Recreational transit service to the California Santa Monica Mountains : final report /

Performed by Crain and Associates, Inc. under contract to the Transportation Systems Center.

A novel venom peptide from an endoparasitoid wasp is required for expression of polydnavirus genes in host hemocytes

Maternal factors introduced into host insects by endoparasitoid wasps are usually essential for successful parasitism. This includes polydnaviruses (PDVs) that are produced in the reproductive organ of female hymenopteran endoparasitoids and are injected, together with venom proteins, into the host hemocoel at oviposition. Inside the host, PDVs enter various tissue cells and hemocytes where viral genes are expressed, leading to developmental and physiological alterations in the host, including the suppression of the host immune system. Although several studies have shown that some PDVs are only effective when accompanied by venom proteins, there is no report of an active venom ingredient(s) facilitating PDV infection and/or gene expression. In this study, we describe a novel peptide (Vn1.5) isolated from Cotesia rubecula venom that is required for the expression of C. rubecula bracoviruses (CrBVs) in host hemocytes (Pieris rapae), although it is not essential for CrBV entry into host cells. The peptide consists of 14 amino acids with a molecular mass of 1598 Da. In the absence of Vn1.5 or total venom proteins, CrBV genes are not expressed in host cells and did not cause inactivation of host hemocytes.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier Ltd. All rights reserved.

Depolymerisation and biodegradation of a synthetic tanning agent by activated sludges, the bacteria Arthrobacter globiformis and Comamonas testosteroni, and the fungus Cunninghamella polymorpha

Degradation of a synthetic tanning agent CNSF (a condensation product of 2-naphthatenesulfonic acid (2-NSA) and formaldehyde) by four activated sludges, two previously characterised bacterial strains, Arthrobacter sp. 2AC and Comamonas sp. 4BC, and the fungus Cunninghamella polymorpha, was studied in batch culture at 25 degrees C by determining the changes in the concentrations of CNSF and its component monomers and oligomers (n2-n11). The loss of individual oligomers was correlated with the length of the NSA-CH2 chain. Approximately 25% of the total CNSF was degraded (i.e. mineralised) by the microbes contained in the four activated sludges and by the two bacterial isolates but with different lag phases and at different overall rates. The decline in CNSF concentration was due almost entirely to the biodegradation of the monomers (34.3% of CNSF) and, in particular, 2-NSA (27% of CNSF). There was no change in the n2-n 11 components. The growth of C. polymorpha, on the other hand, arose from extracellular depolymerisation of CNSF oligomers and the biodegradation of the lower molecular mass products. Between 38% and 42% of total CNSF was degraded by C. polymorpha at 25 degrees C. The order of oligomer degradation was inversely related to degree of polymerisation. Eighty percent and 90% of the n4 and n5 and 100% oligomers n6-n11 were degraded after 120 h. At a higher temperature (37 degrees C) oligomers n4-n11 were degraded completely after 120 h. A combination of biodegradation (75%) and sorption to fungal biomass (25%) accounted for the measured loss of all oligomers from the solution phase. The CNSF degradation rates and the volume of fungal biomass produced (and therefore the extent of biosorption) were dependent on the presence of a second carbon source (both optimum at glucose 5 g/l). This is the first report that identifies and distinguishes between depolymerisation, sorption and biodegradation processes in the removal of CNSF and its component oligomers. The use of combinations of the depolymerising fungus C. polymorpha, and the monomer-degrading bacteria, Arthrobacter sp. 2AC and Comamonas sp. 4BC, have potential for wastewater treatment.

Characterization of polyamine oxidase from the aquatic nitrogen-fixing fern Azolla imbricata

Biochemical properties of a polyamine oxidase (PAO; EC 1.5.3.3) purified from the aquatic nitrogen-fixing fern Azolla imbricata (Roxb.) Nak. were studied. The native molecular mass of the enzyme estimated by Sephadex G 200 get filtration was 66.2 kDa. SDS-PAGE gave a single protein band corresponding to a molecular mass of 65.5 kDa. The light yellow enzyme had absorption maxima at 278, 372 and 454 nm with 1 mol FAD per mole enzyme molecule as its cofactor. The PAO was active on both the triamine Spd and the tetraamine Spm as substrates. However, it was inactive on the diamines Put and Cad. It had a pH optimum of 6.5 for both Spd and Spm. The K-m(S) for Spd and Spm were 6.71 x 10(-2) and 1.13 x 10(-1) nM, respectively. Pre-incubation with 10 mM of K+ (KCl), Ca2(+) (CaCl2) or Mg2+ (MgCl2) had no effect on PAO activity. However, 10 mM Cu2+ (CuCl2), Mn2+ (MnCl2) and Fe2+ (FeSO4) inhibited enzyme activity by 37%, 43% and 58%, respectively. The metal chelator EDTA (10 mM), the carbonyl reagent hydroxylamine (0.5 mM) and the sulfhydryl reagent p-chloro-mercuribenzoate (0.5 mM) had no effect on PAO activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Effects of structural variation in xyloglucan polymers on interactions with bacterial cellulose

