Página 34 dos resultados de 60878 itens digitais encontrados em 0.034 segundos
Resultados filtrados por Publicador: The National Academy of Sciences of the USA

‣ The structure of a CAP–DNA complex having two cAMP molecules bound to each monomer

Passner, J. M.; Steitz, T. A.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 01/04/1997 Português
Relevância na Pesquisa
447.82504%
The 2.2 Å resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer. The second cAMP is in the syn conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a β-hairpin from the regulatory domain and the DNA (via water molecules). The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein. In addition, this site’s close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.

‣ Essential role of the tyrosine kinase substrate phospholipase C-γ1 in mammalian growth and development

Ji, Qun-sheng; Winnier, Glenn E.; Niswender, Kevin D.; Horstman, Debra; Wisdom, Ron; Magnuson, Mark A.; Carpenter, Graham
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 01/04/1997 Português
Relevância na Pesquisa
447.82504%
The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (−/−) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5–E9.0. These results clearly demonstrate that PLC-γ1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

‣ Ras-independent transformation by v-Src

Aftab, Dana T.; Kwan, Joanne; Martin, G. Steven
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 01/04/1997 Português
Relevância na Pesquisa
447.82504%
Signaling by a variety of receptor and nonreceptor tyrosine kinases is mediated by Ras, a membrane-associated GTPase. Expression of v-Src, a transforming nonreceptor tyrosine kinase, results in Ras activation, and inhibition of Ras function in NIH 3T3 cells suppresses transformation by v-Src, indicating that in these cells Ras-dependent signaling pathways are required for v-Src to exert its biological effects. However, we show here that Ras was not activated in Rat-2 fibroblasts transformed by wild-type v-Src, or in chicken embryo fibroblasts transformed by SRX5, a v-Src mutant with a linker insertion at the major site of autophosphorylation. Expression of a dominant-negative mutant of Ras completely inhibited the ability of v-Src to activate the mitogen-activated protein kinase ERK2, which is downstream of Ras. However, dominant-negative Ras did not suppress transformation by v-Src as judged by a variety of criteria. Thus, v-Src can transform at least some cell types in the absence of Ras activation or Ras-stimulated ERK2 activity, and in these cells activation of Ras-independent signaling pathways must therefore be sufficient for transformation.

‣ Refolding chromatography with immobilized mini-chaperones

Altamirano, Myriam M.; Golbik, Ralph; Zahn, Ralph; Buckle, Ashley M.; Fersht, Alan R.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 15/04/1997 Português
Relevância na Pesquisa
447.82504%
Mini-chaperones (e.g., a peptide consisting of residues 191-345 of GroEL) that are immobilized on agarose have very efficient chaperoning activity with several proteins that are otherwise recalcitrant to renaturation by conventional methods. We have used immobilized mini-chaperones both in column chromatography and batchwise to renature an insoluble protein from an inclusion body, to refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Refolding chromatography offers an efficient and simple means to renature proteins in high yield and with biological activity.

‣ Mutagenic effects of a single and an exact number of α particles in mammalian cells

Hei, Tom K.; Wu, Li-Jun; Liu, Su-Xian; Vannais, Diane; Waldren, Charles A.; Randers-Pehrson, Gerhard
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 15/04/1997 Português
Relevância na Pesquisa
447.82504%
One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single α particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1− mutants induced in human–hamster hybrid (AL) cells by either a single or an exact number of α particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1, 2, 4, or 8 α particles at a linear energy transfer of 90 keV/μm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to AL cells (survival fraction ≈ 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 105 survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single α particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.

‣ RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli

Hanada, Katsuhiro; Ukita, Toshiyuki; Kohno, Yuko; Saito, Kazue; Kato, Jun-ichi; Ikeda, Hideo
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 15/04/1997 Português
Relevância na Pesquisa
447.82504%
Bloom syndrome and Werner syndrome are genetic disorders in which an increased rate of chromosomal abnormality is observed. The genes responsible for these diseases, BLM and WRN, have been cloned and identified as homologs of the Escherichia coli recQ genes. We studied the effect of recQ mutations on illegitimate recombination, which is an aberrant biological event related to the chromosomal abnormality in humans, and found that a variety of recQ mutations increased spontaneous illegitimate recombination by 20- to 300-fold and increased UV light-induced illegitimate recombination by 10- to 100-fold. Most λbio or λpro transducing phages are formed by the recombination events at several hot spots, which are enhanced by the recQ mutation. The analysis of nucleotide sequences at the recombination junction in the transducing phages indicates that recombination at the hot spot sites as well as the non-hot spot sites takes place between short homologous sequences. Enhancement of the recombination in the recQ mutants also occurs in the recA, recBC sbcBC, or recBC sbcA backgrounds, indicating that these recombination events are mediated by none of the known recombination pathways, RecBC, RecF, and RecE. We therefore concluded that the RecQ function suppresses illegitimate recombination that depends on short homologous regions. We discuss a model...

