Electrostatic properties of confluent Caco-2 cell layer correlates to their microvilli growth and determines underlying transcellular flow.

Recently, Rajapaksa et al. (2010) showed that the rate of uptake of potential vaccine delivery nanoparticles in the mucosal layer is a function of the electrostatic properties of the corresponding solvent. This fundamentally implies that the dominant driving forces that may be capitalized on for mucosal vaccine strategies are electrostatic in nature. We hypothesize that the driving force normal to the cell (in the direction from apical to basolateral across the cell) is of particular importance. In addition, it has been theoretically shown that the electrostatic properties of mucosal cells are directly related to their development of brush border. Here we correlate the development of brush border on a human mucosal epithelial model (Caco-2) cultured in DMEM on 3.0 µm pore sized polycarbonate membranes to their corresponding electrostatic properties characterized by measuring their normal zeta potential. Properties of normal streaming potential, hydraulic permeability, and brush border development (as determined by size and number) were monitored for 2, 6, and 16 days (after cells were confluent). Human endothelial cells (HECs), which lack brush border, were used as the control. Our results demonstrate that normal zeta potential of Caco-2 cells significantly changed from -5.7 ± 0.11 mV to -3.4 ± 0.11 mV for a period between 2 and 16 days, respectively. The zeta potential of the control cell line, HECs, stayed constant (statistically not different, P > 0.05) for the duration of the experiments. Our results show that the calculated increase in surface area of the Caco-2 cells with microvilli from 6 to 16 days was directly proportional to the corresponding measured zeta potential difference. These results imply that microvilli alter the electrostatic local environment around Caco-2 cells and, hence, enhance the normal electrostatic selective transport of solute across the mucosal barrier.

[Open and endovascular surgery for the treatment of abdominal aortic aneurysm. Review of the available evidence]. / Manejo del aneurisma de la aorta abdominal: estado actual, evidencias y perspectivas para el desarrollo de un programa nacional.

Open and endovascular surgery are therapeutic alternatives for the treatment of abdominal aortic aneurysm. The development of guidelines for its treatment requires a thorough analysis of available evidence to recommend the best treatment for each country's reality. Prospective randomized trials have shown best initial results with endovascular surgery, with higher hospital costs than open surgery. The requirement of anatomical suitability for the placement of endovascular prostheses limits the universal use of endovascular surgery. Moreover, this type of surgery needs a strict imaging and clinical follow up due to the high rates of late complications, which range from 20% to 40%. Many of these complications require further surgical interventions, elevating costs of treatment. The initial benefit of endovascular surgery is lost during long follow up as survival curves become similar to those of open surgery. Even for patients with a high surgical risk, the benefits of endovascular surgery are doubtful.

Filtros de vena cava inferior en posición suprarrenal / Suprarenal inferior vena cava filters: retrospective review of 30 cases

Background: Inferior vena cava (IVC) filters are used to prevent massive pulmonary embolism in cases where anticoagulation is contraindicated or has failed. It is usually implanted below the renal veins. In a few cases it is necessary to deploy the filter above them, with theoretical rísk of secondary renal failure. Aim: To report the experience with filters located above the renal veins. Patients and Methods: Medical records of all patients with percutaneous suprarenal filters are reviewed. Results: Between May 1993 and May 2007, 361 percutaneous IVC filter procedures were performed. In thirty patients aged 19 to 77 years (average 48years, 50 percent males), they were placed in suprarenalposition (8,3 percent). Suprarenal IVC filters were implanted in patients with extensive caval thrombosis, renal vein thrombosis extending to cava, displacement of previous IVC filters and double IVC system. Jugular vein approach was the access of choice. Technical success was 100 percent, no death or pulmonary embolism occurred. Patients were followed from 1 to 165 months (average 57 months). Eight deaths were recorded, five in patients with cáncer No patient had renal failure on follow up (average creatinine 0.90+0,26 mg/dL). Three patients developed a new deep vein thrombosis (10 percent), without pulmonary embolism. Conclusions: In this retrospective analysis of patients, suprarenal placement of IVC filters was not associated to secondary renal failure, and showed good short and long term results.

