Sci USA (sci + usa)

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Kinds of Sci USA

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  • Selected Abstracts

    Strikingly fast microtubule sliding in bundles formed by Chlamydomonas axonemal dynein,

    CYTOSKELETON, Issue 6 2010
    Susumu Aoyama
    Abstract Chlamydomonas axonemal extracts containing outer-arm dynein bundle microtubules when added in the absence of ATP. The bundles dissociate after addition of ATP (Haimo et al., Proc Natl Acad Sci USA 76:5759,5768, 1979). In the present study, we investigated the ATP-induced bundle dissociation process using caged ATP. Application of ,0.5 mM ATP induced microtubule sliding at ,30 ,m·s,1, which was 1.5 times faster than the microtubule sliding observed in protease-treated axonemes and five times faster than microtubule gliding on glass surfaces coated with outer-arm dynein. Bundles formed by mutant dynein molecules that lack one of the three heavy chains (HCs) displayed similar high-speed intermicrotubule sliding. These results suggest that Chlamydomonas outer-arm dynein molecules, when aligned, can translocate microtubules at high speed and that the high-speed sliding under load-free conditions does not require the complete set of the three HCs. It is likely that each of the three HCs has the ability to produce high-speed sliding, which should be an important property for their cooperation. © 2010 Wiley-Liss, Inc. [source]

    Further studies on the interaction of loperamide with capacitative calcium entry in Leukemic HL-60 cells,

    John W. Daly
    Abstract Loperamide at 3,10,µM has augmentative effects on calcium levels elevated by capacitative calcium entry (CCE) in leukemic HL-60 cells after release of intracellular calcium by ATP or thapsigargin (Harper et al. [1997] Proc Natl Acad Sci USA 94:14912,14917). The effect of loperamide on calcium levels was absent at a pH value of 6.8, a pH at which CCE is not active in HL-60 cells. Further investigations of HL-60 cells in recent years revealed a great reduction in the magnitude of the loperamide response. However, when preceded by a CCE blocker, namely N-methylnitrendipine (MRS 1844) or N-propargylnitrendipine (MRS 1845), loperamide caused a significant reversal of the blockade. Six structural analogs of loperamide were synthesized, but only two showed loperamide-like activity. Drug Dev. Res. 67:842,851, 2006. Published 2007 Wiley-Liss, Inc. [source]

    Relative mutagenic potencies of several nucleoside analogs, alone or in drug pairs, at the HPRT and TK loci of human TK6 lymphoblastoid cells,

    Meghan M. Carter
    Abstract Experiments were performed to investigate the impact of didanosine (ddI), lamivudine (3TC), and stavudine (d4T) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using a cell cloning assay for assessing the effects of individual nucleoside analogs (NRTIs)/drug combinations in human TK6 B-lymphoblastoid cells. Three-day treatments with 0, 33, 100, or 300 ,M ddI, 3TC, or ddI-3TC produced positive trends for increased HPRT and TK mutant frequencies. While dose-related trends were too small to reach significance after treatments with d4T or d4T-3TC, pairwise comparisons with control cells indicated that exposure to 100 ,M d4T or d4T-3TC caused significant elevations in HPRT mutants. Measurements of mutagenicity in cells exposed to d4T (or d4T-3TC) were complicated by the cytotoxicity of this NRTI. Enhanced increases in mutagenic responses to combined NRTI treatments, compared with single drug treatments, occurred as additive to synergistic effects in the HPRT gene of cells exposed to 100 ,M ddI-3TC or 100 ,M d4T-3TC, and in the TK gene of cells exposed to 100 or 300 ,M ddI-3TC. Comparisons of these data to mutagenicity studies of other NRTIs in the same system (Meng Q et al. [2000c]: Proc Natl Acad Sci USA 97:12667,126671; Torres SM et al. [2007]: Environ Mol Mutagen) indicate that the relative mutagenic potencies for all drugs tested to date are: AZT-ddI > ddI-3TC > AZT-3TC , AZT-3TC-ABC (abacavir) > AZT ,ddI > d4T-3TC > 3TC > d4T , ABC. These collective data suggest that all NRTIs with antiviral activity against HIV-1 may cause host cell DNA damage and mutations, and impose a cancer risk. Environ. Mol. Mutagen., 2007. © 2007 Wiley-Liss, Inc. [source]

    Molecular analysis of mutations at the HPRT and TK loci of human lymphoblastoid cells after combined treatments with 3,-azido-3,-deoxythymidine and 2,,3,-dideoxyinosine,

