List of Records in Institute for Molecular Bioscience - Publications - UQ eSpace

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

2007-08-15T05:55:24Z

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA ( ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multi-vesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

Aard is specifically up-regulated in Sertoli cells during mouse testis differentiation

2008-02-18T17:25:19Z

Aard (alanine and arginine rich domain) is a gene of unknown function, previously reported to show sexually dimorphic expression in fetal mouse gonads. Here we describe the spatio-temporal expression profile of Aard during gonad development. The period of elevated mRNA expression coincides with early differentiation of the testis and is limited to Sertoli cells of the developing testis cords. Although low levels of Aard transcripts were detected in other tissues by quantitative RT-PCR assays, high levels of Aard expression is specific to the testis in both embryonic and adult mice.

Aard is specifically up-regulated in Sertoli cells during mouse testis differentiation

2009-01-30T09:53:58Z

A Backbone Linker for BOC-Based Peptide Synthesis and On-Resin Cyclization: Synthesis of Stylostatin 1

2008-06-10T15:17:40Z

We have developed a new 4-alkoxybenzyl-derived linker that anchors the C-terminal amino acid to the resin through the alpha-nitrogen atom. The linker allows BOC solid-phase peptide assembly and peptide cleavage using standard HF protocols. This linking strategy provides a versatile on-resin route to cyclic peptides and avoids the diketopiperazine formation that is prominent when using FMOC chemistry on backbone linkers. The linker was prepared by forming the aryl ether fi om 4-hydroxybenzaldehyde and bromovaleric acid. Subsequent reductive amination of the aldehyde with an allyl-protected amino acid eater and acylation of the resulting secondary amine provided the tertiary amide. After linking the amide to the resin, standard BOC SPPS, followed by allyl deprotection, cyclization, and HF cleavage gave cyclic peptides in high purity. To exemplify the strategy, the cytotoxic heptapeptide, stylostatin 1, was synthesized from two linear precursors. For comparison purposes, the yields of the on-resin and solution-phase cyclization were determined and found to be dependent upon the linear precursor. This linker technology provides new solid-phase avenues in accessing libraries of cyclic peptides.

Aberrant dysferlin trafficking in cells lacking caveolin or expressing dystrophy mutants of caveolin-3

2007-09-19T17:44:18Z

Mutations in the dysferlin (DYSF) and caveolin-3 (CAV3) genes are associated with muscle disease. Dysferlin is mislocalized, by an unknown mechanism, in muscle from patients with mutations in caveolin-3 (Cav-3). To examine the link between Cav-3 mutations and dysferlin mistargeting, we studied their localization at high resolution in muscle fibers, in a model muscle cell line, and upon heterologous expression of dysferlin in muscle cell lines and in wild-type or caveolin-null fibroblasts. Dysferlin shows only partial overlap with Cav-3 on the surface of isolated muscle fibers but co-localizes with Cav-3 in developing transverse (T)-tubules in muscle cell lines. Heterologously expressed dystrophy-associated mutant Cav3R26Q accumulates in the Golgi complex of muscle cell lines or fibroblasts. Cav3R26Q and other Golgi-associated mutants of both Cav-3 (Cav3P104L) and Cav-1 (Cav1P132L) caused a dramatic redistribution of dysferlin to the Golgi complex. Heterologously expressed epitope-tagged dysferlin associates with the plasma membrane in primary fibroblasts and muscle cells. Transport to the cell surface is impaired in the absence of Cav-1 or Cav-3 showing that caveolins are essential for dysferlin association with the PM. These results suggest a functional role for caveolins in a novel post-Golgi trafficking pathway followed by dysferlin.

Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators

2007-08-15T05:45:05Z

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were Studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators. (c) 2005 Elsevier Ltd. All rights reserved.

