Potentiometric sensors doped with biomolecules as a new approach to small molecule/biomolecule binding kinetics analysis.

The most successful binding kinetics analysis systems at this moment include surface plasmon resonance (SPR), quartz microcrystal balance (QMB) and surface acoustic wave (SAW). Although these are powerful methods, they generally are complex, expensive and require the use of monolayers. Here, we report on potentiometric sensors as an inexpensive and simple alternative to do binding kinetics analysis between small molecules in solution and biomolecules (covalently) attached in a biopolymer sensor coating layer. As an example, dopamine and an anti-dopamine aptamer were used as the small molecule and the biomolecule respectively. Binding between both follows a Langmuir adsorption type model and creates a surface potential. The system operates in Flow Injection Analysis mode (FIA). Besides being an interesting new binding kinetics tool, the approach allows systematic design of potentiometric biosensors (in the present study a dopamine sensor), and gives new insights into the functioning of ion-selective electrodes (ISE's).

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Exclusion of candidate genes for coat colour phenotypes of the American mink (Neovison vison).

In a previous project, we screened the American mink Bacterial Artificial Chromosome library, CHORI-231, for genes potentially involved in various coat colour phenotypes in the American mink. Subsequently, we 454 sequenced the inserts containing these genes and developed microsatellite markers for each of these genes. Here, we describe a lack of association between three different 'roan-type' phenotypes represented by Cross, Stardust and Cinnamon in American mink and six different genes that we considered to be potentially linked to these phenotypes. Thus, c-KIT (HUGO-approved symbol KIT), ATOH-1 (HUGO-approved symbol ATOH1) and POMC were excluded as potential candidates for these three phenotypes. In addition, MITF and SLC24A5 were excluded for Cross and Cinnamon, and KITL (HUGO-approved symbol KITLG) for Cross and Stardust. Although most of these genes have been implicated as the cause of similar phenotypes in other mammals, including horses, pigs, cows, dogs, cats, mice and humans, they do not appear to be responsible for comparable phenotypes found in American mink.

Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

[The effect of inhibitors of serinethreonine protein kinases on Arabidopsis thaliana root morphology and microtubules organization in its cells].

The effect of different types of serine/thereonine protein kinases inhibitors (cyclin-dependent, Ca2+-calmodulin-dependent and protein kinase C) on microtubules organization in cells of Arabidopsis thaliana main primary root zones were investigated in vivo. The microtubules in epidermal and cortex cells in the transition and elongation zones as well as microtubules in trichoblasts and atrichoblasts in the differentiation zone showed the greatest sensitivity to protein kinases inhibitors studied. It was established that microtubules in these cell types modified their initial transverse/oblique orientation to a chaotic or longitudinal relative to the major axis of primary root as a result of serinethereonine protein kinases inhibition. The microtubules in cells in root meristematic zone as well as in root hairs were less sensitive to influence of protein kinases inhibitors tested. Alterations of microtubules orientation in the cells in primary root zones under the influence of serinethereonine protein kinases inhibitors led to further disturbances in growth and differentiation processes. It was assumed that phosphorylation of microtubules proteins, especially tubulin, might be involved in the regulation of these processes.

Xyloglucan endotransglycosylase/hydrolase (XTH) is encoded by a multi-gene family in the primitive vascular land plant Selaginella kraussiana.

Xyloglucan endotransglycosylase/hydrolases (XTHs) are enzymes that cleave and rejoin xyloglucan chains. To trace the evolutionary origin of XTHs, we used Selaginella kraussiana, a representative of the most primitive land plants (Lycopodiophyta). A Southern blot with a digoxigenin-labeled probe, designed on the conserved catalytic site of XTHs, indicated nine genes. The presence of at least seven functional XTHs was detected by isoelectric focusing (IEF) followed by overlaying the gel with a XET-test paper. Together, these results indicate that XTHs are encoded by a multi-gene family that originated during or even before the colonization of land by plants.

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions.

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.

Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays.

The endotransglucosylase action of the enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was localized in the roots of diverse vascular plants: club-mosses (lycopodiophytes), ferns, gymnosperms, monocots, and dicots. High action was always found in the epidermis cell wall of the elongation zone and in trichoblasts in the differentiation zone. Clearly XTH and its action in root development evolved before the evolutionary divergence of ferns and seed plants and also of the lycopodiophytes and euphyllophytes.