    Quanxin Meng
    Abstract Combinations of antiretroviral drugs that include nucleoside reverse transcriptase inhibitors (NRTIs) are superior to single-agent regimens in treating or preventing HIV infection, but the potential long-term health hazards of these treatments in humans are uncertain. In earlier studies, our group found that coexposure of TK6 human lymphoblastoid cells to 3,-azido-2,,3,-dideoxythymidine (AZT) and 2,,3,-dideoxyinosine (ddI), the first two NRTIs approved by the FDA as antiretroviral drugs, produced multiplicative synergistic enhancement of DNA incorporation of AZT and mutagenic responses in both the HPRT and TK reporter genes, as compared with single-drug exposures (Meng Q et al. [2000a]: Proc Natl Acad Sci USA 97:12667,12671). The purpose of the current study was to characterize the mutational specificity of equimolar mixtures of 100 ,M or 300 ,M AZT + ddI at the HPRT and TK loci of exposed cells vs. unexposed control cells, and to compare the resulting mutational spectra data to those previously found in cells exposed to AZT alone (Sussman H et al. [1999]: Mutat Res 429:249,259; Meng Q et al. [2000b]: Toxicol Sci 54:322,329). Molecular analyses of HPRT mutant clones were performed by reverse transcription,mediated production of cDNA, PCR amplification, and cDNA sequencing to define small DNA alterations, followed by multiplex PCR amplification of genomic DNA to define the fractions of deletion events. TK mutants with complete gene deletions were distinguished by Southern blot analysis. The observed HPRT mutational categories included point mutations, microinsertions/microdeletions, splicing-error mutations, and macrodeletions including partial and complete gene deletions. The only significant difference or shift in the mutational spectra for NRTI-treated cells vs. control cells was the increase in the frequency of complete TK gene deletions following exposures (for 3 days) to 300 ,M AZT,ddI (P = 0.034, chi-square test of homogeneity); however, statistical analyses comparing the observed mutant fraction values (measured mutant frequency × percent of a class of mutation) between control and NRTI-treated cells for each class of mutation showed that the occurrences of complete gene deletions of both HPRT and TK were significantly elevated over background values (0.34 × 10,6 in HPRT and 6.0 × 10,6 in TK) at exposure levels of 100 ,M AZT,ddI (i.e., 1.94 × 10,6 in HPRT and 18.6 × 10,6 in TK) and 300 ,M AZT,ddI (i.e., 5.6 × 10,6 in HPRT and 34.6 × 10,6 in TK) (P < 0.05, Mann,Whitney U -statistic). These treatment-related increases in complete gene deletions were consistent with the spectra data for AZT alone (ibid.) and with the known mode of action of AZT and ddI as DNA chain terminators. In addition, cotreatments of ddI with AZT led to substantial absolute increases in the mutant fraction of other classes of mutations, unlike cells exposed solely to AZT [e.g., the frequency of point mutations among HPRT mutants was significantly increased by 130 and 323% over the background value (4.25 × 10,6) in cells exposed to 100 and 300 ,M AZT,ddI, respectively]. These results indicate that, at the same time that AZT,ddI potentiates therapeutic or prophylactic efficacy, the use of a second NRTI with AZT may confer a greater cancer risk, characterized by a spectrum of mutations that deviates from that produced solely by AZT. Environ. Mol. Mutagen. 39:282,295, 2002. Published 2002 Wiley-Liss, Inc. [source]

    Just how does the cII selection system work in MutaÔMouse?

    Roy R. Swiger
    Abstract The lambda CII protein is an essential component in the lytic vs. lysogeny decision a bacteriophage makes upon infection of a host at low temperatures. The protein interacts with numerous phage promoters modulating the expression of the CI repressor, thus providing the mechanism for lysogenization soon after infection. The Big Blue® and MutaÔMouse are two widely used in vivo mutational model systems. The assays rely on retrievable lambda-based transgenes housing mutational targets (lacI or lacZ, respectively). The transgenes provide an elegant vehicle for the quantification of mutations sustained in virtually any tissue of the rodent. The use of the bacteriophage cII locus as an alternative, or additional mutational target for use with the Big Blue® rodent system was first reported by Jakubczak et al. ([1996]: Proc Natl Acad Sci USA 93:9073,9078). More recently, this selection assay has been applied successfully to the MutaÔMouse (Swiger et al. [1999]: Environ Mol Mutagen 33:201,207). The use of an Hfl bacterial strain and low temperature allows the determination of mutations sustained at the cII locus in either system, with high fidelity. The cII selection assay in the Big Blue® relies on the presence of the lambda repressor protein CI. In contrast, the recombinant construct used to make the MutaÔMouse transgene lacks functional CI protein. Nevertheless, we report an excellent system for quantifying mutations at the cII locus in MutaÔMouse. Just how does cII selection work in the MutaÔMouse? Written in the context of lambda recombinant genetics, this paper explores the question further. Environ. Mol. Mutagen. 37:290,296, 2001 © 2001 Wiley-Liss, Inc. [source]

