An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Computationally efficient solution techniques for adsorption problems involving steep gradients in bidisperse particles

A piecewise uniform fitted mesh method turns out to be sufficient for the solution of a surprisingly wide variety of singularly perturbed problems involving steep gradients. The technique is applied to a model of adsorption in bidisperse solids for which two fitted mesh techniques, a fitted-mesh finite difference method (FMFDM) and fitted mesh collocation method (FMCM) are presented. A combination (FMCMD) of FMCM and the DASSL integration package is found to be most effective in solving the problems. Numerical solutions (FMFDM and FMCMD) were found to match the analytical solution when the adsorption isotherm is linear, even under conditions involving steep gradients for which global collocation fails. In particular, FMCMD is highly efficient for macropore diffusion control or micropore diffusion control. These techniques are simple and there is no limit on the range of the parameters. The techniques can be applied to a variety of adsorption and desorption problems in bidisperse solids with non-linear isotherm and for arbitrary particle geometry.

From Midden to Sieve: The Impact of Differential Recovery and Quantification Techniques on Interpretations of Shellfish Remains in Australian Coastal Archaeology

Experimental mechanical sieving methods are applied to samples of shellfish remains from three sites in southeast Queensland, Seven Mile Creek Mound, Sandstone Point and One-Tree, to test the efficacy of various recovery and quantification procedures commonly applied to shellfish assemblages in Australia. There has been considerable debate regarding the most appropriate sieve sizes and quantification methods that should be applied in the recovery of vertebrate faunal remains. Few studies, however, have addressed the impact of recovery and quantification methods on the interpretation of invertebrates, specifically shellfish remains. In this study, five shellfish taxa representing four bivalves (Anadara trapezia, Trichomya hirsutus, Saccostrea glomerata, Donax deltoides) and one gastropod (Pyrazus ebeninus) common in eastern Australian midden assemblages are sieved through 10mm, 6.3mm and 3.15mm mesh. Results are quantified using MNI, NISP and weight. Analyses indicate that different structural properties and pre- and postdepositional factors affect recovery rates. Fragile taxa (T. hirsutus) or those with foliated structure (S. glomerata) tend to be overrepresented by NISP measures in smaller sieve fractions, while more robust taxa (A. trapezia and P. ebeninus) tend to be overrepresented by weight measures. Results demonstrate that for all quantification methods tested a 3mm sieve should be used on all sites to allow for regional comparability and to effectively collect all available information about the shellfish remains.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Characterization of turbulent jets from high-aspect-ratio rectangular nozzles

Turbulent free jets issuing from rectangular slots with various high aspect ratios (15-120) are characterized. The centerline mean and rms velocities are measured using hot-wire anemometry over a downstream distance of up to 160 slot heights at a slot-height-based Reynolds number of 10000. Experimental results suggest that a rectangular jet with sufficiently high aspect ratio (> 15) may be distinguished between three flow zones: an initial quasi-plane-jet zone, a transition zone, and a final quasi-axisymmetric-jet zone. In the quasi-plane-jet zone, the turbulent velocity field is statistically similar, but not identical, to those of a plane jet. (c) 2005 American Institute of Physics.

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 mu M. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P-Na/P-Cl of 0.034. The halide permeability sequence was P-Cl > P-F > P-I > P-Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate(-), and gluconate(-), with the permeability sequence P-Cl > P-SCN > gluconaie. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 Angstrom.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G(1) (IgG(1)), IgG(2) and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG(3), IgG(4) and IgA. There were no differences in concentrations for IgG(1), IgG(3) and IgM. The IgG(2) concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG(4) concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG(4) and IgA was lowest in group 4, while IgG; seropositivity was almost exclusively seen in healthy patients iii groups 2, 4. These findings suggest that the presence of IgG(3) may reflect non-susceptibility to the disease, while lack of IgG(4) may be indicative of periodontal health and lack of infection.

Functional and structural characterization of isolated perfused stingray liver including effects of ischaemia/reperfusion

The morphological and functional characteristics of stingray liver were studied, including the effect of ischaemia/reperfusion. With an isolated perfused model, it was shown that the stingray liver was more resistant than the rat liver to ischaemia/reperfusion injury; this was consistent with the differing partial oxygen tensions usually present in the two species. This study confirmed that whereas stingray hepatocytes form tubules with central bile canaliculi as in other fish, the stingray liver has portal triads and a lobular architecture as in mammals. Apoptosis of hepatocytes, demonstrated in the normal liver, was only marginally enhanced by ischaemia/reperfusion. Resulting apoptotic bodies were phagocytized by macrophage-like cells in hepatocyte tubules. In contrast to rat liver, the stingray liver showed no necrosis after ischaemia-reperfusion. (C) 1998 W.B. Saunders Company Limited.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

New techniques for biopsy and culture of human olfactory epithelial neurons

Objective: To improve the success of culturing olfactory neurons from human nasal mucosa by investigating the intranasal distribution of the olfactory epithelium and devising new techniques for growing human olfactory epithelium in vitro. Design: Ninety-seven biopsy specimens were obtained from 33 individuals, aged 21 to 74 years, collected from 6 regions of the nasal cavity. Each biopsy specimen was bisected, and 1 piece was processed for immunohistochemistry or electron microscopy while the other piece was dissected further for explant culture. Four culture techniques were performed, including whole explants and explanted biopsy slices. Five days after plating, neuronal differentiation was induced by means of a medium that contained basic fibroblast growth factor. After another 5 days, cultures were processed for immunocytochemical analysis. Results: The probability of finding olfactory epithelium in a biopsy specimen ranged from 30% to 76%, depending on its location. The dorsoposterior regions of the nasal septum and the superior turbinate provided the highest probability, but, surprisingly, olfactory epithelium was also found anteriorly and ventrally on both septum and turbinates. A new method of culturing the olfactory epithelium was devised. This slice culture technique improved the success rate for generating olfactory neurons from 10% to 90%. Conclusions: This study explains and overcomes most of the variability in the success in observing neurogenesis in cultures of adult human olfactory epithelium. The techniques presented here make the human olfactory epithelium a useful model for clinical research into certain olfactory dysfunctions and a model for the causes of neurodevelopmental and neurodegenerative diseases.

Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor

The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB(1-2)), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short Linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of C alpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Pore structure tailoring of pillared clays with cation doping techniques

Techniques and mechanism of doping controlled amounts of various cations into pillared clays without causing precipitation or damages to the pillared layered structures are reviewed and discussed. Transition metals of great interest in catalysis can be doped in the micropores of pillared clay in ionic forms by a two-step process. The micropore structures and surface nature of pillared clays are altered by the introduced cations, and this results in a significant improvement in adsorption properties of the clays. Adsorption of water, air components and organic vapors on cation-doped pillared clays were studied. The effects of the amount and species of cations on the pore structure and adsorption behavior are discussed. It is demonstrated that the presence of doped Ca2+ ions can effectively aides the control of modification of the pillared clays of large pore openings. Controlled cation doping is a simple and powerful tool for improving the adsorption properties of pillared clay.