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1.
Phys Fluids (1994) ; 33(3): 037122, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33897243

RESUMO

This paper presents the Mechanical Ventilator Milano (MVM), a novel intensive therapy mechanical ventilator designed for rapid, large-scale, low-cost production for the COVID-19 pandemic. Free of moving mechanical parts and requiring only a source of compressed oxygen and medical air to operate, the MVM is designed to support the long-term invasive ventilation often required for COVID-19 patients and operates in pressure-regulated ventilation modes, which minimize the risk of furthering lung trauma. The MVM was extensively tested against ISO standards in the laboratory using a breathing simulator, with good agreement between input and measured breathing parameters and performing correctly in response to fault conditions and stability tests. The MVM has obtained Emergency Use Authorization by U.S. Food and Drug Administration (FDA) for use in healthcare settings during the COVID-19 pandemic and Health Canada Medical Device Authorization for Importation or Sale, under Interim Order for Use in Relation to COVID-19. Following these certifications, mass production is ongoing and distribution is under way in several countries. The MVM was designed, tested, prepared for certification, and mass produced in the space of a few months by a unique collaboration of respiratory healthcare professionals and experimental physicists, working with industrial partners, and is an excellent ventilator candidate for this pandemic anywhere in the world.

2.
Phys Rev Lett ; 118(25): 251301, 2017 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-28696731

RESUMO

New results are reported from the operation of the PICO-60 dark matter detector, a bubble chamber filled with 52 kg of C_{3}F_{8} located in the SNOLAB underground laboratory. As in previous PICO bubble chambers, PICO-60 C_{3}F_{8} exhibits excellent electron recoil and alpha decay rejection, and the observed multiple-scattering neutron rate indicates a single-scatter neutron background of less than one event per month. A blind analysis of an efficiency-corrected 1167-kg day exposure at a 3.3-keV thermodynamic threshold reveals no single-scattering nuclear recoil candidates, consistent with the predicted background. These results set the most stringent direct-detection constraint to date on the weakly interacting massive particle (WIMP)-proton spin-dependent cross section at 3.4×10^{-41} cm^{2} for a 30-GeV c^{-2} WIMP, more than 1 order of magnitude improvement from previous PICO results.

3.
Phys Rev Lett ; 118(23): 231301, 2017 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-28644643

RESUMO

A 30-g xenon bubble chamber, operated at Northwestern University in June and November 2016, has for the first time observed simultaneous bubble nucleation and scintillation by nuclear recoils in a superheated liquid. This chamber is instrumented with a CCD camera for near-IR bubble imaging, a solar-blind photomultiplier tube to detect 175-nm xenon scintillation light, and a piezoelectric acoustic transducer to detect the ultrasonic emission from a growing bubble. The time of nucleation determined from the acoustic signal is used to correlate specific scintillation pulses with bubble-nucleating events. We report on data from this chamber for thermodynamic "Seitz" thresholds from 4.2 to 15.0 keV. The observed single- and multiple-bubble rates when exposed to a ^{252}Cf neutron source indicate that, for an 8.3-keV thermodynamic threshold, the minimum nuclear recoil energy required to nucleate a bubble is 19±6 keV (1σ uncertainty). This is consistent with the observed scintillation spectrum for bubble-nucleating events. We see no evidence for bubble nucleation by gamma rays at any of the thresholds studied, setting a 90% C.L. upper limit of 6.3×10^{-7} bubbles per gamma interaction at a 4.2-keV thermodynamic threshold. This indicates stronger gamma discrimination than in CF_{3}I bubble chambers, supporting the hypothesis that scintillation production suppresses bubble nucleation by electron recoils, while nuclear recoils nucleate bubbles as usual. These measurements establish the noble-liquid bubble chamber as a promising new technology for the detection of weakly interacting massive particle dark matter and coherent elastic neutrino-nucleus scattering.

4.
Phys Rev Lett ; 114(23): 231302, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26196790

RESUMO

New data are reported from the operation of a 2 liter C3F8 bubble chamber in the SNOLAB underground laboratory, with a total exposure of 211.5 kg days at four different energy thresholds below 10 keV. These data show that C3F8 provides excellent electron-recoil and alpha rejection capabilities at very low thresholds. The chamber exhibits an electron-recoil sensitivity of <3.5×10(-10) and an alpha rejection factor of >98.2%. These data also include the first observation of a dependence of acoustic signal on alpha energy. Twelve single nuclear recoil event candidates were observed during the run. The candidate events exhibit timing characteristics that are not consistent with the hypothesis of a uniform time distribution, and no evidence for a dark matter signal is claimed. These data provide the most sensitive direct detection constraints on WIMP-proton spin-dependent scattering to date, with significant sensitivity at low WIMP masses for spin-independent WIMP-nucleon scattering.


