Phagotopes derived by antibody screening of phage-displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII-C1, to type II collagen

Antibody screening of phage-displayed random peptide libraries to identify mimotopes of conformational epitopes is promising. However, because interpretations can be difficult, an exemplary system has been used in the present study to investigate whether variation in the peptide sequences of selected phagotopes corresponded with variation in immunoreactivity. The phagotopes, derived using a well-characterized monoclonal antibody, CII-C1, to a known conformational epitope on type II collagen, C1, were tested by direct and inhibition ELISA for reactivity with CII-C1. A multiple sequence alignment algorithm, PILEUP, was used to sort the peptides expressed by the phagotopes into clusters. A model was prepared of the C1 epitope on type II collagen. The 12 selected phagotopes reacted with CII-C1 by both direct ELISA (titres from < 100-11 200) and inhibition ELISA (20-100% inhibition); the reactivity varied according to the peptide sequence and assay format. The differences in reactivity between the phagotopes were mostly in accord with the alignment, by PILEUP, of the peptide sequences. The finding that the phagotopes functionally mimicked the C1 epitope on collagen was validated in that amino acids RRL at the amino terminal of many of the peptides were topographically demonstrable on the model of the C1 epitope. Notably, one phagotope that expressed the widely divergent peptide C-IAPKRHNSA-C also mimicked the C1 epitope, as judged by reactivity in each of the assays used: these included cross-inhibition of CII-C1 reactivity with each of the other phagotopes and inhibition by a synthetic peptide corresponding to that expressed by the most frequently selected phagotope, RRLPFGSQM. Thus, it has been demonstrated that multiple phage-displayed peptides can mimic the same epitope and that observed immunoreactivity of selected phagotopes with the selecting mAb can depend on the primary sequence of the expressed peptide and also on the assay format used.

Rheumatoid arthritis sera react with a phage-displayed peptide selected by a monoclonal antibody to type II collagen that has homology to EBNA-1

Antibodies to type II collagen, and to Epstein Barr virus nuclear antigen-1 (EBNA-1) have been associated with rheumatoid arthritis (RA). In studies involving probing of phage-displayed random peptide libraries with an antibody to type II collagen, CII-C1, we observed that among 17 phagotopes selected 5 expressed peptides with homology with the sequence of EBNA-1. The residues in common were RLPFG. Hence we tested sera from 50 patients with RA, of whom 26 had antibodies to native type II collagen, and 43 healthy controls, for reactivity by ELISA with a phagotope selected 4 times, which expressed the peptide RRLPFGSQM. Eight RA sera (16%) but no normal sera reacted with the phagotope (p = 0.025). This reactivity could not be correlated with reactivity of RA sera with EBNA-1 by semi-quantitative western blot, with which reactivity occurred in 78% of RA patients and 81% of controls. Evidence for molecular mimicry was not found insofar as the phagotope did not inhibit reactivity of RA sera with EBNA-1 and CII-C1 was not reactive with EBNA-1. We conclude that the reactivity of the RA sera with the phagotope is most likely due to the phagotope being a mimic of an epitope of type II collagen for a proportion of RA sera.

Missing in space: An evaluation of imputation methods for missing data in spatial analysis of risk factors for type II diabetes

Background Spatial analysis is increasingly important for identifying modifiable geographic risk factors for disease. However, spatial health data from surveys are often incomplete, ranging from missing data for only a few variables, to missing data for many variables. For spatial analyses of health outcomes, selection of an appropriate imputation method is critical in order to produce the most accurate inferences. Methods We present a cross-validation approach to select between three imputation methods for health survey data with correlated lifestyle covariates, using as a case study, type II diabetes mellitus (DM II) risk across 71 Queensland Local Government Areas (LGAs). We compare the accuracy of mean imputation to imputation using multivariate normal and conditional autoregressive prior distributions. Results Choice of imputation method depends upon the application and is not necessarily the most complex method. Mean imputation was selected as the most accurate method in this application. Conclusions Selecting an appropriate imputation method for health survey data, after accounting for spatial correlation and correlation between covariates, allows more complete analysis of geographic risk factors for disease with more confidence in the results to inform public policy decision-making.

