RESUMEN

Enzymes have become important tools in several industries due to their ability to produce chirally pure and complex molecules with interesting biological properties. The NAD(+)-dependent LDH (lactate dehydrogenase) [bsLDH [Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) LDH] from G. stearothermophilus and the NAD(+)-dependent FDH (formate dehydrogenase) [cmFDH (Candida methylica FDH)] enzyme from C. methylica are particularly crucial enzymes in the pharmaceutical industry and are related to each other in terms of NADH use and regeneration. LDH catalyses the interconversion of pyruvate (oxo acid) and lactate (alpha-hydroxy acid) using the NADH/NAD(+) pair as a redox cofactor. Employing LDH to reduce other oxo acids can generate chirally pure alpha-hydroxy acids of use in the production of pharmaceuticals. One important use of FDH is to regenerate the relatively expensive NADH cofactor that is used by NAD(+)-dependent oxidoreductases such as LDH. Both LDH and FDH from organisms of interest were previously cloned and overproduced. Therefore they are available at a low cost. However, both of these enzymes show disadvantages in the large-scale production of chirally pure compounds. We have applied two routes of protein engineering studies to improve the properties of these two enzymes, namely DNA shuffling and site-directed mutagenesis. Altering the substrate specificity of bsLDH by DNA shuffling and changing the coenzyme specificity of cmFDH by site-directed mutagenesis are the most successful examples of our studies. The present paper will also include the details of these examples together with some other applications of protein engineering regarding these enzymes.

Asunto(s)

Candida/enzimología , Formiato Deshidrogenasas/química , Geobacillus stearothermophilus/enzimología , L-Lactato Deshidrogenasa/química , Ingeniería de Proteínas , Simulación por Computador , Estabilidad de Enzimas , Formiato Deshidrogenasas/aislamiento & purificación , Enlace de Hidrógeno , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/aislamiento & purificación , Especificidad por Sustrato

RESUMEN

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http://www.rubic.rdg.ac.uk/betapairprefsparallel/).

Asunto(s)

Aminoácidos/química , Aminoácidos/metabolismo , Estructura Secundaria de Proteína , Proteínas/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas/metabolismo , Electricidad Estática

RESUMEN

As Plasmodium rely extensively on homolactic fermentation for energy production, Plasmodium falciparum lactate dehydrogenase (PfLDH)--the key enzyme in this process--has previously been suggested as a novel target for antimalarials. This enzyme has distinctive kinetic and structural properties that distinguish it from its human homologues. In this study, we now describe the expression, kinetic characterisation and crystal structure determination of the LDH from Plasmodium berghei. This enzyme is seen to have a similar kinetic profile to its P. falciparum counterpart, exhibiting the characteristic lack of substrate inhibition that distinguishes plasmodial from human LDHs. The crystal structure of P. berghei lactate dehydrogenase (PbLDH) shows a very similar active site arrangement to the P. falciparum enzyme. In particular, an insertion of five amino acid residues in the active site loop creates an enlarged volume in the substrate binding site, and characteristic changes in the residues lining the NADH cofactor binding pocket result in displacement of the cofactor relative to its observed position in mammalian and all other LDH structures. These results imply the special features previously described for PfLDH may be shared across the Plasmodium genus, supporting the universal application of therapeutics targeting this enzyme.

Asunto(s)

L-Lactato Deshidrogenasa/química , Plasmodium berghei/enzimología , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Humanos , Cinética , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Modelos Animales , Modelos Moleculares , Datos de Secuencia Molecular , Análisis de Secuencia de ADN

RESUMEN

An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme-binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the protein's fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations.

Asunto(s)

Cianobacterias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/química , Secuencia de Aminoácidos , Sitios de Unión , Fluorescencia , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADP/metabolismo , Oxidorreductasas/genética , Homología de Secuencia de Aminoácido

RESUMEN

BACKGROUND: The role of planned neck dissection after organ preservation therapy with radiotherapy or chemotherapy/radiotherapy for advanced head and neck cancers presenting with clinically positive neck disease is still being elucidated. The aim of this study is to review the outcomes of such patients treated by organ preservation therapy at our institution. METHODS: A retrospective chart review of 33 patients who underwent planned neck dissections after organ preservation therapy for advanced primary head and neck malignancy. Endpoints measured were disease-free survival and local, regional, and distant control. SETTING: Tertiary metropolitan medical center. RESULTS: Two-year actuarial disease-free survival was 61%, and neck control was 92%, with only two failures in the neck. The use of neoadjuvant chemotherapy and total dose of radiotherapy did not correlate with neck control or disease-free survival. The presence of pathologically positive nodal disease at the time of neck dissection did not correlate with recurrent neck disease, but was a predictor of local recurrence (p = .0086). CONCLUSIONS: Our data suggest that for patients undergoing planned neck dissection after organ preservation therapy, neck control is obtained in almost all cases. The presence of pathologically positive nodal disease at the time of surgery may have implications for the incidence of local recurrence.

