Página 6 dos resultados de 9995 itens digitais encontrados em 0.038 segundos

‣ Dimerization of a selectin and its ligand stabilizes cell rolling and enhances tether strength in shear flow

Ramachandran, Vishwanath; Yago, Tadayuki; Epperson, Terry Kay; Kobzdej, Marcin M. A.; Nollert, Matthias U.; Cummings, Richard D.; Zhu, Cheng; McEver, Rodger P.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Selectins mediate rolling of leukocytes by rapid formation and dissociation of selectin–ligand bonds, which are assumed to require high mechanical strength to prevent premature dissociation by the forces applied in shear flow. This assumption is based largely on the observation that increasing wall shear stress increases only modestly the dissociation of transient leukocyte tethers on very low selectin densities. P-selectin binds to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. Both PSGL-1 and P-selectin are extended homodimers. We perfused transfected cells expressing wild-type dimeric PSGL-1 or a chimeric monomeric form of PSGL-1 on immobilized dimeric or monomeric forms of P-selectin. Cells expressing dimeric or monomeric PSGL-1 tethered to P-selectin at equivalent rates. However, cells expressing dimeric PSGL-1 established more stable rolling adhesions, which were more shear resistant and exhibited less fluctuation in rolling velocities. On low densities of dimeric P-selectin, increasing wall shear stress more rapidly increased transient tether dissociation of cells expressing monomeric PSGL-1 than dimeric PSGL-1. Tether dissociation on low densities of monomeric P-selectin was even more shear sensitive. We conclude that dimerization of both PSGL-1 and P-selectin stabilizes tethering and rolling...

‣ Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model

Hanausek, Malgorzata; Ganesh, Pattabhiraman; Walaszek, Zbigniew; Arntzen, Charles J.; Slaga, Thomas J.; Gutterman, Jordan U.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/09/2001 Português
Relevância na Pesquisa
36.480713%
We tested the ability of avicins, a family of triterpenoid saponins obtained from Acacia victoriae (Bentham) (Leguminosae: Mimosoideae), to inhibit chemically induced mouse skin carcinogenesis. Varying doses of avicins were applied to shaved dorsal skin of SENCAR mice 15 min before application of 100 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) twice a week for 4 weeks (complete carcinogenesis model). The dorsal skin of a second group of mice was treated with one dose of 10 nmol of DMBA. Avicins were then applied 15 min before repetitive doses of 2 μg of phorbol 12-tetradecanoate 13-acetate (TPA) twice a week for 8 weeks (initiation/promotion model). At 12 weeks, avicins produced a 70% decrease in the number of mice with papillomas and a greater than 90% reduction in the number of papillomas per mouse in both protocols. We also observed a 62% and 74% reduction by avicins in H-ras mutations at codon 61 in the DMBA and DMBA/TPA models, respectively, as well as a significant inhibition of the modified DNA base formation (8-OH-dG) in both protocols. Marked suppression of aneuploidy occurred with treatment at 16 weeks in the initiation/promotion experiment. These findings, when combined with the proapoptotic property of these compounds and their ability to inhibit hydrogen peroxide (H2O2) generation...

‣ Singlet exciton binding energy in poly(phenylene vinylene)

Moses, D.; Wang, J.; Heeger, A. J.; Kirova, N.; Brazovski, S.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
The exciton binding energy (Eb) and the band gap energy (Eg) of poly(phenylene vinylene) are determined by high-resolution measurements of the photoconductivity excitation profile as a function of light polarization, applied electric field, and temperature. At high applied electric fields, a peak in the photoconductivity is observed when the sample is pumped at a photon energy just below the onset of the band-to-band π-π* absorption. This peak is interpreted as resulting from field ionization of a weakly bound exciton with Eb ≈ 60 meV. The binding energy is obtained from the energy of the exciton peak relative to the band edge and independently from analysis of the dependence of the exciton dissociation on field and temperature.

‣ Ab initio protein structure prediction on a genomic scale: Application to the Mycoplasma genitalium genome

Kihara, Daisuke; Zhang, Yang; Lu, Hui; Kolinski, Andrzej; Skolnick, Jeffrey
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
An ab initio protein structure prediction procedure, TOUCHSTONE, was applied to all 85 small proteins of the Mycoplasma genitalium genome. TOUCHSTONE is based on a Monte Carlo refinement of a lattice model of proteins, which uses threading-based tertiary restraints. Such restraints are derived by extracting consensus contacts and local secondary structure from at least weakly scoring structures that, in some cases, can lack any global similarity to the sequence of interest. Selection of the native fold was done by using the convergence of the simulation from two different conformational search schemes and the lowest energy structure by a knowledge-based atomic-detailed potential. Among the 85 proteins, for 34 proteins with significant threading hits, the template structures were reasonably well reproduced. Of the remaining 51 proteins, 29 proteins converged to five or fewer clusters. In the test set, 84.8% of the proteins that converged to five or fewer clusters had a correct fold among the clusters. If this statistic is simply applied, 24 proteins (84.8% of the 29 proteins) may have correct folds. Thus, the topology of a total of 58 proteins probably has been correctly predicted. Based on these results, ab initio protein structure prediction is becoming a practical approach.

