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A model for the emergence of cooperation, interdependence, and structure in evolving networks

Jain, Sanjay; Krishna, Sandeep
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Evolution produces complex and structured networks of interacting components in chemical, biological, and social systems. We describe a simple mathematical model for the evolution of an idealized chemical system to study how a network of cooperative molecular species arises and evolves to become more complex and structured. The network is modeled by a directed weighted graph whose positive and negative links represent “catalytic” and “inhibitory” interactions among the molecular species, and which evolves as the least populated species (typically those that go extinct) are replaced by new ones. A small autocatalytic set, appearing by chance, provides the seed for the spontaneous growth of connectivity and cooperation in the graph. A highly structured chemical organization arises inevitably as the autocatalytic set enlarges and percolates through the network in a short analytically determined timescale. This self organization does not require the presence of self-replicating species. The network also exhibits catastrophes over long timescales triggered by the chance elimination of “keystone” species, followed by recoveries.

Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation

Filipp, Dominik; Alizadeh-Khiavi, Kamel; Richardson, Christopher; Palma, Anthony; Paredes, Nethnapha; Takeuchi, Osamu; Akira, Shizuo; Julius, Michael
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/01/2001 Português
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Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

Molecular clocks

Robinson, Noah E.; Robinson, Arthur B.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 30/01/2001 Português
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A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0°C, 0.15 M Tris⋅HCl buffer have been determined; a library of 913 amide-containing peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solutions, the use of this method for quantitative measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as molecular timers of biological events.

Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase

Zhang, Ying; Chang, Chenbei; Gehling, Daniel J.; Hemmati-Brivanlou, Ali; Derynck, Rik
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 30/01/2001 Português
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Smad proteins are key intracellular signaling effectors for the transforming growth factor-β superfamily of peptide growth factors. Following receptor-induced activation, Smads move into the nucleus to activate transcription of a select set of target genes. The activity of Smad proteins must be tightly regulated to exert the biological effects of different ligands in a timely manner. Here, we report the identification of Smurf2, a new member of the Hect family of E3 ubiquitin ligases. Smurf2 selectively interacts with receptor-regulated Smads and preferentially targets Smad1 for ubiquitination and proteasome-mediated degradation. At higher expression levels, Smurf2 also decreases the protein levels of Smad2, but not Smad3. In Xenopus embryos, ectopic Smurf2 expression specifically inhibits Smad1 responses and thereby affects embryonic patterning by bone morphogenetic protein signals. These findings suggest that Smurf2 may regulate the competence of a cell to respond to transforming growth factor-β/bone morphogenetic protein signaling through a distinct degradation pathway that is similar to, yet independent of, Smurf1.

Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: Unexpected functions for ancient enzymes?

MacIntosh, Gustavo C.; Bariola, Pauline A.; Newbigin, Ed; Green, Pamela J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 30/01/2001 Português
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The T2 family of nonspecific endoribonucleases (EC 3.1.27.1) is a widespread family of RNases found in every organism examined thus far. Most T2 enzymes are secretory RNases and therefore are found extracellularly or in compartments of the endomembrane system that would minimize their contact with cellular RNA. Although the biological functions of various T2 RNases have been postulated on the basis of enzyme location or gene expression patterns, the cellular roles of these enzymes are generally unknown. In the present work, we characterized Rny1, the only T2 RNase in Saccharomyces cerevisiae. Rny1 was found to be an active, secreted RNase whose gene expression is controlled by heat shock and osmotic stress. Inactivation of RNY1 leads to unusually large cells that are temperature-sensitive for growth. These phenotypes can be complemented not only by RNY1 but also by both structurally related and unrelated secretory RNases. Additionally, the complementation depends on RNase activity. When coupled with a recent report on the effect of specific RNAs on membrane permeability [Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I. & Yarus, M. (1999) Proc. Natl. Acad. Sci. USA 96, 10649–10654], our work suggests an unexpected role for Rny1 and possibly other secretory RNases. These enzymes may regulate membrane permeability or stability...