Assuntos
Fluorocarbonos/química , Modelos Teóricos , Acústica/instrumentação , Algoritmos , Nêutrons
5.
Phys Rev Lett ; 108(25): 259001; discussion 259002, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-23004668
6.
Phys Rev Lett ; 107(5): 051301, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21867059

RESUMO

We report results of a search for light (≲10 GeV) particle dark matter with the XENON10 detector. The event trigger was sensitive to a single electron, with the analysis threshold of 5 electrons corresponding to 1.4 keV nuclear recoil energy. Considering spin-independent dark matter-nucleon scattering, we exclude cross sections σ(n)>7×10(-42) cm(2), for a dark matter particle mass m(χ)=7 GeV. We find that our data strongly constrain recent elastic dark matter interpretations of excess low-energy events observed by CoGeNT and CRESST-II, as well as the DAMA annual modulation signal.


Assuntos
Radiação Cósmica , Interpretação Estatística de Dados , Elétrons , Física Nuclear , Humanos , Luz , Fótons , Espalhamento de Radiação
7.
Phys Rev Lett ; 106(2): 021303, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21405218

RESUMO

Data from the operation of a bubble chamber filled with 3.5 kg of CF3I in a shallow underground site are reported. An analysis of ultrasound signals accompanying bubble nucleations confirms that alpha decays generate a significantly louder acoustic emission than single nuclear recoils, leading to an efficient background discrimination. Three dark matter candidate events were observed during an effective exposure of 28.1 kg day, consistent with a neutron background. This observation provides strong direct detection constraints on weakly interacting massive particle (WIMP)-proton spin-dependent scattering for WIMP masses >20 GeV/c2.

8.
Phys Rev Lett ; 101(9): 091301, 2008 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-18851599

RESUMO

XENON10 is an experiment to directly detect weakly interacting massive particles (WIMPs), which may comprise the bulk of the nonbaryonic dark matter in our Universe. We report new results for spin-dependent WIMP-nucleon interactions with 129Xe and 131Xe from 58.6 live days of operation at the Laboratori Nazionali del Gran Sasso. Based on the nonobservation of a WIMP signal in 5.4 kg of fiducial liquid xenon mass, we exclude previously unexplored regions in the theoretically allowed parameter space for neutralinos. We also exclude a heavy Majorana neutrino with a mass in the range of approximately 10 GeV/c2-2 TeV/c2 as a dark matter candidate under standard assumptions for its density and distribution in the galactic halo.

9.
Phys Rev Lett ; 100(2): 021303, 2008 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-18232850

RESUMO

The XENON10 experiment at the Gran Sasso National Laboratory uses a 15 kg xenon dual phase time projection chamber to search for dark matter weakly interacting massive particles (WIMPs). The detector measures simultaneously the scintillation and the ionization produced by radiation in pure liquid xenon to discriminate signal from background down to 4.5 keV nuclear-recoil energy. A blind analysis of 58.6 live days of data, acquired between October 6, 2006, and February 14, 2007, and using a fiducial mass of 5.4 kg, excludes previously unexplored parameter space, setting a new 90% C.L. upper limit for the WIMP-nucleon spin-independent cross section of 8.8x10(-44) cm2 for a WIMP mass of 100 GeV/c2, and 4.5x10(-44) cm2 for a WIMP mass of 30 GeV/c2. This result further constrains predictions of supersymmetric models.

10.
Phys Rev Lett ; 97(8): 081302, 2006 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-17026288

RESUMO

We report the first measurements of the absolute ionization yield of nuclear recoils in liquid xenon, as a function of energy and electric field. Independent experiments were carried out with two dual-phase time-projection chamber prototypes, developed for the XENON dark matter project. We find that the charge yield increases with decreasing recoil energy, and exhibits only a weak field dependence. These results are the first unambiguous demonstration of the capability of dual-phase xenon detectors to discriminate between electron and nuclear recoils down to 20 keV, a key requirement for a sensitive dark matter search.

11.
J Biol Chem ; 275(1): 472-8, 2000 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-10617641

RESUMO

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.


Assuntos
DNA Polimerase Dirigida por DNA/química , Exodesoxirribonucleases , Herpesvirus Humano 1/enzimologia , Fragmentos de Peptídeos/química , Proteínas Virais/química , Sequência de Aminoácidos , Temperatura Baixa , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Ultracentrifugação , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
J Biol Chem ; 272(40): 25310-8, 1997 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-9312149

RESUMO

T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta chain (zetaA-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon, respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon, p85alpha, and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon-ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon-ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotirosina , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Anticorpos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Primers do DNA , Humanos , Células Jurkat , Ativação Linfocitária , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Peptídeos/síntese química , Peptídeos/imunologia , Fosfatidilinositol 3-Quinases , Fosforilação , Mutação Puntual , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T
13.
Proc Natl Acad Sci U S A ; 92(5): 1456-60, 1995 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-7878000

RESUMO

The herpes simplex virus DNA polymerase consists of two subunits--a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial alpha-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of alpha-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase delta and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.


