RESUMEN

BACKGROUND AND PURPOSE: Azithromycin has been reported to modify activation of macrophages towards the M2 phenotype. Here, we have sought to identify the mechanisms underlying this modulatory effect of azithromycin on human monocytes, classically activated in vitro. EXPERIMENTAL APPROACH: Human blood monocytes were primed with IFN-γ for 24 h and activated with LPS for 24 h. Azithromycin, anti-inflammatory and lysosome-affecting agents were added 2 h before IFN-γ. Cytokine and chemokine expression was determined by quantitative PCR and protein release by ELISA. Signalling molecules were determined by Western blotting and transcription factor activation quantified with a DNA-binding ELISA kit. KEY RESULTS: Azithromycin (1.5-50 µM) dose-dependently inhibited gene expression and/or release of M1 macrophage markers (CCR7, CXCL 11 and IL-12p70), but enhanced CCL2, without altering TNF-α or IL-6. Azithromycin also enhanced the gene expression and/or release of M2 macrophage markers (IL-10 and CCL18), and the pan-monocyte marker CD163, but inhibited that of CCL22. The Toll-like receptor (TLR) 4 signalling pathway was modulated, down-regulating NF-κB and STAT1 transcription factors. The inhibitory profile of azithromycin differed from that of dexamethasone, the phosphodiesterase-4 inhibitor roflumilast and the p38 kinase inhibitor SB203580 but was similar to that of the lysosomotropic drug chloroquine. Effects of concanamycin and NH4Cl, which also act on lysosomes, differed significantly. CONCLUSIONS AND IMPLICATIONS: Azithromycin modulated classical activation of human monocytes by inhibition of TLR4-mediated signalling and possible effects on lysosomal function, and generated a mediator expression profile that differs from that of monocyte/macrophage phenotypes so far described.

Asunto(s)

Azitromicina/farmacología , Monocitos/efectos de los fármacos , Antiinflamatorios/farmacología , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL22/genética , Quimiocina CCL22/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Therapeutic doses of (131)I administered to thyrotoxic patients may cause thyroid failure. The present study used a rat model to determine thyroid function after the administration of different doses of (131)I (64-277 microCi). Thirty male Fisher rats in the experimental group and 30 in the control group (untreated) were followed for 6 months. The animals were 4 months old at the beginning of the experiment and were sacrificed at an age of 9 months. Hormone concentration was determined before (131)I administration (4-month-old animals) and three times following (131)I administration, when the animals were 7, 8, and 9 months old. The thyroid glands were removed and weighed, their volume was determined and histopathological examination was performed at the end of the experiment. Significant differences in serum triiodothyronine and thyroid-stimulating hormone concentration, measured at the age of 7, 8, and 9 months, were found in the experimental group. During aging of the animals, the concentration of thyroxin fell from 64.8 +/- 8.16 to 55.0 +/- 6.1 nM in the control group and from 69.4 +/- 6.9 to 25.4 +/- 3.2 nM in the experimental group. Thyroid gland volume and weight were significantly lower in the experimental than in the control group. Thyroid glands from the experimental group showed hyaline thickness of the blood vessel wall, necrotic follicles, a strong inflammatory reaction, and peeling of necrotic cells in the follicles. In conclusion, significant differences in hormone levels and histopathological findings indicated prolonged hypothyroidism after (131)I administration to rats, which was not (131)I dose dependent.

Asunto(s)

Radioisótopos de Yodo/administración & dosificación , Glándula Tiroides/efectos de la radiación , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangre , Animales , Peso Corporal , Relación Dosis-Respuesta en la Radiación , Hipertiroidismo/sangre , Masculino , Ratas , Ratas Endogámicas F344 , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiopatología

RESUMEN

Therapeutic doses of 131I administered to thyrotoxic patients may cause thyroid failure. The present study used a rat model to determine thyroid function after the administration of different doses of 131I (64-277 µCi). Thirty male Fisher rats in the experimental group and 30 in the control group (untreated) were followed for 6 months. The animals were 4 months old at the beginning of the experiment and were sacrificed at an age of 9 months. Hormone concentration was determined before 131I administration (4-month-old animals) and three times following 131I administration, when the animals were 7, 8, and 9 months old. The thyroid glands were removed and weighed, their volume was determined and histopathological examination was performed at the end of the experiment. Significant differences in serum triiodothyronine and thyroid-stimulating hormone concentration, measured at the age of 7, 8, and 9 months, were found in the experimental group. During aging of the animals, the concentration of thyroxin fell from 64.8 ± 8.16 to 55.0 ± 6.1 nM in the control group and from 69.4 ± 6.9 to 25.4 ± 3.2 nM in the experimental group. Thyroid gland volume and weight were significantly lower in the experimental than in the control group. Thyroid glands from the experimental group showed hyaline thickness of the blood vessel wall, necrotic follicles, a strong inflammatory reaction, and peeling of necrotic cells in the follicles. In conclusion, significant differences in hormone levels and histopathological findings indicated prolonged hypothyroidism after 131I administration to rats, which was not 131I dose dependent.

Asunto(s)

Animales , Masculino , Ratas , Radioisótopos de Yodo/administración & dosificación , Glándula Tiroides/efectos de la radiación , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangre , Peso Corporal , Relación Dosis-Respuesta en la Radiación , Hipertiroidismo/sangre , Glándula Tiroides/fisiopatología , Glándula Tiroides

RESUMEN

Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics supported by a model describing a dimeric enzyme with conformationally and kinetically non-equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [S. Orhanovic, M. Pavela-Vrancic, Eur. J. Biochem. 270 (2003) 4356-4364]. The significance of interactions at the subunit interface and the involvement of the beta-pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site-directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild-type protein. The T81A mutation, introduced at the subunit interface, significantly affected the protein kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.

Asunto(s)

Fosfatasa Alcalina/química , Fosfatasa Alcalina/genética , Escherichia coli/enzimología , Escherichia coli/genética , Fosfatasa Alcalina/metabolismo , Sustitución de Aminoácidos , Dominio Catalítico/genética , Dimerización , Estabilidad de Enzimas , Calor , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Puntual , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Tyrocidine synthetase 1 (TY1), the initial monomodular constituent of the tyrocidine biosynthetic system, exhibits relaxed substrate specificity, however an efficient editing of the mis-activated amino acid provides for fidelity of product formation. We chose to analyse the consequence of single amino acid substitutions, in the amino acid activation site of apo-TY1, on the editing functions of the enzyme. Discrimination between L-Phe and D-Phe by apo-TY1 depends primarily on the editing reaction. Distraction of unnatural amino acid substrates, such as L-PheSer, implies that editing is not designated to select a specific mis-activated amino acid, but instead to discriminate all mis-activated amino acid analogues. It was shown that active site residues which interact with the adenylate are essential for both activation and editing. Substitution of Lys186 with arginine substantially reduces the editing capacity of the protein. Loss of amino acid discrimination ability by the apo-K186T and apo-R416T mutant proteins suggests a role of active site residues in maintaining the structural determinants for substrate selection. Inadequate conformational changes, induced by non-cognate amino acid substrates, promote ATP breakdown yielding P(i) and ADP. Replacement of residue Lys186 or Arg416 enhances ATP hydrolysis implying a role in binding or adjusting of the triphosphate chain for adenylate formation and pyrophosphate cleavage.

Asunto(s)

Apoproteínas/metabolismo , Péptido Sintasas/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/farmacología , Sustitución de Aminoácidos , Apoproteínas/genética , Arginina/genética , Arginina/metabolismo , Sitios de Unión/genética , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Hidrólisis , Pirofosfatasa Inorgánica/metabolismo , Pirofosfatasa Inorgánica/farmacología , Lisina/genética , Lisina/metabolismo , Péptido Sintasas/genética , Fosfatos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Purina-Nucleósido Fosforilasa/metabolismo , Purina-Nucleósido Fosforilasa/farmacología , Tionucleósidos/metabolismo

RESUMEN

Alkaline phosphatase (AP) displays significant structural changes during metal-ion binding, supporting cooperative interactions between the subunits of the dimeric enzyme. Here, we present data on the dynamic properties of AP from E. coli, and characterize the structural changes that accompany variations in metal-ion content, combining limited proteolysis and MALDI-TOF mass spectrometry. Limited proteolysis revealed an internal cleavage site at Arg-293, reflecting a position of conformational flexibility supporting subunit communication essential for catalysis. A specific shielding of a region distant from the metal-binding site has been demonstrated, implying transmission of conformational changes, induced by metal-ion binding to the adjacent subunit, across the subunit interface.