‣ Shotgun identification of protein modifications from protein complexes and lens tissue

MacCoss, Michael J.; McDonald, W. Hayes; Saraf, Anita; Sadygov, Rovshan; Clark, Judy M.; Tasto, Joseph J.; Gould, Kathleen L.; Wolters, Dirk; Washburn, Michael; Weiss, Avery; Clark, John I.; Yates, John R.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 11/06/2002 Português
Relevância na Pesquisa
36.480713%
Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue...

‣ Identifying genes of agronomic importance in maize by screening microsatellites for evidence of selection during domestication

Vigouroux, Y.; McMullen, M.; Hittinger, C. T.; Houchins, K.; Schulz, L.; Kresovich, S.; Matsuoka, Y.; Doebley, J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Crop species experienced strong selective pressure directed at genes controlling traits of agronomic importance during their domestication and subsequent episodes of selective breeding. Consequently, these genes are expected to exhibit the signature of selection. We screened 501 maize genes for the signature of selection using microsatellites or simple sequence repeats (SSRs). We applied the Ewens–Watterson test, which can reveal deviations from a neutral-equilibrium model, as well as two nonequilibrium tests that incorporate the domestication bottleneck. We investigated two classes of SSRs: those known to be polymorphic in maize (Class I) and those previously classified as monomorphic in maize (Class II). Fifteen SSRs exhibited some evidence for selection in maize and 10 showed evidence under stringent criteria. The genes containing nonneutral SSRs are candidates for agronomically important genes. Because demographic factors can bias our tests, further independent tests of these candidates are necessary. We applied such an additional test to one candidate, which encodes a MADS box transcriptional regulator, and confirmed that this gene experienced a selective sweep during maize domestication. Genomic scans for the signature of selection offer a means of identifying new genes of agronomic importance even when gene function and the phenotype of interest are unknown.

‣ Regulation of transgene expression in plants with polydactyl zinc finger transcription factors

Ordiz, M. Isabel; Barbas, Carlos F.; Beachy, Roger N.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Designer zinc finger transcription factors (TFsZF) have been developed to control the expression of transgenes and endogenous genes in mammalian cells. Application of TFsZF technology in plants would enable a wide range of both basic and applied studies. In this paper, we report the use of TFsZF to target a defined 18-bp DNA sequence to control gene expression in plant cells and in transgenic plants. A β-glucuronidase reporter gene was activated by using the designed six-zinc finger protein 2C7 expressed as a fusion with the herpes simplex virus VP16 transcription factor activation domain. Reporter gene expression was activated 5- to 30-fold by using TFsZF in BY-2 protoplasts, whereas expression was increased as much as 450 times in transgenic tobacco plants. Use of a phloem-specific promoter to drive expression of the TFsZF resulted in activation of the reporter gene in vascular tissues. Transgenic tobacco plants that produce 2C7 transcription factors were phenotypically normal through two generations, suggesting that the factors exerted no adverse effects. This study demonstrates the utility of zinc finger technology in plants, setting the stage for its application in basic and applied agricultural biotechnology.

‣ Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

Mougous, Joseph D.; Leavell, Michael D.; Senaratne, Ryan H.; Leigh, Clifton D.; Williams, Spencer J.; Riley, Lee W.; Leary, Julie A.; Bertozzi, Carolyn R.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell–cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SOdocumentclass[12pt]{minimal} usepackage{amsmath} usepackage{wasysym} usepackage{amsfonts} usepackage{amssymb} usepackage{amsbsy} usepackage{mathrsfs} setlength{oddsidemargin}{-69pt} egin{document} egin{equation*}_{4}^{2-}end{equation*}end{document}). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach...