Tratamiento endovascular de la disección aórtica tipo B mediante endoprótesis / Endovascular treatment of type B aortic dissection

Background: Dissections that involve the ascending aorta are classified as type A, regardless of the site of the primary intimal tear, and all other dissections as type B. Type B dissections can have fatal ischemic and hemorrhagic complications. In the chronic state, dilatation and rupture can be mortal. Endovascular surgery is a therapeutic alternative, considering the high rate of complications of conventional surgery Aim: To report the results of endovascular treatment of type B aortic dissection. Material and methods: Report of 36 treated patients (30 males) aged 43 to 87 years, with a type B aortic dissection. Seventy eight percent were hypertensive and 39 percent smoked. The diagnosis was conñrmed by CAT sean. Acute patients were treated for complications and chronic patients, for dilatation. In the operating room, an endoprothesis was placed through the femoral artery, to cover the tear. The tear was located and the lumens were differentiated using angiography and transesophageal echocardiography. Results: All procedures were successful. In 16 acute dissections the indications were malperfusion syndrome or unmanageable hypertension in seven patients and imminent rupture or persistent pain in nine. Twenty chronic patients were operated due to dilatation (mean 6 cm). One patient died due to cardiac failure. One patient had a transient paraparesia and two had pulmonary embolism. No patient died in a follow up períod ranging from 2.5 to 74 months. Four patients required a new aortic endovascular procedure due to progressive dilatation or endoleak. Conclusión: Endovascular treatment of type B aortic dissection has good immediate andlong term results.

Tratamiento percutaneo de aneurismas aorto-ilíacos / Percutaneous treatment of aorto-iliac aneurysm

Introducción: La reparación endovascular de aneurismas abdominales e ilíacos requiere de la introducción de dispositivos de alto calibre (> 16 F) mediante denudación de arterias femorales. Mediante una variación técnica, el sistema de sutura arterial percutanea Prostar-XL® (Abbott, EEUU) permite el acceso arterial percutaneo evitando la denudación. Objetivo: Analizar la experiencia inicial en el tratamiento percutaneo de aneurismas del territorio aorto-ilíaco. Material y Método: Revisión de las historias clínicas y base de datos de pacientes tratados con sutura arterial percutanea, entre octubre de 2003 y abril de 2008. Resultados: Tratamos 22 pacientes con esta técnica (20 hombres y 2 mujeres). Dieciséis portadores de aneurisma aórtico abdominal, 3 aneurismas ilíacos, 2 reparaciones de endofuga y un aneurisma hipogástrico. La edad promedio fue 72,6 años (rango 56-86). Se utilizó el sistema Prostar XL® para sutura percutanea en 37 arterias femorales. La anestesia más utilizada fue peridural en el 50 por ciento de los pacientes. En 7 casos (31,8 por ciento) se efectuó la operación exclusivamente con anestesia local. El diámetro de los dispositivos de endoprótesis fue de 16 a 23 F. Se obtuvo éxito técnico en 34 cierres (92 por ciento). Tres arterias requirieron reparación quirúrgica tradicional. No hubo mortalidad operatoria. Durante el seguimiento (promedio 12,6 meses, rango 1-53) no se registraron falsos aneurismas femorales ni infección. Discusión: El cierre percutaneo en la reparación endovascular de aneurismas aorto-ilíacos es un procedimiento mínimamente invasivo, seguro y efectivo, que permite eventualmente el uso de anestesia local.

