Bloqueo retrobulbar guiado por ultrasonido: ¿es posible? / Ultrasound-guided retrobulbar blockade

Introducción: La anestesia retrobulbar proporciona excelentes condiciones para la cirugía de cataratas, sin embargo conlleva riesgo de grave daño sobre estructuras nobles. La siguiente serie de casos tiene por objetivo describir el uso de ultrasonografía como guía imagenológica en el bloqueo retrobulbar, para facoeresis e implante de lente intraocular. Método: Se llevó a cabo una serie de 4 casos, en que se utilizó ultrasonido con un transductor lineal de alta frecuencia, para mapeo e inserción de aguja de bloqueo retrobulbar bajo visión ecográfica en tiempo real. Resultados: Se obtuvo visión de la aguja y posicionamiento de ella en el cono muscular retrobulbar e infi ltración con anestésico local en los cuatro pacientes, logrando anestesia total en todos los casos, aquinesia total en tres y parcial en uno. No hubocomplicaciones atribuibles al procedimiento. Conclusión: Creemos que la ultrasonografía no es sólo factible, sino que podría ser un verdadero aporte en la seguridad de la anestesia ocular.

Background: Retrobulbar anesthesia provides excellent surgical conditions for cataract surgery. However, it conveys the risk of serious damage to fragile structures. The aim of this study is to describe the use of ultrasonography as a guide for retrobulbar blockade in surgical cataract patients. Methods: A series offour cases was selected and a high frequency linear ultrasound transducer was used for initial scanning andperforming of a real-time ultrasound-guided retrobulbar blockade. Results: Ultrasound images were used for needle positioning in the muscular cone of the eye and local anesthetic infiltration was performed in four patients. Complete anesthesia was achieved in all cases and complete akinesia was obtained in three, with partial akinesia in the remaining case. There were no complications in this series. Conclusion: We believe that ultrasound guided retrobulbar blockade is a feasible technique and it could be a real improvement on the safety of ophthalmic anesthesia.

Role of protein-solvent interactions in refolding: effects of cosolvent additives on the renaturation of porcine pancreatic elastase at various pHs.

The effects of cosolvent additives on the refolding of porcine pancreatic elastase were studied by comparing the enzymatic activity and the conformation of the enzyme renatured at various pHs with those of the native elastase under the same cosolvent and pH conditions. A lag period was observed before reaching the steady state of the hydrolysis of an amide substrate, and the lag period measured with the refolding enzyme was longer than that measured with the native elastase. Depending on the cosolvent studied (acetonitrile, dimethylsulfoxide, glycerol, methanol) there was or was not a dramatic increase in the duration of the lag period measured with the refolding enzyme, but not in the case of the native elastase. These results and additional kinetic data on inactivation of the enzyme demonstrated that dimethylsulfoxide, glycerol, and methanol enhance the stability of the intermediates able to refold into the native form, contrary to acetonitrile. In neither the case of the native enzyme nor that of the renatured enzyme, did the cosolvents modify the pK(app) of ionization of the amino acids that control enzymatic activity, indicating that they did not penetrate the core of the refolded elastase. Conversely, they shifted toward a more alkaline pH the structural transition of the native elastase, and the amplitude of the shift was comparable to that observed in bulk water with elastase whose Ser 195 has been acylated, suggesting that cosolvents stabilized the structure of the folded molecule by increasing its packing.

Catalytic properties of the two active sites of angiotensin I-converting enzyme on the cell surface.

Angiotensin I converting enzyme is a zinc metallopeptidase that contains two very similar domains, each with an active site. Enzymatic studies of these active sites have always been performed on solubilized enzyme, although angiotensin I converting enzyme is a transmembrane ectopeptidase. The availability of transfected CHO cells expressing wild-type recombinant enzyme and mutants in which one of the two active sites has been inactivated by site-directed mutagenesis allowed the properties of each active site on the cell surface and the effect of anchorage and membrane environment to be studied. Both active centers are catalytically active in the cell membrane-anchored enzyme and convert angiotensin I to angiotensin II. Comparison of the kinetic parameters for the transfected cells with those for the purified enzymes reveals differences in Kcat but suggests that no major conformational changes of these active sites occur upon anchorage of the enzyme to the cell membrane. The chloride activation profiles show that the two domains in the cell-bound enzyme also undergo the same anion-induced conformational changes as in the solubilized enzyme.

Structure-function analysis of angiotensin I-converting enzyme using monoclonal antibodies. Selective inhibition of the amino-terminal active site.

Angiotensin I-converting enzyme (ACE; kininase II) contains two very similar domains (the NH2- and COOH-terminal domains (N and C domains, respectively)), each bearing an active site. These active sites hydrolyze the same peptides, but do not have the same catalytic properties and substrate specificities. In an attempt to develop domain-specific immunological probes, two series of monoclonal antibodies (mAbs), 19 clones in all, were produced and tested against human ACE. These mAbs recognized at least nine different epitopes within three antigenic regions of the ACE molecule. Testing on wild-type recombinant ACE and several mutants with only one intact domain showed that these epitopes were all located in the N domain. None of the mAbs recognized the C domain. This particular specificity and analysis of results obtained with several polyclonal antibodies to human ACE suggest that ACE immunogenicity is determined mainly by the N domain. Two mAbs (3A5 and i2H5) recognizing epitopes from different antigenic regions of ACE inhibited the enzymatic activity of the N (but not of the C) domain. mAb 3A5 had the same inhibitory potency toward hippuryl-His-Leu, benzyloxycarbonyl-Phe-His-Leu, and angiotensin I hydrolysis, with 50% inhibition achieved at a mAb/ACE molar ratio of 6. mAb i2H5 was roughly three times more effective than mAb 3A5 inhibiting the hydrolysis of benzyloxycarbonyl-Phe-His-Leu and the natural substrates angiotensin I and bradykinin (50% inhibition at a molar ratio of 1-2), but was less effective in inhibiting hippuryl-His-Leu cleavage (50% inhibition at a molar ratio of 22-25), indicating that this substrate interacts with a specific subsite. mAb i2H5 almost completely inhibited the hydrolysis of the luteinizing hormone-releasing hormone by the isolated N domain. Both the primary carboxyl- and amino-terminal cleavages of this peptide were suppressed. This antibody suppressed the primary amino-terminal cleavage of the luteinizing hormone-releasing hormone by wild-type ACE by > 90%, indicating that this particular ACE function is mediated mainly by the N domain active site. These data provide evidence for structural differences between the two homologous domains of ACE despite their high degree of sequence homology and show that monoclonal antibodies are able to distinguish between the two active sites in ACE.

