A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as anti-haemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K-i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

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.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Fas ligand and tumour counter-attack in colorectal cancer stratified according to microsatellite instability status

Expression of membrane-bound Fas ligand (FasL) by colorectal cancer cells may allow the development of an immune-privileged site by eliminating incoming tumour-infiltrating lymphocytes (TILs) in a Fas-mediated counter-attack. Sporadic colorectal cancer can be subdivided into three groups based on the level of DNA microsatellite instability (NISI). High-level NISI (NISI-High) is characterized by the presence of TILs and a favourable prognosis, while microsatellite-stable (MSS) cancers are TIL-deficient and low-level MSI (MSI-Low) is associated with an intermediate TIL density. The purpose of this study was to establish the relationship between MSI status and FasL expression in primary colorectal adenocarcinoma. Using immunohistochemistry and a selected series of 101 cancers previously classified as 31 MSI-High, 30 NISI-Low, and 40 MISS, the present study sought to confirm the hypothesis that increased TIL density in MSI-High cancers is associated with low or absent membrane-bound FasL expression, while increased FasL in MSS cancers allows the killing of host TILs. TUNEL/CD3 double staining was also used to determine whether MSS cancers contain higher numbers of apoptotic TILs in vivo than MSI-High or MSI-Low cancers. Contrary to the initial hypothesis, it was found that MSI-High cancers were associated with higher FasL expression (p = 0.04) and a stronger intensity of FasL staining (p = 0.007). In addition, mucinous carcinomas were independently characterized by increased FasL expression (p = 0.03) and staining intensity (p = 0.0005). Higher FasL expression and staining intensity did not correlate with reduced TIL density or increased numbers of apoptotic TILs. However, consistent with the hypothesis that curtailment of the host anti-tumour immune response contributes to the poor prognosis in MSS cancers, it was found that apoptotic TILs were most abundant in MSS carcinomas and metastatic Dukes' stage C or D tumours (p = 0.004; p = 0.046 respectively). This study therefore suggests that MSS colorectal cancers are killing incoming TILs in an effective tumour counter-attack, but apparently not via membrane-bound FasL. Copyright (C) 2003 John Wiley Sons, Ltd.

Electrolytic properties and nanostructural features in the La2O3-CeO2 system

Doped ceria (CeO2) compounds are fluorite-type oxides which show oxide ionic conductivity higher than yttria-stabilized zirconia in oxidizing atmosphere. As a consequence of this, considerable interest has been shown in applications of these materials for low or intermediate temperature operation of solid-oxide fuel cells (SOFCs). In this study, the effective index was suggested to maximize the ionic conductivity in La2O3-CeO2 based oxides. The index considers the fluorite structure, and combines the expected oxygen vacancy level with the ionic radius mismatch between host and dopant cations. Using this approach, the ionic conductivity of this system has been optimized and tested under operating conditions of SOFCs. LaxCe1-xO2-delta (x = 0.125, 0.15, 0.175, and 0.20), (LaxSr1-x)(0.175)Ce0.825O2-delta (x = 0.1, 0.2, and 0.4), and (La1-xSr0.2Bax)(0.175)Ce0.825O2-delta (x 5 0.03, 0.05, and 0.07) were prepared and characterized as the specimens with low, intermediate, and high index, respectively. The ionic conductivity was increased with increasing suggested index. The transmission electron microscopy analysis suggested that partial substitution of alkaline earth elements in place of La into Ce site contributes to a decrease of microdomain size and an improvement of conductivity. (La0.75Sr0.2Ba0.05)(0.175)Ce0.825O1.891 with high index and small microdomains exhibited the highest conductivity, wide ionic domain, and good performance in SOFCs. (C) 2003 The Electrochemical Society.