Introduction: Endovascular repair of aortic (AAA) and iliac artery aneurysms requires introduction and deployment of large bore devices (> 16 F) through surgical exposure of the femoral artery. The Prostar XL ® arterial suture system allows the introduction of such devices without the need for surgical exposure. Aim: To report our initial experience with percutaneous arterial closure during aneurysm endografting. Methods: We reviewed records and database of patients treated with this technique between October2003 and April 2008. Results: We treated 22 patients with this technique (20 men and 2 women, average age 72 years). Sixteen had AAA, 3 iliac artery aneurysm, 1 hypogastric aneurysm and two for endoleak repair. The percutaneous closure device was used in 37 femoral arteries. In 7 patients (31,8 percent) the operation was completed entirely under local anaesthesia. The diameter of the devices ranged between 16 and 23 F. Technical success was obtained in 34 arteries (92 percent). Three arteries required surgical repair due to inadequate haemostasis (sheaths 18, 21, and 21 F). There was no operative mortality. During follow-up (mean 12,6 months, range 1-53) no false aneurysm or infection at the puncture site has been registered and the patients remain free of complications. Discussion: Percutaneous arterial closure in endovascular aneurysm repair is a safe, minimally invasive and effective procedure which allows resolving theses serious conditions in selected patients.

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.

Evidence that histidine-163 is critical for catalytic activity, but not for substrate binding to Escherichia coli agmatinase.

Agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was inactivated by diethyl pyrocarbonate (DEPC) and illumination in the presence of Rose bengal. Protection against photoinactivation was afforded by the product putrescine, and the dissociation constant of the enzyme-protector complex (12 mM) was essentially equal to the K(i) value for this compound acting as a competitive inhibitor of agmatine hydrolysis. Upon mutation of His163 by phenylalanine, the agmatinase activity was reduced to 3-5% of wild-type activity, without any change in K(m) for agmatine or K(i) for putrescine inhibition. The mutant was insensitive to DEPC and dye-sensitized inactivations. We conclude that His163 plays an important role in the catalytic function of agmatinase, but it is not directly involved in substrate binding.

Manganese is essential for catalytic activity of Escherichia coli agmatinase.

Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.

Manganese-dependent inhibition of human liver arginase by borate.

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.

Decreased heterogeneity of CS histone variants after hydrolysis of the ADP-ribose moiety.

Sea urchin CS histone variants are electrophoretically heterogeneous when analyzed in two dimensional polyacrylamide gels (2D-PAGE). Previous results suggested that this heterogeneity is due to the poly (ADP-ribosylation) of these proteins. Consequently, native CS histone variants were subjected to different treatments to remove the ADP-ribose moiety. The incubation in 1 M hydroxylamine was not effective in eliminating the polymers of ADP-ribose from CS variants, and the treatment with sodium hydroxide was deleterious to the proteins. In contrast, the ADP-ribose moiety was successfully removed from the CS variants by incubation with phosphodiesterase (PDE). To eliminate contamination of CS histone variants with PDE extract, the enzyme was covalently bound to Sepharose 4B prior to its utilization. Treatment of native CS histone variants with this immobilized phosphodiesterase removed around 85% of the total ADP-ribose moiety from these proteins. After S-PDE treatment the complex electrophoretic pattern of CS histone variants in 2-D PAGE decreases to five major fractions. From these results we conclude that the electrophoretic heterogeneity of native CS histone variants is mainly due to the extent to which five main CS histone variants are poly(ADP)-ribosylated).

Interaction of arginase with metal ions: studies of the enzyme from human liver and comparison with other arginases.

As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.

An arginine residue at the active site of beta-lactamase from Shigella flexneri UCSF-129.

The role of a residue of arginine at the active site of beta-lactamase from Shigella flexneri UCSF-129 was studied. It is a local pathogenic strain which produces intestinal problems, especially in children. Purified enzymes were obtained by affinity chromatography on phenylboronic acid-agarose gels. The enzyme was serine dependent with a molecular weight of 23.6 kD. It was specifically modified with phenylglyoxal (1/830 molar ratio) and incubated for 20 min in the presence of 50 mM sodium phosphate buffer at pH 8.3. The chemical change was established by isoelectric focusing, since a loss of one positive charge was detected. Protection by cephradine, a substrate, indicated the presence of a residue of arginine at its active site. Controls were conducted by differential spectroscopy. Similar results were obtained with 2,3,butanedione. This vital arginine stabilizes the negative charge of the carboxylic group of C-3 or C-4 from the substrates.

