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KirBac1.1 and 3.1 are bacterial homologues of mammalian inward rectifier K channels. We have performed extended molecular dynamics simulations (five simulations, each of >20 ns duration) of the transmembrane domain of KirBac in two membrane environments, a palmitoyl oleoyl phosphatidylcholine bilayer and an octane slab. Analysis of these simulations has focused on the conformational dynamics of the pore-lining M2 helices, which form the cytoplasmic hydrophobic gate of the channel. Principal components analysis reveals bending of M2, with a molecular hinge at the conserved glycine (Gly134 in KirBac1.1, Gly120 in KirBac3.1). More detailed analysis reveals a dimer-of-dimers type motion. The first two eigenvectors describing the motions of M2 correspond to helix kink and swivel motions. The conformational flexibility of M2 seen in these simulations correlates with differences in M2 conformation between that seen in the X-ray structures of closed channels (KcsA and KirBac) in which the helix is undistorted, and in open channels (e.g. MthK) in which the M2 helix is kinked. Thus, the simulations, albeit on a time scale substantially shorter than that required for channel gating, suggest a gating model in which the intrinsic flexibility of M2 about a molecular hinge is coupled to conformational transitions of an intracellular 'gatekeeper' domain, the latter changing conformation in response to ligand binding.

The 37/67-kDa human laminin receptor (LamR) is a cell surface protein that interacts with molecules located in the extra-cellular matrix. In particular, interactions between LamR and laminins play a major role in mediating changes in the cellular environment that affect cell adhesion, neurite outgrowth, tumor growth and metastasis. The exact interaction mode of laminin-1 and LamR is not fully understood. Laminin-1 is thought to bind to LamR through interaction with the so-called peptide G (residues 161–180) and the C-terminal helix (residues 205–229). Here we performed 100-ns atomistic force field-based molecular dynamics simulations to explore the structure and dynamics of LamR related to laminin-1 interactions. Our main finding is that loop 188–197 in the C-terminal region is highly flexible. It undergoes a major change resulting in a conformational switch that partially solvent exposes the R180 residue in the final part of the G peptide. So, R180 could contribute to laminin-1 binding. Projection of the simulations along the first two principal components also confirms the importance of this conformational switch in the LamR. This may be a basic prerequisite to clarify the key structural determinants of the interaction of LamR with laminin-1.

In this work we propose a parallel graphics pipeline for real-time interactive editing, processing and rendering of smooth surface primitives on the Cell BE. Our approach integrates a special patch-based geometry shader for subdivision surface targeting high-performance single-chip multi-core platforms. We describe a combination of algorithmic, architectural and back-end optimizations that enable us to render smooth subdivision surfaces in real-time and to dynamically deform 3D models represented by subdivision surfaces.

In this work, we investigated the structural and dynamic consequences of two substi- tutions, P58A and G36P, located in two different solvent-exposed loops of cytochrome c551. The re- sults show that both mutations affect regions that are distant from the site of mutation. Here, the two loops appear to be dynamically coupled to each other, because the substitution at one site affects the structure and the dynamics of the other site. However, the substitutions at Gly-36 and Pro-58 presented substantial differences, which were re- lated to the mechanical (rigidity and deformability) properties of the site surrounding the mutation. Although the P58A mutant conserved a significant dynamic similarity to the wild-type protein as the immediate surroundings of position 58 became more rigid, the G36P mutant, which had deformed its flexible surroundings, presented a dynamic behav- ior that was markedly different from that of the wild-type protein. These results suggest that pertur- bation of sterically isolated and flexible regions, such as solvent-exposed loops, can have strong dy- namic consequences on the protein as a whole, raising the possibility that these effects could in turn affect the stability or the function of the pro- tein. Proteins 2003;50:222-229. © 2002 Wiley-Liss, Inc.