A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-kappa B and NFAT families

2009-11-17T12:16:28Z

The Rel/NF-kB family of eukaryotic transcription factors bind DNA with high specificity and affinity as homo- or heterodimers to mediate a diverse range of biological processes. By comparison, the nuclear factor of activated T-cells (NFAT) family has been recognised as Rel homologues due to structural similarities between the DNA-binding domains, yet they bind DNA as lower-affinity monomers. The structural and functional overlap between the NF-kB and NFAT families suggests that they may be evolutionarily divergent from a common, monomeric ancestor but have evolved different mechanisms to achieve high-affinity binding to their target DNA sequences. In order to understand the origin of these mechanistic differences, we constructed two chimeric proteins, based on molecular modelling, comprising the DNA-binding domain of NFAT and the dimerisation domain of NF-kB p50, differing only in the position of the splice site. Biophysical characterisation of the wild-type and chimeric proteins revealed that one of the chimeras bound DNA as a high-affinity, NF-kB-like cooperative dimer, whilst the other bound as a lower-affinity, NFAT-like monomer, demonstrating the importance of the interdomain linker in controlling the intrinsic ability of NFATc to form dimers. In addition, we have studied the rate of exchange of monomers between preformed NF-kB dimers and have determined, for the first time, the intrinsic homodimerisation constant for NF-kB p50. These data support a model in which NF-kB proteins bind DNA both in vitro and in vivo as high-affinity preformed homo- or heterodimers, which in an unbound form can still exchange monomer units on a physiologically relevant timescale in vivo. c 2004 Elsevier Ltd. All rights reserved.

Absolute sterochemistry of puupehenone and related metabolites

Urban, Sylvia og Capon, Robert J. — 2009-10-20T15:21:08Z

Degradative studies have permitted an absolute stereochemistry to be assigned to the marine natural product puupehenone (1). On biosynthetic grounds 10 related co-metabolites were assigned to the same antipodal series.

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

2007-08-14T18:13:47Z

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.

Accelerated chemical synthesis of peptides and small proteins

Miranda, LP og Alewood, PF — 2008-06-10T15:17:18Z

The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10-15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes, Here we report Boc chemistry that employs O-(7-azabenzo-triazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the difficult C-terminal sequence of HIV-1 proteinase (residues 81-99); fragment 65-74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the proinflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation, The benefits of this approach include enhanced ability to identify and characterize difficult couplings, rapid access to peptides for biological and structure-activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods.

Accelerated leap methods for simulating discrete stochastic chemical kinetics

2007-08-23T21:44:21Z

Biologists are increasingly conscious of the critical role that noise plays in cellular functions such as genetic regulation, often in connection with fluctuations in small numbers of key regulatory molecules. This has inspired the development of models that capture this fundamentally discrete and stochastic nature of cellular biology - most notably the Gillespie stochastic simulation algorithm (SSA). The SSA simulates a temporally homogeneous, discrete-state, continuous-time Markov process, and of course the corresponding probabilities and numbers of each molecular species must all remain positive. While accurately serving this purpose, the SSA can be computationally inefficient due to very small time stepping so faster approximations such as the Poisson and Binomial τ-leap methods have been suggested. This work places these leap methods in the context of numerical methods for the solution of stochastic differential equations (SDEs) driven by Poisson noise. This allows analogues of Euler-Maruyuma, Milstein and even higher order methods to be developed through the Itô-Taylor expansions as well as similar derivative-free Runge-Kutta approaches. Numerical results demonstrate that these novel methods compare favourably with existing techniques for simulating biochemical reactions by more accurately capturing crucial properties such as the mean and variance than existing methods.

Accelerating, hyperaccelerating, and decelerating networks

Gagen, M. J. og Mattick, J. S. — 2007-08-15T06:01:18Z

Many growing networks possess accelerating statistics where the number of links added with each new node is an increasing function of network size so the total number of links increases faster than linearly with network size. In particular, biological networks can display a quadratic growth in regulator number with genome size even while remaining sparsely connected. These features are mutually incompatible in standard treatments of network theory which typically require that every new network node possesses at least one connection. To model sparsely connected networks, we generalize existing approaches and add each new node with a probabilistic number of links to generate either accelerating, hyperaccelerating, or even decelerating network statistics in different regimes. Under preferential attachment for example, slowly accelerating networks display stationary scale-free statistics relatively independent of network size while more rapidly accelerating networks display a transition from scale-free to exponential statistics with network growth. Such transitions explain, for instance, the evolutionary record of single-celled organisms which display strict size and complexity limits.

