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Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26

Marguet, Didier; Baggio, Laurie; Kobayashi, Takashi; Bernard, Anne-Marie; Pierres, Michel; Nielsen, Per F.; Ribel, Ulla; Watanabe, Takeshi; Drucker, Daniel J.; Wagtmann, Nicolai
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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45.74%
A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC 3.4.14.5), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26−/− mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26−/−, mice. This inhibitor also improved glucose tolerance in GLP-1 receptor−/− mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.

The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner

Vikis, Haris G.; Li, Weiquan; He, Zhigang; Guan, Kun-Liang
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Semaphorin molecules serve as axon guidance signals that regulate the navigation of neuronal growth cones. Semaphorins have also been implicated in other biological processes, including the immune response. Plexins, acting either alone or in complex with neuropilins, have recently been identified as functional semaphorin receptors. However, the mechanisms of signal transduction by plexins remain largely unknown. We have demonstrated a direct interaction between plexin-B1 and activated Rac. Rac specifically interacts with the cytosolic domain of plexin-B1, but not with that of plexin-A3 or -C1. Neither RhoA nor Cdc42 interacts with plexin-B1, indicating that the Rac/plexin-B1 interaction is highly specific. The binding of GTP and the integrity of the Rac effector domain are required for the interaction with plexin-B1. Furthermore, we have identified that a Cdc42/Rac interactive binding (CRIB) motif in the cytosolic domain of plexin-B1 is essential for its interaction with active Rac. We have also observed that the semaphorin CD100, a ligand for plexin-B1, stimulates the interaction between plexin-B1 and active Rac. Our results support a model by which activated Rac plays a role in mediating semaphorin signals, resulting in reorganization of actin cytoskeletal structure.

RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes

Jiang, Hongping; Kang, Dong-chul; Alexandre, Deborah; Fisher, Paul B.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Human melanoma cells growth-arrest irreversibly and terminally differentiate on treatment with a combination of fibroblast interferon and the protein kinase C activator mezerein. This experimental protocol also results in a loss of tumorigenic potential and profound changes in gene expression. Various cloning and cDNA microarray strategies are being used to determine the complete spectrum of gene expression changes underlying these alterations in human melanoma cells. An efficient approach, Rapid Subtraction Hybridization (RaSH), has been developed that is permitting the identification of genes of potential relevance to cancer growth control and terminal cell differentiation. RaSH cDNA libraries are prepared from double-stranded cDNAs that are enzymatically digested into small fragments, ligated to adapters, and PCR amplified followed by incubation of tester and driver PCR fragments. This subtraction hybridization scheme is technically simple and results in the identification of a high proportion of differentially expressed sequences, including known genes and those not described in current DNA databases. The RaSH approach represents an efficient methodology for identifying and cloning genes displaying differential expression that associate with and potentially regulate complex biological processes.

Potentiation of pathogen-specific defense mechanisms in Arabidopsis by β-aminobutyric acid

Zimmerli, Laurent; Jakab, Gabor; Métraux, Jean-Pierre; Mauch-Mani, Brigitte
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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The nonprotein amino acids γ-aminobutyric acid (GABA) and β-aminobutyric acid (BABA) have known biological effects in animals and plants. Their mode of action has been the object of thorough research in animals but remains unclear in plants. Our objective was to study the mode of action of BABA in the protection of Arabidopis plants against virulent pathogens. BABA protected Arabidopsis against the oomycete pathogen Peronospora parasitica through activation of natural defense mechanisms of the plant such as callose deposition, the hypersensitive response, and the formation of trailing necroses. BABA was still fully protective against P. parasitica in transgenic plants or mutants impaired in the salicylic acid, jasmonic acid, and ethylene signaling pathways. Treatment with BABA did not induce the accumulation of mRNA of the systemic acquired resistance (SAR)-associated PR-1 and the ethylene- and jasmonic acid-dependent PDF1.2 genes. However, BABA potentiated the accumulation of PR-1 mRNA after attack by virulent pathogenic bacteria. As a result, BABA-treated Arabidopsis plants were less diseased compared with the untreated control. In the case of bacteria, BABA protected mutants insensitive to jasmonic acid and ethylene but was not active in plants impaired in the SAR transduction pathway. Thus...

Catalysis of DNA cleavage and nucleoside triphosphate synthesis by NM23-H2/NDP kinase share an active site that implies a DNA repair function

Postel, Edith H.; Abramczyk, Bozena M.; Levit, Mikhail N.; Kyin, Saw
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 19/12/2000 Português
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NM23/NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleoside-diphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme–DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA-linked peptide and by site-directed mutagenesis, we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the ɛ-amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase/lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23/NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally...