Root hair initiation is coupled to a highly localized increase of xyloglucan endotransglycosylase action in Arabidopsis roots.

Root hairs are formed by two separate processes: initiation and subsequent tip growth. Root hair initiation is always accompanied by a highly localized increase in xyloglucan endotransglycosylase (XET) action at the site of future bulge formation, where the trichoblast locally loosens its cell wall. This suggests an important role of XET in the first stages of root hair initiation. The tip of growing root hairs is not marked by localized high XET action. Experiments in which root hair initiation was modulated and observations on root hair mutants support this view. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid shifts both root hair initiation and the local increase in XET action toward the root tip. On the other hand, roots treated with the ethylene inhibitor aminoethoxyvinyl-glycine, as well as roots of mutants affected in root hair initiation (rhl1, rhd6-1, and axr2-1) revealed no localized increases of XET action at all and consequently did not initiate root hairs. Disruption of actin and microtubules did not prevent the localized increase in XET action. Also, the temporal and spatial pattern of action as the specific pH dependence suggest that different isoforms of XET act in different processes of root development.

Ion fluxes, auxin and the induction of elongation growth in Nicotiana tabacum cells.

Immobilized cultured tobacco cells become polarized upon the addition of naphthalene-1-acetic acid and start to elongate from an initial spherical shape. The question as to how a diffuse-growing cell forms a polar axis is addressed here with approaches successfully applied to the study of tip growth. With two kinds of vibrating probes the electric current flow and proton fluxes were mapped around such elongating cells. No consistent polar pattern of ion fluxes, which is typical for actively tip-growing cells, was detected. Therefore, other signals must provide the positional information needed for polar axis formation. Furthermore, neither a specific pattern of intracellular Ca(2+) concentration nor a polar distribution of putative ion-channel antagonist-binding sites were found in elongating tobacco cells. Auxin flux, on the other hand, was found to be important as TIBA, an inhibitor of polar auxin transport, clearly inhibited elongation in a concentration-dependent way. Cross-linking of arabinogalactan-proteins with the beta-Yariv reagent also resulted in inhibition of elongation. A model is proposed for the induction of polar growth where localized auxin efflux starts a signal cascade that triggers molecules that reorient microtubules. These then guide cellulose deposition in the cell wall, which in turn alters cell wall mechanics and leads to elongation. In this scheme, arabinogalactan-proteins are not causal agents but are probably important regulators of growth and survival of the cell.

Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.

Identification of the tumor metastasis suppressor Nm23-H1/Nm23-R1 as a constituent of the centrosome.

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Nucleoside diphosphate kinase beta (Nm23-R1/NDPKbeta) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma.

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKbeta and Nm23-R2/NDPKalpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

In vivo colocalization of xyloglucan endotransglycosylase activity and its donor substrate in the elongation zone of Arabidopsis roots.

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO-SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO-SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide-SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO-SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.

From hormone signal, via the cytoskeleton, to cell growth in single cells of tobacco.

Cultured mesophyll protoplasts of Nicotiana tabacum L. can be hormonally induced into different developmental pathways. In a medium containing auxins (NAA) and cytokinins (BAP) cells divide and eventually give rise to calli. When only auxins are present cells elongate and finally differentiate into very long tubular cells. We focused on the sequence of events leading to elongation. When cultured in a high (1 mg/l) auxin concentration elongating cells seem to pass a certain threshold and increase their nuclear DNA up to about 16C. Cells cultured in a low (0.065 mg/l) auxin concentration only have C-values up to 4C, are unable to pass this threshold and finally fail to elongate. Besides the concentration dependence of the auxin signal, the efflux of auxin seems to be necessary for elongation since addition of TIBA drastically reduces the amount of elongating cells. Concomitant with the changes in nuclear physiology, auxin-induced axiality is seen as sequential rearrangements of microtubules and actin-filaments and of cell wall cellulose microfibrils from 'randomly' arranged in spherical cells to an orientation perpendicular to the long axis of elongating cells.