Theoretical kinetic studies of models for binding myosin subfragment-1 to regulated actin: Hill model versus Geeves model

It was previously shown that a one-dimensional Ising model could successfully simulate the equilibrium binding of myosin S1 to regulated actin filaments (T. L. Hill, E. Eisenberg and L. Greene, Proc. Natl. Acad. Sci. U.S.A. 77:3186-3190, 1980). However, the time course of myosin S1 binding to regulated actin was thought to be incompatible with this model, and a three-state model was subsequently developed (D. F. McKillop and M. A. Geeves, Biophys. J. 65:693-701, 1993). A quantitative analysis of the predicted time course of myosin S1 binding to regulated actin, however, was never done for either model. Here we present the procedure for the theoretical evaluation of the time course of myosin S1 binding for both models and then show that 1) the Hill model can predict the "lag" in the binding of myosin S1 to regulated actin that is observed in the absence of Ca++ when S1 is in excess of actin, and 2) both models generate very similar families of binding curves when [S1]/[actin] is varied. This result shows that, just based on the equilibrium and pre-steady-state kinetic binding data alone, it is not possible to differentiate between the two models. Thus, the model of Hill et al. cannot be ruled out on the basis of existing pre-steady-state and equilibrium binding data. Physical mechanisms underlying the generation of the lag in the Hill model are discussed.     

Oncogenic Ki-ras confers a more aggressive colon cancer phenotype through modification of transforming growth factor-beta receptor III

Transforming growth factor-beta1 (TGF-beta1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. Each of three Ki-ras(G12V) transfectants of HD6-4 colon cancer cells had been shown to be more aggressive in vivo than controls in earlier studies (Yan, Z., Chen, M., Perucho, M., and Friedman, E. (1997) J. Biol. Chem. 272, 30928-30936). We now show that stable expression of oncogenic Ki-ras(G12V) converts the HD6-4 colon cancer cell line from insensitive to TGF-beta1 to growth-promoted by TGF-beta1. Each of three Ki-ras(G12V) transfectants responded to TGF-beta1 by an increase in proliferation and by decreasing the abundance of the Cdk inhibitor p21 and the tumor suppressor PTEN, whereas each of three wild-type Ki-ras transfectants remained unresponsive to TGF-beta1. The wild-type Ki-ras transfectants lack functional TGF-beta receptors, whereas all three Ki-ras(G12V) transfectants expressed functional TGF-beta receptors that bound (125)I-TGF-beta1. The previous studies showed that in cells with wild-type Ki-ras, TGF-beta receptors were not mutated, and receptor proteins were transported to the cell surface, but post-translational modification of TGF-beta receptor III (TbetaRIII) was incomplete. We now show that the betaglycan form of TbetaRIII is highly modified following translation when transiently expressed in Ki-ras(G12V) cells, whereas no such post-translational modification of TbetaRIII occurs in control cells. Antisense oligonucleotides directed to Ki-Ras decreased both TbetaRIII post-translational modification in Ki-ras(G12V) cells and TGF-beta1 down-regulation of p21, demonstrating the direct effect of mutant Ras. Therefore, one mechanism by which mutant Ki-Ras confers a more aggressive tumor phenotype is by enhancing TbetaRIII post-translational modification.     

Linear and spatial organization of polytene chromosomes of the African malaria mosquito Anopheles funestus

Anopheles funestus Giles is one of the major malaria vectors in Africa, but little is known about its genetics. Lack of a cytogenetic map characterized by regions has hindered the progress of genetic research with this important species. This study developed a cytogenetic map of An. funestus using ovarian nurse cell polytene chromosomes. We demonstrate an important application with the cytogenetic map for characterizing various chromosomal inversions for specimens collected from coastal Kenya. The linear and spatial organization of An. funestus polytene chromosomes was compared with the best-studied malaria mosquito, An. gambiae Giles. Comparisons of chromosome morphology between the two species have revealed that the most extensive chromosomal rearrangement occurs in pericentromeric heterochromatin of autosomes. Differences in pericentromeric heterochromatin types correlate with nuclear organization differences between An. funestus and An. gambiae. Attachments of chromosomes to the nuclear envelope strongly depend on the presence of diffusive beta-heterochromatin. Thus, An. funestus and An. gambiae exhibit species-specific characteristics in chromosome-linear and -spatial organizations.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Crystal structure of activated CheY. Comparison with other activated receiver domains

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.     

Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein

DNA replication in higher eukaryotic cells occurs at a large number of discrete sites called replication foci. We have previously purified a protein, focus-forming activity 1 (FFA-1), which is involved in the assembly of putative prereplication foci in Xenopus egg extracts. FFA-1 is the orthologue of the Werner syndrome gene product (WRN), a member of the RecQ helicase family. In this paper we show that FFA-1 colocalizes with sites of DNA synthesis and the single-stranded DNA binding protein, replication protein A (RPA), in nuclei reconstituted in the egg extract. In addition, we show that two glutathione S-transferase FFA-1 fusion proteins can inhibit DNA replication in a dominant negative manner. The dominant negative effect correlates with the incorporation of the fusion proteins into replication foci to form "hybrid foci," which are unable to engage in DNA replication. At the biochemical level, RPA can interact with FFA-1 and specifically stimulates its DNA helicase activity. However, in the presence of the dominant negative mutant proteins, the stimulation is prevented. These results provide the first direct biochemical evidence of an important role for FFA-1 in DNA replication.     

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.     

Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors

CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10-fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium-dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre-treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large-scale generation of dopaminergic neurons for clinical transplantation.