Accelerating networks

Mattick, JS og Gagen, MJ — 2007-08-15T06:24:46Z

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

2009-01-30T14:03:50Z

The decoding release factor (RF) triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre (PTC) of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure: the anticodon loop and the loop at the tip of helix alpha5. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.

AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity

2008-02-18T14:54:47Z

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand- binding domain, to discover a novel alpha-conotoxin (alpha-TxIA) in the venom of Conus textile. alpha-TxIA bound with high affinity to AChBPs from different species and selectively targeted the alpha(3)beta(2) nAChR subtype. A co-crystal structure of Ac- AChBP with the enhanced potency analog TxIA(A10L), revealed a 201 backbone tilt compared to other AChBP - conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.

Acid-mediated conversion of methylene-interrupted bisepoxides to tetrahydrofurans: A biomimetic transformation

Capon, RJ og Barrow, RA — 2007-08-13T10:18:32Z

The acid-mediated transformation of syn and anti methylene interrupted cis,cis and cis,trans bisepoxides to tetrahydrofurans is high yielding, and demonstrates both regioselectivity and stereoselectivity. Trans,trans methylene interrupted bisepoxides do not yield tetrahydrofurans under the same conditions.

A comparison by cDNA microarray of mRNA abundance in Glycine max and Arabidopsis thaliana roots in response to Fusarium solani f.sp. glycines infection

2007-08-24T01:32:17Z

A comparison of booster immunisation with a combination DTPa-IPV vaccine or DTPa plus IPV in separate injections when co-administered with MMR, at age 4–6 years

2009-01-08T16:58:53Z

This study evaluated GSK's combined DTPa-IPV vaccine (Infanrix™-IPV) given as a fifth consecutive acellular pertussis booster dose in conjunction with the second dose of MMR vaccine (Priorix™) in children aged 4–6 years. The immunogenicity and reactogenicity of this vaccine regimen was compared with separate injections of DTPa and IPV when given concomitantly with MMR. A cohort of 362 children previously primed with four doses of DTPa and OPV, and a single dose of MMR were randomized to receive either DTPa-IPV + MMR (N = 181) or DTPa + IPV + MMR (N = 181). Antibody concentrations were measured prior to and 1 month after the booster dose. After immunisation all subjects from both groups had seroprotective antibody levels against diphtheria, tetanus and the three poliovirus serotypes, ≥96% showed vaccine response to PT, FHA and PRN, all were seropositive to mumps and rubella, and all but one subject were seropositive to measles. Immunogenicity results for each component antigen were similar for DTPa-IPV and separately co-administered DTPa and IPV. Local reactions were common with 24.0% and 31.1% of children experiencing swelling >50 mm at the DTPa-IPV and DTPa injection sites, respectively. The DTPa-IPV combination did not increase the incidence or intensity of adverse events compared with separately administered DTPa + IPV. The response to the concomitantly administered MMR vaccine was similar in the two groups and similar to previously reported responses for a second dose of MMR. This combined DTPa-IPV vaccine has a similar reactogenicity profile to DTPa, is immunogenic when given as a booster dose at 4–6 years of age, and has no impact on the immunogenicity of a co-administered second dose of MMR vaccine.

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

2007-08-13T14:02:31Z

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Brinkworth, R. I. og Horne, J. og Kobe, B. — 2007-08-13T12:55:35Z

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates. Copyright (C) 2002 John Wiley Sons, Ltd.

A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis

2007-08-15T04:45:40Z

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae)

2007-08-15T05:45:31Z

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

A Convenient Method for the Synthesis of Cyclic Peptide Libraries

2007-08-15T07:14:55Z

A convergent solution-phase synthesis of the macrocycle Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg], a potent new antiinflammatory drug

2007-08-15T02:45:44Z

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-D-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn-(Boc)-Pro-OH and H-D-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boe-D-Cha-OH was obtained very efficiently via hydrogenation Of D-Phe with PtO2 in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.

