Improved outcomes of islet autotransplant after total pancreatectomy by combined blockade of IL-1ß and TNFα.

The efficacy of islet transplant is compromised by a significant loss of islet mass posttransplant due to an innate inflammatory reaction. We report the use of a combination of etanercept and anakinra (ANA+ETA) to block inflammatory islet damage in 100 patients undergoing total pancreatectomy with islet autotransplant. The patients were divided into 3 groups: no treatment (control [CTL]), etanercept alone (ETA), or a combination of etanercept and anakinra (ANA+ETA). Peritransplant serum samples were analyzed for protein markers of islet damage and for inflammatory cytokines. Graft function was assessed by fasting blood glucose, basal C-peptide, secretory unit of islet transplant objects (SUITO) index, and hemoglobin A1c . Administration of both antiinflammatory drugs was well tolerated without any major adverse events. Reductions in interleukin-6, interleukin-8, and monocyte chemoattractant protein 1 were observed in patients receiving ANA+ETA compared with the CTL group, while also showing a modest improvement in islet function as assessed by basal C-peptide, glucose, hemoglobin A1c , and SUITO index but without differences in insulin dose. These results suggest that double cytokine blockade (ANA+ETA) reduces peritransplant islet damage due to nonspecific inflammation and may represent a promising strategy to improve islet engraftment, leading to better transplant outcomes.

Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation.

AIMS/HYPOTHESIS: Beta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type 1 diabetes and loss of transplanted islets. The nuclear factor κB (NF-κB) signalling pathway is a key regulator of beta cell stress response, survival and apoptosis. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, has been demonstrated to be a potent, safe, anti-inflammatory molecule that can inhibit NF-κB signalling. Therefore, we evaluated the ability of WA to protect mouse and human islets from the damaging effects of pro-inflammatory cytokines in vitro and following intraportal transplantation. METHODS: Mouse and human islets were treated with a cytokine cocktail, and NF-κB activation was measured by immunoblots, p65 nuclear translocation and chromatin immunoprecipitation of p65-bound DNA. Intraportal transplantation of a marginal mass of syngeneic mouse islets was performed to evaluate the in vivo protective effect of WA. RESULTS: Treatment with WA substantially improved islet engraftment of syngeneic islets (83% for infusion with 200 islets + WA; 0% for 200 islets + vehicle) in a mouse model of diabetes, compared with marginal graft controls with superior islet function in WA-treated mice confirmed by glucose tolerance test. Treatment of human and mouse islets with WA prevented cytokine-induced cell death, inhibited inflammatory cytokine secretion and protected islet potency. CONCLUSIONS: WA was shown to be a strong inhibitor of the inflammatory response in islets, protecting against cytokine-induced cell damage while improving survival of transplanted islets. These results suggest that WA could be incorporated as an adjunctive treatment to improve islet transplant outcome.

The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli.

BACKGROUND: N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives. RESULTS: The structure of the enzyme from Escherichia coli has been determined to 2.2 A resolution by X-ray crystallography. The enzyme is shown to be a tetramer, in which each subunit consists of an alpha/beta-barrel domain followed by a carboxy-terminal extension of three alpha-helices. CONCLUSIONS: The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight-stranded beta-barrel. Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate. The sequence of N-acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N-acetylneuraminate lyase.

The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion.