Asunto(s)

Fosfatasa Alcalina/química , Proteínas de Escherichia coli/química , Metales/metabolismo , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Sitios de Unión , Proteínas de Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Iones/metabolismo , Iones/farmacología , Magnesio/metabolismo , Magnesio/farmacología , Metales/farmacología , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tripsina/metabolismo , Zinc/metabolismo , Zinc/farmacología

RESUMEN

This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication. OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv. Cancer. Res. 61 (1993) 143). However, all of its cellular targets have not yet been characterized. The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity. Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP. The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM). The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Ocadaico/farmacología , Animales , Escherichia coli/enzimología , Humanos , Cinética , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 1

RESUMEN

A monitoring program, carried out in 1996 and 1997, has confirmed that toxic compounds, other than the most frequently detected toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1), are involved in DSP phenomena in the Adriatic Sea. Toxicity was assessed by the mouse bioassay; the content and the nature of the toxic components were established through fluorometric HPLC analysis combined with mass spectrometry. A rare pectenotoxin-2 (PTX-2) derivative, 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA), was the exclusive contaminant of samples collected from the central Adriatic in 1996. Contrary to its marked oral toxicity, intraperitoneally 7-epi-PTX-2SA displayed no toxic effects, hampering its detection by the mouse bioassay. In 1997, its concentration and frequency of appearance were lower than in 1996, with concomitant occurrence of OA, DTX-2, and a new unidentified component related to the DSP toxic group of compounds. This is the first report on the occurrence of DTX-2 in Adriatic mussels. A survey of the phytoplankton community in the surrounding seawater has established the presence of Prorocentrum micans and several potentially toxic species from the Dinophysis genus. A case of unexplained toxicity, associated with the occurrence of Gonyaulax polyedra, suggested possible shellfish contamination with yessotoxin (YTX).

Asunto(s)

Bivalvos/metabolismo , Furanos/toxicidad , Fitoplancton , Piranos/toxicidad , Intoxicación por Mariscos , Animales , Cromatografía Líquida de Alta Presión , Croacia , Furanos/administración & dosificación , Furanos/análisis , Inyecciones Intraperitoneales , Macrólidos , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Océanos y Mares , Piranos/administración & dosificación , Piranos/análisis

RESUMEN

A deletion mutant of tyrocidine synthetase 1 (DeltaDeltaTY1), comprising the adenylation domain of TY1 as an independent functional adenylate-forming unit, was used to investigate the ability of the adenylation domain in non-ribosomal peptide synthetases to catalyse peptide bond formation from the aminoacyl adenylate intermediate. The results demonstrate that only one substrate amino acid needs to be activated as an aminoacyl adenylate. In view of the potential exploitation of peptide synthetases for enzymatic synthesis of dipeptides of choice, it is important to note that this does not necessarily require a dimodular construct or an intermediate acyl transfer step.

Asunto(s)

Dipéptidos/biosíntesis , Péptido Sintasas/metabolismo , Adenina/metabolismo , Sitios de Unión , Catálisis , Escherichia coli , Péptido Sintasas/química , Estructura Terciaria de Proteína

Asunto(s)

Anomalías de los Vasos Coronarios , Anciano , Aorta/anomalías , Angiografía Coronaria , Circulación Coronaria , Anomalías de los Vasos Coronarios/patología , Anomalías de los Vasos Coronarios/cirugía , Ecocardiografía , Ecocardiografía Doppler , Femenino , Ventrículos Cardíacos/anomalías , Humanos , Ligadura , Imagen por Resonancia Magnética , Fotograbar , Arteria Pulmonar/anomalías