Asunto(s)

Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/terapia , Disección del Cuello , Terapia Combinada , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento

RESUMEN

Lactate dehydrogenase (LDH) interconverts pyruvate and lactate with concomitant interconversion of NADH and NAD(+). Although crystal structures of a variety of LDH have previously been described, a notable absence has been any of the three known human forms of this glycolytic enzyme. We have now determined the crystal structures of two isoforms of human LDH-the M form, predominantly found in muscle; and the H form, found mainly in cardiac muscle. Both structures have been crystallized as ternary complexes in the presence of the NADH cofactor and oxamate, a substrate-like inhibitor. Although each of these isoforms has different kinetic properties, the domain structure, subunit association, and active-site regions are indistinguishable between the two structures. The pK(a) that governs the K(M) for pyruvate for the two isozymes is found to differ by about 0.94 pH units, consistent with variation in pK(a) of the active-site histidine. The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure. Proteins 2001;43:175-185.

Asunto(s)

Isoenzimas/química , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Cristalización , Humanos , Cinética , Lactato Deshidrogenasa 5 , Modelos Moleculares , Electricidad Estática , Relación Estructura-Actividad

RESUMEN

This study describes a computational method for ab inito protein structure prediction. Protein conformation has been modeled by using six optimized backbone torsion angles and fixed side chains approximating rotationally averaged real side chains. The approximations aim to keep complexity of the structure description to a minimum without seriously compromising the accuracy of the structural representation. An evolutionary Monte Carlo algorithm has been developed to search through this restricted conformational space to locate low-energy protein structures. A simple physicochemical force field has been developed to assess the energies of different conformations within this structural description. The corresponding residue interaction energies are based on hydrophobic, hydrophilic, steric, and hydrogen-bonding potentials. The search procedure has been used to locate native energy minima from primary sequence alone. The 3-D structures of polypeptides up to 38 residues with both beta and alpha secondary structural elements have been accurately predicted. The search procedure has been found to be highly efficient and follows an energetically and structurally plausible pathway to locate native populations. The simple force field described in the study has been compared with a more complex all-atom model and been found to be similarly effective in predicting the structures of proposed independent folding units. Proteins 2001;43:186-202.

Asunto(s)

Conformación Proteica , Algoritmos , Secuencia de Aminoácidos , Animales , Simulación por Computador , Evolución Molecular , Matemática , Modelos Moleculares , Método de Montecarlo , Polipéptido Pancreático/química , Pliegue de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad

RESUMEN

In a recent publication, the structural details of an interaction between the Rhodobacter sphaeroides reaction center and the anionic phospholipid diphosphatidyl glycerol (cardiolipin) were described (K. E. McAuley, P. K. Fyfe, J. P. Ridge, N. W. Isaacs, R. J. Cogdell, and M. R. Jones, 1999, Proc. Natl. Acad. Sci. U.S.A. 96:14706-14711). This was the first crystallographic description of an interaction between this biologically important lipid and an integral membrane protein and was also the first piece of evidence that the reaction center has a specific interaction with cardiolipin. We have examined the extent to which the residues that interact with the cardiolipin are conserved in other species of photosynthetic bacteria with this type of reaction center and discuss the possibility that this cardiolipin binding site is a conserved feature of these reaction centers. We look at how sequence variations that would affect the shape of the cardiolipin binding site might affect the protein-cardiolipin interaction, by modeling the binding of cardiolipin to the reaction center from Rhodopseudomonas viridis.

Asunto(s)

Cardiolipinas/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Rhodobacter sphaeroides/metabolismo , Rhodopseudomonas/metabolismo

RESUMEN

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.