A cellulose/xyloglucan framework is considered to form the basis for the mechanical properties of primary plant cell walls and hence to have a major influence on the biomechanical properties of growing, fleshy plant tissues. In this study, structural variants of xyloglucan have been investigated as components of composites with bacterial cellulose as a simplified model for the cellulose/xyloglucan framework of primary plant cell walls. Evidence for molecular binding to cellulose with perturbation of cellulose crystallinity was found for all xyloglucan types. High molecular mass samples gave homogeneous centimeter-scale composites with extensive cross-linking of cellulose with xyloglucan. Lower molecular mass xyloglucans gave heterogeneous composites having a range of microscopic structures with little, if any, cross-linking. Xyloglucans with reduced levels of galactose substitution had evidence of self-association, competitive with cellulose binding. At comparable molecular mass, fucose substitution resulted in a modest promotion of microscopic features characteristic of primary cell walls. Taken together, the data are evidence that galactose substitution of the xyloglucan core structure is a major determinant of cellulose composite formation and properties, with additional fucose substitution acting as a secondary modulator. These conclusions are consistent with reported structural and mechanical properties of Arabidopsis mutants lacking specific facose and/or galactose residues.

In vivo demonstration of load-induced fluid flow in the rat tibia and its potential implications for processes associated with functional adaptation

Load-induced extravascular fluid flow has been postulated to play a role in mechanotransduction of physiological loads at the cellular level. Furthermore, the displaced fluid serves as a carrier for metabolites, nutrients, mineral precursors and osteotropic agents important for cellular activity. We hypothesise that load-induced fluid flow enhances the transport of these key substances, thus helping to regulate cellular activity associated with processes of functional adaptation and remodelling. To test this hypothesis, molecular tracer methods developed previously by our group were applied in vivo to observe and quantify the effects of load-induced fluid flow under four-point-bending loads. Preterminal tracer transport studies were carried out on 24 skeletally mature Sprague Dawley rats. Mechanical loading enhanced the transport of both small- and larger-molecular-mass tracers within the bony tissue of the tibial mid-diaphysis. Mechanical loading showed a highly significant effect on the number of periosteocytic spaces exhibiting tracer within the cross section of each bone. For all loading rates studied, the concentration of Procion Red tracer was consistently higher in the tibia subjected to pure bending loads than in the unloaded, contralateral tibia, Furthermore, the enhancement of transport was highly site-specific. In bones subjected to pure bending loads, a greater number of periosteocytic spaces exhibited the presence of tracer in the tension band of the cross section than in the compression band; this may reflect the higher strains induced in the tension band compared with the compression band within the mid-diaphysis of the rat tibia. Regardless of loading mode, the mean difference between the loaded side and the unloaded contralateral control side decreased with increasing loading frequency. Whether this reflects the length of exposure to the tracer or specific frequency effects cannot be determined by this set of experiments. These in vivo experimental results corroborate those of previous ex vivo and in vitro studies, Strain-related differences in tracer distribution provide support for the hypothesis that load-induced fluid flow plays a regulatory role in processes associated with functional adaptation.

Synthesis and characterisation of pillared clay catalysts for hydro-cracking of heavy liquid fuels