Variation of bulk properties of anaerobic granules with wastewater type

Development of a granular sludge with high strength, high biological activity and a narrow settling distribution is necessary for optimal operation of high-rate upflow anaerobic treatment systems. Several studies have compared granules produced from different wastewaters but these have largely been from laboratory-fed reactors or compared granules from full-scale reactors fed similar wastewater types. Though two authors have commented on the inferiority of granules produced by a protein-based feed, the properties of these granules have not been characterised. In this paper, granules from full-scale reactors treating fruit and vegetable cannery effluent, two brewery effluents and a pig abattoir (slaughterhouse) were compared in terms of basic composition, size distribution, density, settling velocity, shear strength, and EPS content. The results supported previous qualitative observations by other researchers that indicate granule properties depend more on wastewater type rather than reactor design or operating conditions such as pre-acidification level. The cannery-fed granules bad excellent shear strength, settling distribution and density. Granules from the two brewery-fed reactors had statistically the same bulk properties, which were still acceptable for upflow applications. The protein-grown granule had poor strength and settling velocity. (C) 2001 Elsevier Science Ltd. All rights reserved.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-IBBL into murine DCs, and monitored the ability of these recombinant DO to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DO increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DO. We also report an additive effect of 4-IBBL and receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand coexpression in E7-transduced DC inummogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

Characterization of the acidic properties of mesoporous aluminosilicates synthesized from leached saponite with additional aluminum incorporation

The acidic properties of hexagonal mesoporous aluminosilicates synthesized via a new successful short time synthesis route using leached saponite and a low concentration of surfactant are thoroughly investigated. The resulting aluminosilicate mesoporous materials with high Si/Al ratios of around 11 have a maximal surface area of 1130 m(2)/g, a pore volume of 0.92 cm(3)/g, and a narrow pore size distribution at around 3 nm. The replacement of the sodium ions, present as counterions in the synthesized aluminosilicates, with protons imparts useful catalytic acidity. This acidity is extensively studied with FTIR spectroscopy after adsorption of ammonia and cyclohexylamine, while deuterated acetonitrile differentiates between Bronsted and Lewis acidity. Al-27 NMR spectroscopy determined the coordination of the aluminum in the FSM materials. Simultaneously the effect of an additional Al incorporation, utilizing sodium aluminate, aluminum nitrate, and aluminum isopropoxide is studied. From an acidic point of view, the incorporation with Al(NO3)(3) appears to be the most optimal, as the sample has a very high amount of acid sites (1.3 mmol/g). Investigating the nature of the acid sites it is found that in all samples except the one incorporated with Al(NO3)(3), more Bronsted than Lewis sites are present, both sites being quite acidic as they resist desorption temperatures up to 300 degreesC. Probing the coordination and location of the Al atoms, all the catalysts appeared to have mostly tetrahedral aluminum, up to 95% of the total Al amount for the proton exchanged AI(NO3)(3) incorporated sample.

Actively shielded multi-layer gradient coil designs with improved cooling properties

In standard cylindrical gradient coils consisting of a single layer of wires, a limiting factor in achieving very large magnetic field gradients is the rapid increase in coil resistance with efficiency. This is a particular problem in small-bore scanners, such as those used for MR microscopy. By adopting a multi-layer design in which the coil wires are allowed to spread out into multiple layers wound at increasing radii, a more favourable scaling of resistance with efficiency is achieved, thus allowing the design of more powerful gradient coils with acceptable resistance values. Previously this approach has been applied to the design of unshielded, longitudinal, and transverse gradient coils. Here, the multi-layer approach has been extended to allow the design of actively shielded multi-layer gradient coils, and also to produce coils exhibiting enhanced cooling characteristics. An iterative approach to modelling the steady-state temperature distribution within the coil has also been developed. Results indicate that a good level of screening can be achieved in multi-layer coils, that small versions of such coils can yield higher efficiencies at fixed resistance than conventional two-layer (primary and screen) coils, and that performance improves as the number of layers of increases. Simulations show that by optimising multi-layer coils for cooling it is possible to achieve significantly higher gradient strengths at a fixed maximum operating temperature. A four-layer coil of 8 mm inner diameter has been constructed and used to test the steady-state temperature model. (C) 2003 Elsevier Inc. All rights reserved.