Asunto(s)

Amidas/química , Apamina/química , Asparagina/química , Hidrógeno/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Cisteína/química , Deuterio/química , Disulfuros/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Termodinámica

RESUMEN

BACKGROUND: Fine-needle aspiration represents a critical diagnostic test in determining proper management of thyroid disease and the use of ultrasound-guided fine-needle aspiration (USGFNA) has increased over the years. METHODS: A retrospective chart review of patients undergoing USGFNA. Two hundred fifteen patients underwent 234 procedures with 362 nodules aspirated within a 2 (1/2)-year period. RESULTS: The mean ages of women and men were 51.9 and 57.8, respectively. The average size of nodules was 2.1 cm. A difficult to assess gland or nodule was the most common indication for USGFNA (33%). The sensitivity was 88.2%, specificity was 80.0%, the PPV was 65.2%, the negative predictive value was 94.1%, and the accuracy was 82.5%. The cancer yield, inadequacy, and complication rates were 44%, 10.5%, and 8.5%, respectively. CONCLUSIONS: USGFNA aspiration is a safe and effective diagnostic modality in the management of thyroid disease, especially for nodules that are difficult to palpate.

Asunto(s)

Biopsia con Aguja/métodos , Enfermedades de la Tiroides/diagnóstico por imagen , Enfermedades de la Tiroides/patología , Ultrasonografía Intervencional/métodos , Biopsia con Aguja/efectos adversos , Biopsia con Aguja/economía , Reacciones Falso Negativas , Reacciones Falso Positivas , Femenino , Técnicas Histológicas , Humanos , Masculino , Persona de Mediana Edad , Palpación , Selección de Paciente , Estudios Retrospectivos , Sensibilidad y Especificidad , Enfermedades de la Tiroides/terapia , Ultrasonografía Intervencional/efectos adversos , Ultrasonografía Intervencional/economía

RESUMEN

Respiratory rates involving the alternative oxidase (AO) were studied in mitochondria from Tapesia acuformis. There was no evidence for regulation by pyruvate, in contrast with plant AO. The site of interaction of pyruvate with the plant AO is a conserved cysteine. The primary sequence was obtained for AO from Magnaporthe grisea and compared with four published sequences for fungal AO. In all cases this cysteine was absent. Sequence data were obtained for the C-terminal domain of a further five fungal AOs. In this region the fungal sequences were all consistent with a four-helix, di-iron binding structure as in the ferritin-fold family. A molecular model of this domain was deduced from the structure of Delta-9 desaturase. This is in general agreement with that developed for plant AOs, despite very low sequence identity between the two kingdoms. Further modelling indicated an appropriate active site for binding of ubiquinol, required in the AO redox reaction.

Asunto(s)

Hongos/enzimología , Mitocondrias/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Ácido Pirúvico/farmacología , Ubiquinona/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Secuencia Conservada/genética , Cisteína/genética , Cisteína/metabolismo , Dimerización , Hongos/genética , Holoenzimas/química , Holoenzimas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Oxidación-Reducción , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Oxígeno/metabolismo , Proteínas de Plantas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ácido Pirúvico/metabolismo , Alineación de Secuencia , Ubiquinona/metabolismo

RESUMEN

It can be argued from the principle of solvent exclusion that the introduction of hydrophobic residues onto the surface of a protein will not destabilize the folded state because the nonpolar side chain will be at least as exposed in the unfolded state as it is when the protein chain is folded. A comparison of the folding pathway of wild type and 11 site-directed mutants of CD2.d1 shows this to be true. In fact, owing to partial burial of nonpolar groups as folding proceeds, we find that the rapidly formed intermediate state and, to a greater extent, the transition state are generally stabilized by hydrophobic surface mutations. This effect is slightly moderated in the folded state presumably by the perturbation of van der Waals' contacts and/or local electrostatic interactions that have a greater influence in this fully compact structure. The fact that in all but one case we find that stabilization of the rapidly collapsed intermediate is accompanied by a faster acquisition of the folded state refutes the argument that I states are generally "off pathway" conformations or ensembles that lead to the inhibition of otherwise more rapid folding trajectories.

