[Minimally invasive management of Choledochal Cyst in pediatric age]. / Manejo mínimamente invasivo de Quiste de Colédoco en edad pediátrica.

INTRODUCTION: The choledochal cyst (also bile duct cyst) is a rare condition. It is important to know its clinical presentation, diagnosis, and treatment alternatives, which allow a resolution with low morbidity. OBJECTIVE: to report the clinical diagnosis together with the laparoscopic techniques for the mana gement of the bile duct cyst. CLINICAL CASES: Case 1: 4-year-old preschooler with history of recurrent abdominal pain. Abdominal ultrasound showed a choledochal cyst. Blood amylase levels 111 IU / L. Other tests were normal. Case 2: 5-year-old preschooler with a 5-days history of abdominal pain, vomiting, and diarrhea. He was admitted due to acute pancreatitis (blood lipase 947 IU / L, blood amylase 217 IU / L). Abdominal CT scan reported a lobulated cystic lesion in the hilum of the liver. Case 3: 3-year-old preschooler with recurrent abdominal pain and a 3-day history of epigastric pain and vomiting. Blood amylase and lipase levels were 248 IU / L and 253 IU / L, respectively, diagnosing acute pancreatitis. Abdominal CT scan showed a finding suggestive of a common bile duct cyst. In all 3 cases, the magnetic resonance cholangiopancreatography reported a type I choledochal cyst. All pa tients underwent laparoscopic surgery, performing cyst resection, and hepaticoduodenostomy. One case presented pneumobilia without requiring specific management, the other two did not present incidents and all remain asymptomatic in the follow-up period that was longer than one year after surgery. CONCLUSIONS: In the choledochal cyst, clinical suspicion and timely diagnosis with imaging studies and minimally invasive surgery are important, which allow optimal results in the medium- and long term.

Mucormicosis Rinocerebral asociado a Cetoacidosis diabética / Rhinocerebral Mucormycosis associated to diabetic ketoacidosis

Mucormycosis is an infrequent fungal infection This infection is difficult to diagnose and treat and have a high morbility and mortality and affects immunocompromised patients, especially those patients with decompensated diabetes mellitus. We report the case of a 60 years old diabetic patient with poor metabolic control who was admitted for diabetic ketoacidosis and days later present right periorbital swelling and pain, is diagnosed of mucomycosis and is successfully treated with amphoterin B and surgery.

Protein models in drug discovery.

Over the last few years, the utilization of protein structural information in drug discovery research has matured and is today applied throughout the process, ranging from genomics-derived target identification and selection to the final design of suitable drug candidates. An especially powerful methodology has arisen from the clear synergies of the combination of target structural information with combinatorial chemistry. Several structural genomics initiatives have recently been started and are now generating 3-D structures of target molecules at an unprecedented rate that will provide a wealth of novel information that can be utilized for rational drug design.

Characterization of neutralizing sites in the second variable and fourth variable region in gp125 and a conserved region in gp36 of human immunodeficiency virus type 2.

Several determinants of human immunodeficiency virus (HIV) have been suggested to harbor sites important for neutralization. The third variable region (V3) of the envelope glycoprotein (gp) is an important neutralizing determinant for both serotypes of HIV. The localization of additional neutralizing regions is an urgent task because the virus appears to mutate to phenotypes that escape neutralizing antibodies. Therefore, we have focused on the possibility of finding other immunodominant regions in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2). By immunization of guinea pigs with peptides corresponding to different selected regions of gp125 and gp36, we have found three antigenic determinants located in the V2 and V4 regions of the envelope protein gp125, and one region in the glycoprotein gp36, which are important for human antibody binding and also as targets for neutralization. The peptide representing the V2 region had the most pronounced capacity to induce neutralizing anti-HIV-2 antibodies in guinea pigs. Neutralizing activity was also detected in an antipeptide guinea pig sera representing a linear site in gp36, amino acids 644-658. A substitution set of peptides representing the conserved antigenic site in the central part of gp36 was used to identify the role of individual amino acids important for human antibody binding.

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD.

Protein engineering of the cholera toxin A1 subunit (CTA1) fused to a dimer of the Ig-binding D-region of Staphylococcus aureus protein A (DD) was employed to investigate the effect of specific amino acid changes on solubility, stability, enzymatic activity and capacity to act as an adjuvant in vivo. A series of CTA1-DD analogues were selected by a rational modeling approach, in which surface-exposed hydrophobic amino acids of CTA1 were exchanged for hydrophilic counterparts modeled for best structural fit. Of six different mutants initially produced, two analogues, CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated to have 50 and 70% increased solubility, respectively, at neutral pH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at least threefold more soluble, demonstrating an additive effect of the two mutations. Only the Phe132Ser analogue retained full biological activity and stability compared with the native CTA1-DD fusion protein. Two mutants, Pro185Gln and Phe31His mutations, exhibited unaltered ADP-ribosyltransferase activity in vitro, but demonstrated markedly reduced adjuvant function. Since the Pro185 and Phe31 amino acids are located in close vicinity on the distal side of the molecule relative to the enzymatically active cleft, it is conceivable that this region is involved in mediating a biological function, separate from the enzymatic activity but intrinsic to the adjuvant activity of CTA1.

Fine characterization of a V3-region neutralizing epitope in human immunodeficiency virus type 2.

