Compatibility with Jet A-1 of a GCL Subjected to Freeze–Thaw Cycles

Needle-punched geosynthetic clay liner (GCL) specimens subjected to 0, 5, and 12 freeze–thaw cycles in the laboratory, and GCL specimens recovered from a composite barrier wall in the Canadian Arctic after 1 and 3 years were examined to assess the hydraulic conductivity/permeability with respect to both deionized deaired water and Jet A-l. The GCL specimens recovered from the field after 3 years had a hydraulic conductivity with respect to water that was approximately 30% less than that of the GCL specimens subjected to 12 initial freeze–thaw cycles in the laboratory, suggesting that the laboratory conditions are more severe than field conditions. The combined effects of both the freeze–thaw cycles and Jet A-l permeation increased the permeability. This increase is attributed to the creation of macropores in the GCL due to freezing and to an expansion of free-pore space due to contraction of the double layer caused by permeation of Jet A-l. Although there was an increase in permeability due to the combined effect of freeze–thaw and permeation by Jet A-l, the effect was relatively small and the results suggest that the GCL continued to exhibit good performance as a hydraulic barrier when subject to extreme climatic conditions and hydrocarbons both in the laboratory and in the field.     

Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle

L-Asparaginase derived from Erwinia chrysanthemi which is being investigated as an alternative to E. coli for the treatment of lymphoblastic leukaemia has been found in our laboratory to lose activity upon exposure to consecutive freeze-thaw cycles. An investigation was undertaken using several techniques to characterize fully the physicochemical changes L-Asparaginase is undergoing during freeze-thaw cycling leading to the loss of its activity. A total protein assay suggested that the loss of some enzyme activity was a result of protein precipitation. Circular dichroism (CD) studies showed a decrease of alpha-helical structure with a concomitant increase in beta sheet and random coil content, suggesting alterations in the secondary structure leading to unfolding, the first step of denaturation processes. The elution profiles obtained from size-exclusion chromatography (SEC) studies indicated the formation of several species during the process of freezing and thawing. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed bands corresponding to 1-3 kDa and 32 kDa, suggesting that some of the species are fragments and shortened monomers resulting from cleavage of monomers. The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of polydispersed fragments ranging from 900 Da to 3 kDa and a small fraction of aggregates corresponding to 300 kDa. A scheme was proposed to explain the cascade of events leading to the loss of soluble protein and accompanying loss of enzyme activity. Tetramers of the enzyme dissociate into monomers some of which are cleaved into small fragments. The shortened monomers then aggregate and precipitate.     

Simulating the Effect of Liquid CO2 on Cryptosporidium parvum Oocysts in Aquifer Material

The effects of liquid CO2 injection on the viability of Cryptosporidium parvum oocysts were evaluated. A laboratory study was designed to test the effects of saturated CO2, freeze–thaw cycles and different freezing protocols on C. parvum oocysts in aquifer material. Oocysts were exposed to a saturated solution of CO2 at room temperature for 1-, 4-, 8-, and 12-h intervals and their viability was compared with controls. One- and three-cycle freeze–thaw experiments on oocyst survival were conducted. Inactivation of oocysts was assessed for: (1) rapid freezing and rapid thawing and (2) gradual freezing and rapid thawing. Exposure to 1 atm of CO2 in water at room temperature had a negligible effect on oocyst viability. Average oocyst viability after the one- and three-cycle freeze–thaw experiments was 24.7 and 2.7%, repsectively. The average oocyst viability associated with the rapid freeze–thaw and gradual freeze–thaw experiments was 11.3 and 26.2%, respectively. Freezing associated with injection of liquid CO2 into aquifers would be the factor inactivating oocysts; to cause a 3-log decrease in oocyst viability multiple injections may be required.     