A range of chromia pillared montmorillonite and tin oxide pillared laponite clay catalysts, as well as new pillared clay materials such as cerium and europium oxide pillared montmorillonites were synthesised. Methods included both conventional ion exchange techniques and microwave enhanced methods to improve performance and/or reduce preparation time. These catalytic materials were characterised in detail both before and after use in order to study the effect of the preparation parameters (starting material, preparation method, pillaring species, hydroxyl to metal ratio etc.) and the hydro cracking procedure on their properties. This led to a better understanding of the nature of their structure and catalytic operation. These catalysts were evaluated with regards to their performance in hydrocracking coal derived liquids in a conventional microbomb reactor (carried out at Imperial College). Nearly all catalysts displayed better conversions when reused. The chromia pillared montmorillonite CM3 and the tin oxide pillared laponite SL2a showed the best "conversions". The intercalation of chromium in the form of chromia (Cr203) in the interlayer clearly increased conversion. This was attributed to the redox activity of the chromia pillar. However, this increase was not proportional to the increase in chromium content or basal spacing. In the case of tin oxide pillared laponite, the catalytic activity might have been a result of better access to the acid sites due to the delaminated nature of laponite, whose activity was promoted by the presence of tin oxide. The manipulation of the structural properties of the catalysts via pillaring did not seem to have any effect on the catalysts' activity. This was probably due to the collapse of the pillars under hydrocracking conditions as indicated by the similar basal spacing of the catalysts after use. However, the type of the pillaring species had a significant effect on conversion. Whereas pillaring with chromium and tin oxides increased the conversion exhibited by the parent clays, pillaring with cerium and europium oxides appeared to have a detrimental effect. The relatively good performance of the parent clays was attributed to their acid sites, coupled with their macropores which are able to accommodate the very high molecular mass of coal derived liquids. A microwave reactor operating at moderate conditions was modified for hydro cracking coal derived liquids and tested with the conventional catalyst NiMo on alumina. It was thought that microwave irradiation could enable conversion to occur at milder conditions than those conventionally used, coupled with a more effective use of hydrogen. The latter could lead to lower operating costs making the process cost effective. However, in practice excessive coke deposition took place leading to negative total conversion. This was probably due to a very low hydrogen pressure, unable to have any hydro cracking effect even under microwave irradiation. The decomposition of bio-oil under microwave irradiation was studied, aiming to identify the extent to which the properties of bio-oil change as a function of time, temperature, mode of heating, presence of char and catalyst. This information would be helpful not only for upgrading bio-oil to transport fuels, but also for any potential fuel application. During this study the rate constants of bio-oil's decomposition were calculated assuming first order kinetics.

Purification and characterisation of two cancer cachexia factors

Cachexia is a wasting phenomenon that often accompanies malignant disease. Its manifestation is associated with shortened survival and reduced responsiveness to anti-tumour therapy and as yet there is no established, effective amelioratory treatment. The MAC 16 model of cancer cachexia has been shown by many studies to closely mirror the human condition. Thus, cachexia is mediated by the presence of a small, slow-growing solid tumour that is mainly resistant to chemotherapy. In addition, the condition is largely attributable to aberrations in metabolic processes, while weight loss due to anorexia is negligible. Cachexia induced by the MAC 16 tumour, has been shown to be mediated by the production of tumour-derived circulatory catabolic factors, and the further elucidation of the structure of these molecules contributes towards the main content of this report. Thus, a factor with in vitro lipid-mobilising activity has been purified from the MAC 16 tumour, and has been found to have similarities to tumour-derived lipolytic factors published to date. Further work demonstrated that this factor was also purifiable from the urine of a patient with pancreatic cancer, and that it was capable of inducing weight loss in non tumour-bearing mice. Sequence analysis of the homogeneous material revealed an identity to Zn-α-2-glycoprotein, the significance of which is discussed. An additional factor, first detected as a result of its specific reactivity with a monoclonal antibody produced by fusion of splenocytes from MAC 16 tumour-bearing mice with mouse BALB/c myeloma cells, was identified as a co-purificant during studies to isolate the lipolytic factor. Subsequent purification of this material to homogeneity resulted in the determination of 18 of the N-terminal amino acids and revealed the highly glycosylated nature of its structure. Thus, this material (P24) was found to have an apparent molecular mass of 24kD of which 2kD was due to protein, while the remainder (92%) was due to the presence of carbohydrate groups. Sequence analysis of the protein core of P24 revealed an identity with Streptococcal pre-absorbing antigen (PA-Ag) in 11 of the amino acids, and the significance of this is discussed. P24 was shown to induce muscle protein breakdown in vitro and to induce cachexia in vivo, as measured by the depletion of fat (29%) and muscle (14%) tissue in the absence of a reduction of food and water intake. Further studies revealed that the same material was purifiable from the urine of patients with pancreatic cancer and was found to be detectable in the urine of cancer patients with weight loss greater than l.Skg/month. Thus, cachexia induced by the MAC 16 tumour in mice and by malignant disease in humans may be induced by similar mediators. Attempts to isolate the gene for P24 using information provided by the N-terminal protein sequence were unsuccessful. This was probably due to the low abundance o[ the material, as determined by protein purification studies; and the nature of the amino acids of the N-terminal sequence, which conferred a high degree o[ degeneracy to the oligonucleotides designed for the polymerase chain reaction.

