From one well to 9000: using high-density streptavidin-coated membranes for kinase detection

The study of kinases and their role in cellular regulation continues to expand as the human genome is sequenced and new kinases are identified as expression products of newly discovered genes. Reagents and assay systems that allow for sensitive, accurate, and high-throughput analysis of both purified kinases as well as crude extracts will enhance the characterization of these important cellular components and will speed the identification of appropriate therapeutic targets and the development of new and more effective treatments.     

The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.     

Inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway induces apoptosis in human endothelial cells

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.     

Cellular and molecular aspects of thiamin uptake by human liver cells: studies with cultured HepG2 cells

The liver is an important site for thiamin metabolism, utilization, and storage. Little is known about the mechanism of thiamin uptake by the human liver. In this study, we examined cellular and molecular aspects of the human liver thiamin uptake process using the human-derived liver HepG2 cells as a model system. Our studies showed that the initial rate of thiamin uptake to be: (1) Na(+)-independent and occurs with no detectable metabolic alterations in the transported substrate, (2) highly pH-dependent with diminished uptake upon decreasing incubation buffer pH from 8.0 to 5.0, (3) higher following cell acidification compared to unacidified control cells, (4) saturable as a function of concentration with an apparent K(m) of 7.7+/-1.6 microM, (5) inhibited by the thiamin structural analogues oxythiamin and amprolium but not by the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN), and (6) inhibited in a concentration-dependent manner by the membrane transport inhibitor amiloride. Both of the recently cloned human thiamin transporters, i.e., SLC19A2 and SLC19A3, were found to be expressed in liver HepG2 cells with the former being the predominant form. High promoter activity of the predominant form, i.e., SLC19A2, was detected in HepG2 cells, and the minimal region of the SLC19A2 promoter required for its basal activity in these cells was found to be encoded in a sequence between -356 and -36 and has multiple putative cis-regulatory elements. Mutation of a number of these putative cis-elements diminished promoter activity of the SLC19A2 minimal region. These results show the involvement of a specialized carrier-mediated mechanism for thiamin uptake by human liver HepG2 cells. In addition, SLC19A2 was found to be the predominant thiamin uptake carrier expressed in these cells and its promoter displays a high level of activity in them.     

Acidification of freshwaters by red soil in a subtropical silicate rock area,Okinawa, Japan

Two samples of red soil, one from Gushikawa Recreation Center (GRC) and one from Okinawa Royal Golf Club (ORGC), were examined for particle size distribution, textures, minerals, and chemical compositions. The effects of particle size and grinding of clay minerals on pH, electrical conductivity (EC), and dissolved chemical species were studied in deionized water and river water. The results of red soil solutions were compared with those of acidic waters found in red soil dominated areas. The minimum pH values of soil solutions extracted by deionized water were 4.38–5.36 and 5.16–5.89 and the maximum values of EC were 4.91–16.98mSm^–1 and 3.54–11.23mSm^–1 for GRC and ORGC, respectively. In the river water samples equilibrated with red soils, the minimum pH values were 4.48–5.10 and 4.77–5.91 and the maximum EC values were 19.6–34.2mSm^–1 and 17.5–25.0mSm^–1 for GRC and ORGC, respectively. The values of pH and EC varied with the soil–solution ratio and the particle size. The chemical composition of river water without mixing with red soil shows Na⁺K⁺ and Ca²⁺Mg²⁺. After mixing with red soil, the trend of the concentrations changed to Na⁺K⁺ and Mg²⁺Ca²⁺, which is the same as that of soil solutions in deionized water as well as that of acidic waters found in the red soil area. The pH of the acidic waters was 4.95–5.81 and EC was 7.76–30.0mSm^–1. Laboratory experimental results agreed well with those found in the field in terms of trend of concentrations of the chemical species and pH. Therefore, the results of this study suggest that the low pH and trend of the concentrations of chemical species of the acidic waters found in the red soil dominated areas were the result of the interaction of natural water and red soil.     

Identification of non-TIR-NBS-LRR markers linked to the <Emphasis Type="Italic">Pl5</Emphasis>/<Emphasis Type="Italic">Pl8</Emphasis> locus for resistance to downy mildew in sunflower

The resistance of sunflower, Helianthus annuus L., to downy mildew, caused by Plasmopara halstedii, is conferred by major genes denoted by Pl. Using degenerate and specific primers, 16 different resistance gene analogs (RGAs) have been cloned and sequenced. Sequence comparison and Southern-blot analysis distinguished six classes of RGA. Two of these classes correspond to TIR-NBS-LRR sequences while the remaining four classes correspond to the non-TIR-NBS-LRR type of resistance genes. The genetic mapping of these RGAs on two segregating F2 populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked to the Pl5/ Pl8 locus for resistance to downy mildew in sunflower. These and other results indicate that different Pl loci conferring resistance to the same pathogen races may contain different sequences.     

The low molecular weight (LMW) isoforms of cyclin E deregulate the cell cycle of mammary epithelial cells

Cyclin E in complex with CDK2 is a positive regulator of the G1 to S phase transition of the cell cycle and is responsible for cells passing the restriction point, committing the cell to another round of cell division. Cyclin E is overexpressed and proteolytically cleaved into low molecular weight (LMW) isoforms in breast cancer cell lines and tumor tissues compared to normal cells and tissues. These alterations in cyclin E are linked to poor prognosis in breast cancer patients. In order to evaluate the biological effects of the LMW cyclin E, immortalized mammary epithelial cells, 76NE6, were stably transfected with each of the three cyclin E constructs. Our results reveal that the LMW forms of cyclin E (T1 and T2) are biologically functional, as their overexpression in the immortalized cells increases the ability of these cells to enter S and G2/M phase by 2 fold over full length or vector-alone transfected cells, concomitant with an increased rate of cell proliferation. In addition, these LMW isoforms are biochemically hyperactive, shown by their ability to phosphorylate substrates such as histone H1 4 fold more in cells transfected with T1 or T2 versus cells transfected with the full length form. These results suggest that overexpression of the LMW forms of cyclin E is mitogenic, stimulating the cells to progress through the cell cycle much more efficiently than the full length cyclin E.