Performance Evaluation of Reinforced Concrete Bridge Columns Wrapped with Fiber Reinforced Polymers

This study investigates the performance of new bridge columns wrapped with fiber reinforced polymers (FRP) when exposed to aggressive environmental conditions. This has been accomplished through field monitoring and laboratory tests. As part of the field monitoring, temperature data were collected at various locations of bridge columns. In addition, visual inspection of two bridges was performed periodically for over a period of two years. No evidence of deterioration of the FRP wraps was detected during that period. Laboratory tests were performed to investigate how FRP wraps protect reinforced concrete columns from corrosion, and freeze–thaw laboratory tests were conducted to study the impact of temperature cycles on the mechanical behavior of FRP-wrapped columns. From the corrosion experimental tests, it was found that FRP provides excellent protection against aggressive agents (salty water or moisture) even when a single layer is used. Compression tests were conducted on specimens subjected to freeze–thaw cycles. It was found that minor thermal cycles have no effect on the performance of FRP-wrapped concrete specimens. However, for large thermal cycles, some degradation of ductility in the axial and the hoop directions was observed.     

Partial purification of box jellyfish (Chironex fleckeri) nematocyst venom isolated at the beachside

Chironex fleckeri, the northern Australian box jellyfish produces one of, if not, the most potent animal venoms. Study of the venom has been hampered by the limits of the animals' range and the venom's thermolability. Using retained lethality and native polyacrylamide gel electrophoresis (NPAGE), we show that lyophilization of autolysis isolated nematocysts is an effective method of transporting the venom. In addition, Sephadex G-200 chromatography, spin concentration, and NPAGE fail to demonstrate the presence of a 600 kDa protein to which the bulk of the lethal activity has been ascribed. Sodium dodecyl sulfate capillary electrophoresis of crude venom yields several protein bands with a molecular weight range of 30-200 kDa. Freeze-thaw studies show a loss of activity and NPAGE bands after two freeze thaw cycles.     

Low-Cycle Fatigue Testing of High-Performance Concrete Bonded Overlay–Bridge Deck Slab Systems

Plain and fibrous latex modified concrete and microsilica concrete overlays with acceptable laboratory strength and durability characteristics were installed on a full-scale prototype bridge deck for field performance evaluation. After 1?year of exposure to drying shrinkage, temperature variations, and freeze–thaw cycles, no cracking or debonding were observed in the overlays. The bond strengths at 28?days and at 1?year were acceptable for all overlay types due to the excellent curing and surface preparation using water-jet blasting. The prototype bridge was then statically tested before and after applying low-cycle fatigue loading simulating AASHTO HS20 truck service load, overload, and ultimate load. Minor bond strength deterioration at the maximum negative moment region and slight bridge stiffness degradation were observed after each load case. Significant enhancement in the bridge stiffness was observed after installation of the overlays. Also, the overlay with synthetic fibers showed better crack bridging action than the overlay with steel fibers.     

Bond Performance of Near-Surface-Mounted FRP Bars

Near-surface-mounted (NSM) reinforcement has become a well-known method for strengthening existing concrete structures. The bond between the NSM reinforcing bars and concrete is the key factor in the NSM technique. In the NSM technique, there are two bond interfaces: one between the NSM bar and the adhesive, and the other between the adhesive and the concrete. For this technique to perform efficiently, these two interfaces need to be investigated. On the other hand, concrete structures that require rehabilitation are often exposed to aggressive environments. Many of these environments are related to cold-climate conditions as can be found in Canada. Environmental factors including freeze/thaw action, exposure to deicing salts, and sustained low temperatures combine to attack the integrity of repaired structures. Consequently, repair materials for the Canadian infrastructure must be able to withstand these harsh conditions for prolonged periods of time. A total of 80 NSM-fiber-reinforced polymer (FRP) bars installed in C-shaped concrete specimens were tested in pull-out setup to failure. Sixty specimens were tested at normal room temperature, while the remaining 20 specimens were tested after conditioning in an environmentally controlled chamber for 200 freeze/thaw cycles. The dimensions of the specimens were designed, upon a preliminary phase of testing, to ensure that no transverse cracking would occur in the specimen before bond failure of the NSM bar. The results are presented in term of failure load, average bond stress, strains in FRP bar, end slip, and mode of failure. A bond-slip model was proposed for the used FRP bars.     