Ultrasensitive magnetic biosensor for homogeneous immunoassay

Chemla, Y. R.; Grossman, H. L.; Poon, Y.; McDermott, R.; Stevens, R.; Alper, M. D.; Clarke, J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 19/12/2000 Português
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A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a “microscope” based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 μm from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Néel relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 ± 2) × 104 magnetic nanoparticles to register a reproducible signal.

Electrophoresis in lyotropic polymer liquid crystals

Rill, Randolph L.; Locke, Bruce R.; Liu, Yingjie; Van Winkle, David H.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 17/02/1998 Português
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Excellent electrophoretic separations of a variety of biological molecules can be accomplished by using uncharged, triblock copolymers as the “gel” media. These copolymers form uncrosslinked, lyotropic liquid crystalline phases of large micelles between which molecules must travel. Unlike crosslinked hydrogels in common use, these alternative media have highly ordered internal structures. Pluronic F127, representative of the copolymer class, contains poly(ethylene oxide) (EO) and poly(propylene oxide) (PO) units with an approximate molecular formula (EO)106(PO)70(EO)106. Concentrated (18–30%) solutions of Pluronic F127 are freely flowing liquids at low temperature (0–5°C) but form gel-like, cubic liquid crystals of large, spherical micelles when warmed. The utility of these media is illustrated by separations of linear, double-stranded DNA up to 3,000 bp long by conventional electrophoresis, and of single-stranded DNAs from 4 to 60 nt long by capillary electrophoresis. Extraordinary separations of supercoiled DNAs were also obtained by capillary electrophoresis. The versatility, availability, and ease of use of Pluronic polymers offer major advantages over conventional media for preparative and high performance analytical separations of nucleic acids and other biomolecules. Mechanisms of molecular transport and separation operating in polymer liquid crystals must differ in fundamental ways from those in crosslinked gels. Lyotropic polymer liquid crystals are unique systems for elucidating mechanisms of macromolecule migration in ordered...

The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP

Skowronek, Markus H.; Hendershot, Linda M.; Haas, Ingrid G.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 17/02/1998 Português
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Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER). Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, κNS1 and λFS62, are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells. Furthermore, the BiP/λFS62 Ig L chain complex appears to be more stable than the BiP/κNS1 complex. Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of κNS1 and λFS62 Ig L chains. Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains. Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain...

Synthesis and biological evaluation of cytotoxic analogs of somatostatin containing doxorubicin or its intensely potent derivative, 2-pyrrolinodoxorubicin

Nagy, Attila; Schally, Andrew V.; Halmos, Gábor; Armatis, Patricia; Cai, Ren-Zhi; Csernus, Valér; Kovács, Magdolna; Koppán, Miklós; Szepesházi, Károly; Kahán, Zsuzsanna
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 17/02/1998 Português
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To create cytotoxic hybrid analogs of somatostatin (SST), octapeptides RC-160 (d-Phe-Cdocumentclass[12pt]{minimal} usepackage{amsmath} usepackage{wasysym} usepackage{amsfonts} usepackage{amssymb} usepackage{amsbsy} usepackage{mathrsfs} setlength{oddsidemargin}{-69pt} egin{document} egin{equation*}overline{{mathrm{ys-Tyr- extsc{d}-Trp-;Lys-Val-Cy}}}end{equation*}end{document}s-Trp-NH2) and RC-121 (d-Phe-Cdocumentclass[12pt]{minimal} usepackage{amsmath} usepackage{wasysym} usepackage{amsfonts} usepackage{amssymb} usepackage{amsbsy} usepackage{mathrsfs} setlength{oddsidemargin}{-69pt} egin{document} egin{equation*}overline{{mathrm{ys-Tyr- extsc{d}-Trp-;Lys-Val-Cy}}}end{equation*}end{document}s-Thr-NH2) were linked to doxorubicin (DOX) or its superactive derivative, 2-pyrrolino-DOX (AN-201). The conjugation was performed by coupling N-9-fluorenylmethoxycarbonyl (N-Fmoc)-DOX-14-O-hemiglutarate or 2-pyrrolino-DOX-14-O-hemiglutarate to the amino terminus of [Lys(Fmoc)5]RC-160 yielding AN-163 and AN-258, respectively, after deprotection. The respective cytotoxic conjugates of RC-121 (AN-162 and AN-238) were prepared similarly. In vitro tests on human cancer cell lines—MKN-45 gastric cancer, MDA-MB-231 breast cancer, PC-3 prostate cancer...

