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‣ Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

McDaniel, Robert; Thamchaipenet, Arinthip; Gustafsson, Claes; Fu, Hong; Betlach, Melanie; Betlach, Mary; Ashley, Gary
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/03/1999 Português
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The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

‣ Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen

Zhang, Gang; Gibbs, Emma; Kelman, Zvi; O’Donnell, Michael; Hurwitz, Jerard
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/03/1999 Português
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Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase δ (or ɛ)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36–55 and 196–215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase δ-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase δ-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase δ-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase δ in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

‣ Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly

Matsuzono, Yuji; Kinoshita, Naohiko; Tamura, Shigehiko; Shimozawa, Nobuyuki; Hamasaki, Maho; Ghaedi, Kamran; Wanders, Ronald J. A.; Suzuki, Yasuyuki; Kondo, Naomi; Fujiki, Yukio
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/03/1999 Português
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At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box...

‣ Interferon γ expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity

Bukreyev, Alexander; Whitehead, Stephen S.; Bukreyeva, Natalya; Murphy, Brian R.; Collins, Peter L.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/03/1999 Português
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Interferon γ (IFN-γ) has pleiotropic biological effects, including intrinsic antiviral activity as well as stimulation and regulation of immune responses. An infectious recombinant human respiratory syncytial virus (rRSV/mIFN-γ) was constructed that encodes murine (m) IFN-γ as a separate gene inserted into the G-F intergenic region. Cultured cells infected with rRSV/mIFN-γ secreted 22 μg mIFN-γ per 106 cells. The replication of rRSV/mIFN-γ, but not that of a control chimeric rRSV containing the chloramphenicol acetyl transferase (CAT) gene as an additional gene, was 63- and 20-fold lower than that of wild-type (wt) RSV in the upper and lower respiratory tract, respectively, of mice. Thus, the attenuation of rRSV/mIFN-γ in vivo could be attributed to the activity of mIFN-γ and not to the presence of the additional gene per se. The mice were completely resistant to subsequent challenge with wt RSV. Despite its growth restriction, infection of mice with rRSV/mIFN-γ induced a level of RSV-specific antibodies that, on day 56, was comparable to or greater than that induced by infection with wt RSV. Mice infected with rRSV/mIFN-γ developed a high level of IFN-γ mRNA and an increased amount of interleukin 12 p40 mRNA in their lungs...

‣ Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA

Morse, Daniel P.; Bass, Brenda L.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

‣ A peroxisome proliferator-activated receptor γ ligand inhibits adipocyte differentiation

Oberfield, Jennifer L.; Collins, Jon L.; Holmes, Christopher P.; Goreham, Donna M.; Cooper, Joel P.; Cobb, Jeffery E.; Lenhard, James M.; Hull-Ryde, Emily A.; Mohr, Christopher P.; Blanchard, Steven G.; Parks, Derek J.; Moore, Linda B.; Lehmann, Jürgen M
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARγ subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2,4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPARγ ligand that was a weak partial agonist of PPARγ transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.

‣ DNA methylation is a reversible biological signal

Ramchandani, Shyam; Bhattacharya, Sanjoy K.; Cervoni, Nadia; Szyf, Moshe
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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The pattern of DNA methylation plays an important role in regulating different genome functions. To test the hypothesis that DNA methylation is a reversible biochemical process, we purified a DNA demethylase from human cells that catalyzes the cleavage of a methyl residue from 5-methyl cytosine and its release as methanol. We show that similar to DNA methyltransferase, DNA demethylase shows CpG dinucleotide specificity, can demethylate mdCpdG sites in different sequence contexts, and demethylates both fully methylated and hemimethylated DNA. Thus, contrary to the commonly accepted model, DNA methylation is a reversible signal, similar to other physiological biochemical modifications.

‣ Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

Wang, Guangshun; Wylie, Gregory P.; Twigg, Pamela D.; Caspar, Donald L. D.; Murphy, John R.; Logan, Timothy M.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β-strands and three helices arranged into a partially orthogonal, two-sheet β-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2...

‣ Laser-mediated, site-specific inactivation of RNA transcripts

Grate, Dilara; Wilson, Charles
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and in vitro characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction.

‣ Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals

Elfgang, Claudia; Rosorius, Olaf; Hofer, Lotte; Jaksche, Herbert; Hauber, Joachim; Bevec, Dorian
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.