In recent years, increased interest in the origin of protein thermal stability has gained attention both for its possible role in understanding the forces governing the folding of a protein and for the design of new highly stable engineered biocatalysts. To study the origin of thermostability, we have performed molecular dynamics simulations of two rubredoxins, from the mesophile Clostridium pasteurianum and from the hyperthermophile Pyrococcus furiosus. The simulations were carried out at two temperatures, 300 and 373 K, for each molecule. The length of the simulations was within the range of 6-7.2 ns. The rubredoxin from the hyperthermophilic organism was more flexible than its mesophilic counterpart at both temperatures; however, the overall flexibility of both molecules at their optimal growth temperature was the same, despite 59% sequence homology. The conformational space sampled by both molecules was larger at 300 K than at 373 K. The essential dynamics analysis showed that the principal overall motions of the two molecules are significantly different. On the contrary, each molecule showed similar directions of motion at both temperatures.

The ABC transporter superfamily is among the largest and most broadly expressed protein families. Members of this family use the energy stored in ATP molecules to actively extrude a variety of substrates from cells, including exogenous compounds such as drugs, metabolites, peptides, steroids, ions and phospholipids. So far, the best known and characterized major drugs transporters, have been studied in details with respect to their structure and function. It has been showed that ATP-dependent transporters can cause resistance in cancer cells by actively extruding the clinically relevant chemotherapic drugs. There is accumulating evidence that active export of anticancer drugs from cells by means of specific transporters is one of the major mechanism of drug resistance. Camptothecin (CPT) and its derivatives has been proven to be effective against a broad set of tumors. The CPT target is the human DNA topoisomerase I, an enzyme that changes the topological state of the DNA double helix during biological activity of the cell. Here we report a computaional study of the interaction mechanism between a set of biochemically and clinically relevant campthotecin ligands. A series of multiple docking simulations were carried out using the topotecan, gimatecan and irinotecan CPT derivatives as possible ligands, and a homology model of ABCG2 transporter was used as target molecule. Our results show that the camptothecin derivatives dock to distinct sites located in the trans-membrane region of the transporter molecule. The chemical nature of the substitutions at position A of the CPT analogues used in this study is also analyzed to identify the structural prerequisites responsible for the relative selectivity of the ligand. The structural basis of ligand binding may help design new CPT analogues with reduced side effects and higher affinity.

This chapter focuses on homology modeling and molecular dynamics (MD) simulations studies of ion channels for the range of single cell organisms from prokaryotes to eukaryotes. The glutamate receptor channels (GluRs) share some distant homology in their transmembrane (TM) domains with K channels but possess distinct extracellular ligand-binding domains for which several structures, of both bacterial and mammalian homologs, are known. It can be seen that molecular modeling and simulations can contribute to studies of ion channels in two respects. Modeling studies enable extrapolation from experimental structures of prokaryotic ion channels to molecular models of eukaryotic homologs, thus aiding design and interpretation of, for example, mutation experiments for dissecting structure-function relationships. Ion channel structures and ion channel models may also be used as the basis of multinanosecond MD simulations. Finally, it will become increasingly important to run multiple simulations on multiple channels to allow comparative analysis of simulation results, which in turn will enable the formulation of more general hypotheses concerning the relationship between the conformational dynamics of channel proteins and their physiological functions.

From a specific definition of the roto-translational (external) and intramolecular (internal) coordinates, a constrained dynamics algorithm is derived for removing the roto-translational motions during molecular dynamics simulations, within the leap-frog integration scheme. In the paper the theoretical basis of this new method and its statistical mechanical consistency are reported, together with two applications.

The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.

Gambierol is a ladder-shaped polyether toxin that acts as a gating modifier to inhibit Kv3.1 channels with nanomolar affinity. Binding determinants for gambierol have been identified at an interface between S5 and S6, located outside the permeation pathway. However, the high gambierol sensitivity of Kv3.1 channels could not be fully transplanted to the insensitive Kv2.1 channel by introducing the S5-S6 determinants. To explore whether also the voltage-sensing domain (VSD) is a determinant for gambierol sensitivity, we exchanged the complete VSD (S1-S4), parts of the VSD (the S1-S3a region and the Sb-S4 paddle), and the electromechanical coupling (L45+S6c) between Kv3.1 and Kv2.1. Our results show that the L45+S6c and the S1-S3a region did not alter the affinity of Kv3.1 channels for gambierol. In contrast, the distal part of the VSD, the S3b-S4 paddle, displayed a 100-fold decrease in affinity compared to WT Kv3.1. Since all VSD chimeras displayed similar biophysical properties and remained sensitive to well-known pore blockers, the loss in gambierol sensitivity in the S3b-S4 paddle chimera is most likely not the result of allosteric effects. Molecular-Dynamics simulations indicated that the S3b-S4 paddle motif resides in proximity of gambierol and that the structure and position of the VSD may regulate the space of the binding site between the pore domain (S5 and S6) and the gating machinery. Hence, our results suggest that the VSD, and especially the S3b-S4 paddle motif, contributes to the structure and/or the accessibility of the binding site for gambierol. (This research was supported by FWO grant G0433.12N to DJS and JT).