Synthesis and complexation properties of some novel lariat-crown ethers

Several new lariat-crown ethers bearing either bridged bisdioxine or tetraoxaadamantane units as chiral substituents are prepared by reacting the corresponding amino-crown ether derivatives with the dimeric alpha-oxoketene, the latter obtained by flash vacuum pyrolysis of a furan-2,3-dione precursor. Complexation properties towards differently charged metal ions are investigated by H-1 NMR titration to obtain complexation constants (K-c-values for potassium/ sodium rhodanides: 480-1100 mol dm(-3)), as well as extraction experiments to explore the metal ion transportation abilities of the new lariat crown derivatives. In particular, a significantly increased ability to transport metal ions from water into chloroform was found with spherical tetraoxaadamantyl derivatives when compared with the free amino-benzocrown ethers.

The influence of key chemical constituents in activated sludge on surface and flocculating properties

This paper examines the influence of the chemical constituents of activated sludge and extracted extracellular polymeric substances (EPS) on the surface properties, hydrophobicity, surface charge (SC) and flocculating ability (FA) of activated sludge floes. Activated sludge samples from 7 different full-scale wastewater treatment plants were examined. Protein and humic substances were found to be the dominant polymeric compounds in the activated sludges and the extracted EPS, and they significantly affected the FA and surface properties, hydrophobicity and SC, of the sludge floes. The polymeric compounds proteins, humic substances and carbohydrates in the sludge floes and the extracted EPS contributed to the negative SC, but correlated negatively to the hydrophobicity of sludge floes. The quantity of protein and carbohydrate within the sludge and the extracted EPS was correlated positively to the FA of the sludge floes, while increased amounts of humic substances resulted in lower FA. In contrast, increased amounts of total extracted EPS had a negative correlation to FA. The results reveal that the quality and quantity of the polymeric compounds within the sludge floes is more informative, with respect to understanding the mechanisms involved in flocculation, than if only the extracted EPS are considered. This is an important finding as it indicates that extracting EPS may be insufficient to characterise the EPS. This is due to the low extraction efficiency and difficulties involved in the separation of EPS from other organic compounds. Correlations were observed between the surface properties and FA of the sludge floes., This confirms that the surface properties of the, sludge flocs play an important role in the bioflocculation process but that also other interactions like polymer entanglement are important. (C) 2002 Elsevier Science Ltd. All rights reserved.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

The preparation of zinc(II) and cadmium(II) complexes of the pentadentate N3S2 ligand formed from 2,6-diacetylpyridine and S-benzyldithiocarbazate (H2SNNNS) and the X-ray crystal structure of the novel dimeric [Zn2(SNNNS)2] complex

The pentadentate chelating agent, 2,6-diacetylpyridinebis(S-benzyldithiocarbazate) (H2SNNNS) reacts with zinc(II) and cadmium(II) ions forming stable complexes of empirical formula, [M(SNNNS)] (M=Zn2+, Cd2+; SNNNS2 =doubly deprotonated anionic form of the Schiff base). These complexes have been characterized by a variety of physico-chemical techniques. IR and H-1 NMR spectral evidence indicate that the Schiff base coordinates to the zinc(II) and cadmium(II) ions via the pyridine nitrogen atoms, the azomethine nitrogen atoms and the mercaptide sulfur atoms. The crystal and molecular structure of the zinc(II) complex has been determined by X-ray diffraction. The complex is a dimer in which the pyridine nitrogen atom,the azomethine nitrogen atom and the thiolate sulfur atom from one ligand coordinate to one of the zinc(II) ions whereas the azomethine and thiolate sulfur atoms from another ligand complete pentacoordination around the zinc(II) ion, the ligands being coordinated in their deprotonated forms. The coordination geometry about each zinc(II) can be considered as intermediate between a square-pyramid and trigonal-bipyramid. The cadmium(II) complex is also assigned with a dimeric structure. (C) 2003 Elsevier Ltd. All rights reserved.