    TP53 mutation signature supports involvement of aristolochic acid in the aetiology of endemic nephropathy-associated tumours

    Tatiana Nedelko
    Abstract The proposal has been put forward that the primary cause of Balkan endemic nephropathy (BEN) is exposure to food crops contaminated with seeds of Aristolochia spp, which contain high levels of aristolochic acids (AA). Recently, tumour DNA samples from patients with BEN were found to harbour principally A to T mutations in the TP53 tumour suppressor gene (Grollman et al., Proc Natl Acad Sci USA 2007;104:12129,34). Using a novel mutation assay in which we can induce and select mutations in human TP53 sequences in vitro by exposure of cultured cells to a mutagen, we found that A to T mutations were elicited by aristolochic acid at sites in TP53 rarely mutated in human cancers in general, but which were observed in the BEN patients. This concordance of specific mutations in patient tumours and aristolochic acid I-exposed cultures supports the argument that AA has a direct role in the aetiology of BEN-associated cancer. © 2008 Wiley-Liss, Inc. [source]

    Inverse Monte Carlo procedure for conformation determination of macromolecules

    Mark Bathe
    Abstract A novel numerical method for determining the conformational structure of macromolecules is applied to idealized biomacromolecules in solution. The method computes effective inter-residue interaction potentials solely from the corresponding radial distribution functions, such as would be obtained from experimental data. The interaction potentials generate conformational ensembles that reproduce thermodynamic properties of the macromolecule (mean energy and heat capacity) in addition to the target radial distribution functions. As an evaluation of its utility in structure determination, we apply the method to a homopolymer and a heteropolymer model of a three-helix bundle protein [Zhou, Y.; Karplus, M. Proc Natl Acad Sci USA 1997, 94, 14429; Zhou, Y. et al. J Chem Phys 1997, 107, 10691] at various thermodynamic state points, including the ordered globule, disordered globule, and random coil states. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 876,890, 2003 [source]

    Detection of human bocavirus in respiratory, fecal, and blood samples by real-time PCR,

    Sarah J. Tozer
    Abstract Human bocavirus (HBoV) has been detected worldwide in respiratory samples. Two real-time PCR assays, targeting the non-structural protein (NP-1) and viral protein (VP-1) genes, were designed and validated to detect HBoV in patients with respiratory disease, gastroenteritis, or systemic illness. Sensitivity of the NP-1 and VP-1 assays were equal to the conventional PCR assay previously described by Allander et al. [2005: Proc Natl Acad Sci USA 102: 12891,12896] being 100%, and giving specificity of 94% and 93%, respectively. There was no cross-reaction identified with unrelated respiratory agents, or to human DNA. The limits of detection were 10 copies of genomic DNA equivalents per reaction for both assays. The assays were used to screen three different sample populations, combined nose, and throat swabs (n,=,96) from children with acute respiratory disease, fecal samples (n,=,375) from adults, and children with gastroenteritis and whole blood (n,=,229) collected from 31 immunocompromised children taken over an 18-month period. In total 17 (18%) respiratory samples and 18 (4.8%) fecal samples were identified as having HBoV present. Of the pediatric whole blood specimens investigated, HBoV was detected in six (2.6%) samples from four patients. In summary, two real-time PCR assays targeting different genes were designed and validated for use as screening methods for the detection of HBoV. HBoV was found in three different specimen types: parent-collected combined nose,throat swabs, fecal samples collected from symptomatic individuals and whole blood from immunocompromised children. J. Med. Virol. 81:488,493, 2009. © 2009 Wiley-Liss, Inc. [source]

    Antitumor activity and mechanism of action of the iron chelator, Dp44mT, against leukemic cells,

