RESUMO
Antibiotic administration is crucial to ensure the health and productivity of dairy cattle. Mastitis is a disease that is typically a result of an intramammary infection (IMI), and antibiotic regimens are implemented to aid in curing IMI. Diagnosis is usually by detection of elevated milk somatic cell counts (SCC) and/or presence of culturable pathogens in the milk. Antibiotic treatment success is associated with the SCC at the time of treatment, though this correlation is still poorly understood. The objective of this project was to evaluate pre-treatment SCC and its association with IMI cure incidence following a standard antibiotic treatment. We hypothesized that pre-treatment SCC would be significantly lower in cases where the IMI ultimately cured compared to cases where the IMI failed to cure. Milk samples were collected aseptically from lactating cow quarters experiencing clinical or subclinical mastitis (n = 52). Clinical mastitis was diagnosed by a trained milking technician and subclinical mastitis was diagnosed at the quarter level as a SCC > 200,000 cells/mL and presence of bacterical growth in milk at time of treatment. After collection of the day 0 (D0) milk samples, the SCC was enumerated, and the milk sample cultured. Intramammary antibiotic therapy Cetftiofur hydrochloride (Spectramast® LC) was administered once/day for 5 days. Post-treatment samples were collected 14 d (D14) and 28 d (D28) later. A bacteriological cure was confirmed when both the D14 and D28 samples were free of culturable pathogens. The overall cure rate was 46.2%. Interestingly, the cure rates of antibiotic therapy decreased as pre-treatment SCC increased. Quarters that experienced bacteriological cure demonstrated a lower pre-treatment SCC (507,041 cells/mL ± 127.86 SEM, P = 0.01) compared to cows that did not cure, which had high pre-treatment SCC (1,640,392 cells/mL ± 333.28 SEM). Quarters that failed to cure had higher SCC values 28 days post-treatment in comparison to quarters that cured (P < 0.001). Future studies should investigate whether we can develop unique SCC-dependent mastitis treatment protocols which increase mastitis cure rates and enhance overall mammary health.
Assuntos
Doenças dos Bovinos , Mastite Bovina , Bovinos , Animais , Feminino , Mastite Bovina/diagnóstico , Mastite Bovina/tratamento farmacológico , Mastite Bovina/epidemiologia , Lactação , Glândulas Mamárias Animais , Contagem de Células/veterinária , Leite , Antibacterianos/uso terapêutico , Doenças dos Bovinos/tratamento farmacológicoRESUMO
The role of nitric oxide in the immune response to allogeneic tissue was explored in an in vivo cardiac transplant model in the rat. Nitric oxide production during organ rejection was demonstrated by elevations in systemic serum nitrite/nitrate levels and by electron paramagnetic resonance spectroscopy. Messenger RNA for the inducible nitric oxide synthase enzyme was detected in the rejecting allografted heart, but not in the nonrejecting isografted heart. The enzyme was demonstrated to be biologically active by the in vitro conversion of L-arginine to L-citrulline and was immunohistochemically localized to the infiltrating inflammatory cells. Treatment with aminoguanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, prevented the increased nitric oxide production in the transplanted organ and significantly attenuated the pathogenesis of acute rejection. Aminoguanidine treatment prolonged graft survival, improved graft contractile function, and significantly reduced the histologic grade of rejection. These results suggest an important role for nitric oxide in mediating the immune response to allogeneic tissue. Inhibition of inducible nitric oxide synthase may provide a novel therapeutic modality in the management of acute transplant rejection and of other immune-mediated processes.