Patterns of spread in biological invasions dominated by long-distance jump dispersal: Insights from Argentine ants

Suarez, Andrew V.; Holway, David A.; Case, Ted J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 30/01/2001 Português
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45.74%
Invading organisms may spread through local movements (giving rise to a diffusion-like process) and by long-distance jumps, which are often human-mediated. The local spread of invading organisms has been fit with varying success to models that couple local population growth with diffusive spread, but to date no quantitative estimates exist for the relative importance of local dispersal relative to human-mediated long-distance jumps. Using a combination of literature review, museum records, and personal surveys, we reconstruct the invasion history of the Argentine ant (Linepithema humile), a widespread invasive species, at three spatial scales. Although the inherent dispersal abilities of Argentine ants are limited, in the last century, human-mediated dispersal has resulted in the establishment of this species on six continents and on many oceanic islands. Human-mediated jump dispersal has also been the primary mode of spread at a continental scale within the United States. The spread of the Argentine ant involves two discrete modes. Maximum distances spread by colonies undergoing budding reproduction averaged 150 m/year, whereas annual jump-dispersal distances averaged three orders of magnitude higher. Invasions that involve multiple dispersal processes...

Lack of type XV collagen causes a skeletal myopathy and cardiovascular defects in mice

Eklund, Lauri; Piuhola, Jarkko; Komulainen, Jyrki; Sormunen, Raija; Ongvarrasopone, Chalermporn; Fässler, Reinhard; Muona, Anu; Ilves, Mika; Ruskoaho, Heikki; Takala, Timo E. S.; Pihlajaniemi, Taina
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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45.74%
Type XV collagen occurs widely in the basement membrane zones of tissues, but its function is unknown. To understand the biological role of this protein, a null mutation in the Col15a1 gene was introduced into the germ line of mice. Despite the complete lack of type XV collagen, the mutant mice developed and reproduced normally, and they were indistinguishable from their wild-type littermates. However, Col15a1-deficient mice showed progressive histological changes characteristic for muscular diseases after 3 months of age, and they were more vulnerable than controls to exercise-induced muscle injury. Despite the antiangiogenic role of type XV collagen-derived endostatin, the development of the vasculature appeared normal in the null mice. Nevertheless, ultrastructural analyses revealed collapsed capillaries and endothelial cell degeneration in the heart and skeletal muscle. Furthermore, perfused hearts showed a diminished inotropic response, and exercise resulted in cardiac injury, changes that mimic early or mild heart disease. Thus, type XV collagen appears to function as a structural component needed to stabilize skeletal muscle cells and microvessels.

Prolonging the half-life of human interferon-α2 in circulation: Design, preparation, and analysis of (2-sulfo-9-fluorenylmethoxycarbonyl)7- interferon-α2

Shechter, Yoram; Preciado-Patt, Liana; Schreiber, Gideon; Fridkin, Mati
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 30/01/2001 Português
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45.74%
Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-α2. The derivative thus obtained (FMS7–IFN-α2) has ≈4% the biological potency and 33 ± 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS7–IFN-α2 undergoes time-dependent spontaneous hydrolysis, generating active interferon with t1/2 values of 24 ± 2 h at pH 8.5 and 98 ± 10 h at pH 7.4. When native IFN-α2 is intravenously administered to mice, circulating antiviral activity is maintained for a short duration and then declines with t1/2 = 4 ± 0.5 h, reaching undetectable values at ≈18 h after administration. With intravenously administered FMS7–IFN-α2, there is a lag period of 2 h, followed by a progressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t1/2 = 35 ± 4 h. FMS7–IFN-α2 is resistant to α-chymotrypsin digest and to proteolytic inactivation by human serum proteases in vitro. We have thus introduced here an inactive IFN-α2 derivative, which is resistant to in situ inactivation and has the capability of slowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes...

