Objective pain assessment after ureteral reimplantation: comparison of open versus robotic approach.

INTRODUCTION: While open ureteral reimplantation is the gold standard of surgical intervention for vesicoureteral reflux (VUR), minimally invasive approaches offer the potential benefits of decreased postoperative pain, improved cosmesis, and shorter hospital stay and convalescence. Studies comparing open and minimally invasive surgery with respect to postoperative pain in children have been inconclusive. OBJECTIVE: We sought to compare postoperative pain in children undergoing open versus robotic ureteral reimplantation by using age-appropriate, validated pain assessment scales. METHODS: A prospective cohort of all patients enrolled in an Institutional Review Board-approved VUR surgery registry between July 2010 and February 2013 was analyzed. Patients who underwent endoscopic treatment or who received caudal or epidural anesthesia were excluded. Age-appropriate, validated pain scales ranging from 0 to 10 were utilized for pain assessment. Pain scores and narcotic doses administered on the first postoperative day were analyzed. RESULTS: Of the 34 subjects included, 11 underwent open intravesical reimplantation, while 23 patients underwent robotic extravesical reimplantation. Table 1 displays patient characteristics and results of pain assessment. Robotic surgery was associated with lower narcotic requirement compared to open surgery (P < 0.05). The difference in pain scores between the two cohorts approached, but did not reach, statistical significance (P = 0.12). However, the percentage of patients with mild or no pain (57% robotic, 27% open) versus severe pain (9% robotic, 45% open) was notably different between the two cohorts. DISCUSSION: Previous studies addressing the effect of surgical modality on pediatric postoperative pain are limited by their reliance on narcotic administration as an indirect surrogate for measuring pain. In the present study, postoperative pain was assessed with narcotic requirements and consistently collected validated pain scores, which more accurately reflect a patient's perceived pain. Although there was no significant difference in subjective pain scores between patients undergoing open versus robotic reimplantation, the percentage of patients with mild or no pain (57% robotic, 27% open) versus severe pain (9% robotic, 45% open) was notably different between the two cohorts. This study was limited by a lack of randomization as well as small sample size, which did not allow for age sub-group analysis or small differences to be statistically significant. CONCLUSIONS: In the present study, robotic ureteral reimplantation was associated with lower narcotic requirement compared to open surgery, and lower intensity of postoperative pain according to a direct pain assessment tool. Larger sample sizes are necessary to strengthen statistical comparisons.

Application of a static fluorescence-based cytometer: the CellScan in clinical immunology.

The CellScan system is a laser scanning cytometer which enables repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. By monitoring FP changes in peripheral blood lymphocytes (PBL) following exposure to antigenic stimuli, the CellScan may have a role in the diagnosis of autoimmune diseases. Monitoring changes in FI and FP in PBLs from patients with atherosclerosis following exposure to various stimuli, has illustrated the role of the immune system in the atherosclerotic process. The CellScan has also been evaluated as a diagnostic tool for drug-induced allergy, based on FP reduction in PBLs following incubation with the suspected drugs. FI and FP changes in cancer cells have been found to correlate with the cytotoxic effect of different anti-neoplastic drugs, illustrating the potential role of the CellScan system in clinical oncology. In conclusion, the CellScan is a promising new tool with a variety of applications in cell biology, immunology, cancer research and clinical pharmacology.

Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II.

Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.

Specific chemical and structural damage to proteins produced by synchrotron radiation.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target.

BACKGROUND: Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. RESULTS: The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 A resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (approximately 2000 A2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. CONCLUSIONS: Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin.

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.

The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation.

Lim and Spirin [25] proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation. Spirin and Lim [26] excluded the possibilities of the side-by-side model proposed by Johnson et al [13] and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus [3]. However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge. With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al [19]. In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA. The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation. The A76 of A site tRNA overlaps the binding region of puromycin. The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA [14] and 17 A from the 5' end of the anticodon of A site tRNA [15]. It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally [6]. A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model.

Gamma-chymotrypsin is a complex of alpha-chymotrypsin with its own autolysis products.

The determination of three separate gamma-chymotrypsin structures at different temperatures and resolutions confirmed the presence of electron density in the active site, which could be interpreted as an oligopeptide as had previously been suggested by Dixon and Matthews [(1989) Biochemistry 28, 7033-7038]. HPLC analyses of the enzyme before and after crystallization demonstrated the presence of a wide variety of oligopeptides in the redissolved crystal, most with COOH-terminal aromatic residues, as expected of the products of chymotrypsin cleavage, which appeared to arise from extensive autolysis of the enzyme under the crystallization conditions. The refined structures agree well with the conformation of both gamma-chymotrypsin and alpha-chymotrypsin. The electron density in the active site is thus interpreted as arising from a repertoire of autolysed oligopeptides produced concomitantly with crystallization. The COOH-terminal carbons of the polypeptide(s) display short contact distances (1.97, 2.47, and 2.13 A, respectively) to Ser195 O gamma in all three refined structures, but the electron density is not continuous between these two atoms in any of them. This suggests that some sequences are covalently bound as enzyme intermediates while others are noncovalently bound as enzyme-product complexes.

Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C.

A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2.0 A (1 A = 0.1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163.4(+/- 0.2) A, b = 112.1(+/- 0.2) A, c = 81.3(+/- 0.1) A.

Prediction of three-dimensional structure of Escherichia coli ribosomal RNA.

A model for the tertiary structure of 23S, 16S and 5S ribosomal RNA molecules interacting with three tRNA molecules is presented using the secondary structure models common to E. coli, Z. mays chloroplast, and mammalian mitochondria. This ribosomal RNA model is represented by phosphorus atoms which are separated by 5.9 A in the standard A-form double helix conformation. The accumulated proximity data summarized in Table 1 were used to deduce the most reasonable assembly of helices separated from each other by at least 6.2 A. Straight-line approximation for single strands was adopted to describe the maximum allowed distance between helices. The model of a ribosome binding three tRNA molecules by Nierhaus (1984), the stereochemical model of codon-anticodon interaction by Sundaralingam et al. (1975) and the ribosomal transpeptidation model, forming an alpha-helical nascent polypeptide, by Lim & Spirin (1986), were incorporated in this model. The distribution of chemically modified nucleotides, cross-linked sites, invariant and missing regions in mammalian mitochondrial rRNAs are indicated on the model.