Differences in the properties and enzymatic specificities of the two active sites of angiotensin I-converting enzyme (kininase II). Studies with bradykinin and other natural peptides.

Angiotensin I-converting enzyme (ACE, E.C.3.4.15.1) has been recently shown to contain two very similar domains, each of which bears a functional active site hydrolyzing Hip-His-Leu or angiotensin I (AI). The substrate specificity of the two active sites of ACE was compared using wild-type recombinant ACE and mutants, where one active site is suppressed by deletion or inactivated by mutations of 2 histidines coordinating an essential zinc atom. Both active sites converted bradykinin (BK) to BK1-7 and BK1-5 with similar kinetics and with Kappm at least 30 times lower and kcat/kappm 10 times higher than for AI. The carboxyl-terminal active site, but not the amino-terminal site, was activated by chloride; however, chloride activation was minimal compared with AI. Both domains also hydrolyzed substance P and cleaved a carboxyl-terminal protected dipeptide and tripeptide. The carboxyl-terminal active site was more readily activated by chloride and hydrolyzed substance P faster. Luteinizing-hormone releasing hormone was hydrolyzed by both active sites, but hydrolysis by the amino-terminal active site was faster. It performed the endoproteolytic amino-terminal cleavage of this peptide at least 30 times faster than the carboxyl-terminal active site. Both active sites cleaved a carboxyl-terminal tripeptide from luteinizing hormone-releasing hormone. Thus, both active sites of ACE possess dipeptidyl carboxypeptidase and endopeptidase activities. However, only the carboxyl-terminal active site can undergo a chloride-induced alteration that greatly enhances the hydrolysis of AI or substance P, and the amino-terminal active site possesses an unusual amino-terminal endoproteolytic specificity for a natural peptide. This suggests physiologically important differences between the subsites of the two active centers, and different substrate specificity, despite the high degree of sequence homology.

The angiotensin I-converting enzyme (kininase II): molecular organization and regulation of its expression in humans.

Protein sequencing and molecular cloning of human endothelial angiotensin I-converting enzyme (ACE; kininase II), have led to a description of the structure of the enzyme and to several questions concerning the intracellular maturation of ACE and the mechanisms of enzyme action. With the help of recombinant ACE expression in mammalian cells and site-directed mutagenesis, a model for the maturation of ACE in endothelial cells has been proposed. This model comprises transmembrane anchoring of the membrane-bound ACE near its carboxyterminal extremity, and post-translational cleavage of the anchor in the secreted form. The endothelial ACE displays a high degree of internal homology between two large peptidic domains that each bears a consensus sequence for zinc binding and therefore a putative active site. The testicular ACE, however, encoded from the same gene by a shorter mRNA, contains only the carboxyterminal half of endothelial ACE and therefore a single active site. Expression of ACE mutants with only one intact homologous domain, however, indicates that in endothelial ACE both domains are enzymatically active. Further characterization of these two active sites of endothelial ACE is in progress. In humans, population studies have indicated that the large interindividual variability in plasma ACE levels is partly genetically determined and under the influence of a major gene effect. This was later confirmed and extended by the observation of an insertion-deletion polymorphism of the ACE gene that is associated with the level of ACE in plasma. The clinical implications of these observations are discussed.

[Angiotensin converting enzyme (kininase II). Molecular and physiological aspects]. / L'enzyme de conversion de l'angiotensine (kininase II). Aspects moléculaires et physiologiques.

The angiotensin I-converting enzyme (kininase II, ECA) is a membrane bound enzyme anchored to the cell membrane through a single transmembrane domain located near its carboxyterminal extremity. Secretion of ACE by the cell occurs most likely as a result of a posttranslational cleavage of the membrane anchor and intracellular region. The ACE molecule is organized into two large highly homologous domains, each bearing consensus sequences for zinc binding in metallopeptidases. Site directed mutagenesis allowed to establish that both domains bear in fact a functional active site, able to convert angiotensin I into angiotensin II and to hydrolyze bradykinin or substance P. The two active sites of ACE, however, do not display the same sensitivity to anion activation (the C terminal active site being more chloride activatable) and also differs in kinetic parameters for peptide hydrolysis. The C terminal active site can hydrolyze faster angiotensin I and substance P and the N terminal active site is able to perform a peculiar endoproteolytic cleavage of an in vitro substrate of ACE, the luteinizing hormone releasing hormone. Both active sites bind with a high affinity, competitive inhibitors but the Kd of the reaction can vary up to 10 between the two active sites. All together, these observations suggest that ACE contains two active sites, whose structure is not exactly identical. They may have a different substrate specificity, however this remains speculative at the present time. Concerning the regulation of ACE gene expression in man, population studies indicated that the large interindividual variability in plasma ACE levels is genetically determined. An insertion/deletion polymorphism located in an intron of ACE gene is associated with differences in the level of ACE in plasma and cells. The physiological and clinical implications of these observations is discussed.