The oncogenic transforming potential of the passage of single α particles through mammalian cell nuclei

Miller, Richard C.; Randers-Pehrson, Gerhard; Geard, Charles R.; Hall, Eric J.; Brenner, David J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 05/01/1999 Português
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45.74%
Domestic, low-level exposure to radon gas is considered a major environmental lung-cancer hazard involving DNA damage to bronchial cells by α particles from radon progeny. At domestic exposure levels, the relevant bronchial cells are very rarely traversed by more than one α particle, whereas at higher radon levels—at which epidemiological studies in uranium miners allow lung-cancer risks to be quantified with reasonable precision—these bronchial cells are frequently exposed to multiple α-particle traversals. Measuring the oncogenic transforming effects of exactly one α particle without the confounding effects of multiple traversals has hitherto been unfeasible, resulting in uncertainty in extrapolations of risk from high to domestic radon levels. A technique to assess the effects of single α particles uses a charged-particle microbeam, which irradiates individual cells or cell nuclei with predefined exact numbers of particles. Although previously too slow to assess the relevant small oncogenic risks, recent improvements in throughput now permit microbeam irradiation of large cell numbers, allowing the first oncogenic risk measurements for the traversal of exactly one α particle through a cell nucleus. Given positive controls to ensure that the dosimetry and biological controls were comparable...

Mechanisms by which IκB proteins control NF-κB activity

Simeonidis, Simos; Stauber, Deborah; Chen, Guoying; Hendrickson, Wayne A.; Thanos, Dimitris
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 05/01/1999 Português
Relevância na Pesquisa
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The biological activity of the transcription factor NF-κB is differentially controlled by three IκB proteins, IκBα, IκBβ, and IκBɛ. We have examined the molecular basis for the differential inhibitory strengths of IκB proteins by constructing hybrid IκBs and found that the first ankyrin repeat of IκBα is responsible for its strong inhibitory effect. Swapping a putative β-turn within the first ankyrin repeat between the strong IκBα and the weak IκBβ inhibitors switches their in vivo inhibitory activity on NF-κB. The qualitatively distinct contacts made by this β-turn in IκBα and IκBβ with NF-κB determine the efficiency by which IκBs sequester NF-κB to the cytoplasm, thus explaining their distinct effects on gene activity.

Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101

Datta, Hirock J.; Khatri, Ghan Shyam; Bastia, Deepak
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 05/01/1999 Português
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Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA–DnaB interaction cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo. Although, interaction of DnaA with DnaB could not substitute for RepA–DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.

Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted in vivo activities

Varga, József L.; Schally, Andrew V.; Csernus, Valér J.; Zarándi, Márta; Halmos, Gábor; Groot, Kate; Rékási, Zoltán
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 19/01/1999 Português
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45.74%
Some antagonists of human growth hormone-releasing hormone (hGH-RH) synthesized previously were shown to inhibit in vivo proliferation of various human cancers in nude mice. However, the activity of these analogs requires an increase to assure clinical efficacy. In an attempt to prepare hGH-RH antagonists with a high and protracted activity, we synthesized and biologically tested 22 antagonistic analogs of hGH-RH(1–29)NH2. The ability of the antagonists to inhibit hGH-RH-induced GH release was evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinity of the peptides to GH-RH receptors also was determined. All antagonistic analogs had the common core sequence [PhAc-Tyr1,d-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (α-aminobutyric acid),Nle27]hGH-RH(1–29)NH2 and contained Arg, d-Arg, homoarginine (Har), norleucine (Nle), and other substitutions. The following analogs were determined to have a high and/or protracted antagonistic activity: [PhAc-Tyr1,d-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27,d-Arg29]hGH-RH(1–29)NH2 (JV-1–10), [PhAc-Tyr1,d-Arg2,Phe(4-Cl)6,Abu15,Nle27,d-Arg28,Har29]hGH-RH(1–29)NH2 (MZ-6–55), [PhAc-Tyr1,d-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27...

Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization

Watari, Hidemichi; Blanchette-Mackie, E. Joan; Dwyer, Nancy K.; Glick, Jane M.; Patel, Shutish; Neufeld, Edward B.; Brady, Roscoe O.; Pentchev, Peter G.; Strauss, Jerome F.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/02/1999 Português
Relevância na Pesquisa
45.74%
Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.