Assuntos
Aminoácido Oxirredutases/antagonistas & inibidores , Rejeição de Enxerto , Transplante de Coração , Óxido Nítrico/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/química , Indução Enzimática/efeitos dos fármacos , Expressão Gênica , Guanidinas/farmacologia , Dados de Sequência Molecular , Óxido Nítrico Sintase , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos LewRESUMO
The influence of several metabolic inhibitors and pharmacologic agents on macrophage deformation (induced by fluid shear stress) was examined in relationship to changes in ATP content and phagocytosis of latex beads. Two relatively specific inhibitors of glycolysis (iodoacetate [IA], and sodium fluoride [NaF]) and a sulfhydryl-binding agent (N-ethylmaleimide [NEM] markedly inhibited phagocytosis and reduced cell deformability. A microtubule-disrupting agent (vinblastine) and a highly specific inhibitor of glycolysis (2-deoxyglucose) markedly inhibited phagocytosis without influencing cell deformability. An organomercurial sulfhydryl binding agent p-chloromercuribenzene (PCMBS) and a microfilament-disrupting agent (cytochalasin B) inhibited phagocytosis and increased cell deformability. The effects of these agents on phagocytosis and cell deformability bore no consistent relationship to alterations in cellular content of ATP. The observation that 2-deoxyglucose, the most specific inhibitor of glycolysis examined, reduced ATP content to levels far lower (15 percent of control values) than those achieved by any other agent examined and inhibited phagocytosis without altering cell deformability, suggests that alterations in cell deformability induced by NaF, IA, NEM, PCMBS, and cytochalasin B are not due to inhibition of glycolysis per se, but instead result from direct or indirect effects of these agents on cell constituents, possibly contractile proteins, which are determinants of cell deformability. The finding that cytochalasin B, NEM, PCMBS, and IA interfere with phagocytosis and alter cell deformability, together with evidence that these agents interact with isolated actin and myosin, suggests that contractile proteins are important both in phagocytosis and as determinants of cell deformability. The observation that vinblastine, colchicines, and heavy water (D(2)O) did not alter cell deformability, even though vinblastine caused formation of intracellular crystals of microtubular protein, indicates that microtubules are not major determinants of cell deformability. The observations that beads adhered normally to surfaces of cytochalasin B- and of PCMBS-treated cells and that shear-stress induced deformation was increased whereas phagocytosis was markedly inhibited, suggest that deformation of cells around beads associated with ingestion depends on some form of cellular (contractile?) activity, whereas deformation of cells by fluid shear stress is a passive phenomenon.
Assuntos
Macrófagos/citologia , Fagocitose , 4-Cloromercuriobenzenossulfonato/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Colchicina/farmacologia , Citocalasina B/farmacologia , Deutério/farmacologia , Etilmaleimida/farmacologia , Fluoretos/farmacologia , Cobaias , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Estresse Mecânico , Vimblastina/farmacologiaRESUMO
Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4-bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6-bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5-trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction.
Assuntos
Líquidos Corporais/metabolismo , Cálcio/metabolismo , Fosfatos de Inositol/farmacologia , Líquido Intracelular/metabolismo , Óvulo/crescimento & desenvolvimento , Fosfatos Açúcares/farmacologia , Animais , Cálcio/biossíntese , Eletrofisiologia , Feminino , Fertilização/efeitos dos fármacos , Inositol 1,4,5-Trifosfato , Líquido Intracelular/efeitos dos fármacos , Iontoforese/instrumentação , Microeletrodos , Microinjeções , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Xenopus laevisRESUMO
The folding pathways of large, highly structured RNA molecules are largely unexplored. Insight into both the kinetics of folding and the presence of intermediates was provided in a study of the Mg(2+)-induced folding of the Tetrahymena ribozyme by hybridization of complementary oligodeoxynucleotide probes. This RNA folds via a complex mechanism involving both Mg(2+)-dependent and Mg(2+)-independent steps. A hierarchical model for the folding pathway is proposed in which formation of one helical domain (P4-P6) precedes that of a second helical domain (P3-P7). The overall rate-limiting step is formation of P3-P7, and takes place with an observed rate constant of 0.72 +/- 0.14 minute-1. The folding mechanism of large RNAs appears similar to that of many multidomain proteins in that formation of independently stable substructures precedes their association into the final conformation.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/química , RNA de Protozoário/química , Tetrahymena/genética , Animais , Sequência de Bases , Íntrons , Cinética , Magnésio/metabolismo , Magnésio/farmacologia , Modelos Químicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA Catalítico/metabolismo , Ribonuclease H/metabolismo , TemperaturaRESUMO
The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.
Assuntos
Arginina/metabolismo , HIV-1/genética , RNA Mensageiro/química , RNA Viral/química , Proteínas de Ligação a RNA/química , Sequência de Bases , Sítios de Ligação , Produtos do Gene tat/metabolismo , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
In the magnesium ion-dependent folding of the Tetrahymena ribozyme, a kinetic intermediate accumulates in which the P4-P6 domain is formed, but the P3-P7 domain is not. The kinetic barriers to P3-P7 formation were investigated with the use of in vitro selection to identify mutant RNA molecules in which the folding rate of the P3-P7 domain was increased. The critical mutations disrupt native tertiary interactions within the P4-P6 domain and increase the rate of P3-P7 formation by destabilizing a kinetically trapped intermediate. Hence, kinetic traps stabilized by native interactions, and not simply by mispaired nonnative structures, can present a substantial barrier to RNA folding.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/química , Animais , Sítios de Ligação , Cinética , Magnésio/metabolismo , Modelos Moleculares , Mutação , RNA Catalítico/genética , RNA Catalítico/metabolismo , Tetrahymena/químicaRESUMO
The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.