BACKGROUND: Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the "F", or fusion, protein. Membrane fusion is assumed to involve a series of structural transitions of F from a metastable (prefusion) state to a highly stable (postfusion) state. No detail is available at the atomic level regarding the metastable form of these proteins or regarding the transitions accompanying fusion. RESULTS: The three-dimensional structure of the fusion protein of Newcastle disease virus (NDV-F) has been determined. The trimeric NDV-F molecule is organized into head, neck, and stalk regions. The head is comprised of a highly twisted beta domain and an additional immunoglobulin-like beta domain. The neck is formed by the C-terminal extension of the heptad repeat region HR-A, capped by a four-helical bundle. The C terminus of HR-A is encased by a further helix HR-C and a 4-stranded beta sheet. The stalk is formed by the remaining visible portion of HR-A and by polypeptide immediately N-terminal to the C-terminal heptad repeat region HR-B. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface. CONCLUSION: We propose that prior to fusion activation, the hydrophobic fusion peptides in NDV-F are sequestered within the radial channels within the head, with the central HR-A coiled coil being only partly formed. Fusion activation then involves, inter alia, the assembly of a complete HR-A coiled coil, with the fusion peptides and transmembrane anchors being brought into close proximity. The structure of NDV-F is fundamentally different than that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane.

The crystal structure of pneumococcal surface antigen PsaA reveals a metal-binding site and a novel structure for a putative ABC-type binding protein.

BACKGROUND: . The surface protein PsaA of the pathogenic bacterium Streptococcus pneumoniae plays an essential role in its virulence. PsaA is a putative ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of Mn2+ and possibly Zn2+ and is considered to be both a potential drug target and and a candidate vaccine component. RESULTS: . The structure of PsaA has been determined to 2.0 A resolution using X-ray crystallography and is the first structure obtained for an ABC-type binding protein from a Gram-positive organism. The protein consists of two (beta/alpha)4 domains linked together by a single helix. A metal-binding site is formed in the domain interface by the sidechains of His67, His139, Glu205 and Asp280 and is occupied in the structure. CONCLUSIONS: . The structural topology of PsaA is fundamentally different from that of other ABC-type binding proteins determined thus far in that PsaA lacks the characteristic 'hinge peptides' involved in conformational change upon solute uptake and release. In our structure, the metal-binding site is probably occupied by Zn2+. The site seems to be well conserved amongst related receptors from both Gram-positive and Gram-negative bacteria.

Determining the structural stability of UiO-67 with respect to time: a solid-state NMR investigation.

The stability of UiO-67 has been questioned for some time. We have used solid-state NMR to investigate the temporal stability of this MOF. Proper activation is necessary to achieve optimal surface area. However, even with proper activation, the long-term (30+ days) fate of UiO-67 is hydrolysis of the linker-metal bonds and, ultimately, pore collapse.

Shape complementarity at protein/protein interfaces.

A new statistic Sc, which has a number of advantages over other measures of packing, is used to examine the shape complementarity of protein/protein interfaces selected from the Brookhaven Protein Data Bank. It is shown using Sc that antibody/antigen interfaces as a whole exhibit poorer shape complementarity than is observed in other systems involving protein/protein interactions. This result can be understood in terms of the fundamentally different evolutionary history of particular antibody/antigen associations compared to other systems considered, and in terms of the differing chemical natures of the interfaces.

Crystallographic structure of allosterically inhibited phosphofructokinase at 7 A resolution.

The structure of the allosterically inhibited form of phosphofructokinase from Bacillus stearothermophilus has been determined by X-ray crystallography to 7 A resolution by molecular replacement using the known structure of the active state as a starting model. Comparing the inhibited state with the active state, the tetramer is twisted about its long axis such that one pair of subunits in the tetramer rotates relative to the other pair by about 8 degrees around one of the molecular dyad axes. This rotation partly closes the binding site for the co-operative substrate fructose-6-phosphate, explaining its weaker binding to this conformational state. Within the subunit, one domain rotates relative to the other by 4.5 degrees, which further closes the fructose-6-phosphate site, without closing the cleft between the domains of the same subunit: this motion causes little change to the catalytic site. This T-state model is consistent with the simple allosteric kinetic scheme in which the active and the inhibited conformations differ in their affinities for fructose-6-phosphate, but not in their catalytic rates. It does not explain the heterotropic allosteric effects.

Structure of phaseolin at 2.2 A resolution. Implications for a common vicilin/legumin structure and the genetic engineering of seed storage proteins.