Displacement of insulin-like growth factors from their binding proteins as a potential treatment for stroke

Loddick, Sarah A.; Liu, Xin-Jun; Lu, Zi-Xian; Liu, Changlu; Behan, Dominic P.; Chalmers, Derek C.; Foster, Alan C.; Vale, Wylie W.; Ling, Nicholas; De Souza, Errol B.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 17/02/1998 Português
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45.74%
Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role in normal growth and brain development and protect brain cells from several forms of injury. The effects of IGFs are mediated by type-I and type-II receptors and modulated by potentially six specific binding proteins that form high-affinity complexes with IGFs in blood and cerebrospinal fluid (CSF) and under most circumstances inactivate them. Because brain injury is commonly associated with increases in IGFs and their associated binding proteins, we hypothesized that displacement of this large “pool” of endogenous IGF from the binding proteins would elevate “free” IGF levels to elicit neuroprotective effects comparable to those produced by administration of exogenous IGF. A human IGF-I analog [(Leu24, 59, 60, Ala31)hIGF-I] with high affinity to IGF-binding proteins (Ki = 0.3–3.9 nM) and no biological activity at the IGF receptors (Ki = >10,000 nM) increased the levels of “free, bioavailable” IGF-I in the CSF. Intracerebroventricular administration of this analog up to 1h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to IGF-I. This novel strategy for increasing “free” IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases.

From oligonucleotide shapes to genomic SELEX: Novel biological regulatory loops

Gold, Larry; Brown, David; He, Yi-yuan; Shtatland, Timur; Singer, Britta S.; Wu, Yan
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 07/01/1997 Português
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45.74%
The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 1014–1015 sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust “linkage map” between their oligonucleotides and proteins and/or small molecules that enriches life?

Expression of p24, a novel p21Waf1/Cip1/Sdi1-related protein, correlates with measurement of the finite proliferative potential of rodent embryo fibroblasts

Mazars, G. Raoul; Jat, Parmjit S.
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 07/01/1997 Português
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45.74%
Normal mammalian fibroblasts undergo a limited number of divisions when cultured in vitro before entering a state of replicative senescence. The molecular basis for the determination of the finite mitotic potential is not known. Nevertheless, simian virus 40 T antigen, among other oncogenes, is able to prevent senescence in rodent embryo fibroblasts. T antigen immortalized cells are dependent upon this protein for maintaining growth once their normal mitotic life span has elapsed. Even though the mechanism that measures the finite mitotic potential of rodent fibroblasts is not known, it has been shown that it continues to function normally in the presence of this immortalizing gene. Accumulation of cyclin-dependent kinase inhibitors such as p21Waf1/Cip1/Sdi1 could potentially be a component of the mechanism that determines the finite life span. Here we show that accumulation of p21Waf1/Cip1/Sdi1 does not correlate with this biological counting mechanism, but we have identified p24, a p21Waf1/Cip1/Sdi1-related protein, whose accumulation does correlate with the measurement of the finite proliferative potential of rodent embryo fibroblasts and suggest that sequestration might be a mechanism by which its activity is regulated.

Evolutionary matches of enzyme and transporter capacities to dietary substrate loads in the intestinal brush border

Weiss, Stacey L.; Lee, Eric A.; Diamond, Jared
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 03/03/1998 Português
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45.74%
Safety factors of enzymes and transporters are defined as the ratio of Vmax (maximal reaction rates at high substrate concentrations) to the reaction rate under actual physiological conditions. Although corresponding safety factors have been measured for macroscopic biological structures and for human-engineered structures, safety factors have been little studied at the molecular level. Some evolutionary considerations suggest that safety factors should be modestly in excess of 1.0 (“enough but not too much”) and should tend to be similar for the various steps of a pathway consisting of two or more elements arranged in series. Hence we used a preparation of intact mouse small intestine to measure Vmax values (capacities) of brush-border sucrase (yielding glucose plus fructose) and of the brush-border glucose transporter, for comparison with each other and with dietary sucrose loads. Load was manipulated by varying dietary sucrose level or by studying lactating mice with increased energy requirements. Capacities both of sucrase and the glucose transporter increased with sucrose load (i.e., both proteins are inducible) and remained approximately matched to each other except on a carbohydrate-free diet. Their safety factors decreased from ca. 2.7 at low load to 1.0 at high load. Thus...