Assuntos
RNA Ribossômico/química , Ribonucleoproteínas/química , Proteínas Ribossômicas/química , Ribossomos/química , Thermus thermophilus/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Ribonucleoproteínas/metabolismo , Proteína S6 Ribossômica , Proteínas Ribossômicas/metabolismo , Thermus thermophilus/ultraestruturaRESUMO
C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na+- and K+-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.
Assuntos
Peptídeo C/química , Peptídeo C/uso terapêutico , Angiopatias Diabéticas/prevenção & controle , Neuropatias Diabéticas/prevenção & controle , Sequência de Aminoácidos , Animais , Circulação Sanguínea/efeitos dos fármacos , Glicemia/metabolismo , Peptídeo C/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Dicroísmo Circular , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Humanos , Masculino , Dados de Sequência Molecular , Condução Nervosa/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/metabolismo , EstereoisomerismoRESUMO
The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side chains make base-specific contacts, and an asparagine residue contacts a G.A base pair. The phosphate backbone adjacent to a G.G base pair adopts an unusual structure that allows the peptide to access a widened major groove. The structure formed by the two purine-purine base pairs of the RRE creates a distinctive binding pocket that the peptide can use for specific recognition.
Assuntos
Proteínas de Ligação a DNA , Produtos do Gene rev/química , Produtos do Gene rev/metabolismo , Genes env , HIV-1/química , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Viral/química , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Arginina/química , Asparagina/química , Composição de Bases , Sequência de Bases , Proteínas Fúngicas/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Quinases/química , RNA Viral/genética , RNA Viral/metabolismo , Treonina/química , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Normal red cells deform markedly as they pass through the spleen and the peripheral capillaries. In these studies, the effects of Plasmodium falciparum infection and maturation on the deformability of parasitized red cells exposed to fluid shear stress in vitro were examined by means of a rheoscope. Red cells containing the early (ring) erythrocytic stage of the parasite have impaired deformability at physiologic shear stresses, and recover their normal shape more slowly. Red cells containing more mature parasites (trophozoites or schizonts) exhibit no deformation under the same conditions. These results provide a mechanism to explain the ability of the spleen to remove parasitized red cells from the circulation of both immune and nonimmune hosts.
Assuntos
Eritrócitos/parasitologia , Malária/sangue , Plasmodium falciparum/fisiologia , Animais , Viscosidade Sanguínea , Elasticidade , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Humanos , Malária/parasitologia , Malária/fisiopatologia , Reologia , Baço/fisiopatologia , Propriedades de SuperfícieRESUMO
Hereditary pyropikilocytosis is a hemolytic anemia in which the erythrocytes show increased sensitivity to heat-induced fragmentation. Circular dichroism measurements were employed to study the effect of heat on the secondary structure of pyropoikilocyte membrane proteins. The magnitude of the ellipticity at 222 nm over the temperature range from 25 degrees to 70 degrees C was determined for erythrocyte ghosts, spectrin, and ghost residue after extraction. In pyropoikilocyte ghosts, protein denaturation began at a lower temperature and the midpoint of the structural transition was displaced from 49 degrees C (the value for normal ghosts) to 44 degrees C. This thermal transition was present in spectrin, but not in the ghost residue after extraction. We conclude that an abnormality in the spectrin molecule alters the physical and morphologic properties of the erythrocyte membrane in pyropoikilocytosis.
Assuntos
Anemia Hemolítica Congênita/sangue , Temperatura Alta , Proteínas de Membrana/análise , Espectrina/análise , Anemia Hemolítica Congênita/genética , Dicroísmo Circular , Membrana Eritrocítica/análise , HumanosRESUMO
Exposure of skin chamber granulation tissue vessels in nondiabetic rats to 11 or 15 mM D-glucose (but not L-glucose or 3-O-methylglucose) twice daily for 10 d induces vascular functional changes (increased albumin permeation and blood flow) identical to those in animals with mild or severe streptozotocin diabetes, respectively. These vascular changes are strongly linked to increased metabolism of glucose via the sorbitol pathway and are independent of nonenzymatic glycosylation as well as systemic metabolic and hormonal imbalances associated with the diabetic milieu. (J. Clin. Invest. 1990. 85:1167-1172.)