Assuntos
Exodesoxirribonucleases/antagonistas & inibidores , Inibidores da Síntese de Ácido Nucleico , Simplexvirus/enzimologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA , Substâncias Macromoleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Proteínas Virais/química , Replicação Viral
14.
Proc Natl Acad Sci U S A ; 91(5): 1942-5, 1994 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8127911

RESUMO

Octapeptides synthesized from D amino acids were absorbed from the intestine and excreted in urine of normal rats drinking 5% glucose/1% creatinine containing the 125I-labeled peptides at 0.1-25 mg/dl. The rats ingested fluid at the rate of about 20 ml/hr and produced urine at 15 ml/hr for several hours during the nocturnal feeding period. Sixty-one +/- 4% of the ingested creatinine and 50 +/- 3% of a lipid-insoluble D octapeptide (EASASYSA, 784 Da) were excreted intact in the urine. The steady-state molar rate of absorption-excretion of creatinine equaled or exceeded the maximum rate of carrier-mediated intestinal transport of glucose, suggesting that both the creatinine and the D octapeptide were transported paracellularly by solvent drag through absorptive cell junctions that were dilated by the glucose. More than 70% of the ingested glucose was also absorbed paracellularly. The results demonstrate that intact oligopeptides can be absorbed efficiently from the intestine when they are not hydrolyzed by membrane-bound peptidases of the brush border. The results also provide support for recent theories proposing that coupling of membrane digestion with paracellular solvent drag accounts for a major fraction of normal intestinal absorption of nutrients.


Assuntos
Absorção Intestinal , Oligopeptídeos/farmacocinética , Sequência de Aminoácidos , Aminoácidos/química , Animais , Transporte Biológico Ativo , Creatinina/farmacocinética , Creatinina/urina , Fezes/química , Feminino , Glucose/farmacocinética , Masculino , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/urina , Ratos , Estereoisomerismo
15.
J Immunol ; 152(1): 193-200, 1994 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-7504709

RESUMO

Schistosoma mansoni triose-phosphate isomerase (TPI), a glycolytic enzyme, was originally identified as the target of the mAb M.1 that conferred protection when the antibody was administered in vivo. In this study we increase the evidence that schistosome TPI is a potential vaccine Ag by showing that it also a potent inducer of IL-2 and IFN-gamma production (Th1 responses), driving production of these cytokines in the same cell populations of infected animals that have high Th2 responses directed at other parasite egg Ag. With the goal of synthetic peptide vaccine design, rTPI was used to determine specific T and B cell epitopes recognized by two strains of mice representing high and moderate responders (C57Bl/6J and CBA/J). All selected epitopes were from nonconserved regions of TPI and thus parasite-specific. We then defined minimal size immunoreactive epitopes and synthesized four-armed multiple antigenic peptides (MAP) consisting of T and B cell epitopes that could be recognized by both strains of mice in the same molecule. Characterization of the immunoreactivity of the MAP showed that higher antibody recognition of the MAP was attained when the B cell epitope was placed on the amino termini relative to the T cell epitope, whereas equivalent immunoreactivity occurred for the T cell epitopes when located at either position. Most interesting was the finding that one of the minimal T cell epitopes, when incorporated into the MAP, required enlarging to retain immunoreactivity. Finally we showed that both the full-length TPI molecule and the final version of the MAP were immunogenic to T cells in naive animals and induced cross-recognition in the form of IL-2 and IFN-gamma production.


Assuntos
Antígenos de Helmintos/química , Linfócitos B/imunologia , Schistosoma mansoni/imunologia , Linfócitos T/imunologia , Triose-Fosfato Isomerase/imunologia , Vacinas Sintéticas/química , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/imunologia , Epitopos/análise , Feminino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Proteínas Recombinantes/imunologia , Schistosoma mansoni/enzimologia , Especificidade da Espécie , Triose-Fosfato Isomerase/química , Vacinas Sintéticas/imunologia
16.
Proc Natl Acad Sci U S A ; 89(12): 5462-6, 1992 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-1608956

