Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

In the article 'Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis' (Milinovich et al., 2007), it is found that with reference to Horse 1, the histological signs of laminitis were first observed at 12 h post-oligofructose administration, and not 30 h as was indicated in the Results section under the subheading 'Induction of Laminitis' and in Fig. 1.

Studies on tryptophan residues of Abrus agglutinin Stopped-flow kinetics of modification and fluorescence-quenching studies

The presence of two essential tryptophan residues/molecule was implicated in the binding site of Abrus agglutinin [Patanjali, Swamy, Anantharam, Khan & Surolia (1984) Biochem. J. 217, 773-781]. A detailed study of the stopped-flow kinetics of the oxidation of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues into two phases was observed upon ligand binding. The heterogeneity of tryptophan exposure was substantiated by quenching studies with acrylamide, succinimide and Cs+. Our study revealed the microenvironment of tryptophan residues to be hydrophobic, and also the presence of acidic amino acid residues in the vicinity of surface-localized tryptophan residues.

Analysis of Daphnane Orthoesters in Poisonous Australian Pimelea Species by Liquid Chromatography-Tandem Mass Spectrometry.

Cattle grazing in arid rangelands of Australia suffer periodic extensive and serious poisoning by the plant species Pimelea trichostachya, P. simplex, and P. elongata. Pimelea poisoning (also known as St. George disease and Marree disease) has been attributed to the presence of the diterpenoid orthoester simplexin in these species. However, literature relating to previous studies is complicated by taxonomic revisions, and the presence of simplexin has not previously been verified in all currently recognized taxa capable of inducing pimelea poisoning syndrome, with no previous chemical studies of P. trichostachya (as currently classified) or P. simplex subsp. continua. We report here the isolation of simplexin from P. trichostachya and the development of a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to measure simplexin concentrations in pimelea plant material. Simplexin was quantified by positive-ion atmospheric pressure chemical ionization (APCI) LC-MS/MS with selected reaction monitoring (SRM) of the m/z 533.3 > 253.3 transition. LC-MS/MS analysis of the four poisonous taxa P. trichostachya, P. elongata, P. simplex subsp. continua, and P. simplex subsp. simplex showed similar profiles with simplexin as the major diterpenoid ester component in all four taxa accompanied by varying amounts of related orthoesters. Similar analyses of P. decora, P. haematostachya, and P. microcephala also demonstrated the presence of simplexin in these species but at far lower concentrations, consistent with the limited reports of stock poisoning associated with these species. The less common, shrubby species P. penicillaris contained simplexin at up to 55 mg/kg dry weight and would be expected to cause poisoning if animals consumed sufficient plant material.

Bilirubin binding to polypeptides and chiral amines. Induced circular dichroism and fluorescence studies

Induced Cotton effects have been observed in the visible region on interaction of bilirubin with chiral mono- and diamines and poly-l-lysine. At alkaline pH distinct CD spectra are observed for bilirubin bound to the α-helical and β-sheet conformation of poly-l-lysine, which differ from that observed for the pigment bound to human serum albumin. The CD pattern observed on binding to N-acetyl-Lys-N1-methylamide in CH2Cl2 and dioxane is different from that observed in the presence of l-Ala-NH-(CH2)6-NH-l-Ala in dioxane. The latter case resembles the spectrum observed in the presence of human serum albumin. Binding to the helical polypeptide melittin and the antiparallel β-sheet peptide, gramicidin S, in aqueous solutions results in opposite signs of the bilirubin CD bands. The quenching of tryptophan fluorescence in melittin, in aqueous solution and enhancement of bilirubin fluorescence in dioxane on binding to gramicidin S have been used to monitor pigment-peptide interactions. The results suggest the utility of bilirubin as a conformational probe.

Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double- stranded DNA, thus behaving like a DNA-melting protein.

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl α-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59 · 105 M−1 at 25° C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of d-galactose. Studies with other sugars indicate that a hydrophobic substituent with α-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.

Fluorescence And Nmr-Studies Of The Binding Of Cholinergic Fluorescent-Probes To Horse Serum-Cholinesterase

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

7-Alkoxy coumarins as fluorescence probes for microenvironments

7-Alkoxy and 4-methyl-7-alkoxy coumarins show solvent-dependent fluorescence emission. The monomeric fluorescence emission of these alkoxy coumarins was exploited as a probe to measure the surface polarity of the micelles formed by ionic (sodium dodecylsulphate and cetyltrimethyl-ammonium bromide) and non-ionic (Triton X-100) detergents. By comparing the solvent-dependent fluorescence of these alkoxy coumarins in various homogeneous solvents, the polarity of the micelles was determined qualitatively. All three micelles are more polar than hydrocarbon solvents but are less polar than water.