Probabilistic assessment of herbicide runoff from Japanese rice paddies: The effects of local meteorological conditions and site-specific water management

Uncertainty assessments of herbicide losses from rice paddies in Japan associated with local meteorological conditions and water management practices were performed using a pesticide fate and transport model, PCPF-1, under the Monte Carlo (MC) simulation scheme. First, MC simulations were conducted for five different cities with a prescribed water management scenario and a 10-year meteorological dataset of each city. The effectiveness of water management was observed regarding the reduction of pesticide runoff. However, a greater potential of pesticide runoff remained in Western Japan. Secondly, an extended analysis was attempted to evaluate the effects of local water management and meteorological conditions between the Chikugo River basin and the Sakura River basin using uncertainty inputs processed from observed water management data. The results showed that because of more severe rainfall events, significant pesticide runoff occurred in the Chikugo River basin even when appropriate irrigation practices were implemented. © Pesticide Science Society of Japan.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Catalysis and mechanism of malonyl transferase activity in type II fatty acid biosynthesis acyl carrier proteins

One of the unexplored, yet important aspects of the biology of acyl carrier proteins (ACPs) is the self-acylation and malonyl transferase activities dedicated to ACPs in polyketide synthesis. Our studies demonstrate the existence of malonyl transferase activity in ACPs involved in type II fatty acid biosynthesis from Plasmodium falciparum and Escherichia coli. We also show that the catalytic malonyl transferase activity is intrinsic to an individual ACP. Mutational analysis implicates an arginine/lysine in loop II and an arginine/glutamine in helix III as the catalytic residues for transferase function. The hydrogen bonding properties of these residues appears to be indispensable for the transferase reaction. Complementation of fabD(Ts) E. coli highlights the putative physiological role of this process. Our studies thus shed light on a key aspect of ACP biology and provide insights into the mechanism involved therein.

Photo-induced double-strand DNA and site-specific protein cleavage activity of L-histidine (mu-oxo)diiron(III) complexes of heterocyclic bases

Three oxo-bridged diiron(III) complexes of L-histidine and heterocyclic bases [Fe-2(mu-O)(L-his)(2)(B)(2)](ClO4)(2) (1-3), where B is 2,2'-bipyridine (bpy),1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), were prepared and characterized. The bpy complex 1 was structurally characterized by X-ray crystallography. The molecular structure showed a {Fe-2(mu-O)} core in which iron(III) in a FeN4O2 coordination is bound to tridentate monoanionic L-histidine and bidentate bpy ligands. The Fe center dot center dot center dot Fe distance is similar to 3.5 angstrom. The Fe-O-Fe unit is essentially linear, giving a bond angle of similar to 172 degrees. The complexes showed irreversible cyclic voltammetric cathodic response near -0.1 V vs. SCE in H2O-0.1 M KCl. The binuclear units displayed antiferromagnetic interaction between two high-spin (S = 5/2) iron(III) centers giving a -J value of -110 cm(-1). The complexes showed good DNA binding propensity giving a binding constant value of similar to 10(5) M-1. Isothermal titration calorimetric data indicated single binding mode to the DNA. The binding was found to be driven by negative free energy change and enthalpy. The dpq complex 3 showed oxidative double-strand DNA cleavage on exposure to UV-A and visible light. The phen complex 2 displayed single-strand photocleavage of DNA. The DNA double-strand breaks were rationalized from theoretical molecular docking calculations. Mechanistic investigations showed formation of hydroxyl radicals as the reactive species through photodecarboxylation of the L-histidine ligand. The complexes exhibited good binding propensity to bovine serum albumin (BSA) protein in Tris-HCl/NaCl buffer medium. The dpq complex 3 showed UV-A light-induced site-specific oxidative BSA cleavage forming fragments of similar to 45 kDa and similar to 20 kDa molecular weights via SOH pathway.