Cellular signalling pathways involved in muscle atrophy in cancer cachexia

Cancer cachexia encompases severe weight loss, characterised by the debilitating atrophy of adipose and skeletal muscle mass. Skeletal muscle proteolysis in cancer cachexia is mediated by a sulphated glycoprotein with a relative molecular mass of 24kDa, termed Proteolysis-Inducing Factor (PIF). PIF induced a significant increase in protein degradation, peaking at 4.2nM PIF (p<0.001), ‘chymotrypsin-like’ activity of the proteasome (p<0.001) and increased expression of components of the ATP-ubiquitin dependent proteolytic pathway. This was attenuated in vitro by pre-incubation with the PKC inhibitor calphostin C (100µM) and NF-kB the inhibitors SN50 (18µM), curcumin (50µM) and resveratrol (30µM), 2 hours prior to the addition of PIF. In vivo studies found the IKK inhibitor resveratrol (1mg/kg) to be successful in attenuating protein degradation (p<0.001) and upregulation of ubiquitin-dependent proteolysis in MAC16 tumour bearing mice. C2C12 myoblasts transfected with mutant IkBα and PKCα inserts did not elicit a PIF-induced response, suggesting the importance of the transcription factor NF-kB and PKC  involvement in PIF signal transduction. 15(S)-HETE acts as an intracellular mediator of PIF and exerts an effect through the activation of PKC and subsequently IKK, which phosphorylates IkBα and allows NF-kB to migrate to the nucleus. This effect was negated with the PKC inhibitor calphostin C (300nM). A commercially produced PIF receptor antibody was raised in rabbits immunised with a peptide containing the partial N-terminal sequence of the PIF receptor. The PIF receptor antibody was successful in attenuating the PIF-induced increase in skeletal muscle catabolism and protein degradation in vitro at 10µg/ml (p<0.001) and 3.47mg/kg in vivo (p<0.001). The data suggest great potential in the development of this antibody as a therapy against cancer cachexia.

Regulation of mucus secretion by cells isolated from the rat gastric mucosa

     This study was undertaken to further understanding of the mechanisms which regulate mucus secretion by rat stomach cells. Particular objectives were: (i) to develop and use a radiochemical assay to estimate the secretion of mucin by a suspension of gastric mucosal cells in vitro, (ii) to develop and use a solid-phase enzyme immunoassay (EIA) to study the regulation of the release of bulk gastric mucin from the isolated cells and (iii) to compare the results obtained with the two procedures.      Cells were isolated by exposure of gastric mucosa to pronase and EDTA. Cell suspensions were preincubated with D-[6-3H]glucosamine. [3H]-labelled material of high molecular mass released into the incubation medium, was purified by Fast Protein Liquid Chromatography, and appeared to be gastric mucin. Some unidentified [3H]-labelled material of lower molecular mass was also found in the medium. Release of [3H]-labelled high molecular mass material was essentially linearly related to time. Secretin, isoprenaline and carbachol stimulated release of [3H]-labelled high molecular mass material. The half-maximally effective concentrations of secretin and isoprenaline were 2.3nM and 34nM respectively. Histamine, gastrin and epidermal growth factor were without effect.      A rabbit polyclonal antibody was raised by using purified 'native' rat gastric mucin as immunogen. The antibody preparation appeared specific for rat gastric mucin and was used to establish a quantitative solid-phase EIA. Release of bulk mucin was essentially linearly related to time. Phorbol-12-myristate-13-acetate (PMA), forskolin and A23187 dose-dependently stimulated bulk mucin release. Synergistic interactions were observed between PMA and forskolin, and PMA and A23187. Secretin and isoprenaline were confirmed as mucin secretogogues.      In conclusion gastric mucin release was investigated for the first time by using a suspension of gastric mucosal cells. Two different assay procedures were developed. Some pathways and agents responsible for controlling mucin secretion were identified.

Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis

Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.

Phagocyte oxidation of phospholipids: comparison of hydroperoxide, chlorohydrin and epoxyisoprostane formation by LC-MS

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by isolated human polymorphonuclear and mononuclear leukocytes, to provide a model of lipid oxidation in the absence of competing protein. PMA-stimulated cells were incubated with phospholipid vesicles contammg dipalmitoyl phosphatidylcholine (DPPC), palmitoyl-arachidonoyl phosphatidylcholine (PAPC), and stearoyl-oleoyl phosphatidylcholine (SOPC), before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. In this system, oxidized phosphatidylcholines elute earlier than the native lipids owing to their decreased hydrophobicity, and can be identified according to their molecular mass. The formation of monohydroperoxides of P APC was observed routinely, together with low levels of hydroxides, but no chlorohydrin derivatives of P APC or SOPC were detected. However, the major oxidized product occurred at 828 m/z, and was identified as I-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. These results show that phagocytes triggered by PMA cause oxidative damage to lipids predominantly by free radical mechanisms, and that electrophilic addition involving HOCl is not a major mechanism of attack. The contribution of myeloperoxidase and metal ions to the oxidation process is currently being investigated, and preliminary data suggest that myeloperoxidase-derived oxidants are responsible for the epoxyisoprostane phospholipid formation. The identification of an epoxyisoprostane phospholipid as the major product following phagocyte-induced phospholipid oxidation is novel and has implications for phagocyte involvement in atherogenesis.

Transglutaminases:nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.