Le simulazioni di dinamica molecolare consentono di analizzare la struttura e la flessibilità di macromolecole importanti dal punto di vista biomedico, come proteine e DNA. Questo permette di studiare le cause di molte malattie genetiche, spesso provocate da una mutazione di un gene che codifica per una proteina, ma anche di progettare nuovi farmaci più efficaci. In particolare, lo studio delle mutazioni che insorgono su proteine di membrana come i canali ionici voltaggio-dipendenti permettono di delucidare i meccanismi molecolari alla base delle più importanti patologie neurologiche e muscolari. Le moderne risorse di calcolo parallelo, basate sui nuovi processori GPU ad architettura multi-core, consentono di estendere la simulazione di questi sistemi biologici su scale di tempo molto maggiori e quindi di modellare funzioni biologiche più complesse. Ma lo sviluppo non è solo nelle tecnologie. Nuovi algoritmi e campi di forza che regolano le interazioni inter-atomiche vengono continuamente proposti e implementati nell’ambiente di calcolo del CASPUR.

The inwardly-rectifying potassium channel Kir4.1 is a major player in the astrocyte-mediated regulation of [K(+)](o) in the brain, which is essential for normal neuronal activity and synaptic functioning. KCNJ10, encoding Kir4.1, has been recently linked to seizure susceptibility in humans and mice, and is a possible candidate gene for Autism Spectrum Disorders (ASD). In this study, we performed a mutational screening of KCNJ10 in 52 patients with epilepsy of "unknown cause" associated with impairment of either cognitive or communicative abilities, or both. Among them, 14 patients fitted the diagnostic criteria for ASD. We identified two heterozygous KCNJ10 mutations (p.R18Q and p.V84M) in three children (two unrelated families) with seizures, ASD, and intellectual disability. The mutations replaced amino acid residues that are highly conserved throughout evolution and were undetected in about 500 healthy chromosomes. The effects of mutations on channel activity were functionally assayed using a heterologous expression system. These studies indicated that the molecular mechanism contributing to the disorder relates to an increase in either surface-expression or conductance of the Kir4.1 channel. Unlike previous syndromic associations of genetic variants in KCNJ10, the pure neuropsychiatric phenotype in our patients suggests that the new mutations affect K(+) homeostasis mainly in the brain, by acting through gain-of-function defects. Dysfunction in astrocytic-dependent K(+) buffering may contribute to autism/epilepsy phenotype, by altering neuronal excitability and synaptic function, and may represent a new target for novel therapeutic approaches.

Voltage-gated K+ channels (Kv) are composed of four subunits, each of which contains six trans-membrane domains (TMs), S1 through S6. The S1-S4 segments comprise the voltage-sensing domain (VS), which senses membrane potential and controls the gating of the pore domain (PD). Although still controversial, the voltage-sensing domain undergoes conformational changes within the membrane electric field, upon membrane depolarization, that is mechanically transferred via the S4-S5 linker to the intracellular gate of the channel. MD simulations of portions of PD and VS regions highlighted the importance of their flexibility for proper channel function. Nevertheless, a comprehensive description of the dynamics of both domains at atomic level has not been provided yet. Here we report the analysis of all-atom multiple molecular dynamics simulations (∼200 ns) of the entire Kv1.2/2.1 chimera, consisting of the α and β chain embedded in a 549 monomer POPC bilayer, and immersed in a box of 135K explicit SPC water molecules at 300 K. We used principal components analysis (PCA) of the Cα atomic fluctuations covariance matrix to analyze the essential subspace that characterizes the channel internal dynamics. Briefly, we observed an up to 4.5 A conformational drift of VS from its starting position. The average RMSF of the S3b-S4 domain was between 1.6 and 3.0 A. Relative to the pore region, i) the second principal component shows that all four VS domains fluctuate in a concerted manner and affect the flexibility of the intracellular gates; ii) the first principal component reveals that T1 domain moves approximately 3.5 A downwards, influencing the local structure and dynamics of the neighboring intracellular gate. Protein-lipids interactions are crucial for channel structure/function. Thus, the contributions of H-bonds and salt-bridges between channel atoms and lipid head-groups on global channel dynamics will be illustrated.