‣ Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Dong, Jianying; Opresko, Lee K.; Dempsey, Peter J.; Lauffenburger, Douglas A.; Coffey, Robert J.; Wiley, H. Steven
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor α and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor α release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

‣ Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

Rondon, Michelle R.; Raffel, Sandra J.; Goodman, Robert M.; Handelsman, Jo
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

‣ Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor

Siragy, Helmy M.; Inagami, Tadashi; Ichiki, Toshihiro; Carey, Robert M.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 25/05/1999 Português
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The vast majority of the known biological effects of the renin–angiotensin system are mediated by the type-1 (AT1) receptor, and the functions of the type-2 (AT2) receptor are largely unknown. We investigated the role of the AT2 receptor in the vascular and renal responses to physiological increases in angiotensin II (ANG II) in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (P < 0.0001). In AT2-null mice, infusion of ANG II (4 pmol/kg/min) for 7 days produced a marked and sustained increase in SBP [from 116 ± 0.5 to 208 ± 1 mmHg (P < 0.0001) (1 mmHg = 133 Pa)] and reduction in urinary sodium excretion (UNaV) [from 0.6 ± 0.01 to 0.05 ± 0.002 mM/day (P < 0.0001)] whereas neither SBP nor UNaV changed in WT mice. AT2-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3′,5′-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3′,5′-monophosphate by ≈4-fold (P < 0.0001) whereas no changes were observed in AT2-null mice. These results demonstrate that the AT2 receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT2 receptor leads to vascular and renal hypersensitivity to ANG II...

‣ Sustained activation of Ras/Raf/mitogen-activated protein kinase cascade by the tumor suppressor p53

Lee, Sam W.; Fang, Li; Igarashi, Makoto; Ouchi, Toru; Lu, Kun Ping; Aaronson, Stuart A.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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The p53 tumor suppressor gene can inhibit proliferation transiently, induce permanent cell-cycle arrest/senescence, or cause apoptosis depending on the cellular context. The mitogen-activated protein kinase (MAPK) cascade is known to play a crucial role in cell proliferation and differentiation. Moreover, the duration and intensity of MAPK activation can profoundly influence the biological response observed. We demonstrated that a sustained activation of MAPK cascade could be induced by wild-type p53 expression but not by p21Waf1/Cip1. Furthermore, exposure of normal cells to DNA-damaging agents induced MAPK activation in a p53-dependent manner. Tumor-derived p53 mutants defective in DNA binding failed to activate MAPK, implying that p53 transcriptional activity is essential for this function. Finally, activation of MAPK by p53 was inhibited by expression of dominant-negative Ras (N17Ras) and Raf1 mutants, indicating that MAPK activation by p53 is mediated at a level upstream of Ras. All of these findings establish a biochemical link between p53 signaling and the Ras/Raf/MAPK cascade.

‣ Activation of p53 in cervical carcinoma cells by small molecules

Hietanen, Sakari; Lain, Sonia; Krausz, Eberhard; Blattner, Christine; Lane, David P.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 18/07/2000 Português
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In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21WAF1/CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells...

‣ Parent–progeny recognition as a function of MHC odortype identity

Yamazaki, K.; Beauchamp, G. K.; Curran, M.; Bard, J.; Boyse, E. A.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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The several linked polymorphic genes of the MHC, which has been proposed as a prime determinant of sensed genetic individuality within species, is known to operate in mice by olfactory recognition in aspects of reproductive behavior that concern mate selection, thereby favoring outbreeding and heterozygosity, and also concern the maintenance of pregnancy. A single base-change can alter an individual MHC odortype, and the potential range of combinatorial MHC-determined odortypes is clearly vast. Following our findings that newborn mice already express their MHC odortype (which is detectable at 9 days of gestational age), we sought to determine whether MHC is involved in behavioral aspects of early development, such as rearing. In the studies presented herein, we report the ability and proclivity of mothers to recognize and preferentially retrieve syngeneic (genetically identical) pups from other pups differing only for MHC. Reciprocally, we report the ability of pups to recognize their familial environment, regardless of whether they had been nursed by their biological mothers or by foster mothers. Early learning experiences of the MHC environment are apparently a key element in survival, assuring maternal protection and promoting outbreeding.

