A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alpha beta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alpha beta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alpha beta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alpha beta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin las an allosterically acting 'competitive' antagonist) binds to this residue.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Mutations in exon 3 of the beta-catenin gene are rare in melanoma cell lines

Mutations in exon 3 of the CTNNB1 gene encoding beta-catenin have been reported in colorectal cancer cell lines and tumours. Although one study reported mutations or deletions affecting beta-catenin in 20% of melanoma cell lines, subsequent reports detected a much lower frequency of aberrations in uncultured melanomas. To determine whether this difference in mutation frequency reflected an in vitro culturing artefact, exon 3 of CTNNB1 was screened in a panel of 62 melanoma cell lines. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect intragenic deletions affecting exon 3. One out of 62 (1.6%) cell lines was found to carry a mutation, indicating that aberration of the Wnt-l/wingless pathway through activation of beta-catenin is a rare event, even in melanoma cell lines. (C) 2002 Lippincott Williams Wilkins.

2,6,,9-trioxabicyclo[3.3.1]nona-3,7-dienes and 2,4,6,8-tetraoxaadamantanes: Novel chiral spacer units in macrocyclic polyethers

The unusual chiral heterocyclic systems, trioxabicyclo[3.3.1]nona-3,7-dienes (bridged bisdioxines), are incorporated as novel spacer molecules into macrocyclic polyether ring systems of various sizes (8, 9 as well as 11-15) by cyclocondensation reaction of the! bisacid chloride 4b or bisesters 6,7 and 10, with several ethylene glycols. The 2:2 macrocycles 12-14 are obtained in approximately 50:50 mixtures of diastereomers. These conclusions are mainly based on HPLC data presented in Table I as well as X-ray analyses of (1R,5R)-8c (space group Pbca, a = 10.163(3) Angstrom, b = 18.999(4) Angstrom, c = 36.187(10) Angstrom, V = 6987(3) Angstrom(3), Z = 8, d(calc) = 1.218 g cm(-3), 6974 reflections, R = 0.0553.), mesolrac-11 (space group P (1) over bar, a = 10.472(5) Angstrom, b = 16.390(5) Angstrom, c = 17.211(5) Angstrom, alpha = 98.69(2)degrees, beta = 93.04(2)degrees, gamma = 98.52(2)degrees, V = 2879.3(18) Angstrom(3), Z = 2, d(calc) = 1.173 g cm(-3), 11,162 reflections, R = 0.0945) and meso-12 (space group P2(1)/c, a = 9.927(2), b = 18.166(3), c = 17.820(3) Angstrom, beta = 96.590(10)degrees, V = 3192.3(10)Angstrom(3), Z = 4, D-c = 1.109 g cm(-3), 3490 reflections, R = 0.0646). The 1:1 macrocycles 8b,c are also formed by intramolecular transesterification of the open-chain bisesters 7b,c and their formation is favored by the use of metal ions as templates. The bridged bisdioxine moieties in 8b and 12 are converted into the corresponding chiral tetra-oxaadamantane spacers to afford macrocycles 16 and 17. Preliminary metal ion complexation studies with selected species (8c, 11-14) were also performed.

EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins-A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon-axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli. (C) 2002 Elsevier Science B.V. All rights reserved.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Human peripheral blood V alpha 24(+) V beta 11(+) NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells

Background and Objectives We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. Materials and Methods Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. Results No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24(+) Vbeta11(+) NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. Conclusions Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24(+) Vbeta11(+) NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24(+) Vbeta11(+) NKT cells, or for autoimmune diseases.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Osteonectin/SPARC induction by ectopic beta(3) integrin in human radial growth phase primary melanoma cells

Expression of the beta(3) integrin subunit in melanoma in situ has been found to correlate with tumor thickness, the ability to invade and metastasize, and poor prognosis. Transition from the radial growth phase (RGP) to the vertical growth phase (VGP) is a critical step in melanoma progression and survival and is distinguished by the expression of beta(3), integrin. The molecular pathways that operate in melanoma cells associated with invasion and metastasis were examined by ectopic induction of the beta(3), integrin subunit in RGP SBcl2 and WM1552C melanoma cells, which converts these cells to a VGP phenotype. We used cDNA representational difference analysis subtractive hybridization between beta(3)-Positive and -negative melanoma cells to assess gene expression profile changes accompanying RGP to VGP transition. Fourteen fragments from known genes including osteonectin (also known as SPARC and BM-40) were identified after three rounds of representational difference analysis. Induction of osteonectin was confirmed by Northern and Western blot analysis and immunohistochemistry and correlated in organotypic cultures with the beta(3)-induced progression from RGP to VGP melanoma. Expression of osteonectin was also associated with reduced adhesion to vitronectin, but not to fibronectin. Osteonectin expression was not blocked when melanoma cells were cultured with anti-alpha(v)beta(3) LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through a(v)beta(3) integrin during conversion from RGP to VGP.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.