Shaker-like (KV1.1) channels contain a highly conserved Pro-Val-Pro (PVP) motif at the base of S6 that produces a kink in the S6 helices and provides a flexible element thought to be essential for channel gating. The role of proline-induced kinks in transmembrane helices is well known, but the contribution of the small hydrophobic valine between these two prolines is not known, and interestingly, Shab-like (KV2.1) channels possess an isoleucine at this position (PIP). Here we show that the exact nature of this central hydrophobic residue within the PXP motif confers unique functional properties to KV1 channels, including changes in activation and deactivation kinetics, voltage-dependent properties and open probabilities, but single-channel conductance and cell expression levels are not affected. In support of these functional changes, molecular dynamic simulations demonstrate that valine and isoleucine contribute differently to S6 flexibility within this motif. These results therefore indicate that the nature of the central hydrophobic residue in the PXP motif is an important functional determinant of KV channel gating by contributing, at least in part, to the relative flexibility of this motif.

The energetic profile of an ion translated along the axis of an ion channel should reveal whether the structure corresponds to a functionally open or closed state of the channel. In this study, we explore the combined use of Poisson-Boltzmann electrostatic calculations and evaluation of van der Waals interactions between ion and pore to provide an initial appraisal of the gating state of a channel. This approach is exemplified by its application to the bacterial inward rectifier potassium channel KirBac3.1, where it reveals the closed gate to be formed by a ring of leucine (L124) side chains. We have extended this analysis to a comparative survey of gating profiles, including model hydrophobic nanopores, the nicotinic acetylcholine receptor, and a number of potassium channel structures and models. This enables us to identify three gating regimes, and to show the limitation of this computationally inexpensive method. For a (closed) gate radius of 0.4 nm < R < 0.8 nm, a hydrophobic gate may be present. For a gate radius of 0.2 nm < R < 0.4 nm, both electrostatic and van der Waals interactions will contribute to the barrier height. Below R = 0.2 nm, repulsive van der Waals interactions are likely to dominate, resulting in a sterically occluded gate. In general, the method is more useful when the channel is wider; for narrower channels, the flexibility of the protein may allow otherwise-unsurmountable energetic barriers to be overcome.

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.

Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. Examples of such simulations for ion channels and for bacterial outer membrane proteins are described. Comparison of simulations of KcsA (an alpha-helical bundle) and OmpA (a beta-barrel) reveals the importance of two classes of side chains in stabilizing interactions with the head groups of lipid molecules: (i) tryptophan and tyrosine; and (ii) arginine and lysine. Arginine residues interacting with lipid phosphate groups play an important role in stabilizing the voltage-sensor domain of the KvAP channel within a bilayer. Simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid-binding site at the interface between adjacent protein monomers. A combination of molecular modelling and simulation reveals a potential phosphatidylinositol 4,5-bisphosphate-binding site on the surface of Kir6.2.

Molecular modeling and simulations enable extrapolation for the structure of bacterial potassium channels to the function of their mammalian homologues. Molecular dynamics simulations have revealed the concerted single-file motion of potassium ions and water molecules through the selectivity filter of K channels and the role of filter flexibility in ion permeation and in "fast gating." Principal components analysis of extended K channel simulations suggests that hinge-bending of pore-lining M2 (or S6) helices plays a key role in K channel gating. Based on these and other simulations, a molecular model for gating of inward rectifier K channel gating is presented.