Primary structure of a cadmium-induced metallothionein from the insect Orchesella cincta (Collembola)

The induction of metallothionein was studied in the springtail Orchesella cincta (Collembola), a species of insect living in forest soils. Upon dietary exposure to Cd, two Cd-binding, cysteine-rich peptides were isolated from whole-body homogenates, using gel filtration and reversed-phase FPLC. Mass spectrometric analysis revealed that the molecular masses of these peptides were 2989 Da and 4139 Da, respectively. Amino acid sequencing of the 2989-Da peptide resulted in a sequence typical for a metallothionein. Sequencing of the 4139-Da protein was unsuccessful, probably due to N-terminal blockage. Using different PCR techniques (3' and 5' RACE) with (degenerate) primers based on the identified amino acid sequence of the 2989 Da peptide, a metallothionein cDNA was isolated. The sequence of this cDNA potentially codes for a protein of 77 amino acids. The 2989 Da peptide corresponds to the C-terminal part of this protein. The 4139-Da protein is probably encoded by the N-terminal part of this protein. These results suggest that the identified peptides are products of one gene, and that the primary gene product is subject to post-translational processing. The deduced amino acid sequence of the O. cincta metallothionein shows low sequence similarity with metallothioneins from Drosophila. The similarity between O. cincta MT and MTs of invertebrates is not higher than that between O. cincta and vertebrates.     

Antigenic activity of angiotensin II and its fragments

The antigenic activity of angiotensin and its seven fragments has been studied in cross-reaction with specific antibodies, elicited to angiotensin and its fragments: C-terminal hexapeptide and middle tetrapeptide. It has been found that all the fragments studied possess certain affinity for antibodies elicited to angiotensin, C-terminal hexapeptide and middle tetrapeptide. The middle tetrapeptide was identified to be the immunologically active centre of the angiotensin molecule.     

Location of the complement factor H binding site on streptococcal M6 protein

The surface M protein of group A streptococci binds factor H, a regulatory protein of the alternative complement pathway, which may contribute to the antiphagocytic activity of the M molecules. To locate the factor H binding domain in the alpha-helical coiled-coil structure of the M molecule, the M protein was cleaved with pepsin at pH 5.8, which separates the molecule approximately in half. Western blot (immunoblot), amino acid sequence, and mass spectrometric analyses revealed that factor H bound to a 14.6-kDa C-terminal fragment of the M molecule. Competitive inhibition of factor H binding to the 14.6-kDa fragment with M protein peptides localized the binding site to amino acids 256 to 292. This segment is located within the surface-exposed region of the M6 protein, identified as the C-repeat region, whose sequence is conserved among heterologous M and M-like molecules. These studies also identified a second pepsin-susceptible site with the sequence ELAK located within the cell wall-associated region of the M molecule.     

Alpha1-antichymotrypsin gene polymorphism and susceptibility to Parkinson''s disease

A clinical strain of Vibrio cholerae non-O1 non-O139 isolated in France produced a new beta-lactamase with a pI of 5.35. The purified enzyme, with a molecular mass of 33,000 Da, was characterized. Its kinetic constants show it to be a carbenicillin-hydrolyzing enzyme comparable to the five previously reported CARB beta-lactamases and to SAR-1, another carbenicillin-hydrolyzing beta-lactamase that has a pI of 4.9 and that is produced by a V. cholerae strain from Tanzania. This beta-lactamase is designated CARB-6, and the gene for CARB-6 could not be transferred to Escherichia coli K-12 by conjugation. The nucleotide sequence of the structural gene was determined by direct sequencing of PCR-generated fragments from plasmid DNA with four pairs of primers covering the whole sequence of the reference CARB-3 gene. The gene encodes a 288-amino-acid protein that shares 94% homology with the CARB-1, CARB-2, and CARB-3 enzymes, 93% homology with the Proteus mirabilis N29 enzyme, and 86.5% homology with the CARB-4 enzyme. The sequence of CARB-6 differs from those of CARB-3, CARB-2, CARB-1, N29, and CARB-4 at 15, 16, 17, 19, and 37 amino acid positions, respectively. All these mutations are located in the C-terminal region of the sequence and at the surface of the molecule, according to the crystal structure of the Staphylococcus aureus PC-1 beta-lactamase.     