RESUMEN

In response to nutritional stress conditions, Bacillus brevis produces the cyclodecapeptide antibiotic tyrocidine via tyrocidine synthetase, a multifunctional non-ribosomal peptide synthetase. The apo-form of tyrocidine synthetase 1 forms adenosine (5')tetraphospho(5')adenosine, when incubated with MgATP(2-), amino acid and inorganic pyrophosphatase. The synthesis is an intrinsic property of the adenylation domain, is strictly dependent upon the amino acid, and proceeds from a reverse reaction of adenylate formation involving a second ATP molecule. In the presence of tri- or tetrapolyphosphate preferential synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate occurs, respectively. A potential involvement of adenosine (5')-n-phospho(5')adenosine in the regulation of the biosynthetic process has been suggested.

Asunto(s)

Fosfatos de Dinucleósidos/metabolismo , Péptido Sintasas/metabolismo , Adenosina Trifosfato/metabolismo , Fosfatos de Dinucleósidos/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Péptido Sintasas/genética , Plásmidos , Pirofosfatasas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Okadaic acid (OA) and 7-epi-pectenotoxin-2 seco acid (7-epi-PTX-2SA) were identified as the toxic determinants in mussels from the central Adriatic Sea. The nature of the pectenotoxin-2 derivative was confirmed by chromatographic comparison with toxins present in algae extracts of Dinophysis acuta from Ireland, and by mass spectrometric analysis. The origin of shellfish toxicity has been associated with the occurrence of the Dinophysis species. This is the first report on the incidence of 7-epi-PTX-2SA in the Adriatic region.

Asunto(s)

Furanos/metabolismo , Toxinas Marinas/análisis , Piranos/metabolismo , Animales , Cromatografía Liquida , Dinoflagelados/química , Enfermedades Transmitidas por los Alimentos , Furanos/química , Macrólidos , Toxinas Marinas/toxicidad , Ratones , Ratones Endogámicos BALB C , Ácido Ocadaico/análisis , Ácido Ocadaico/toxicidad , Piranos/química , Agua de Mar , Espectrometría de Fluorescencia

Asunto(s)

Aneurisma/cirugía , Aneurisma Coronario/cirugía , Vena Safena/trasplante , Anciano , Puente de Arteria Coronaria , Humanos , Masculino , Complicaciones Posoperatorias/cirugía

RESUMEN

The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PP(i) generation using active site titration measurements with [gamma-(32)P]ATP. The initial 'burst' of product formation can be correlated to the generation of the aminoacyl adenylate:enzyme complexes at the four amino acid activation domains and the subsequent aminoacylation of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amino acid substrates at a saturating concentration displayed a consumption of 8.3 ATP/GS2. In the presence of single amino acids, a binding stoichiometry higher than the anticipated two ATP per active site was obtained, implying misactivation at non-cognate domains. Breakdown of acyladenylate intermediates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation.

Asunto(s)

Isomerasas de Aminoácido/metabolismo , Complejos Multienzimáticos/metabolismo , Biosíntesis de Péptidos , Péptido Sintasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Catálisis , Difosfatos/metabolismo

RESUMEN

How genes are expressed and translated into proteins (using mRNA, codons and tRNAs as adaptor molecules) forms the basis of the 'genetic code'. Many peptides are synthesized nonribosomally, however, by large protein complexes that also serve as templates. Recent advances have shed light on what the nonribosomal code is and how it can be read.