Further experimental and computational studies are required before we reach a complete structural understanding of the mechanisms of voltage-sensing and voltage-gating of Kv channels. Progress is likely to be made using a range of indirect techniques to complement the structural data that have come from X-ray crystallography. In particular, spectroscopic [7xMolecular architecture of the KvAP voltage-dependent K+ channel in a lipid bilayer. Cuello, L.G., Cortes, D.M., and Perozo, E. Science. 2004; 306: 491–495Crossref | PubMed | Scopus (173)See all References[7] and chemical modification [8xMolecular mechanism of voltage sensor movements in a potassium channel. Elliott, D.J.S., Neale, E.J., Aziz, Q., Dunham, J.P., Munsey, T.S., Hunter, M., and Sivaprasadarao, A. EMBO J. 2005; in pressSee all References[8] studies of Kv channels in situ in lipid bilayers will help to resolve the structure of the resting (closed state) channel. These methods will then have to be deployed in combination with a transmembrane voltage difference to reveal the change in sensor conformation and orientation during the transition from the closed to the open state.It is likely that computational methods will be used to integrate data from these diverse sources in a molecular mechanism. Given the proposed changes in conformation of the voltage sensor upon activation of Kv channels, it is important to characterize the intrinsic flexibility of this domain. Molecular dynamics simulations offer one possibility for exploring the conformational dynamics of the sensor domain (our unpublished data).A number of other techniques may also yield information on the location of the voltage sensor relative to the membrane. For example, toxins that interact with the sensor, for example Vstx1 [9xA membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom. Lee, S.Y. and MacKinnon, R. Nature. 2004; 430: 232–235Crossref | PubMed | Scopus (169)See all References[9], provide valuable probes. Their location – and hence by extension the location of the voltage-sensor – relative to a lipid bilayer may be established via molecular simulations. Similarly, electron microscopy [10xElectron microscopic analysis of KvAP voltage-dependent K+ channels in an open conformation. Jiang, Q.X., Wang, D.N., and MacKinnon, R. Nature. 2004; 430: 806–810Crossref | PubMed | Scopus (82)See all References[10] may reveal the overall shape of Kv molecules trapped in different conformational states. Thus, by combining information from these disparate sources, a complete mechanism of Kv voltage-sensing and gating may emerge.

In a previous article (Zbilut et al., Biophys J 2003;85:3544-3557), we demonstrated how an aggregation versus folding choice could be approached considering hydrophobicity distribution and charge. In this work, our aim is highlighting the mutual interaction of charge and hydrophobicity distribution in the aggregation process. Use was made of two different peptides, both derived from a transmembrane protein (amyloid precursor protein; APP), namely, Abeta(1-28) and Abeta(1-40). Abeta(1-28) has a much lower aggregation propensity than Abeta(1-40). The results obtained by means of molecular dynamics simulations show that, when submitted to the most "aggregation-prone" environment, corresponding to the isoelectric point and consequently to zero net charge, both peptides acquire their maximum flexibility, but Abeta(1-40) has a definitely higher conformational mobility than Abeta(1-28). The absence of a hydrophobic "tail," which is the most mobile part of the molecule in Abeta(1-40), is the element lacking in Abeta(1-28) for obtaining a "fully aggregating" phenotype. Our results suggest that conformational flexibility, determined by both hydrophobicity and charge effect, is the main mechanistic determinant of aggregation propensity.

Although the use of surgical staples is a well established practice in intestinal tract surgery on adults, their role in biliodigestive anastomoses in adults and children has been more limited. The Authors describe a 12-year-old girl with a type-IV choledochal cyst, who was successfully treated with cyst excision and Roux-en-Y hepaticojejunostomy created with a surgical stapler.

Toxins that block voltage-gated potassium (Kv) channels provide a possible template for improved homology models of the Kv pore. In assessing the interactions of Kv channels and their toxins it is important to determine the dynamic flexibility of the toxins. Multiple 10 ns duration molecular dynamics simulations combined with essential dynamics analysis have been used to explore the flexibility of four different Kv channel-blocking toxins. Three toxins (Tc1, AgTx and ChTx) share a common fold. They also share a common pattern of conformational dynamics, as revealed by essential dynamics analysis of the simulation results. This suggests that some aspects of dynamic behaviour are conserved across a single protein fold class. In each of these three toxins, the residue exhibiting minimum flexibility corresponds to a conserved lysine residue that is suggested to interact with the filter domain of the channel. Thus, comparative simulations reveal functionally important conservation of molecular dynamics as well as protein fold across a family of related toxins.