    Egarit Noulsri
    Iron chelators have been reported to induce apoptosis and cell cycle arrest in cancer cells. Recent studies suggest broad and selective antitumor activity of the new iron chelator, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Whitnall et al., Proc Natl Acad Sci USA 2006;103:14901,14906). However, little is known concerning its effects on hematological malignancies. Using acute leukemia cells, the effect of Dp44mT on apoptosis, cell cycle, caspase-3 activation, and mitochondrial trans-membrane potential has been examined by flow cytometry. Dp44mT acted to induce a G1/S arrest in NB4 promyelocytic leukemia cells at low concentrations (0.5,2.5 ,M), being far more effective than the clinically used chelator, desferrioxamine (DFO). Moreover, Dp44mT induced apoptosis of NB4 cells in a dose- and time-dependent manner with markedly less effect on nonproliferating cells. The apoptosis-inducing activity of Dp44mT was significantly more effective than DFO. Furthermore, this study also showed that Dp44mT had broad activity, inducing apoptosis in several types of acute leukemia and also multiple myeloma cell lines. Additional studies examining the cytotoxic mechanisms of Dp44mT showed that a reduction in the mitochondrial trans-membrane potential and caspase-3 activation could be involved in the mechanism of apoptosis. Our results suggest that Dp44mT possesses potential as an effective cytotoxic agent for the chemotherapeutic treatment of acute leukemia. Am. J. Hematol. 2009. © 2008 Wiley-Liss, Inc. [source]

    Childhood, adolescence, and longevity: A multilevel model of the evolution of reserve capacity in human life history

    Barry Bogin
    The grandmother hypothesis (GH) of Hawkes et al. ([1998]: Proc Natl Acad Sci USA 95: 1336,1339) finds that selection for lower adult mortality and greater longevity allow for the evolution of prolonged growth in human beings. In contrast, other researchers propose that the evolution of the human childhood and adolescent stages of life history prolonged the growth period and allowed for greater biological resilience and longevity compared with apes. In this article, the GH model is reanalyzed using new values for some of its key variables. The original GH set the age at human feeding independence at 2.8 years of age (weaning) and used demographic data from living foragers to estimate average adult lifespan after first birth at 32.9 years. The reanalysis of the GH uses age 7.0 years (end of the childhood stage) as the minimum for human feeding independence and uses data from healthier populations, rather than foragers, to derive an estimate of 48.9 years for average adult life span. Doing so finds that selection operated to first shorten the infancy stage (wean early compared with apes), then prolong the growth period, and finally result in greater longevity. The reanalysis provides a test of the reserve capacity hypothesis as part of a multilevel model of human life history evolution. Am. J. Hum. Biol. 2009. © 2009 Wiley-Liss, Inc. [source]

    Engineering antibody fragments to fold in the absence of disulfide bonds

    PROTEIN SCIENCE, Issue 2 2009
    Min Jeong Seo
    Abstract Disulfide bonds play a critical role in the stabilization of the immunoglobulin ,-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193,9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues. [source]

    Folding and binding cascades: Dynamic landscapes and population shifts

    PROTEIN SCIENCE, Issue 1 2000
    Sandeep Kumar
    Abstract Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713,720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181,1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970,9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three-dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings. [source]

    The phylogenetic affinities of the Pondaung tali

    Marian Dagosto
    Abstract The phylogenetic affinities of the primates of the late middle Eocene Pondaung Formation of Myanmar have long been disputed. The discovery of the NMMP 39 talus (Marivaux et al.: Proc Natl Acad Sci USA 100 (2003) 13173,13178) provided the first clear evidence from the postcranium that a relatively large-bodied haplorhine primate is represented in the Pondaung fauna. Another talus (NMMP 82; Marivaux et al., 2010). Talar morphology, phylogenetic affinities and locomotor adaptation of a large-bodied amphipithecid primate from the late middle Eocene of Myanmar, Am J Phys Anthropol DOI: 10.1002/ajpa.21307) has been recently recovered which also pertains to Haplorhini. The metric and nonmetric features supporting the hypothesis of anthropoid affinities for NMMP 39 have been criticized by Gunnell and Ciochon (Gunnell GF, Ciochon RL. 2008. Revisiting primate postcrania from the Pondaung Formation of Myanmar. In: Fleagle JG, Gilbert CC, editors. Elwyn Simons: a search for origins. New York: Springer. p 211,228). Their analysis, however, was based on a very limited choice of variables, taxa, and individuals. Based on an extended sample, we are able to produce both principal components and discriminant functions that yield a rather clear separation of extant haplorhine and strepsirhine tali. Both principal components and discriminant function scores of the Pondaung tali fall with those of haplorhine primates. In addition, the Pondaung tali lack all the derived nonmetric features characteristic of strepsirhine primates, but exhibit all the features characteristic of haplorhine primates. We dispute the features Gunnell and Ciochon (2008) claim are uniquely shared by the Pondaung tali and adapiforms. Their rejection of the phylogenetic significance of the features shared by these tali and haplorhines is unwarranted by the evidence. Based on both metric and nonmetric features, the Pondaung tali are structurally most similar to the tali of haplorhines, particularly anthropoids. Am J Phys Anthropol 143:223,234, 2010. © 2010 Wiley-Liss, Inc. [source]