‣ Single-cell nonphotochemical hole burning of ovarian surface epithelial carcinoma and normal cells

Walsh, R. J.; Matsuzaki, S.; Reinot, T.; Hayes, J. M.; Kalli, K. R.; Hartmann, L. C.; Small, G. J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Persistent spectral nonphotochemical hole-burning (NPHB) spectroscopy has recently been applied to dye molecules in cells. The sensitivity of NPHB to the nanoenvironment of the probe is well established. It has been shown that NPHB applied to bulk suspensions of cultured human cells can distinguish between normal and cancer cells. Thus, NPHB has potential as a diagnostic cancer tool. For this reason, the methodology is referred to as hole-burning imaging, by analogy with MRI. The optical dephasing time (T2) of the dye in hole-burning image replaces the proton T1 relaxation time in MRI. In addition to the T2 mode of operation, there are four other modes including measurement of the spectral hole growth kinetics (HGK). Reported here is that the selectivity and sensitivity of NPHB operating in the HGK mode allow for distinction between normal and carcinoma cells at the single-cell level. The ovarian cell lines are ovarian surface epithelial cells with temperature-sensitive large T antigens (analogously normal) and ovarian surface epithelial carcinoma (OV167) cells. The mitochondrial specific dye used was rhodamine 800 (Molecular Probes). This carbocationic dye is highly specific for the outer and inner membranes of mitochondria. In line with the results for bulk suspensions of the two cell lines...

‣ DNA unzipped under a constant force exhibits multiple metastable intermediates

Danilowicz, Claudia; Coljee, Vincent W.; Bouzigues, Cedric; Lubensky, David K.; Nelson, David R.; Prentiss, Mara
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single λ phage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min.

‣ Probing the kinesin reaction cycle with a 2D optical force clamp

Block, Steven M.; Asbury, Charles L.; Shaevitz, Joshua W.; Lang, Matthew J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADP/Pi release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.

‣ Caspase 8 small interfering RNA prevents acute liver failure in mice

Zender, Lars; Hütker, Sebastian; Liedtke, Christian; Tillmann, Hans Ludger; Zender, Steffen; Mundt, Bettina; Waltemathe, Morlen; Gösling, Thomas; Flemming, Peer; Malek, Nisar Peter; Trautwein, Christian; Manns, Michael Peter; Kühnel, Florian; Kubicka,
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
A major concern in therapy of acute liver failure is protection of hepatocytes to prevent apoptosis and maintain liver function. Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells. To evaluate the therapeutic efficacy of siRNA in vivo we used different mouse models of acute liver failure. We directed 21-nt siRNAs against caspase 8, which is a key enzyme in death receptor-mediated apoptosis. Systemic application of caspase 8 siRNA results in inhibition of caspase 8 gene expression in the liver, thereby preventing Fas (CD95)-mediated apoptosis. Protection of hepatocytes by caspase 8 siRNA significantly attenuated acute liver damage induced by agonistic Fas (CD95) antibody (Jo2) or by adenovirus expressing Fas ligand (AdFasL). However, in a clinical situation the siRNAs most likely would be applied after the onset of acute liver failure. Therefore we injected caspase 8 siRNA at a time point during AdFasL- and adenovirus wild type (Adwt)-mediated liver failure with already elevated liver transaminases. Improvement of survival due to RNA interference was significant even when caspase 8 siRNA was applied during ongoing acute liver failure. In addition, it is of particular interest that caspase 8 siRNA treatment was successful not only in acute liver failure mediated by specific Fas agonistic agents (Jo2 and AdFasL) but also in acute liver failure mediated by Adwt...

‣ Regulation of zinc homeostasis by inducible NO synthase-derived NO: Nuclear metallothionein translocation and intranuclear Zn2+ release

Spahl, Daniela U.; Berendji-Grün, Denise; Suschek, Christoph V.; Kolb-Bachofen, Victoria; Kröncke, Klaus-D.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.

‣ Phytotoxicity of foliar-applied urea

Krogmeier, Michael J.; McCarty, Gregory W.; Bremner, John M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1989 Português
Relevância na Pesquisa
36.480713%
Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH2CONH2 + H2O → 2NH3 + CO2) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as phenylphosphorodiamidate. Because the leaf-tip necrosis often observed after foliar fertilization of plants with urea is usually attributed to ammonia formed through hydrolysis of urea by plant urease, we studied the possibility that this necrosis could be eliminated or reduced by adding phenylphosphorodiamidate to the urea fertilizer. We found that, although addition of this urease inhibitor to foliar-applied urea increased the urea content and decreased the ammonia content and urease activity of soybean [Glycine max. (L.) Merr.] leaves fertilized with urea, it increased the leaf-tip necrosis observed after fertilization. We conclude that this necrosis resulted from accumulation of toxic amounts of urea rather than from formation of toxic amounts of ammonia. This conclusion was supported by our finding that the necrotic areas of soybean leaves treated with urea or with urea and phenylphosphorodiamidate contained much higher concentrations of urea than did the nonnecrotic areas.