Background. Bladder autoaugmentation uses partial detrusorectomy to create a diverticular bulge in the bladder mucosa. This technique has eliminated certain serious complications of cystoplasty with gastrointestinal tissues (e.g., fluid/electrolyte/acid-base imbalances, mucous hypersecretion), but the exposed mucosa is subject to fibrosis and, sometimes, to perforation, which can annul the benefits of surgery. Methods. We have developed an original technique based on traditional autoaugmentation with protection of the herniated mucosa by split-thickness pedunculated rectus abdominis muscle flaps that are sutured to the incised margins of the detrusor. Preliminary testing was done on 30 adult Wistar rats. A control group of 15 rats underwent laparotomy alone. Bladder capacity was measured via suprapubic cystography before and after (4 weeks, 8 weeks, 1 year) surgery, just before sacrifice. Sections of the reconstructed bladder were examined histologically. Results. Twenty-three bladder-augmented rats and 13 controls survived. In the experimental group, bladder capacity increased by 38% (mean). None of the rats experienced urinary retention, although one developed bladder stones. Histology revealed no pathologic changes (other than chronic inflammatory infiltrates at suture sites) in the mucosa, detrusor, or muscle flaps, which were all viable and well integrated by the fourth postoperative week. There were no signs of mucosal or muscle fibrosis. Conclusions. Preliminary results in a rat model suggest that this new technique can produce an enlarged bladder that is fully functional and less vulnerable to fibrotic retraction and rupture. Residual contractility in the muscle flaps might theoretically be exploited to facilitate paraphysiologic micturition.

Several studies indicate that in young patients (less than 21 years of age at the time of diagnosis), the prognosis of thyroid carcinoma (TC) is more favorable than in older patients. However, a more radical treatment approach is recommended in children and adolescents due to the higher prevalence of local lymph-node involvement in these cases. Since the extent of primary surgical treatment is closely related to the overall prognosis, preoperative diagnosis becomes essential in the management of thyroid neoplasms in young patients. In this retrospective study (1987-1998), we analyzed a surgical series of 50 children and adolescents with thyroid nodules in an attempt to establish the role of diagnostic studies in detecting malignant lesions prior to surgery. Our diagnostic protocol for evaluating thyroid nodules was based on clinical evaluation, measurement of thyroid-hormone and thyroglobulin (TG) levels, anti-TG and anti-TPO antibody titers, calcitonin, CEA, and TPA levels, sonography, scintigraphy, and fine-needle aspiration cytology (FNAC) of the thyroid nodules and any enlarged lymph nodes. Eleven of the 15 cases of histologically confirmed carcinoma were preoperatively identified as malignant lesions with the aid of FNAC. The authors conclude that the preoperative work-up of children and adolescents with thyroid nodules requires the collaboration of an experienced team of professionals, and recommend FNAC as the initial test.

The effects of core-packing on the structure, function and mechanics of the RNA-binding 4-helix-bundle Rop have been studied by molecular dynamics simulations. The structural, dynamical and geometrical properties of the Rop homodimer, (formed by the antiparallel juxtaposition of two helix-turn-helix motifs), have been compared with those of three protein variants described by Munson et al. (Protein Sci, 5:1584-1593, 1996), where the core of the native protein has been systematically repacked using a two-amino acid alphabet: Ala(2)Leu(2)-8, Ala(2)Leu(2)-8-rev, and Leu(2)Ala(2)-8. The results showed that it was possible to readily distinguish the inactive protein Leu(2)Ala(2)-8 from the other functionally active systems based on tertiary and quaternary structure criteria. Structural properties such as native secondary structure content did not correlate with biological activity. Biological activity was related in part to the relative arrangement of the residues within the binding site. But, more global aspects, related to the overall topology of the helical bundle, accounted for the small functional differences between Ala(2)Leu(2)-8 and Ala(2)Leu(2)-8-rev. Mechanically, the 4-helix-bundle absorbed core mutations by altering the local structure at the sequence termini and in the turns that join the two helices of each monomer, and by changing the overall orientation and separation of the extremely rigid helices. Proteins 1999;36:436-446.

The 28-residue peptide thymosin alpha1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin alpha1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin alpha1 has a structure characterized by two regions. A beta-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an alpha helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin alpha1.

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.