The refinement to 2.2 A resolution of the three-dimensional structure of the seed storage protein phaseolin from the French bean (Phaseolus vulgaris) via an alternative crystal form is described. The refined structure reveals details of the molecule hitherto unobserved and in particular we identify the structural role of conserved residues within the broader 7 S (vicilin) family of seed storage proteins. On this basis we are able to postulate a canonical model for the structure of the 7 S proteins. This model in turn provides a means for interpreting the structure of the 11 S (legumin) family of seed storage proteins, for which no X-ray diffraction data are available. The 11 S proteins are shown to bear a much closer relationship to the 7 S proteins than was previously recognized. The canonical model of the 7 S protein structure also provides a basis for proposing engineered mutations of these proteins with the goal of enhancing nutritional and functional properties.

Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase.

We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase. Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase. These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates. In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate). From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family. A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear.

Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.

The N-acetylneuraminate lyase (NAL) sub-family of (beta/alpha)(8) enzymes share a common catalytic step but catalyse reactions in different biological pathways. Known examples include NAL, dihydrodipicolinate synthetase (DHDPS), d-5-keto-4-deoxyglucarate dehydratase, 2-keto-3-deoxygluconate aldolase, trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase and trans-2'-carboxybenzalpyruvate hydratase-aldolase. Little is known about the way in which the three-dimensional structure of the respective active sites are modulated across the sub-family to achieve cognate substrate recognition. We present here the structure of Haemophilus influenzae NAL determined by X-ray crystallography to a maximum resolution of 1.60 A, in native form and in complex with three substrate analogues (sialic acid alditol, 4-deoxy-sialic acid and 4-oxo-sialic acid). These structures reveal for the first time the mode of binding of the complete substrate in the NAL active site. On the basis of the above structures, that of substrate-complexed DHDPS and sequence comparison across the sub-family we are able to propose a unified model for active site modulation. The model is one of economy, allowing wherever appropriate the retention or relocation of residues associated with binding common substrate substituent groups. Our structures also suggest a role for the strictly conserved tyrosine residue found in all active sites of the sub-family, namely that it mediates proton abstraction by the alpha-keto acid carboxylate in a substrate-assisted catalytic reaction pathway.

Regulation of insulin gene transcription by a Ca(2+)-responsive pathway involving calcineurin and nuclear factor of activated T cells.

Immunosuppressants such as FK506 (tacrolimus), the primary cellular target of which is calcineurin, decrease beta-cell insulin content and preproinsulin mRNA expression. This study offers an explanation for this effect by establishing that calcineurin is an important regulator of insulin gene expression through the activation of a transcription factor, nuclear factor of activated T cells. Three putative nuclear factor of activated T cells binding sites were located within the proximal region of the rat insulin I gene promoter (-410 to +1 bp). Expression of nuclear factor of activated T cells in both clonal (INS-1) and primary (islet) beta-cells was confirmed by immunoblot and immunocytochemical analyses. Moreover, nuclear factor of activated T cells DNA-binding activity was detected in INS-1 and islet nuclear extracts by EMSAs. Activation of the insulin gene promoter by glucose or elevated extracellular K(+) (to depolarize the beta-cell) was totally prevented by FK506 (5-10 microM). K(+)-induced promoter activation was suppressed (>65%) by a 2-bp mutation of a single nuclear factor of activated T cells binding site in -410 rInsI. Both stimulants also activated a minimal promoter-reporter construct containing tandem nuclear factor of activated T cells consensus sequences. The effects of FK506 on K(+)-induced nuclear factor of activated T cells reporter or insulin gene promoter activity were not mimicked by rapamycin, indicating specificity toward calcineurin. These findings suggest that the activation of calcineurin by beta-cell secretagogues that elevate cytosolic Ca(2+) plays a fundamental role in maintenance of insulin gene expression via the activation of nuclear factor of activated T cells.

The nature of heme-aniline interactions during hemin-mediated oxygen activation and insertion reactions.

