the blood flow ratio of the deep femoral artery to external iliac artery, indicating that collateral flow was significantly restored in the thigh muscles (P < 0.01). Reduction of neointimal hyperplasia was also observed in the VGs treated by SeV-hFGF2; these grafts demonstrated significant restoration of endothelium-dependent vasorelaxation. These findings thus extend the indications of therapeutic angiogenesis using SeV-hFGF2 to include not only limb salvage but also prevention of late graft failure.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Crystal structure of an ADP-dependent glucokinase from Pyrococcus furiosus: implications for a sugar-induced conformational change in ADP-dependent kinase

ADP-dependent kinases are used in the modified Embden-Meyerhoff pathway of certain archaea. Our previous study has revealed a mechanism for ADP-dependent phosphoryl transfer by Thermococcus litoralis glucokinase (tlGK), and its evolutionary relationship with ATP-dependent ribokinases and adenosine kinases (PFKB carbohydrate kinase family members). Here, we report the crystal structure of glucokinase from Pyrococcus furiosus (pfGK) in a closed conformation complexed with glucose and AMP at 1.9A resolution. In comparison with the tlGK structure, the pfGK structure shows significant conformational changes in the small domain and a region around the hinge, suggesting glucose-induced domain closing. A part of the large domain next to the hinge is also shifted accompanied with domain closing. In the pfGK structure, glucose binds in a groove between the large and small domains, and the electron density of O1 atoms for both the alpha and beta-anomer configurations was observed. The structural details of the sugar-binding site of ADP-dependent glucokinase were firstly clarified and then site-directed mutagenesis analysis clarified the catalytic residues for ADP-dependent kinase, such as Arg205 and Asp451 of tlGK. Homology search and multiple alignment of amino acid sequences using the information obtained from the structures reveals that eucaryotic hypothetical proteins homologous to ADP-dependent kinases retain the residues for the recognition of a glucose substrate.     

Reduction of natural adenovirus tropism to the liver by both ablation of fiber-coxsackievirus and adenovirus receptor interaction and use of replaceable short fiber

The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and alphav integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.     

Human immunodeficiency virus type 1 Vif is efficiently packaged into virions during productive but not chronic infection

Packaging of the human immunodeficiency virus type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. Here, we compared the efficiency of packaging of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion), while packaging into virions from chronically infected H9 cells was near the limit of detection (four to six copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild-type virus was readily packaged (30 to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild-type infectivity. The results from our study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.     

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.