Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated “core” sequences (GACA, GATA, and GCAC). The principal objectives of this investigation were to determine whether: (1) the previously reported almost complete lack of polymorphism at allozyme loci in this species was indicative of a reduced amount of genetic diversity at VNTR loci as well; (2) VNTR markers were informative about possible clonal propagation; and (3) significant differences in genetic structure of sampling sites were associated with differences in environmental levels of pollutants at those sites. Previously, widespread sampling across the eastern United States, surveying across ten allozyme loci, has detected only two genotypes, involving a difference at a single locus, among 104 populations. In this study, the amount of genetic diversity detected at VNTR loci: (1) among ramets (N = 40; 40 genotypes detected) collected at ∼8-km intervals along a 320-km transect; (2) among ramets (N = 220; 117 genotypes detected) from five study sites separated by 50–3000 m; and (3) even among ramets within each study site [N = 44 per site; from 13 to 34 genotypes detected per site (270 m²)] exceeds that previously found in those more geographically widespread allozyme surveys. Among the 260 ramets analyzed here, the mean number of bands scored per individual was 48.61 (SD = 2.80). Mean genetic similarity among ramets collected along the 320-km transect was 0.91, which was within the range of mean genetic similarity within the five study sites (range: 0.89–0.95). Among the five study sites, 61% of the samples analyzed appeared to be clonal ramets, with up to 12 clones detected for 44 ramets sampled within a site. Clones grew intermingled and ranged up to 39 m in extent. Permutation tests of genetic similarity revealed significant genetic differentiation between each of the five study sites. Consistent with the previous allozyme studies, T. latifolia was characterized by extremely low genetic variation relative to levels of polymorphism detected at VNTR loci in other plant species. Estimated heterozygosity among ramets along the 320-km transect ranged from 0.11 to 0.13, while that within the five study sites ranged from 0.05 to 0.12. Estimates of F_st (0.32–0.41) also indicated considerable genetic subdivision among these stands. Significantly higher genetic diversity was detected at the two study sites that chemistry and toxicity data indicate to be the most severely impacted by pollutants. Although this correlation does not establish cause and effect, the results of this study indicate that the analysis of genetic diversity at VNTR loci may be a useful tool for monitoring anthropogenic-induced changes in the genetic structure of natural populations of plants.     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells

The objective ofthis study was to assess the effects of two structurally distinct yetselective proteasome inhibitors (PS-341 and lactacystin) on leukocyteadhesion, endothelial cell adhesion molecule (ECAM) expression, andnuclear factor-B (NF-B) activation in tumor necrosisfactor (TNF)--stimulated human umbilical vein endothelial cells(HUVEC) and the transformed, HUVEC-derived, ECV cell line. We foundthat TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclearneutrophil (PMN) adhesion to HUVEC but not to ECV; TNF alsosignificantly enhanced surface expression of vascular cell adhesionmolecule 1 and E-selectin (in HUVEC only), as well as intercellularadhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVECwith lactacystin completely blocked TNF-stimulated PMN adhesion,partially blocked U-937 adhesion, and completely blocked TNF-stimulatedECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1expression in ECV. Pretreatment of HUVEC with PS-341 partially blockedTNF-stimulated leukocyte adhesion and ECAM expression. These effects oflactacystin and PS-341 were associated with inhibitory effects onTNF-stimulated NF-B activation in both HUVEC and ECV. Our resultsdemonstrate the importance of the 26S proteasome in TNF-inducedactivation of NF-B, ECAM expression, and leukocyte-endothelialadhesive interactions in vitro.     

Actin filament organization is required for proper cAMP-dependent activation of CFTR

Previous studieshave indicated a role of the actin cytoskeleton in the regulation ofthe cystic fibrosis transmembrane conductance regulator (CFTR) ionchannel. However, the exact molecular nature of this regulation isstill largely unknown. In this report human epithelial CFTR wasexpressed in human melanoma cells genetically devoid of the filaminhomologue actin-cross-linking protein ABP-280 [ABP()]. cAMP stimulation of ABP() cells orcells genetically rescued with ABP-280 cDNA [ABP(+)] waswithout effect on whole cell Cl currents. InABP() cells expressing CFTR, cAMP was also without effect onCl conductance. In contrast, cAMP induced a 10-foldincrease in the diphenylamine-2-carboxylate (DPC)-sensitive whole cellCl currents of ABP(+)/CFTR(+) cells. Further, incells expressing both CFTR and a truncated form of ABP-280 unable tocross-link actin filaments, cAMP was also without effect on CFTRactivation. Dialysis of ABP-280 or filamin through the patch pipette,however, resulted in a DPC-inhibitable increase in the whole cellcurrents of ABP()/CFTR(+) cells. At the single-channel level,protein kinase A plus ATP activated single Clchannels only in excised patches from ABP(+)/CFTR(+) cells.Furthermore, filamin alone also induced Cl channelactivity in excised patches of ABP()/CFTR(+) cells. The presentdata indicate that an organized actin cytoskeleton is required forcAMP-dependent activation of CFTR.

     

Accelerated response of the myogenin gene to denervation in mutant mice lacking phosphorylation of myogenin at threonine 87

Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation.     

Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.     

Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein

Tumor cells resist the apoptotic stimuli associated with invasion and metastasis by activating survival signals that suppress apoptosis. Focal adhesion kinase (FAK), a tyrosine kinase that is overexpressed in a variety of human tumors, mediates one of these survival signals. Attenuation of FAK expression in tumor cells results in apoptosis that is mediated by caspase 8- and FADD-dependent pathways, suggesting that death receptor pathways are involved in the process. Here, we report a functional link between FAK and death receptors. We have demonstrated that FAK binds to the death domain kinase receptor-interacting protein (RIP). RIP is a major component of the death receptor complex and has been shown to interact with Fas and tumor necrosis factor receptor 1 through its binding to adapter proteins. We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK. We thus propose that FAK overexpression in human tumors provides a survival signal function by binding to RIP and inhibiting its interaction with the death receptor complex.