Asunto(s)

Biosíntesis de Péptidos/fisiología , Acilación , Adenosina Monofosfato/metabolismo , Animales , Humanos , Biblioteca de Péptidos , Alineación de Secuencia , Moldes Genéticos

RESUMEN

Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide families. Activation of the amino acid substrate proceeds in analogy to the ribosomal biosynthetic mechanism generating aminoacyl adenylate and acyl intermediates. To approach the mechanism of fidelity of amino acid selection, the stability of the aminoacyl adenylates was studied by employing a continuous coupled spectrophotometric assay. The apo-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an l-phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylation domain. Adenylates of substrate analogues have shown variable and reduced degrees of stability, thus leading to an enhanced generation of pyrophosphate due to hydrolysis and continuous adenylate formation. Discrimination of the non-aromatic amino acids l-Leu and l-Met, or l-Phe analogues such as p-amino- and p-chloro-l-Phe derivatives, as well as the stereospecific selection of l-Phe, is supported by less-stable adenylate intermediates exhibiting elevated susceptibility to hydrolysis. Breakdown of the l-phenylalanyl intermediate utilizing 2'-deoxy-ATP as the nucleotide substrate was significantly enhanced compared with the natural analogue. Apo-TY1 engineered at positions involved in adenylate formation showed variable protection against hydrolysis. The results imply that stability of the aminoacyl intermediates may act as an essential factor in substrate selection and fidelity of non-ribosomal-peptide-forming systems.

Asunto(s)

Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Péptido Sintasas/metabolismo , Apoproteínas/metabolismo , Bacillus , Catálisis , Escherichia coli , Pirofosfatasas/metabolismo

RESUMEN

The boundaries of the structural domains in peptide synthetases and the conformational changes related to catalysis were investigated by limited proteolysis of tyrocidine synthetase 1 (TY1). Four regions sensitive to proteolysis were detected (cleavage site at Arg13, Arg424, Arg509 and Arg602) that, in addition to an N-terminal extension, accurately delineate the domain boundaries of the adenylate-forming domain, the aminoacyl carrier domain, and the epimerisation domain. Limited proteolysis of an active N-terminal truncated deletion mutant, His6DeltaTY1, generated two stable and structurally independent subunits, corresponding to the subdomains of the adenylation domain. The structural integrity of the carrier domain was substantiated by its resistance to proteolytic degradation. Evidence is provided that the C-terminal "spacer" region with epimerising and/or condensing activity folds into an autonomous domain stable against degradation by limited proteoly sis. In the presence of substrates, reduced susceptibility to proteolysis was observed in the linker region connecting the subdomains of the adenylation domain, and corresponding to a peptide stretch of low electron density in the X-ray structure of the homologous firefly luciferase. Sequence analysis has shown that the respective linker contains conserved residues, whereas the linker regions connecting the structural domains are of low homology with a significant content of Pro, Ala, Glu and polar residues. A combination of kinetic and proteolytic studies using ATP analogues with substitutions in the phosphate chain, AMP-PcP, AMP-PNP and AMP-cPP, strongly suggests that the generation of a productive complex is associated with the ability of the beta, gamma-pyrophosphate moiety of ATP to adopt the proper active-site conformation. These data substantiate the observation that peptide synthetases undergo a series of conformational changes in the process of adenylate formation and product release.

Asunto(s)

Péptido Sintasas/química , Conformación Proteica , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/fisiología , Adenilil Imidodifosfato/química , Regulación Alostérica , Sitio Alostérico , Sitios de Unión , Catálisis , Difosfatos/química , Cinética , Ligandos , Fragmentos de Péptidos/química , Péptido Sintasas/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína , Eliminación de Secuencia

RESUMEN

Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recently become available, providing evidence for the localization of the active site at the interface between two subdomains separated by a distorted linker region [Conti, E., Franks, N. P. & Brick, P. (1996) Structure 4, 287-298]. The functional importance of two flexible loops, corresponding to the linker region of firefly luciferase and the highly conserved (S/T)GT(T/S)GXPKG core sequence, has been studied in view of the proposed conformational changes by the use of mutant analysis, limited proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg416, residing in the loop separating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested significant structural changes as manifested by lack of protection to degradation in the presence of substrates, revealing a probable disturbance of the induced-fit mechanism regulating the transformation from an open to a closed conformation. Mutants, obtained by replacement of the conserved Lys186 from the (S/T)GT(T/S)GXPKG core sequence, displayed only minor differences in substrate-binding affinity despite significant reduction of catalytic efficiency. Residue Lys186 appears to play an important role in either stabilization of the bound substrate through charge-charge-interactions, and/or fixing of the loop for maintainance of the active-site conformation.