    Brief communication: "Pathological" deformation in the skull of LB1, the type specimen of Homo floresiensis

    Yousuke Kaifu
    Abstract If the holotype of Homo floresiensis, LB1, suffered from a severe developmental pathology, this could undermine its status as the holotype of a new species. One of the proposed pathological indicators that still remains untested is asymmetric distortion in the skull of LB1 (Jacob et al.: Proc Natl Acad Sci USA 103 (2006) 13421,13426). Here, we present evidence that LB1 exhibits antemortem craniofacial deformities that are consistent with posterior deformational (positional) plagiocephaly. This is a relatively common condition in modern people with no serious associated health problems and does not represent a severe developmental abnormality in LB1. Am J Phys Anthropol 2009. © 2009 Wiley-Liss, Inc. [source]

    Ribosome motions modulate electrostatic properties

    BIOPOLYMERS, Issue 6 2004
    Joanna Trylska
    Abstract The electrostatic properties of the 70S ribosome of Thermus thermophilus were studied qualitatively by solving the Poisson,Boltzmann (PB) equation in aqueous solution and with physiological ionic strength. The electrostatic potential was calculated for conformations of the ribosome derived by recent normal mode analysis (Tama, F., et al. Proc Natl Acad Sci USA 2003 100, 9319,9323) of the ratchet-like reorganization that occurs during translocation (Frank, J.; Agrawal, R. K. Nature 2000 406, 318,322). To solve the PB equation, effective parameters (charges and radii), applicable to a highly charged backbone model of the ribosome, were developed. Regions of positive potential were found at the binding site of the elongation factors G and Tu, as well as where the release factors bind. Large positive potential areas are especially pronounced around the L11 and L6 proteins. The region around the L1 protein is also positively charged, supporting the idea that L1 may interact with the E-site tRNA during its release from the ribosome after translocation. Functional rearrangement of the ribosome leads to electrostatic changes which may help the translocation of the tRNAs during the elongation stage. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004 [source]

    Thermal stabilization of the DNA duplex by adducts of aflatoxin B1

    BIOPOLYMERS, Issue 3 2002
    Indrajit Giri
    Abstract The trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 cationic guanine N7 adduct of aflatoxin B1 thermally stabilizes the DNA duplex, as reflected in increased Tm values upon adduction. The magnitude of the increased Tm value is characteristically 2,3°C. The major rotamer of the neutral guanine N7 adduct trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B1 (the FAPY major adduct) exhibits a 15°C increase in Tm in 5,-d(CTATFAPYGATTCA)-3,-5,-d(TGAATCATAG)-3,. Site-specific mutagenesis experiments reveal the FAPY major adduct induces G,T mutations in Escherichia coli at a frequency six times higher than that of the cationic adduct (Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc Natl Acad Sci USA, 99, 6655,6660). Thus, the FAPY major lesion may account substantially for the genotoxicity of AFB1. Structural studies for cationic and FAPY adducts of aflatoxin B1 suggest both adducts intercalate above the 5,-face of the modified deoxyguanosine and that in each instance the aflatoxin moiety spans the DNA helix. Intercalation of the aflatoxin moiety, accompanied by favorable stacking with the neighboring base pairs, is thought to account for the increased thermal stability of the aflatoxin cationic guanine N7 and the FAPY major adducts. However, the structural basis for the large increase in thermal stability of the FAPY major adduct in comparison to the cationic guanine N7 adduct of aflatoxin B1 is not well understood. In light of the site-specific mutagenesis studies, it is of considerable interest. For both adducts, the intercalation structures are similar, although improved stacking with neighboring base pairs is observed for the FAPY major adduct. In addition, the presence of the formamido group in the aflatoxin B1 FAPY major adduct may enhance duplex stability, perhaps via intrastrand sequence-specific hydrogen bonding interactions within the duplex. © 2002 Wiley Periodicals, Inc. Biopolymers (Nucleic Acid Sci) 65: 190,201, 2002 [source]