Asunto(s)

Inmunoglobulinas/química , Pliegue de Proteína , Sustitución de Aminoácidos , Disulfuros , Fluorescencia , Guanidina/farmacología , Inmunoglobulinas/genética , Isomerismo , Cinética , Modelos Moleculares , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica/efectos de los fármacos , Renaturación de Proteína , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína , Electricidad Estática , Termodinámica

Asunto(s)

Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Proteínas de Escherichia coli , Proteínas Virales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Dimerización , Integrasas/química , Integrasas/metabolismo , Represoras Lac , Modelos Moleculares , Neisseria gonorrhoeae/enzimología , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismo

RESUMEN

Twenty-four cases of the tall cell variant (TCV), a subset of papillary thyroid carcinoma, were identified in a group of 624 patients with thyroid cancer. All pathology specimens were reviewed, and each patient's carcinoma was categorized according to characteristics on presentation, local recurrence, distant metastases, follow-up, and tumor-related mortality. The TCV group was compared with a historical control group (Mazzaferri and Jhiang: 1355 patients). The TCV group had a statistically higher percentage of stage 3 and 4 carcinoma, extrathyroidal invasion, and tumor size less than 1.5 cm than the control group. There was no statistical relationship between age greater than 50 years and stage in the TCV group. No relationship could be found between TCV histology and recurrence or mortality. These findings, combined with those of studies that link stage on presentation to poor outcomes, have led to our conclusion that TCV is an aggressive malignancy warranting appropriate treatment and close follow-up.

Asunto(s)

Carcinoma Papilar/patología , Neoplasias de la Tiroides/patología , Adulto , Anciano , Carcinoma Papilar/clasificación , Carcinoma Papilar/mortalidad , Femenino , Estudios de Seguimiento , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Tasa de Supervivencia , Glándula Tiroides/patología , Neoplasias de la Tiroides/clasificación , Neoplasias de la Tiroides/mortalidad

RESUMEN

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.

Asunto(s)

Proteínas de Plantas/química , Albuminas 2S de Plantas , Secuencia de Aminoácidos , Antígenos de Plantas , Dicroismo Circular , Helianthus/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metionina/química , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/aislamiento & purificación , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Semillas/química , Alineación de Secuencia , Ultracentrifugación

RESUMEN

We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.

Asunto(s)

Antígenos CD2/química , Antígenos CD2/genética , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Animales , Guanidina/química , Isoleucina/genética , Cinética , Leucina/genética , Modelos Químicos , Desnaturalización Proteica , Ratas , Proteínas Recombinantes de Fusión/química , Sulfatos/química , Temperatura , Termodinámica , Valina/genética

RESUMEN

BACKGROUND: The prognostic importance of vascular invasion has not been extensively studied in patients with papillary thyroid cancer. OBJECTIVE: To determine whether the presence of vascular invasion in papillary thyroid carcinoma, even within the thyroid gland, is associated with more aggressive disease at diagnosis and a higher incidence of tumor recurrence. PATIENTS AND METHODS: We identified 410 patients who had been diagnosed with papillary thyroid cancer since 1986 who had a follow-up period of longer than 1 year (median follow-up, 5.5 years). Pathology reports were reviewed and patients were separated into 3 groups: no vascular invasion, intrathyroidal vascular invasion, and extrathyroidal vascular invasion. MAIN OUTCOME MEASURES: Statistical comparison was performed by univariate and multivariate analysis. RESULTS: Patients with intrathyroidal vascular invasion were more likely to have distant metastasis at the time of diagnosis (26.1% vs 2.2%, P = .001). Similarly, patients with extrathyroidal vascular invasion had a higher incidence of distant metastases at diagnosis (40% vs 4.4%, P = .02). Patients with tumors identified to have intrathyroidal vascular invasion were more likely to develop distant recurrence (20% vs 3%, P = .002). CONCLUSIONS: These associations were found to be independent by multiple regression analysis. Patient age, sex, palpable or fixed lymph nodes, radiation exposure, and race did not differ between the patient group with and those without vascular invasion. Preliminary analysis of our data suggests that the presence of vascular invasion in papillary, thyroid carcinoma, even within the thyroid gland, is associated with more aggressive disease at diagnosis and with a higher incidence of tumor recurrence.

Asunto(s)

Carcinoma Papilar/patología , Células Neoplásicas Circulantes , Neoplasias de la Tiroides/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Arterias/patología , Carcinoma Papilar/mortalidad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia/patología , Pronóstico , Tasa de Supervivencia , Glándula Tiroides/irrigación sanguínea , Neoplasias de la Tiroides/mortalidad

Asunto(s)

Pigmentación , Glándula Tiroides/patología , Adulto , Antibacterianos/efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Minociclina/efectos adversos , Pigmentación/efectos de los fármacos , Glándula Tiroides/efectos de los fármacos , Neoplasias de la Tiroides/patología

RESUMEN

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.

Asunto(s)

Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Mutación , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Glutamina/genética , Glutamina/fisiología , Modelos Moleculares , Organofosfatos/metabolismo , Conformación Proteica