RESUMO

The salient pathological feature of Alzheimer disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of victims. The plaques are predominantly composed of human beta-amyloid peptide (beta A4), a 40-mer whose neurotoxicity is related to its aggregation. Radioiodinated human beta A4 is rapidly deposited in vitro from a dilute (less than 10 pM) solution onto neuritic and diffuse plaques and cerebrovascular amyloid in AD brain tissue, whereas no deposition is detectable in tissue without performed plaques. This growth of plaques by deposition of radiolabeled beta A4 to plaques is reversible, with a dissociation half-time of approximately 1 h. The fraction of grey matter occupied by plaques that bind radiolabeled beta A4 in vitro is dramatically larger in AD cortex (23 +/- 11%) than in age-matched normal controls (less than 2%). In contrast to the human peptide, rat/mouse beta A4 (differing at three positions from human beta A4) does not affect the deposition of radiolabeled human beta A4. beta A4 has no detectable interaction with tachykinin receptors in rat or human brain. The use of radioiodinated beta A4 provides an in vitro system for the quantitative evaluation of agents or conditions that may inhibit or enhance the growth or dissolution of AD plaques. This reagent also provides an extremely sensitive method for visualizing various types of amyloid deposits and a means for characterizing and locating sites of amyloid peptide binding to cells and tissues.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Idoso , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/síntese química , Animais , Sítios de Ligação , Encéfalo/patologia , Feminino , Lobo Frontal/metabolismo , Humanos , Cinética , Masculino , Dados de Sequência Molecular , Ratos , Homologia de Sequência do Ácido Nucleico , Lobo Temporal/metabolismo
17.
J Immunol ; 147(7): 2368-76, 1991 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-1717557

RESUMO

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.


Assuntos
Deltaretrovirus/imunologia , Epitopos/análise , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Citotoxicidade Celular Dependente de Anticorpos , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Plasmídeos , Conformação Proteica , Coelhos , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/análise
18.
Biochemistry ; 30(14): 3371-86, 1991 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-1849423

RESUMO

ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, we have undertaken biochemical and structural studies of fragments of ZFY. We describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal beta--hairpin and N-terminal alpha-helix (beta beta alpha motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the "jumping-linker" model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX4HX3-hydrophobic residue-X3. Analogous use of a hydrophobic residue in a linker that spans the minor groove has recently been described in crystallographic and 3D NMR studies of homeodomain-DNA complexes. The proposed model of ZFY is supported in part by the hydroxyl radical footprint of the TFIIIA-DNA complex [Churchill, M.E.A., Tullius, T.D., & Klug, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5528-5532].


Assuntos
Proteínas de Ligação a DNA/química , Dedos de Zinco/genética , Sequência de Aminoácidos , Gráficos por Computador , Humanos , Hidróxidos , Radical Hidroxila , Fatores de Transcrição Kruppel-Like , Espectroscopia de Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Solubilidade , Marcadores de Spin , Fator de Transcrição TFIIIA , Fatores de Transcrição/genética , Zinco/química
19.
Biochemistry ; 29(24): 5660-4, 1990 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-2116899

RESUMO

ZFY, a putative transcription factor encoded by the human Y chromosome, contains a distinctive two-finger repeat: odd-numbered and even-numbered CC/HH metal-binding motifs exhibit systematic alternation in sequence pattern. Such alternation, which is not generally observed in zinc-finger proteins, has also been described in an extensive family of Kruppel-like genes in Xenopus laevis and in the AIDS-associated human DNA-binding protein HIV-EP1. The strict conservation of a two-finger repeat among ZFY-, Kruppel- and HIV-related zinc-finger proteins suggests distinct mechanisms of protein-nucleic acid recognition. To test whether this sequence pattern reflects an underlying alternation in domain structure, we have synthesized and characterized single-finger peptides from the human ZFY gene. Remarkably, systematic differences in metal-dependent folding are observed in the circular dichroism spectra of even- and odd-numbered domains. Our results suggest the existence of distinct CC/HH finger submotifs, which may play different roles in nucleic acid recognition.


Assuntos
Proteínas de Ligação a DNA , Metaloproteínas , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Galinhas , Dicroísmo Circular , Cobalto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Espectroscopia de Ressonância Magnética , Masculino , Metaloproteínas/metabolismo , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/metabolismo , Zinco/metabolismo
20.
Biochem Biophys Res Commun ; 133(3): 844-50, 1985 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-3002372

RESUMO

The effect of ergosterol on cell division and phospholipid metabolism was investigated in Saccharomyces cerevisiae strain GL7, a sterol and unsaturated fatty acid auxotroph. Cells growing poorly on cholesterol were stimulated to grow more rapidly by supplementing the medium with 100 ng of ergosterol per ml. Within 10 min after ergosterol addition to cells prelabeled with 32Pi or [3H]inositol the isotope content of the polyphosphoinositides increases markedly followed by an equally striking and rapid decrease. Subsequently upon continuous labeling, 32P incorporation into phosphatidylinositol and, to a lesser degree, other phospholipids increased. Finally 3h after ergosterol addition the growth rate increased. Only stimulation of the first process, i.e. polyphosphoinositide metabolism, upon ergosterol addition is resistant to inhibition by cycloheximide.


Assuntos
Ergosterol/farmacologia , Fosfatidilinositóis/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Colesterol/metabolismo , Fosfatos de Fosfatidilinositol , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
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