A cryo-electron facility for sub-tropical Southeast Queensland

McDowall, A. W. — 2008-06-06T13:13:05Z

A CSF-1 receptor kinase inhibitor targets effector functions and inhibits pro-inflammatory cytokine production from murine macrophage populations

2007-08-15T09:40:46Z

CSF-1 regulates macrophage differentiation, survival, and function, and is an attractive therapeutic target for chronic inflammation and malignant diseases. Here we describe the effects of a potent and selective inhibitor of CSF-1R -CYC10268 -on CSF1R-dependent signaling. In in vitro kinase assays, CYC10268 was active in the low nanomolar range and showed selectivity over other kinases such as Ab1 and Kit. CYC10268 blocked survival mediated by CSF-1R in primary murine bone marrow-derived macrophages (BMM) and in the factor-dependent cell line Ba/ F3, in which the CSF-1R was ectopically expressed. CYC10268 also inhibited CSF-1 regulated signaling (Akt, ERK-1/ 2), gene expression (urokinase plasminogen activator, toll-like receptor 9, and apolipoprotein E), and priming of LPS-inducible cytokine production in BMM. In thioglycollate-elicited peritoneal macrophages (TEPM), which survive in the absence of exogenous CSF-1, CYC10268 impaired LPS-induced cytokine production and regulated expression of known CSF-1 target genes. These observations support the conclusion that TEPM are CSF-1 autocrine and that CSF-1 plays a central role in macrophage effector functions during inflammation. CSF-1R inhibitors such as CYC10268 provide a powerful tool to dissect the role of the CSF-1/ CSF-1R signaling system in a range of biological systems and have potential for a number of therapeutic applications.

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons

2009-01-15T16:17:54Z

Abstract2We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential ¢ring under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10^20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (IADP) in the presence of TEA (10^20 mM) reversed at 338 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the IADP to 358 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Naþ and Kþ to this current were estimated to be about 1:5. Substitution of external Naþ with N-methyl D-glucamine blocked the current while replacement of internal Cl3 with gluconate did not block the IADP. The IADP was also inhibited when CsCl-¢lled patch pipettes were used. The IADP was blocked or substantially decreased in amplitude in the presence of N-type Ca2þ channel antagonists, g-conotoxin GVIA and g-conotoxin MVIIC, respectively, and was eliminated by external Cd2þ, indicating that it was dependent on Ca2þ entry. The IADP was also inhibited by ryanodine (10^20 WM), indicating that Ca2þ-induced Ca2þ release was involved in its activation. Ni£umic acid consistently inhibited the IADP with an IC50 of 63 WM. Using antibodies against the poreforming subunits of L-, N- and P/Q-type voltage-gated Ca2þ channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca2þ channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca2þ-channel blocker, is generated by the opening of Ca2þ-activated non-selective cation channels following action potential-mediated Ca2þ entry mainly through N-type Ca2þ channels. Ca2þ release from ryanodine-sensitive stores triggered by Ca2þ entry contributes signi¢cantly to the activation of this current. F 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

2007-08-15T05:50:01Z

alpha-Melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected REK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent. (c) 2005 Elsevier Inc. All rights reserved.

Activation of the growth hormone receptor

Pelekanos, Rebecca A. og Waters, Michael J. — 2009-01-16T09:59:05Z

Growth hormone (GH) is a major regulator of postnatal growth and metabolism. There are extensive clinical applications for GH or its antagonists, including treatments for dwarfism, cancer and metabolic wasting. Owing to this, there is considerable interest in the mechanisms of GH receptor (GHR) activation. It is conventionally thought that GH induces dimerization of two GHR monomers, which initiates intracellular signaling cascades. However, recent studies have provided evidence for a ligand-induced conformational change within constitutively dimerized GHRs being responsible for activating signaling pathways. This review will relate the new model of GHR activation to the activation of related cytokine receptors and discuss the implication of this new model for the design of small GH mimetics and antagonists for therapeutic use

Activation of the insert negative human calcitonin receptor: Phosphorylation of MAP kinase and inhibition of cellular proliferation

2009-10-09T13:06:10Z

Activation of the peroxisome proliferator-activated receptor pathway potentiates interleukin-1 receptor antagonist production in cytokine-treated chondrocytes