‣ Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore

Gu, Li-Qun; Cheley, Stephen; Bayley, Hagan
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
The flux of solvent water coupled to the transit of ions through protein pores is considerable. The effect of this electroosmotic solvent flow on the binding of a neutral molecule [β-cyclodextrin (βCD)] to sites within the staphylococcal α-hemolysin pore was investigated. Mutant α-hemolysin pores were used to which βCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of the applied potential. The Kd values for βCD for individual mutant pores varied by >100-fold with the applied potential over a range of –120 to +120 mV. In all cases, the signs of the changes in binding free energy and the influence of potential on the association and dissociation rate constants for βCD were consistent with an electroosmotic effect.

‣ A simulation method for calculating the absolute entropy and free energy of fluids: Application to liquid argon and water

White, Ronald P.; Meirovitch, Hagai
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
The hypothetical scanning (HS) method is a general approach for calculating the absolute entropy and free energy by analyzing Boltzmann samples obtained by Monte Carlo (MC) or molecular dynamics techniques. With HS applied to a fluid, each configuration i of the sample is reconstructed by adding its atoms gradually to the initially empty volume, i.e., by placing them in their positions at i using transition probabilities (TPs). At each step of the process, the volume is divided into two parts, the already visited part (the “past”) and the “future” part, where obtaining the TP requires calculating partition functions over the future part in the presence of the frozen past. In recent publications, the TPs were calculated approximately by taking into account only partial future. Here we present a “complete HSMC” procedure, where the TPs are calculated from MC simulations carried out over the complete future. The complete HSMC method is applied to systems of liquid argon and the TIP3P model of water, and very good results for the free energy are obtained, as compared with results obtained by thermodynamic integration.

‣ Cardioprotective effects of thioredoxin in myocardial ischemia and the reperfusion role of S-nitrosation

Tao, Ling; Gao, Erhe; Bryan, Nathan S.; Qu, Yan; Liu, Hui-Rong; Hu, Aihua; Christopher, Theodore A.; Lopez, Bernard L.; Yodoi, Junji; Koch, Walter J.; Feelisch, Martin; Ma, Xin L.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Apoptosis contributes to myocardial ischemia/reperfusion (MI/R) injury, and both thioredoxin (Trx) and nitric oxide have been shown to exert antiapoptotic effects in vitro. Recent evidence suggests that this particular action of Trx requires S-nitrosation at Cys-69. The present study sought to investigate whether or not exogenously applied Trx reduces MI/R injury in vivo and to which extent this effect depends on S-nitrosation. Adult mice were subjected to 30 min of MI and treated with either vehicle or human Trx (hTrx, 2 mg/kg, i.p.) 10 min before reperfusion. Native hTrx was incorporated into myocardial tissue as shown by immunostaining, and reduced MI/R injury as evidenced by decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, DNA fragmentation, caspase-3 activity, and infarct size. When hTrx was partially S-nitrosated by preincubation with S-nitrosoglutathione, its cardioprotective effect was markedly enhanced. Treatment with hTrx significantly reduced p38 mitogen-activated protein kinase (MAPK) activity, and this effect was also potentiated by S-nitrosation. To further address the role of S-nitrosation for the overall antiapoptotic effect to Trx, the action of Escherichia coli Trx (eTrx) was investigated in the same model. Whereas eTrx inhibited MI/R-induced apoptosis to a degree similar to hTrx...

‣ Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Jonas, Elizabeth A.; Hickman, John A.; Chachar, Mushtaque; Polster, Brian M.; Brandt, Teresa A.; Fannjiang, Yihru; Ivanovska, Iva; Basañez, Gorka; Kinnally, Kathleen W.; Zimmerberg, Joshua; Hardwick, J. Marie; Kaczmarek, Leonard K.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Neuronal death is often preceded by functional alterations at nerve terminals. Anti- and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. N-truncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC...

‣ Heptameric (L12)6/L10 rather than canonical pentameric complexes are found by tandem MS of intact ribosomes from thermophilic bacteria

Ilag, Leopold L.; Videler, Hortense; McKay, Adam R.; Sobott, Frank; Fucini, Paola; Nierhaus, Knud H.; Robinson, Carol V.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these high-resolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7/L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover...

‣ DNA shuffling as a tool for protein crystallization

Keenan, Robert J.; Siehl, Daniel L.; Gorton, Rebecca; Castle, Linda A.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
36.480713%
The success of structural studies performed on an individual target in small scale or on many targets in the systemwide scale of structural genomics depends critically on three parameters: (i) obtaining an expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two variants that crystallized readily. One of these was used to determine the high-resolution structure of the enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal packing space by modifying the surface properties of the enzyme. The approach demonstrated here does not require guidance as to the type of mutation necessary for improvements in expression...