Assuntos
Aldeído Redutase/antagonistas & inibidores , Angiopatias Diabéticas/etiologia , Glucose/farmacologia , Microcirculação/efeitos dos fármacos , Naftalenos/farmacologia , Desidrogenase do Álcool de Açúcar/antagonistas & inibidores , Aldeído Redutase/fisiologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Glicosilação , Masculino , Ratos , Ratos Endogâmicos , Albumina Sérica/metabolismo , Sorbitol/metabolismo , EstereoisomerismoRESUMO
The present experiments were undertaken to examine the hypothesis that glucose-induced increased de novo synthesis of 1,2-diacyl-sn-glycerol (which has been observed in a number of different tissues, including retinal capillary endothelial cells exposed to elevated glucose levels in vitro) and associated activation of protein kinase C may play a role in mediating glucose-induced vascular functional changes. We report here that twice daily instillation of 30 mM glucose over 10 d in a rat skin chamber granulation tissue model induces approximately a 2.7-fold increase in diacylglycerol (DAG) levels (versus tissues exposed to 5 mM glucose) in association with marked increases in vascular clearance of albumin and blood flow. The glucose-induced increase in DAG levels as well as the vascular functional changes are prevented by addition of 3 mM pyruvate. Pharmacological activation of protein kinase C with the phorbol ester TPA in the presence of 5 mM glucose increases microvascular albumin clearance and blood flow, and similar effects are observed with 1-monoolein (MOG), a pharmacological inhibitor of the catabolism of endogenous DAG. A pharmacological inhibitor of protein kinase C (staurosporine) greatly attenuates the rise in microvascular albumin clearance (but not the rise in blood flow) induced by glucose or by MOG. These findings are compatible with the hypothesis that elevated concentrations of glucose increase tissue DAG content via de novo synthesis, resulting in protein kinase C activation, and that these biochemical events are among the factors that generate the increased microvascular albumin clearance.
Assuntos
Diglicerídeos/metabolismo , Glucose/farmacologia , Microcirculação/efeitos dos fármacos , Alcaloides/farmacologia , Animais , Permeabilidade Capilar , Angiopatias Diabéticas/etiologia , Glicerídeos/farmacologia , Tecido de Granulação/metabolismo , Masculino , NAD/metabolismo , Proteína Quinase C/fisiologia , Ratos , Ratos Endogâmicos , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In view of the similarity of cataracts and neuropathy in galactose-fed and diabetic rats, the present experiments were undertaken to determine whether consumption of galactose-enriched diets (10, 25, or 50% by weight) also increases collagen crosslinking and permeation of vessels by 125I-albumin analogous to that observed in diabetic rats. The observations in these experiments: demonstrate that consumption of galactose-enriched diets for 3 wk selectively increases 125I-albumin permeation of the same vascular beds affected in diabetic rats and by diabetic vascular disease in humans (i.e., the aorta and vessels in the eye, kidney, sciatic nerve, and new tissue formed in the diabetic milieu); demonstrate that the susceptibility of the vasculature to aldose reductase-linked injury (increased permeability) varies greatly in different tissues; indicate that collagen solubility (crosslinking) changes in galactose-fed rats differ sharply from those in diabetic rats; and provide new evidence that consumption of galactose-enriched diets induces a hypogonadal state in male rats.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Colágeno/metabolismo , Galactose/farmacologia , Imidazolidinas , Aldeído Redutase/antagonistas & inibidores , Animais , Peso Corporal/efeitos dos fármacos , Imidazóis/farmacologia , Masculino , Naftalenos/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Próstata/anatomia & histologia , Ratos , Ratos Endogâmicos , Soroalbumina Bovina/metabolismo , Solubilidade , Testosterona/sangue , Distribuição TecidualRESUMO
The purpose of these experiments was to investigate a potential role for vascular endothelial growth factor (VEGF) in mediating vascular dysfunction induced by increased glucose flux via the sorbitol pathway. Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 wk later, granulation tissue in the chamber was exposed twice daily for 7 d to 5 mM glucose, 30 mM glucose, or 1 mM sorbitol in the presence and absence of neutralizing VEGF antibodies. Albumin permeation and blood flow were increased two- to three-fold by 30 mM glucose and 1 mM sorbitol; these increases were prevented by coadministration of neutralizing VEGF antibodies. Blood flow and albumin permeation were increased approximately 2.5-fold 1 h after topical application of recombinant human VEGF and these effects were prevented by nitric oxide synthase (NOS) inhibitors (aminoguanidine and N(G)-monomethyl L-arginine). Topical application of a superoxide generating system increased albumin permeation and blood flow and these changes were markedly attenuated by VEGF antibody and NOS inhibitors. Application of sodium nitroprusside for 7 d or the single application of a calcium ionophore, A23187, mimicked effects of glucose, sorbitol, and VEGF on vascular dysfunction and the ionophore effect was prevented by coadministration of aminoguanidine. These observations suggest a potentially important role for VEGF in mediating vascular dysfunction induced by "hypoxia-like" cytosolic metabolic imbalances (reductive stress, increased superoxide, and nitric oxide production) linked to increased flux of glucose via the sorbitol pathway.