TOXCAT: A measure of transmembrane helix association in a biological membrane

Russ, William P.; Engelman, Donald M.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/02/1999 Português
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45.74%
The noncovalent association of transmembrane α-helices is a fundamental event in the folding of helical membrane proteins. In this work, a system (TOXCAT) is developed for the study of transmembrane helix–helix oligomerization in a natural membrane environment. This assay uses a chimeric construct composed of the N-terminal DNA binding domain of ToxR (a dimerization-dependent transcriptional activator) fused to a transmembrane domain (tm) of interest and a monomeric periplasmic anchor (the maltose binding protein). Association of the tms results in the ToxR-mediated activation of a reporter gene encoding chloramphenicol acetyltransferase (CAT). The level of CAT expression indicates the strength of tm association. The assay distinguishes between a known dimerizing tm and a mutant in which dimerization is disrupted. In addition, modulation of the chimera concentration shows that the dimerization exhibits concentration dependence in membranes. TOXCAT also is used to select oligomeric tms from a library of randomized sequences, demonstrating the potential of this system to reveal novel oligomerization motifs. The TOXCAT system has been used to investigate glycophorin A tm-mediated dimerization. Although the overall sensitivity of glycophorin A tm dimerization to mutagenesis is found to be similar in membranes and in detergent micelles...

FOG-2: A novel GATA-family cofactor related to multitype zinc-finger proteins Friend of GATA-1 and U-shaped

Tevosian, Sergei G.; Deconinck, Anne E.; Cantor, Alan B.; Rieff, Heather I.; Fujiwara, Yuko; Corfas, Gabriel; Orkin, Stuart H.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/02/1999 Português
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45.74%
GATA factors are transcriptional regulatory proteins that play critical roles in the differentiation of multiple cell types in both vertebrates and invertebrates. Recent evidence suggests that the biological activities of both mammalian and Drosophila GATA factors are controlled in part by physical interaction with multitype zinc-finger proteins, Friend of GATA-1 (FOG) and U-shaped (Ush), respectively. Here we describe a new FOG-related polypeptide, designated FOG-2, that is likely to participate in differentiation mediated by GATA factors in several tissues. Expression of FOG-2 mRNA differs from that of FOG and is largely restricted to heart, neurons, and gonads in the adult. Somewhat broader expression is evident during mouse embryonic development. Similar to FOG and Ush, FOG-2 protein interacts specifically with the amino finger of GATA factors in the yeast two-hybrid system and in mammalian cells. Remarkably, though FOG-2 is quite divergent from FOG in its primary sequence, forced expression of FOG-2 rescues terminal erythroid maturation of FOG−/− hematopoietic cells. Thus, members of the FOG family of cofactors share highly specific association with GATA factors and are substantially interchangeable with respect to some aspects of function in vivo. The interaction of GATA and FOG family members constitutes an evolutionarily conserved paradigm for transcriptional control in differentiation and organogenesis.

Species realities and numbers in sexual vertebrates: Perspectives from an asexually transmitted genome

Avise, John C.; Walker, DeEtte
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/02/1999 Português
Relevância na Pesquisa
45.74%
A literature review is conducted on the phylogenetic discontinuities in mtDNA sequences of 252 taxonomic species of vertebrates. About 140 of these species (56%) were subdivided clearly into two or more highly distinctive matrilineal phylogroups, the vast majority of which were localized geographically. However, only a small number (two to six) of salient phylogeographic subdivisions (those that stand out against mean within-group divergences) characterized individual species. A previous literature summary showed that vertebrate sister species and other congeners also usually have pronounced phylogenetic distinctions in mtDNA sequence. These observations, taken together, suggest that current taxonomic species often agree reasonably well in number (certainly within an order-of-magnitude) and composition with biotic entities registered in mtDNA genealogies alone. In other words, mtDNA data and traditional taxonomic assignments tend to converge on what therefore may be “real” biotic units in nature. All branches in mtDNA phylogenies are nonanastomose, connected strictly via historical genealogy. Thus, patterns of historical phylogenetic connection may be at least as important as contemporary reproductive relationships per se in accounting for microevolutionary unities and discontinuities in sexually reproducing vertebrates. Findings are discussed in the context of the biological and phylogenetic species concepts.