Asunto(s)

Péptido Sintasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cartilla de ADN , Hidrólisis , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptido Sintasas/química , Péptido Sintasas/genética , Estructura Secundaria de Proteína , Relación Estructura-Actividad

RESUMEN

Con el objeto de conocer las indicaciones, complicaciones, control y evolución de los accesos venosos centrales, se confeccionó un protocolo de trabajo prospectivo para el análisis de los mismos. Del total de las vías efectuadas se escogió una muestra de 100 casos. La relación hombre: mujer fué de 1,17: 1; el promedio de edad de 52,8 años (max. 91, min. 15). Del total de vías centrales 68 fueron por punción (67,6 por ciento yugular interna por vía media, 20,5 por ciento por vía posterior, 8,8 por ciento por subclavia infraclavicular y 2,9 por ciento por otras vías. Por disección se utilizaron la vena basílica en el 93 por ciento de los casos, la cefálica y la safena en 3,1 por ciento respectivamente. Treita y dos casos fueron controlados radiológicamente luego del procedimiento, pudiendo detectarse 4 neumotórax, que junto a 3 hematomas, fueron las complicaciones inherentes a la función. Un hematoma 2 infecciones de la herida y una trombosis de la vena ilíaca, fueron secundarias al acceso por disección en 2 pacientes. Las complicaciones secundarias al cateter fueron: sepsis en 2 casos y dolor torácico en 1 caso. Se observó que las complicaciones se vieron con una frecuencia mayor pero no significativa (p: 0,68) en aquellos operadores con menor experiencia. Cuarenta y un cateteres fueron enviados a cultivos luego de su extracción, 16 fueron positivos, el 50 por ciento a estafilococo aureus. Concluímos que salvo contraindicación absoluta los accesos vasculares centrales deben realizarse por punción, el control radiológico se realizará ante sospecha de lesión pleural o disfunción del cateter y, como todo acto quirúrgico, debe ir precedido de una correcta antisepsia:

Asunto(s)

Humanos , Persona de Mediana Edad , Cateterismo , Cateterismo Venoso Central , Venas

RESUMEN

Con el objeto de conocer las indicaciones, complicaciones, control y evolución de los accesos venosos centrales, se confeccionó un protocolo de trabajo prospectivo para el análisis de los mismos. Del total de las vías efectuadas se escogió una muestra de 100 casos. La relación hombre: mujer fué de 1,17: 1; el promedio de edad de 52,8 años (max. 91, min. 15). Del total de vías centrales 68 fueron por punción (67,6 por ciento yugular interna por vía media, 20,5 por ciento por vía posterior, 8,8 por ciento por subclavia infraclavicular y 2,9 por ciento por otras vías. Por disección se utilizaron la vena basílica en el 93 por ciento de los casos, la cefálica y la safena en 3,1 por ciento respectivamente. Treita y dos casos fueron controlados radiológicamente luego del procedimiento, pudiendo detectarse 4 neumotórax, que junto a 3 hematomas, fueron las complicaciones inherentes a la función. Un hematoma 2 infecciones de la herida y una trombosis de la vena ilíaca, fueron secundarias al acceso por disección en 2 pacientes. Las complicaciones secundarias al cateter fueron: sepsis en 2 casos y dolor torácico en 1 caso. Se observó que las complicaciones se vieron con una frecuencia mayor pero no significativa (p: 0,68) en aquellos operadores con menor experiencia. Cuarenta y un cateteres fueron enviados a cultivos luego de su extracción, 16 fueron positivos, el 50 por ciento a estafilococo aureus. Concluímos que salvo contraindicación absoluta los accesos vasculares centrales deben realizarse por punción, el control radiológico se realizará ante sospecha de lesión pleural o disfunción del cateter y, como todo acto quirúrgico, debe ir precedido de una correcta antisepsia: (AU)

Asunto(s)

Humanos , Persona de Mediana Edad , Venas/cirugía , Venas/patología , Cateterismo/estadística & datos numéricos , Cateterismo Venoso Central/métodos