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Glucose/metabolismo , Linfocinas/fisiologia , Fluxo Sanguíneo Regional , Albumina Sérica/metabolismo , Pele/irrigação sanguínea , Animais , Calcimicina/farmacologia , Permeabilidade da Membrana Celular , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/metabolismo , Feminino , Guanidinas/farmacologia , Humanos , Linfocinas/antagonistas & inibidores , Linfocinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nitroprussiato/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Sorbitol/metabolismo , Superóxidos/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , ômega-N-Metilarginina/farmacologiaRESUMO
Large RNAs often have rugged folding energy landscapes that result in severe misfolding and slow folding kinetics. Several interdependent parameters that contribute to misfolding are now well understood and examples of large RNAs and ribonucleoproteins that avoid kinetic traps have been reported. These advances have facilitated the exploration of fundamental RNA folding processes that were previously inaccessible.
Assuntos
RNA/química , Éxons , Cinética , Conformação de Ácido Nucleico , RNA/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMO
Large ribozymes fold on a 'glacial' timescale compared to the folding of their protein counterparts. The sluggish folding exhibited by large RNAs results from the formation of kinetically trapped, misfolded intermediates, which are nonessential features of the folding mechanism. Newly developed mutant ribozymes that avoid kinetic traps should facilitate the study of the RNA folding problem.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Animais , Sequência de Bases , Endorribonucleases/química , Cinética , Dados de Sequência Molecular , Peso Molecular , RNA Bacteriano/química , RNA Catalítico/química , RNA de Protozoário/química , Ribonuclease P , Tetrahymena/enzimologia , Tetrahymena/genéticaRESUMO
Hydrogen bond networks stabilize RNA secondary and tertiary structure and are thus essentially important for protein recognition. During structure refinements using either NMR or X-ray techniques, hydrogen bonds were usually inferred indirectly from the proximity of donor and acceptor functional groups. Recently, quantitative heteronuclear J(N,N)-HNN COSY NMR experiments were introduced that allowed the direct identification of donor and acceptor nitrogen atoms involved in hydrogen bonds. However, protons involved in base pairing interactions in nucleic acids are often not observable due to exchange processes. The application of a modified quantitative J(N,N)-HNN COSY pulse scheme permits observation of(2h)J(N,N) couplings via non-exchangeable protons. This approach allowed the unambiguous identification of the A27.U23 reverse Hoogsteen base pair involved in a U-A.U base triple in the HIV-2 transactivation response element-argininamide complex. Despite a wealth of NOE information, direct evidence for this interaction was lacking due to the rapid exchange of the U23 imino proton. The ability to directly observe hydrogen bonds, even in D(2)O and in the presence of rapid exchange, should facilitate structural studies of RNA.
Assuntos
RNA Viral/química , Pareamento de Bases , Sequência de Bases , Deutério , Repetição Terminal Longa de HIV , HIV-2/química , HIV-2/genética , Ligação de Hidrogênio , Iminas/química , Espectroscopia de Ressonância Magnética , Nitrogênio/química , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/genéticaRESUMO
The helix-loop-helix structure formed in the pre-mRNA and the mRNA of L30, a ribosomal protein from the yeast Saccharomyces cerevisiae, serves as an auto-regulatory binding site for the protein to suppress the L30 synthesis upon overproduction. Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were investigated using nuclear magnetic resonance (NMR) spectroscopy. Homonuclear and(13)C/(15)N-based resonance assignments and spectral comparisons indicated that the purine-rich internal loop is dynamic in the free RNA but becomes ordered in the presence of L30 protein. Although the resonances in the loop region are sharper and more disperse in the bound RNA, their assignment was extremely challenging, due to spectral complexity and broadened resonances caused by local dynamics. Two strategies, namely selective(13)C/(15)N-labeling and NMR analyses of five complexes with RNA mutants, were used to overcome these difficulties. Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleotide conformations within the internal loop be made. In the case of structural determination of the L30-mRNA complex, it was critical to be able to take advantage of the available biochemical information in order to complete the structure determination.