2009-02-11T12:54:27Z

Objective To determine whether peroxisome proliferator-activated receptor (PPAR) agonists protect chondrocytes against the effects of interleukin-1 (IL-1). Methods PPAR expression and function in cultured rabbit articular chondrocytes were studied by Northern blotting, electrophoretic mobility shift assay, and transient expression of a luciferase reporter construct bearing the human IL-1 receptor antagonist (Il-1Ra) gene promoter. Chondrocytes were incubated in vitro with IL-1 alone or in combination with CloFibrate (CloF) or other PPAR ligands. Proteoglycans were evaluated by 35S-sulfate incorporation, matrix metalloproteinase (MMP) levels were assessed by zymography and enzyme-linked immunosorbent assay (ELISA), and MMP messenger RNA (mRNA) levels were measured by Northern blotting and real-time reverse transcriptase-polymerase chain reaction. IL-1 and IL-1Ra soluble contents were measured by ELISA. Results CloF counteracted IL-1-induced 35S-proteoglycan degradation, gelatinolytic activity, and MMP-1, -3, and -13 mRNA expression. CloF also maximized IL-1-induced endogenous production of soluble IL-1Ra (sIL-1Ra). This stimulating effect on IL-1Ra gene expression was shown, by transient expression assay, to be transcriptional. Inhibition of sIL-1Ra expression by a specific small interfering RNA suppressed the effect of CloF on IL-1-induced MMP expression. The stimulatory effect of CloF was enhanced by cotransfection with wild-type PPAR and abolished by a dominant-negative PPAR mutant. Fenofibrate and WY-14643 displayed a similar stimulating effect on the IL-1Ra promoter, while rosiglitazone did not. Two PPAR response elements, an NF-B-binding site, and a CCAAT/enhancer binding protein-binding site were identified in the IL-1Ra promoter. All 4 sites were necessary for mediation of the effects of CloF. Conclusion Our findings support the notion that there is a PPAR-dependent mechanism that inhibits IL-1 function in chondrocytes, which operates via an increase in sIL-1Ra production.

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3

2007-08-15T05:10:02Z

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site.

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

2007-08-14T16:38:58Z

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

Ac Transposase Induces Methylation of a Ds Transposon in Trausgenic Tomato

2009-04-22T10:06:25Z

When a maize Dissociation (Ds) transposon was introduced into tomato, marker genes within the element were expressed until exposure to the transacting transposase source stabilised Activator (sAc). sAc was consistently found to induce transcriptional silencing of nos:BAR and nos:SPEC markers within Ds elements, and this silencing correlated with increased methylation of marker sequences. We identified one transposed Ds line in which a nos:BAR marker was reactivated and in which marker gene methylation was reduced.

Acute ischemic cardiac dysfunction is attenuated via gene transfer of a peptide inhibitor of the beta-adrenergic receptor kinase

2009-02-10T16:51:27Z

Acute myocardial ischemia is a critical adverse effect potentially occurring during cardiac procedures. A peptide inhibitor of the -adrenergic receptor kinase (ARK1), ARKct, has been successful in rescuing chronic myocardial ischemia. The present study focused on the effects of adenoviral-mediated ARKct (Adv-ARKct) delivery on left ventricle (LV) dysfunction induced by acute coronary occlusion. Rabbits received intracoronary delivery of phosphate-buffered saline (PBS) (n = 9) or 5 × 1011 viral particles of ARKct (n = 8). A loose prolene 5-0 Potz-loop suture was placed around the circumflex coronary artery (LCx) with both ends buried under the skin. Four days later, the suture was retrieved and pulled to occlude the LCx. Ischemia was confirmed by immediate ECG changes. LV function was continuously recorded for 45 min. Contractility (LVdP/dtmax), relaxation (LVdP/dtmin) and end diastolic pressure (EDP) were less impaired in the ARKct group as compared to PBS (P < 0.05, two-way ANOVA). AR density was higher in the ischemic area of the LV in the ARKct group (ARKct: 71.9 ± 4.6 fmol/mg protein, PBS: 54.5 ± 4.0 fmol/mg protein, P < 0.05). Adenylyl cyclase activity was also improved basally and in response to AR stimulation. ARK1 activation was less in the ARKct group (P < 0.05). Therefore, inhibition of myocardial ARK1 may represent a new strategy to prevent LV dysfunction induced by acute coronary ischemia.