Type II beta-turn conformation of pivaloyl-L-prolyl-alpha-aminoisobutyryl-N-methylamide: Theoretical, spectroscopic, and X-ray studies

Pivaloyl-L-Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in (CD3)2SO solution, by 1H-nmr methods, suggesting the existence of beta -turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II beta-turn conformations are about 2 kcal mol-1 more stable than Type III structures. A crystallographic study has established the Type II beta-turn in the solid state. The molecule crystallizes in the space group P21 with a = 5.865 Å, b = 11.421 Å, c = 12.966 Å, beta = 97.55°, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II -turn conformation is stabilized by an intramolecular 4 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are Pro = -57.8°, Pro = 139.3°, Aib = 61.4°, and Aib = 25.1°. The Type II beta-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.

Modification of chemical reactivity by cyclodextrins. Observation of moderate effects on Norrish type I and type II photobehavior

The photochemistry and photophysics of organic molecules in organized assemblies are being studied with great interest in order to understand the features controlling the selectivity in the photoreactions brought about by these media.l These studies have paved the way to an intriguing number of possibilities by which photoreactivity can be modified. In this connection, we have investigated the photobehavior of a number of phenyl alkyl ketones and cu,cu-dimethylphenyl alkyl ketones (Scheme I) incorporated in the hydrophobic interior of cyclodextrin cavities.

Modeling population growth and site specific control of the invasive Lantana camara L. (Verbenaceae) under differing fire regimes

It is at the population level that an invasion either fails or succeeds. Lantana camara L. (Verbenaceae) is a weed of great significance in Queensland Australia and globally but its whole life-history ecology is poorly known. Here we used 3 years of field data across four land use types (farm, hoop pine plantation and two open eucalyptus forests, including one with a triennial fire regime) to parameterise the weed’s vital rates and develop size-structured matrix models. Lantana camara in its re-colonization phase, as observed in the recently cleared hoop pine plantation, was projected to increase more rapidly (annual growth rate, λ = 3.80) than at the other three sites (λ 1.88–2.71). Elasticity analyses indicated that growth contributed more (64.6 %) to λ than fecundity (18.5 %) or survival (15.5 %), while across size groups, the contribution was of the order: juvenile (19–27 %) ≥ seed (17–28 %) ≥ seedling (16–25 %) > small adult (4–26 %) ≥ medium adult (7–20 %) > large adult (0–20 %). From a control perspective it is difficult to determine a single weak point in the life cycle of lantana that might be exploited to reduce growth below a sustaining rate. The triennial fire regime applied did not alter the population elasticity structure nor resulted in local control of the weed. However, simulations showed that, except for the farm population, periodic burning could work within 4–10 years for control of the weed, but fire frequency should increase to at least once every 2 years. For the farm, site-specific control may be achieved by 15 years if the biennial fire frequency is tempered with increased burning intensity.

Should I stay or should I go? : Reproductive and dispersal strategies of site-specific liverwort species