‣ Polymorphism of murine Fas ligand that affects the biological activity

Kayagaki, Nobuhiko; Yamaguchi, Noriko; Nagao, Fumiko; Matsuo, Seishi; Maeda, Hiroaki; Okumura, Ko; Yagita, Hideo
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 15/04/1997 Português
Relevância na Pesquisa
447.82504%
Fas ligand (FasL) is a member of the tumor necrosis factor family and induces apoptosis in Fas (CD95)-bearing target cells. In this study, we generated several mAbs that react with mouse FasL (mFasL) and characterized their functional properties. One of these mAbs, K10, specifically reacted with mFasL derived from C57BL/6 (B6) mice, but not that from BALB/c mice as estimated by surface staining and blocking of cytotoxic activities of mFasL transfectants, suggesting a polymorphism of mFasL. Sequence analysis of mFasL cDNA from several strains revealed that BALB/c and DBA/2 mice have three nucleotide differences from the known B6 and C3H sequences, which result in two amino acid substitutions (Thr-184 → Ala-184 and Glu-218 → Gly-218) in the extracellular region. Analysis of the K10 reactivity and genotyping by PCR-restriction fragment length polymorphism revealed that inbred mice segregate into the following two allotypes: mFasL.1 (B6, C3H, MRL, SJL, NOD, NZB, NZW) and mFasL.2 (BALB/c, DBA/1, DBA/2). Interestingly, COS7 cells expressing BALB/c FasL lysed Fas-bearing target cells more efficiently than those expressing B6 FasL. Furthermore, BALB/c-derived CD8-FasL fusion protein, which is composed of the extracellular domains of human CD8α and mFasL...

‣ Dynamic electron microscopy of ATP-induced myosin head movement in living muscle thick filaments

Sugi, Haruo; Akimoto, Tsuyoshi; Sutoh, Kazuo; Chaen, Shigeru; Oishi, Noboru; Suzuki, Suechika
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 29/04/1997 Português
Relevância na Pesquisa
447.82504%
Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M⋅ADP⋅Pi) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction...

‣ Inositol hexakisphosphate stimulates non-Ca2+-mediated and primes Ca2+-mediated exocytosis of insulin by activation of protein kinase C

Efanov, Alexandre M.; Zaitsev, Sergei V.; Berggren, Per-Olof
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 29/04/1997 Português
Relevância na Pesquisa
447.82504%
d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), formed via complex pathways of inositol phosphate metabolism, composes the main bulk of inositol polyphosphates in the cell. Relatively little is known regarding possible biological functions for InsP6. We now show that InsP6 can modulate insulin exocytosis in permeabilized insulin-secreting cells. Concentrations of InsP6 above 20 μM stimulated insulin secretion at basal Ca2+-concentration (30 nM) and primed Ca2+-induced exocytosis (10 μM), both effects being due to activation of protein kinase C. Our results suggest that InsP6 can play an important modulatory role in the regulation of processes such as exocytosis in insulin-secreting cells. The specific role for InsP6 can then be to recruit secretory granules to the site of exocytosis.

‣ An apportionment of human DNA diversity

Barbujani, Guido; Magagni, Arianna; Minch, Eric; Cavalli-Sforza, L. Luca
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 29/04/1997 Português
Relevância na Pesquisa
447.82504%
It is often taken for granted that the human species is divided in rather homogeneous groups or races, among which biological differences are large. Studies of allele frequencies do not support this view, but they have not been sufficient to rule it out either. We analyzed human molecular diversity at 109 DNA markers, namely 30 microsatellite loci and 79 polymorphic restriction sites (restriction fragment length polymorphism loci) in 16 populations of the world. By partitioning genetic variances at three hierarchical levels of population subdivision, we found that differences between members of the same population account for 84.4% of the total, which is in excellent agreement with estimates based on allele frequencies of classic, protein polymorphisms. Genetic variation remains high even within small population groups. On the average, microsatellite and restriction fragment length polymorphism loci yield identical estimates. Differences among continents represent roughly 1/10 of human molecular diversity, which does not suggest that the racial subdivision of our species reflects any major discontinuity in our genome.

‣ Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin

Kato, Takashi; Oda, Atsushi; Inagaki, Yoshimasa; Ohashi, Hideya; Matsumoto, Atsushi; Ozaki, Katsutoshi; Miyakawa, Yoshitaka; Watarai, Hiroshi; Fuju, Kazumi; Kokubo, Atsuko; Kadoya, Toshihiko; Ikeda, Yasuo; Miyazaki, Hiroshi
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 29/04/1997 Português
Relevância na Pesquisa
447.82504%
A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin-cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.

‣ The second finger of Urbs1 is required for iron-mediated repression of sid1 in Ustilago maydis

An, Zhiqiang; Zhao, Qin; McEvoy, James; Yuan, Walter M.; Markley, John L.; Leong, Sally A.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 27/05/1997 Português
Relevância na Pesquisa
447.82504%
The urbs1 gene encodes a transcriptional regulator of siderophore biosynthesis in Ustilago maydis. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated urbs1 alleles from three previously described N′-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in urbs1–1 and P-491 to L in urbs1–3), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a urbs1–minus phenotype. The third NTG urbs1 mutant (urbs1–2) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a urbs1–minus phenotype. A second frame shift mutation was identified in urbs1–2 and is necessary for the urbs1–minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a urbs1 null mutant strain. By contrast...

‣ The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2

Sin, Ny; Meng, Lihao; Wang, Margaret Q. W.; Wen, James J.; Bornmann, William G.; Crews, Craig M.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 10/06/1997 Português
Relevância na Pesquisa
447.82504%
The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.