We have previously identified two distinct antigenic sites in the third variable region (V3) of human immunodeficiency virus type 2 (HIV-2) corresponding to the principal neutralizing determinant (PND) of HIV-1, the conserved Phe-His-Ser-Gln and Trp-Cys-Arg motifs (positions 315-318 and 329-331), which possibly interact to form a discontinuous antigenic site. The aim of this study was to further identify and characterize the immunogenic sites in the V3-loop of HIV-2 that are important in the binding of neutralizing antibodies and to study in detail the importance of different configurations of peptides corresponding to this region. Peptides representing modifications of the V3-region of HIV-2(SBL6669-ISY) were used for immunization of guinea pigs. With one exception, both the Phe-His-Ser-Gln and the Trp-Cys-Arg motifs were required in the peptide sequences to obtain neutralizing hyperimmune guinea pig sera, and the highest titers were obtained after immunization with 20-27 amino acids (aa) long peptides. Neither substitutions nor deletions of residues between the two motifs, nor the addition of peptide sequences representing a T-helper epitope improved the induction of neutralizing antibodies. Computer simulation modeling revealed that the Phe-315, His-316, Trp-329 and Cys-330 are likely to participate in the formation of a discontinuous epitope. Taken together, these data support the hypothesis that the well conserved motifs FHSQ (positions 315-318) and WCR (positions 329-331) of the HIV-2(SBL6669) V3 region are important targets for neutralizing antibodies, and this may have implications for the design of a future HIV-2 vaccine.

Identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in Escherichia coli.

The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli. The product is a putative agent for immunotherapy of non-small cell lung cancer. During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/liter. This level was notably low compared with similar products containing identical CH1, CL, and superantigen moieties, and the Fv framework was therefore engineered. Using hybrid molecules, the light chain was found to limit high expression levels. Substituting five residues in VL increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 --> Ser, Thr-45 --> Lys, Thr-77 --> Ser, and Leu-78 --> Val were most powerful. In addition, replacing four VH residues diminished cell lysis during fermentation. Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter. All engineered products retained a high affinity for the tumor-associated antigen. It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E. coli.

Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols.

Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.

Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa.

The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme. Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.

Molecular dynamics simulations of an enzyme surrounded by vacuum, water, or a hydrophobic solvent.

We report on molecular dynamics simulations of a medium-sized protein, a lipase from Rhizomucor miehei, in vacuum, in water, and in a nonpolar solvent, methyl hexanoate. Depending on force field and solvent, the molecular dynamics structures obtained as averages over 150 ps had root-mean-square deviations in the range of 1.9 to 3.6 A from the crystal structure. The largest differences between the structures were in hydrogen bonding and exposed surface areas of the protein. The surface area increased in both solvents and became smaller in vacuum. The change of surface exposure varied greatly between different residues and occurred in accordance with the hydrophobicity of the residue and the nature of the solvent. The fluctuations of the atoms were largest in the external loops and agreed well with crystallographic temperature factors. Root-mean-square fluctuations were significantly smaller in the nonpolar solvents than they were in water, which is in accordance with the notion that proteins become more rigid in nonpolar solvents. In methyl hexanoate a partial opening of the lid covering the active site occurred, letting a methyl hexanoate molecule approach the active site.

Theoretical studies of Rhizomucor miehei lipase activation.

Computational methods have been used to study the extensive conformational change of Rhizomucor miehei lipase upon activation. The present study considers the possible activation route, the energies involved and molecular interactions during the conformational change of the lipase in a hydrophobic environment. The conformational change was studied by conventional molecular dynamics methods and with a combined molecular dynamics and mechanics protocol, in which the conformational change was simulated by restraining C alpha pseudotorsional angles in small steps between the two crystallographically observed positions of the lid. In the closed conformer of the enzyme the active site is completely buried under a short helical loop, 'the lid'. The activation of the lipase consists of a movement of the lid, which results in an open conformer with an exposed active site. From the results of the simulations in the present work we suggest that the lipase in a hydrophobic environment is stabilized in the open form by electrostatic interactions.

The role of arginines in stabilizing the active open-lid conformation of Rhizomucor miehei lipase.

Molecular dynamics simulations for the lid covering the active site of Rhizomucor miehei lipase [EC 3.1.1.3] postulated that, among other interactions, Arg86 in the lid stabilized the open-lid conformation of the protein by multiple hydrogen bonding to the protein surface. Chemical modification of arginine residues in R. miehei lipase with 1,2-cyclohexanedione or phenylglyoxal resulted in residual activities in the hydrolysis of tributyrin of 66 and 46%, respectively. Tryptic maps of native and phenylglyoxal-reacted R. miehei lipase showed that Arg86 was the residue modified most, when the lipase was inhibited to the greatest extent. Guanidine, a structural analog to an arginine side chain, inhibited both the native enzyme and the arginine-modified enzymes, resulting in residual activities of 26% as compared to the native enzyme. The inhibition was not an effect of enzyme denaturation. The native enzyme was also inhibited by 1-ethylguanidine, benzamidine and urea, but to a lesser degree than by guanidine. Lipases from Humicola lanuginosa and porcine pancreas in 100 mM guanidine showed residual activities of 88 and 70%, respectively. The lipases from Candida antarctica, C. rugosa, Pseudomonas cepacia and P. fluorescens were not inhibited by guanidine. The inhibition of R. miehei lipase by structural analogs of the arginine side chain and after chemical modification of arginine residues suggest a role of an arginine residue in stabilizing the active open-lid conformation of the enzyme.