The area of intensively managed forests, in which required conditions for several liverwort species are seldom found, has expanded over the forest landscape during the last century. Liverworts are very sensitive to habitat changes, because they demand continuously moist microclimate. Consequently, about third of the forest liverworts have been classified as threatened or near threatened in Finland. The general objective of this thesis is to increase knowledge of the reproductive and dispersal strategies of the substrate-specific forest bryophytes. A further aim was to develop recommendations for conservation measures for species inhabiting unstable and stable habitats in forest landscape. Both population ecological and genetic methods have been applied in the research. Anastrophyllum hellerianum inhabits spatially and temporally limited substrate patches, decaying logs, which can be considered as unstable habitats. The results show that asexual reproduction by gemmae is the dominant mode of reproduction, whereas sexual reproduction is considerably infrequent. Unlike previously assumed, not only spores but also the asexual propagules may contribute to long-distance dispersal. The combination of occasional spore production and practically continuous, massive gemma production facilitates dispersal both on a local scale and over long distances, and it compensates for the great propagule losses that take place preceding successful establishment at suitable sites. However, establishment probability of spores may be restricted because of environmental and biological limitations linked to the low success of sexual reproduction. Long-lasting dry seasons are likely to result in a low success of sexual reproduction and decreased release rate of gemmae from the shoots, and consequent fluctuations in population sizes. In the long term, the substratum limitation is likely to restrict population sizes and cause local extinctions, especially in small-sized remnant populations. Contrastingly, larger forest fragments with more natural disturbance dynamics, to which the species is adapted, are pivotal to species survival. Trichocolea tomentella occupies stable spring and mesic habitats in woodland. The relatively small populations are increasingly fragmented with a high risk for extinction for extrinsic reasons. The results show that T. tomentella mainly invests in population persistence by effective clonal growth via forming independent ramets and in competitive ability, and considerably less in sexuality and dispersal potential. The populations possess relatively high levels of genetic diversity regardless of population size and of degree of isolation. Thus, the small-sized populations inhabiting stable habitats should not be neglected when establishing conservation strategies for the species and when considering the habitat protection of small spring sites. Restricted dispersal capacity, also on a relatively small spatial scale, is likely to prevent successful (re-)colonization in the potential habitat patches of recovering forest landscapes. By contrast, random short-range dispersal of detached vegetative fragments within populations at suitable habitat seems to be frequent. Thus, the restoration actions of spring and streamside habitats close to the populations of T. tomentella may contribute to population expansion. That, in turn, decreases the harmful effects of environmental stochasticity.

Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase

DNA topoisomerases are ubiquitous nuclear enzymes that govern the topological interconversions of DNA by transiently breaking/rejoining the phosphodiester backbone of one (type I) or both (type II) strands of the double helix. Consistent with these functions, topoisomerases play key roles in many aspects of DNA metabolism. Type II DNA topoisomerase (topo II) is vital for various nuclear processes, including DNA replication, chromosome segregation, and maintenance of chromosome structure. Topo II expression is regulated at multiple stages, including transcriptional, posttranscriptional, and posttranslational levels, by a multitude of signaling factors. Topo II is also the cellular target for a variety of clinically relevant anti-tumor drugs. Despite significant progress in our understanding of the role of topo II in diverse nuclear processes, several important aspects of topo II function, expression, and regulation are poorly understood. We have focused this review specifically on eukaryotic DNA topoisomerase II, with an emphasis on functional and regulatory characteristics.

Type II beta-turn conformation of pivaloyl-L-prolyl-a-aminoiso-butyryl-N-methylamide: Theoretical, spectroscopic and X-ray studies

Pivaloyl-L-Pro-Aib-N-methylamihdaes been shown to possess one intramolecular hydrogen bond in (CD&SO solution, by 'H-nmr methods, suggesting the existence of p-turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II P-turn conformations are about 2 kcal mol-' more stable than Type 111 structures. A crystallographic study has established the Type I1 /%turn in the solid state. The molecule crystallizes in the space group P21 with a = 5.865 8, b = 11.421 A, c = 12.966 A, /3 = 97.55", and 2 = 2. The structure has been refined to a final R value of 0.061. The Type I1 p-turn conformation is stabilized by an intramolecular 4 - 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are @pro= -57.8", $pro = 139.3', @Aib = 61.4', and $Ajb = 25.1'. The Type 11 /%turn conformation for Pro-Aib in this peptide is compared with the Type I11 structures observed for the same segment in larger peptides.

Self-acylation properties of type II fatty acid biosynthesis acyl carrier protein

Acyl carrier protein (ACIP) plays a central role in many metabolic processes inside the cell, and almost 4% of the total enzymes inside the cell require it as a cofactor. Here, we report self-acylation properties in ACPs from Plasmodium falciparum and Brassica napus that are essential components of type II fatty acid biosynthesis (FAS II), disproving the existing notion that this phenomenon is restricted only to ACPs involved in polyketide biosynthesis. We also provide strong evidence to suggest that catalytic self-acylation is intrinsic to the individual ACP. Mutational analysis of these ACPs revealed the key residue(s) involved in this phenomenon. We also demonstrate that these FAS 11 ACPs exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mechanism for the self-acylation reaction is also proposed.