A cyclic metallopeptide induces alpha helicity in short peptide fragments of thermolysin

2006-04-03T12:30:59Z

Acyclic permutants of naturally occurring cyclic proteins

Daly, N. L. og Craik, D. J. — 2008-06-10T10:29:19Z

Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Daly, NL og Craik, DJ — 2007-08-13T11:49:18Z

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

Adams-type methods for the numerical solution of stochastic ordinary differential equations

2008-06-10T10:47:22Z

Adaptors for Clathrin Coats: Structure and Function

2007-09-19T18:14:19Z

Clathrin-coated vesicles (CCVs) are responsible for the transport of proteins between various compartments of the secretory and endocytic systems. Clathrin forms a scaffold around these vesicles that is linked to membranes by clathrin adaptors. The adaptors simultaneously bind to clathrin and to transmembrane proteins and/or phospholipids and can also interact with each other and with other components of the CCV formation machinery. The result is a collection of proteins that can make multiple, moderate strength (muM K-d) interactions and thereby establish the dynamic regulatable networks to drive vesicle genesis at the correct time and place in the cell. This review focuses on the structure of clathrin adaptors and how these structures provide functional information on the mechanism of CCV formation.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

2007-08-14T16:49:58Z

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

A dimerization-dependent conformational change in the extracellular domain of the growth hormone receptor (GHR) is required for full signalling

2008-06-06T14:46:07Z

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes

2008-06-10T12:21:30Z

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-2-ol blocked adipsin secretion and resulted in accumulation of adipsin in Trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane. whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.

A distributed systems approach to secure Internet mail

Machanick, P. — 2007-08-15T07:15:51Z

One of the obstacles to improved security of the Internet is ad hoc development of technologies with different design goals and different security goals. This paper proposes reconceptualizing the Internet as a secure distributed system, focusing specifically on the application layer. The notion is to redesign specific functionality, based on principles discovered in research on distributed systems in the decades since the initial development of the Internet. Because of the problems in retrofitting new technology across millions of clients and servers, any options with prospects of success must support backward compatibility. This paper outlines a possible new architecture for internet-based mail which would replace existing protocols by a more secure framework. To maintain backward compatibility, initial implementation could offer a web browser-based front end but the longer-term approach would be to implement the system using appropriate models of replication. (C) 2005 Elsevier Ltd. All rights reserved.

β-Adrenergic signaling regulates NR4A nuclear receptor and metabolic gene expression in multiple tissues

2009-10-30T13:13:29Z

Adsorption of aromatic compounds onto activated carbons: Effects of the orientation of the adsorbates

2007-08-14T18:18:05Z

Adsorption of model aromatic compounds onto two untreated activated carbons with similar physical and chemical properties is investigated. The solution pH of all experiments was lowered so that all solutes were in their molecular forms. It is shown that the difference in the maximum adsorption capacities of the solutes was mainly attributed to the difference in the sizes of the molecules. This new experimental finding is significant to gaining insight into the orientation of the adsorbed phase and hence the adsorption mechanism of aromatic compounds in aqueous solutions. It is shown that the adsorption of aromatic compounds in a stacked motif for pi-pi interactions is unlikely, and in the absence of physical restrictions such as pore width, a T-shaped motif is the preferred orientation.

Advanced electron microscopy applications on inorganic nanomaterials

Zou, J. — 2009-02-02T17:49:34Z

Advances in Application of Alloying Additions for Promoting the Glass-Forming Ability of Bulk Metallic Glass

2009-02-03T13:38:38Z

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation

2007-08-14T15:44:30Z

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7, MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Aerosolization of protein solutions using thermal inkjet technology

2007-08-14T19:02:57Z

Vapotronics Inc. is developing the thermal inkjet (TIJ) technology used extensively in the printer industry to create a digital aerosol inhaler for the inhalation of therapeutics for local and systemic delivery. The operation of thermal inkjet printers requires generation of high temperatures and vaporization of the liquid formulation to effect droplet ejection. A study was conducted to develop formulations that would permit the generation of aerosols of therapeutic proteins without damage to the inkjet system or degradation of the proteins. Two proteins, human growth hormone and insulin, were formulated and aerosolized. The aerosol was collected and subjected to assays to compare the physicochemical and biological activities of these proteins before and after aerosolization. In each case, there was no significant changes to the proteins as a result of the aerosolization, providing evidence that TIJ can be used for aerosolizing solutions of protein therapeutics.