Fatigue Performance of CFRP Strengthened RC Beams under Environmental Conditioning and Sustained Load

Carbon fiber-reinforced polymers (CFRPs) have become increasingly important in recent years in bridge rehabilitation. Significant research has been done on the static behavior of CFRP-strengthened reinforced concrete (RC) structures; however, the fatigue behavior of such structures with interface defects subjected to harsh environmental conditions still needs to be investigated. Hence, an experimental program has been carried out to investigate the fatigue behavior, under a load range, which generates service load stress levels, of RC beams strengthened with CFRP fabrics. The effect of aggressive environments was studied by subjecting the test members to freeze–thaw, extreme temperature, ultraviolet light exposure, and relative humidity cycles. All beams survived 2 million fatigue cycles without showing significant bond degradation between composite and substrate. However, significant flexural stiffness degradation was observed in the conditioned specimens. The presence of defects also affected specimen stiffness; however, limited growth in defect size was observed due to fatigue cycling.     

Anatomy of the pleura

Earlier studies involving water-mediated transformations in lysozyme and ribonuclease A have shown that the overall movements in the protein molecule consequent to the reduction in the amount of surrounding water are similar to those that occur during enzyme action, thus highlighting the relationship among hydration, plasticity, and action of these enzymes. Monoclinic lysozyme retains its crystallinity even when the level of hydration is reduced further below that necessary for activity (about 0.2 gram of water per gram of protein). In order to gain insights into the role of water in the stability and the plasticity of the protein molecule and the geometrical basis for the loss of activity that accompanies dehydration, the crystal structures of monoclinic lysozyme with solvent contents of 17.6%, 16.9%, and 9.4% were determined and refined. A detailed comparison of these forms with the normally hydrated forms show that the C-terminal segment (residues 88-129) of domain I and the main loop (residues 65-73) in domain II exhibit large deviations in atomic positions when the solvent content is reduced, although the three-dimensional structure is essentially preserved. Many crucial water bridges between different regions of the molecule are conserved in spite of differences in detail, even when the level of hydration is reduced well below that required for activity. The loss of activity that accompany dehydration appears to be caused by the removal of functionally important water molecules from the active-site region and the reduction in the size of the substrate binding cleft.     

A site of interaction between pleckstrin's PH domains and G beta gamma

Pleckstrin is a 40 kDa substrate for protein kinase C found in platelets and neutrophils. Based upon its sequence, pleckstrin contains two of the recently-described PH domains that are thought to be binding motifs for phosphatidyl 4,5-bisphosphate (PIP2) and/or G protein beta gamma heterodimers (G beta gamma). In the present studies we have examined the interaction between pleckstrin and G beta gamma by incubating pleckstrin fusion proteins with lysates from human platelets. In this analysis, both the N-terminal and C-terminal PH domains from pleckstrin bound G beta gamma in vitro, as did peptides containing as little as the first 30 residues of the C-terminal pleckstrin PH domain. Introduction of a point mutation into this region, analogous to the mutation in the Btk PH domain that causes X-linked immunodeficiency disease (XID) in mice, dramatically disrupted this interaction. We propose that pleckstrin may interact with G beta gamma, and that one potential site for this interaction involves the first 30 residues of pleckstrin's C-terminal PH domain.     

DNA ploidy analysis in breast carcinoma. Comparison of unfixed and fixed tissue analyzed by image and flow cytometry

We present here the isolation and characterization of four antimicrobial peptides produced by a European bumblebee Bombus pascuorum. A 51-residue insect defensin was characterized which, like the Apis mellifera defensins, had a highly conserved 12-residue extension to its C-terminal compared to defensins from other insects. Monoisotopic mass analysis of the C-terminal of B. pascuorum defensin confirmed that this molecule was C-terminally amidated. This defensin showed strong anti-Gram-positive activity and some anti-fungal activity; also, in contrast to other insect defensins, it showed anti-Gram-negative activity. A 17-residue apidaecin was characterized, showing anti-Gram-negative activity, and differing by a single amino acid substitution from the A. mellifera apidaecin. A 39-residue abaecin was isolated, the largest proline-rich antimicrobial peptide characterized to date, which showed activity against both Gram-negative and Gram-positive bacteria. Finally, we isolated an N-terminally blocked molecule, with a molecular mass of 10,122 Da, which showed activity against Gram-negative bacteria only. These characteristics are reminiscent of hymenoptaecin from the honeybee A. mellifera, but a definitive characterization of this molecule awaits further work. No evidence of lysozyme activity was found in the haemolymph of challenged or naive B. pascuorum.     

Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.     

Membrane topology and glycosylation of the human multidrug resistance-associated protein

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.     

Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.