Aggregation of the human high affinity immunoglobulin G receptor (FcγRI) activates both tyrosine kinase and G protein-coupled phosphoinositide 3-kinase isoforms

Melendez, Alirio J.; Gillooly, David J.; Harnett, Margaret M.; Allen, Janet M.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 03/03/1998 Português
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Phosphoinositide 3-kinases (PI3-kinases) play an important role in the generation of lipid second messengers and the transduction of a myriad of biological responses. Distinct isoforms have been shown to be exclusively activated either by tyrosine kinase-coupled or G protein-coupled receptors. We show here, however, that certain nonclassical receptors can couple to both tyrosine kinase- and G protein-dependent isoforms of PI3-kinase: thus, aggregation of FcγRI, the human high affinity IgG receptor, on monocytes unusually leads to activation of both of these types of PI3-kinase. After aggregation of FcγRI, phosphatidylinositol 3,4,5-triphosphate (PIP3) levels rise rapidly in interferon γ-primed cells, reaching a peak within 30 sec. Moreover, and in contrast to the situation observed after stimulation of these cells with either insulin or ATP, which exclusively activate the tyrosine kinase- and G protein-coupled forms of PI3-kinase, respectively, PIP3 levels remain elevated up to 15 min after receptor aggregation. We show here that although the initial peak results from transient activation of the p85-dependent p110 isoform of PI-3kinase, presumably through recruitment of tyrosine kinases by the γ chain, the later sustained rise of PIP3 results from activation of the G protein βγ subunit-sensitive isoform...

Newborn primate infants are entrained by low intensity lighting

Rivkees, Scott A.; Hofman, Paul L.; Fortman, Jeffrey
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 07/01/1997 Português
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45.74%
At the present time we do not know when the circadian timing system of human infants becomes responsive to light. Because of human study limitations, it is not currently possible to address this issue in clinical studies. Therefore, to provide insights into when the circadian system of humans becomes responsive to light, baboons were studied. We first assessed if the biological clock located in suprachiasmatic nuclei (SCN) is responsive to light at birth. When term newborn infants were exposed to bright light at night (5000 lux), SCN metabolic activity and c-fos mRNA expression increased, indicating the presence of photic responsiveness. When photic entrainment of developing rhythmicity was examined in infants, low intensity (200 lux) cycled lighting was sufficient to entrain circadian phase. However, low intensity lighting was not sufficient to induce changes in SCN metabolic activity or c-fos mRNA expression. Phase–response studies indicated that light exposure (200 lux) before the onset of activity most effectively shifted circadian phase. These data provide direct evidence that the SCN are responsive to visually mediated light information in a primate at birth. Further consideration of lighting conditions that infants are exposed to is therefore warranted.

Ectopic expression of N-acetylglucosaminyltransferase III in transgenic hepatocytes disrupts apolipoprotein B secretion and induces aberrant cellular morphology with lipid storage

Ihara, Yoshito; Yoshimura, Masafumi; Miyoshi, Eiji; Nishikawa, Atsushi; Sultan, Ahmed S.; Toyosawa, Satoru; Ohnishi, Akio; Suzuki, Misao; Yamamura, Ken-ichi; Ijuhin, Naokuni; Taniguchi, Naoyuki
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 03/03/1998 Português
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45.74%
N-Acetylglucosaminyltransferase III (GnT-III) produces “bisecting-GlcNAc” and regulates the branching of N-glycans. GnT-III activity is elevated during hepatocarcinogenesis, which is in contrast to the undetectable level found in normal hepatocytes. To determine the biological significance of GnT-III in hepatocytes, transgenic mice that specifically express GnT-III in the liver were established and characterized. The transgenic hepatocytes had a swollen oval-like morphology, with many lipid droplets. Apolipoprotein B, which contained increased level of bisecting-GlcNAc accumulated in the transgenic hepatocytes. In the transgenic serum, triglycerides, the β- and pre-β-lipoprotein fractions, and apolipoprotein B100 were significantly decreased, compared with levels in nontransgenic serum. These abnormal phenotypes were more prominent in the mice with more copies of the transgene and a resulting high GnT-III activity. We demonstrate that aberrant glycosylation, as the direct result of the formation of bisecting-GlcNAc, disrupts the function of apolipoprotein B, leading to the generation of fatty liver. This observation suggests a novel mechanism for the pathogenesis of fatty liver.

Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: Implications for the mechanism of StAR action

Arakane, Futoshi; Sugawara, Teruo; Nishino, Hideaki; Liu, Zhiming; Holt, John A.; Pain, Debkumar; Stocco, Douglas M.; Miller, Walter L.; Strauss, Jerome F.
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 26/11/1996 Português
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45.74%
Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone biosynthesis, presumably by facilitating the delivery of cholesterol to P450scc in the inner mitochondrial membranes. StAR is synthesized as a 37-kDa preprotein that is processed to a 30-kDa mature form by cleavage of an N-terminal mitochondrial import sequence. To identify structural features required for StAR biological activity, we mutated the human StAR cDNA, including the deletion of N- and C-terminal sequences, and examined the ability of the mutants to promote steroidogenesis and enter the mitochondria of transfected COS-1 cells. Deletion of up to 62 residues from the N terminus (N-62) did not significantly affect steroidogenesis-enhancing activity. The N-terminal deletion mutants were associated with mitochondria-enriched fractions, but import and processing were progressively impaired with increasing length of the deletion. Immunogold electron microscopy and in vitro import assays showed that the active N-62 mutant was not imported into the mitochondria. Removal of the 28 C-terminal amino acids (C-28) inactivated StAR. Deletion of the C-terminal 10 amino acids (C-10) reduced steroidogenic activity by 53%, while truncation of the last 4 amino acids had no effect. The C-28 mutant StAR was not efficiently imported into mitochondria or processed...

Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction

Yablonski, Deborah; Marbach, Irit; Levitzki, Alexander
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 26/11/1996 Português
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The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response pathway is organized on the Ste5 protein, which binds each of the kinases of the cascade prior to signaling. In this study, a structure–function analysis of Ste5 deletion mutants uncovered new functional domains of the Ste5 protein and revealed that Ste5 dimerizes during the course of normal signal transduction. Dimerization, mediated by two regions in the N-terminal half of Ste5, was first suggested by intragenic complementation between pairs of nonfunctional Ste5 mutants and was confirmed by using the two-hybrid system. Coimmunoprecipitation of differently tagged forms of Ste5 from cells in which the pathway has been activated by Ste5 overexpression further confirmed dimerization. A precise correlation between the biological activity of various Ste5 fragments and dimerization suggests that dimerization is essential for Ste5 function.

Visualization of supercoiled DNA with atomic force microscopy in situ

Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.
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 21/01/1997 Português
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Tertiary structure of supercoiled DNA is a significant factor in a number of genetic functions and is apparently affected by environmental conditions. We applied atomic force microscopy (AFM) for imaging the supercoiled DNA deposited at different ionic conditions. We have employed a technique for the sample preparation that permits high-resolution AFM imaging of DNA bound to the surface in buffer solutions without drying the sample (AFM in situ). The AFM data show that at low ionic strength, DNA molecules are loosely interwound supercoils with an irregular shape. Plectonemic superhelices are formed in high-concentration, near-physiological salt solutions. At such ionic conditions, superhelical loops are typically separated by regions of close helix–helix contacts. The data obtained show directly and unambiguously that overall geometry of supercoiled DNA depends dramatically on ionic conditions. This fact and the formation of close contacts between DNA helices are important features of supercoiled DNA related to its biological functions.

Carbon dioxide stimulates peroxynitrite-mediated nitration of tyrosine residues and inhibits oxidation of methionine residues of glutamine synthetase: Both modifications mimic effects of adenylylation

Berlett, Barbara S.; Levine, Rodney L.; Stadtman, Earl R.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 17/03/1998 Português
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The activity of glutamine synthetase (EC 6.3.1.2) from Escherichia coli is regulated by the cyclic adenylylation and deadenylylation of Tyr-397 in each of the enzyme’s 12 identical subunits. The nitration of Tyr-397 or of the nearby Tyr-326 by peroxynitrite can convert the unadenylylated enzyme to a form exhibiting regulatory characteristics similar to the form obtained by adenylylation. The adenylylated conformation can also be elicited by the oxidation of surface-exposed methionine residues to methionine sulfoxide. However, the nitration of tyrosine residues and the oxidation of methionine residues are oppositely directed by the presence and absence of CO2. At physiological concentrations of CO2, pH 7.4, nitration occurs but oxidation of methionine residues is inhibited. Conversely, in the absence of CO2 methionine oxidation is stimulated and nitration of tyrosine is prevented. It was further established that adenylylation of Tyr-397 precludes its nitration by peroxynitrite. Furthermore, nitration of Tyr-326 together with either nitration or adenylylation of Tyr-397 leads to inactivation of the enzyme. These results demonstrate that CO2 can alter the course of peroxynitrite-dependent reactions and serve notice that (i) the reactions have physiological significance only if they are shown to occur at physiological concentrations of CO2 and physiological pH; and (ii) the peroxynitrite-dependent nitration of tyrosine residues or the oxidation of methionine residues of metabolically regulated proteins can seriously compromise their biological function.