The interaction of aniline with hemin during oxygen activation and insertion mediated by the metalloporphyrin has been studied by a combined theoretical, semiempirical, and experimental approach. Results indicate that association is via a planar pi bonding interaction between the aromatic pi electrons of the amine and tetrapyrrole ring system. The effect of very high concentrations of hydroxyl radical scavengers on the system is discussed.

A method for monitoring the collapse of plastic sections as a function of electron dose.

We present a method for monitoring the collapse of plastic sections when irradiated in the electron microscope. The two surfaces of the section are separately coated with colloidal gold particles. The section is then tilted to an angle of 45 degrees in the microscope and a series of micrographs recorded, corresponding to increasing total electron dose. The collapse of the specimen normal to the plane of the section causes a relative movement in the image of the two sets of particles marking the two surfaces. By measuring the positions of a few gold particles on each side of the section in each exposure of the series, the collapse and also the in-plane shrinkage can be computed. The sections exhibit a rapid initial collapse, followed by a much slower phase of thinning. These effects should be taken into account when producing quantitative three-dimensional maps from tilt series of sectioned material.

The application of the maximum entropy method to electron microscopic tomography.

The maximum entropy method has been applied to single axis tilt electron microscopic tomography. Its application requires that the problem be correctly formulated and that the model for the noise in electron micrographs be developed. A suitable noise model was determined empirically. The maximum entropy method was applied to a reconstruction of a test object from projections to which noise had been added. These reconstructions were superior to those obtained by reciprocal space weighted back protection. The method was also robust towards the incorrect specification of the noise, the penalty being an increase in the time required for convergence rather than degradation of the quality of the reconstructed image. In the reconstruction of negatively stained chromatin fibres it was possible to obtain satisfactory images utilizing all the information in the projections, in contrast to conventional methods in which high resolution data are removed by the application of Fourier space filters.

Modelling the surface lattice of alpha-keratin filaments.

A recently-proposed model for the distribution of scattering material on the surface lattice of alpha-keratin intermediate filaments in dry porcupine quill is examined in detail. It is shown that, while retaining the basic form of the model (namely a dislocated helix with finite lattice spacing of 198.2 A), alternative meridonal distributions of scattering material within the finite lattice unit cell can be obtained which are consistent with the low-angle meridional X-ray pattern. The Gaussian shape function used to demonstrate the finite lattice in the model is questioned. The meridional diffraction pattern from hydrated porcupine quill is also examined and, apart from the intense fifth order reflection, can be modelled by distortion of the dry species scattering material distribution.

CLIX: a search algorithm for finding novel ligands capable of binding proteins of known three-dimensional structure.

A computer algorithm, CLIX, capable of searching a crystallographic data-base of small molecules for candidates which have both steric and chemical likelihood of binding a protein of known three-dimensional structure is presented. The algorithm is a significant advance over previous strategies which consider solely steric or chemical requirements for binding. The algorithm is shown to be capable of predicting the correct binding geometry of sialic acid to a mutant influenza-virus hemagglutinin and of proposing a number of potential new ligands to this protein.

Four universal forms of chlorophyll a.

We have matched the red absorption band measured at -196 C in a variety of chloroplast preparations with four major component curves representing forms of chlorophyll a having peaks at 661.6, 669.6, 677.1, and 683.7 nanometers. Chloroplast fractions enriched in one or the other of the two photochemical systems both contain these four major components, but system 1 preparations contain relatively more chlorophyll a 684. Chlorophyll a 677 and chlorophyll a 684 have greater bandwidths in system 1. Bands at longer wavelengths near 693 and 704 nanometers also often occur, but with far smaller heights than the above major bands. The longer wavelength bands are more common in system 1 than in system 2. In system 1 the half-widths of the four major bands in typical spectra average 11.3, 10.0, 10.3, and 10.8 nanometers while in system 2 they are 11.6, 9.8, 9.4, and 9.6 nanometers. Some spectra with sharper and some with wider bands were found, but the wavelengths were identical.