RESUMEN
OBJECTIVE: To evaluate the efficacy of combined infiltrative bupivacaine with low intraperitoneal pressure insufflation in reducing the post-laparoscopic pain in patients undergoing laparoscopic cholecystectomy (LC). METHODS: This randomized prospective single-blind study included 473 patients undergoing LC. The study took place at University Hospital Center Mother Teresa, Tirana, Albania between January 2006 to September 2009. The patients were divided in 4 groups: Group 1 (n=120) with intra-abdominal insufflation pressure 15 mm Hg and no infiltrative bupivacaine (HPNBG); Group 2 (n=122) with intra-abdominal insufflation pressure 15 mm Hg and with 5 ml infiltrative bupivacaine 0.5% in abdominal minincisions (HPBG); Group 3 (n=110) with intra-abdominal insufflation pressure under 10 mm Hg and no infiltrative bupivacaine (LPNBG); and Group 4 (n=121) with intra-abdominal insufflation pressure under 10 mm Hg and infiltrative bupivacaine (LPBG). RESULTS: There were statistically significant differences (p=0.003) between groups regarding incisional pain intensity, between LPBG and HPNBG (p=0.001), between LPBG and HPBG (p=0.037), between LPBG and LPNBG (p=0.001), as well the shoulder-tip pain intensity (p=0.001); between LPBG and HPNBG (p=0.001), between LPBG and HPBG (p=0.001), and between LPBG and LPNBG (p=0.031). We found statistically significant differences related to pain beginning time (ANOVA test, p=0.027); between LPBG and HPNBG (p=0.041), between LPBG and HPBG (p=0.031), and between LPBG and LPNBG (p=0.05). CONCLUSION: The combination of infiltrative bupivacaine with low intraperitoneal pressure insufflation shows to be more efficient in reducing the post-laparoscopic pain, compared with other regimens.
Asunto(s)
Anestésicos Locales/uso terapéutico , Bupivacaína/uso terapéutico , Colecistectomía Laparoscópica/métodos , Insuflación/métodos , Dolor Postoperatorio/prevención & control , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Presión , Estudios Prospectivos , Dolor de Hombro/prevención & control , Método Simple CiegoRESUMEN
Hydrophobins are a class of small proteins that fulfill a wide spectrum of functions in fungal growth and development. They do so by self-assembling into an amphipathic membrane at hydrophilic-hydrophobic interfaces. The SC3 hydrophobin of Schizophyllum commune is the best-studied hydrophobin. It assembles at the air-water interface into a membrane consisting of functional amyloid fibrils that are called rodlets. Here we examine the dynamics of SC3 assembly at an oil-water and air-water interface and the permeability characteristics of the assembled layer. Hydrophobin assembled at an oil-water interface is a dynamic system capable of emulsifying oil. It accepts soluble-state SC3 oligomers from water in a unidirectional process and sloughs off SC3 vesicles back into the water phase enclosing a portion of the oil phase in their hydrophobic interior. The assembled layer is impermeable to solutes >200 Da from either the water phase or the oil phase; however, due to the emulsification process, oil and the hydrophobic marker molecules in the oil phase can be transferred into the water phase, thus giving the impression that the assembled layer is permeable to the marker molecules. By contrast, the layer assembled at an air-water interface is permeable to water vapor from either the hydrophobic or hydrophilic side.
Asunto(s)
Biofisica/métodos , Proteínas Fúngicas/química , Membranas/química , Tiazoles/química , Aire , Péptidos beta-Amiloides/química , Benzotiazoles , Proteínas de la Membrana/química , Membranas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Octoxinol/farmacología , Aceites/química , Parafina , Permeabilidad , Conformación Proteica , Estructura Secundaria de Proteína , Schizophyllum/metabolismo , Factores de Tiempo , Agua/químicaRESUMEN
The physiochemical nature of surfaces can be changed by small proteins which are secreted by filamentous fungi. These proteins, called hydrophobins, are characterized by the presence of eight conserved cysteine residues and a typical hydropathy pattern. Upon contact with a hydrophilic-hydrophobic interface they self-assemble into highly insoluble amphipathic membranes. As a result, hydrophobic surfaces become hydrophilic and vice versa. Genetic engineering of hydrophobins was used to study structure-function relationships. In addition, engineered hydrophobins were constructed to increase the biocompatibility of surfaces. The glycosylated N-terminal region of the mature SC3 hydrophobin was deleted and the cell-binding domain of human fibronectin was introduced at the N-terminus. The gross properties of the hydrophobins were not affected. However, the physiochemical properties of the hydrophilic side of the assembled protein did change. Growth of fibroblasts on Teflon could be improved by coating the solid with the engineered hydrophobins. Thus, by changing the N-terminal part of hydrophobins, the physiochemical nature of the hydrophilic side of the assembled form can be altered and a variety of new functionalities introduced. The fact that hydrophobins self-assemble at any hydrophilic-hydrophobic interface, irrespective of the chemical nature of the surface, therefore provides a generic approach to modify surfaces and make them interesting candidates for the use in various technical and medical applications.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Fibroblastos/efectos de los fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Ingeniería de Proteínas/métodos , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Proteínas Fúngicas/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/farmacología , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Propiedades de SuperficieRESUMEN
We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.
Asunto(s)
Técnicas Genéticas , Recombinación Genética , Alelos , Secuencia de Aminoácidos , Intercambio Genético , ADN Complementario/metabolismo , Biblioteca de Genes , Datos de Secuencia Molecular , Mutagénesis , Mutación , Nocardia/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Rhodococcus/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50,000-Mr activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Bacillus/enzimología , Cinética , Estructura Molecular , Peso Molecular , NAD/química , Oxidación-Reducción , Conformación Proteica , EspectrofotometríaRESUMEN
The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.