Subcellular localization and kinetic properties of arginase from the liver of Genypterus maculatus.

1. From the liver of the teleost fish Genypterus maculatus, a partially purified preparation of arginase was obtained and characterized. 2. The Km value for arginine was found to be 9.1 mM at pH 7.5 and 11.5 mM at the optimum pH of 9.5. At both pH values, competitive inhibition was caused by ornithine and lysine, whereas proline, leucine, valine and isoleucine caused a non-competitive inhibitory effect. Branched chain amino acids were more inhibitory than proline. 3. The enzyme was found localized in the mitochondrial matrix of the liver of Genypterus maculatus. It is suggested that this localization would be of importance in the use of arginine as an energy source.

Kinetics of inhibition of rat liver and kidney arginases by proline and branched-chain amino acids.

The effects of proline, leucine, isoleucine and valine on kidney and liver arginases were studied. At pH 7.5 and at nearly physiological concentrations, the branched-chain amino acids caused a significant inhibition of liver arginase A1 and only minor effects on kidney arginase A4. Kidney arginase was, however, much more sensitive to inhibition by proline than the liver enzyme. The inhibition of liver and kidney arginases by branched-chain amino acids was partial, indicating the existence of allosteric sites on both enzymes. The function of kidney arginase in proline biosynthesis and a possible role of branched-chain amino acids in the hydrolysis of arginine in liver is discussed.

Comparative kinetic studies of Mn2+-activated and fructose-1,6-P-modified Mg2+-activated pyruvate kinase from Concholepas concholepas.

Initial velocity and product inhibition studies of Mn2+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzyme-PEP-ATP.

Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas.

In contrast to the Mg2+-activated enzyme, in the presence of Mn2+ pyruvate kinase exhibits hyperbolic kinetics with respect to the substrate phosphoenolpyruvate and is insensitive to fructose 1,6-biphosphate, phenylalanine and alanine. However, with both metal activated species inhibition by excess ADP is observed. In contrast with Mg2+, which affords significant protection against inactivation caused by 5,5'-dithiobis (2-nitrobenzoic acid), the rate of inactivation by this reagent is increased in the presence of Mn2+. Differences in conformational changes induced by combination of pyruvate kinase with Mg2+ or Mn2+ were indicated by u.v. difference spectra.

Hybrid, immobilised dimers of human liver arginase.

The activity of matrix-bound monomers of arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was not changed by incubation with p-hydroxymercuribenzoate. When the chemically modified, matrix-bound monomers were incubated with soluble subunits in the presence of Mn2+, dimers were obtained. These dimers were hybrids between modified and native monomers. The results obtained are in accord with a D2-symmetry where two dimers meet to form the tetrameric enzyme. From kinetic studies it is concluded that the structure of the active sites of arginase is not affected by the chemical modification with p-hydroxymercuribenzoate.

Negative cooperativity in human liver argininosuccinase.

The kinetic properties of argininosuccinase (L-argininosuccinate arginine-lyase, EC 4.3.2.1.) were investigated. Negative cooperativity was observed in the response of the enzyme to the substrate argininosuccinate and GTP behaved as a positive allosteric effector. These effects were observed in 60 mM potassium phosphate but not in 50 mM Tris-HCl. Structural changes in the protein molecule are suggested to explain previous observations of Michaelis-Menten kinetics for this enzyme.

Evidence for cooperative effects in human liver arginase.

The reaction kinetics of human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) in terms of arginine concentration is strikingly altered by varying the pH. Lowering the pH from the optimum (9.5) toward a more physiological value (7.5) there is a transition from hyperbolic to sigmoidal kinetics. The cooperative effects are observed in the presence and absence of the product ornithine. Dimers of arginase exhibit typical Michaelis-Menten kinetics even in the presence of ornithine. Dimer-dimer interactions are suggested to explain the kinetic properties of arginase at pH 7.5.