‣ Opposing roles of pRB and p107 in adipocyte differentiation

Classon, Marie; Kennedy, Brian K.; Mulloy, Roseann; Harlow, Ed
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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The retinoblastoma (RB) family of proteins, pRB, p107, and p130, have been postulated to be partially redundant in their ability to regulate progression through the G1 phase of the cell cycle. However, pRB appears to be unique in its capacity as a classical tumor suppressor, possibly because of a specialized role in maintaining the balance between proliferation and differentiation. A variety of studies have in fact revealed an apparent role for pRB in cellular differentiation and development. However, roles for p107 and p130 in differentiation have not yet been established, and knockout mouse studies have indicated that they may be functionally redundant during development, and possibly perform a role in differentiation distinct from that of pRB. Using adipogenesis as a model, we have indeed found distinct roles for the pRB family proteins in regulating differentiation. 3T3 fibroblasts deficient in p107 and p130 differentiate with high efficiency, whereas pRB−/− 3T3 cells exhibit defects in their differentiation potential. Moreover, over-expression of pRB in wild-type cells promotes differentiation, whereas over-expression of p107 antagonizes differentiation. The seemingly opposing roles of pRB family members in adipocyte differentiation can be explained...

‣ Modulation of Akt kinase activity by binding to Hsp90

Sato, Saori; Fujita, Naoya; Tsuruo, Takashi
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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Serine/threonine kinase Akt/PKB is a downstream effector molecule of phosphoinositide 3-kinase and is thought to mediate many biological actions toward anti-apoptotic responses. We found that Akt formed a complex with a 90-kDa heat-shock protein (Hsp90) in vivo. By constructing deletion mutants, we identified that amino acid residues 229–309 of Akt were involved in the binding to Hsp90 and amino acid residues 327–340 of Hsp90β were involved in the binding to Akt. Inhibition of Akt-Hsp90 binding led to the dephosphorylation and inactivation of Akt, which increased sensitivity of the cells to apoptosis-inducing stimulus. The dephosphorylation of Akt was caused by an increase in protein phosphatase 2A (PP2A)-mediated dephosphorylation and not by a decrease in 3-phosphoinositide-dependent protein kinase-1-mediated phosphorylation. These results indicate that Hsp90 plays an important role in maintaining Akt kinase activity by preventing PP2A-mediated dephosphorylation.

‣ Fully competent dendritic cells as inducers of T cell anergy in autoimmunity

Quaratino, Sonia; Duddy, Linda P.; Londei, Marco
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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Mature immunologically competent dendritic cells are the most efficient antigen-presenting cells that powerfully activate T cells and initiate and sustain immune responses. Indeed, dendritic cells are able to efficiently capture antigens, express high levels of costimulatory molecules, and produce the combination of cytokines required to create a powerful immune response. They are also considered to be important in initiating autoimmune disease by efficiently presenting autoantigens to self-reactive T cells that, in this case, will mount a pathogenic autoimmune reaction. Triggering T cells is not a simple on–off procedure, as T cell receptor responds to minor changes in ligand with gradations of T cell activation and effector functions. These “misfit” peptides have been called Altered Peptide Ligands, and have been shown to have important biological significance. Here, we show that fully capable dendritic cells may present, upon natural antigen processing, a self-epitope with Altered Peptide Ligands features that can unexpectedly induce anergy in a human autoreactive T cell clone. These results indicate that presentation of a self-epitope by immunologically competent dendritic cells does not always mean “danger” and show a mechanism involved in the fine balance between activation and tolerance induction in humans.

‣ Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma

Tang, Xao X.; Zhao, Huaqing; Robinson, Marjorie E.; Cohen, Brian; Cnaan, Avital; London, Wendy; Cohn, Susan L.; Cheung, Nai-Kong V.; Brodeur, Garrett M.; Evans, Audrey E.; Ikegaki, Naohiko
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
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Neuroblastoma (NB) is a common pediatric tumor that exhibits a wide range of biological and clinical heterogeneity. EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ligand ephrins play pivotal roles in neural and cardiovascular development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6) and its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is associated with low-stage NB (stages 1, 2, and 4S) and high TrkA expression. In this study, we showed that EFNB2 and TrkA expressions were associated with both tumor stage and age, whereas EPHB6 and EFNB3 expressions were solely associated with tumor stage, suggesting that these genes were expressed in distinct subsets of NB. Kaplan-Meier and Cox regression analyses revealed that high-level expression of EPHB6, EFNB2, and EFNB3 predicted favorable NB outcome (P < 0.005), and their expression combined with TrkA expression predicted the disease outcome more accurately than each variable alone (P < 0.00005). Interestingly, if any one of the four genes (EPHB6, EFNB2, EFNB3, or TrkA) was expressed at high levels in NB, the patient survival was excellent (>90%). To address whether a good disease outcome of NB was a consequence of high-level expression of a “favorable NB gene...