Specific chemical and structural damage to proteins produced by synchrotron radiation

Weik, Martin; Ravelli, Raimond B. G.; Kryger, Gitay; McSweeney, Sean; Raves, Maria L.; Harel, Michal; Gros, Piet; Silman, Israel; Kroon, Jan; Sussman, Joel L.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 18/01/2000 Português
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45.74%
Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative “weak links” in a given biological macromolecule, which may be of structural and functional significance.

Identification of SSF1, CNS1, and HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90 loss-of-function mutation

Nathan, Debra F.; Vos, Melissa Harju; Lindquist, Susan
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/02/1999 Português
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45.74%
Hsp90 functions in a multicomponent chaperone system to promote the maturation and maintenance of a diverse, but specific, set of target proteins that play key roles in the regulation of cell growth and development. To identify additional components of the Hsp90 chaperone system and its targets, we searched for multicopy suppressors of various temperature-sensitive mutations in the yeast Hsp90 gene, HSP82. Three suppressors were isolated for one Hsp90 mutant (glutamate → lysine at amino acid 381). Each exhibited a unique, allele-specific pattern of suppression with other Hsp90 mutants and had unique structural and biological properties. SSF1 is a member of an essential gene family and functions in the response to mating pheromones. CNS1 is an essential gene that encodes a component of the Hsp90 chaperone machinery. The role of HCH1 is unknown; its sequence has no strong homology to any protein of known function. SSF1 and CNS1 were weak suppressors, whereas HCH1 restored wild-type growth rates at all temperatures tested to cells expressing the E381K mutant. Overexpression of CNS1 or HCH1, but not SSF1, enhanced the maturation of a heterologous Hsp90 target protein, p60v-src. These results suggest that like Cns1p...

Single-target molecule detection with nonbleaching multicolor optical immunolabels

Schultz, Sheldon; Smith, David R.; Mock, Jack J.; Schultz, David A.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 01/02/2000 Português
Relevância na Pesquisa
45.74%
We introduce and demonstrate the use of colloidal silver plasmon-resonant particles (PRPs) as optical reporters in typical biological assays. PRPs are ultrabright, nanosized optical scatterers, which scatter light elastically and can be prepared with a scattering peak at any color in the visible spectrum. PRPs are readily observed individually with a microscope configured for dark-field microscopy, with white-light illumination of typical power. Here we illustrate the use of PRPs, surface coated with standard ligands, as target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, such as those based on radioactivity, fluorescence, chemiluminescence, or enzymatic/colorimetric detection that are used routinely in biochemistry, cell biology, and medical diagnostic applications. Moreover, because PRP labels are nonbleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection is now practical. We also present the results of a model sandwich immunoassay for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal.

Multinuclear solid-state three-dimensional MRI of bone and synthetic calcium phosphates

Wu, Yaotang; Chesler, David A.; Glimcher, Melvin J.; Garrido, Leoncio; Wang, Jinxi; Jiang, Hong J.; Ackerman, Jerome L.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/02/1999 Português
Relevância na Pesquisa
45.74%
Multinuclear three-dimensional solid-state MRI of bone, tooth, and synthetic calcium phosphates is demonstrated in vitro and in vivo with a projection reconstruction technique based on acquisition of free induction decays in the presence of fixed amplitude magnetic field gradients. Phosphorus-31 solid-state MRI provides direct images of the calcium phosphate constituents of bone substance and is a quantitative measurement of the true volumetric bone mineral density of the bone. Proton solid-state MRI shows the density of bone matrix including its organic constituents, which consist principally of collagen. These solid-state MRI methods promise to yield a biological picture of bone richer in information concerning the bone composition and short range-crystalline order than the fluid-state images provided by conventional proton MRI or the density images produced by radiologic imaging techniques. Three-dimensional solid-state projection reconstruction MRI should be readily adaptable to both human clinical use and nonmedical applications for a variety of solids in materials science.