Ionophore-mediated transmembrane movement of divalent cations in small unilamellar liposomes: An evaluation of the chlortetracycline fluorescence technique and correlations with black lipid membrane studies

Conceptual advances in the field of membrane transport have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest,viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537 A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof — on X537 A concentration, has been observed. An exponential dependence of ldquocurrentrdquo on ldquotransmembrane potentialrdquo in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.

Studying the diurnal and seasonal acclimation of photosystem Ii using chlorophyll-a fluorescence

A small fraction of the energy absorbed in the light reactions of photosynthesis is re-emitted as chlorophyll-a fluorescence. Chlorophyll-a fluorescence and photochemistry compete for excitation energy in photosystem II (PSII). Therefore, changes in the photochemical capacity can be detected through analysis of chlorophyll fluorescence. Chlorophyll fluorescence techniques have been widely used to follow the diurnal (fast), and the seasonal (slow) acclimation in the energy partitioning between photochemical and non-photochemical processes in PSII. Energy partitioning in PSII estimated through chlorophyll fluorescence can be used as a proxy of the plant physiological status, and measured at different spatial and temporal scales. However, a number of technical and theoretical limitations still limit the use of chlorophyll fluorescence data for the study of the acclimation of PSII. The aim of this Thesis was to study the diurnal and seasonal acclimation of PSII in field conditions through the development and testing of new chlorophyll fluorescence-based tools, overcoming these limitations. A new model capable of following the fast acclimation of PSII to rapid fluctuations in light intensity was developed. The model was used to study the rapid acclimation in the electron transport rate under fluctuating light. Additionally, new chlorophyll fluorescence parameters were developed for estimating the seasonal acclimation in the sustained rate constant of thermal energy dissipation and photochemistry. The parameters were used to quantitatively evaluate the effect of light and temperature on the seasonal acclimation of PSII. The results indicated that light environment not only affected the degree but also the kinetics of response of the acclimation to temperature, which was attributed to differences in the structural organization of PSII during seasonal acclimation. Furthermore, zeaxanthin-facilitated thermal dissipation appeared to be the main mechanisms modulating the fraction of absorbed energy being dissipated thermally during winter in field Scots pine. Finally, the integration between diurnal and seasonal acclimation mechanisms was studied using a recently developed instrument MONI-PAM (Walz GmbH, Germany) capable of continuously monitoring the energy partitioning in PSII.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Immuno-fluorescence staining patterns of leukocyte subsets in the skin of taurine and indicine cattle

The immuno-staining patterns of skin leukocytes were investigated in three breeds of cattle: Holstein–Friesian, Brahman and Santa Gertrudis of similar age before and after tick infestation. The antibodies specific for CD45 and CD45RO reacted with cells in the skin of all Holstein–Friesian cattle but did not react with cells in the skin of any Brahman cattle. The same antibodies reacted with cells from the skin of four (CD45) and seven (CD45RO) of twelve Santa Gertrudis cattle. The antibodies specific for T cells and γδ subset of T cells recognized cells from all three breeds of cattle. The antibody specific for MHC class II molecules labelled cells of mostly irregular shape, presumably dermal dendritic cells and/or macrophages and Langerhans cells. The antibody specific for granulocytes (mAb CH138) reacted with cells only in sections cut from skin with lesions. The antibody specific for CD25+ cells labelled regularly shaped cells that showed a wide range of intensities of staining.

Multiangular Spectrometry and Optical Properties of Debris Covered Surfaces

Due to the recent development in CCD technology aerial photography is now slowly changing from film to digital cameras. This new aspect in remote sensing allows and requires also new automated analysis methods. Basic research on reflectance properties of natural targets is needed so that computerized processes could be fully utilized. For this reason an instrument was developed at Finnish Geodetic Institute for measurement of multiangular reflectance of small remote sensing targets e.g. forest understorey or asphalt. Finnish Geodetic Institute Field Goniospectrometer (FiGIFiGo) is a portable device that is operated by 1 or 2 persons. It can be reassembled to a new location in 15 minutes and after that a target's multiangular reflectance can be measured in 10 - 30 minutes (with one illumination angle). FiGIFiGo has effective spectral range approximately from 400 nm to 2000 nm. The measurements can be made either outside with sunlight or in laboratory with 1000 W QTH light source. In this thesis FiGIFiGo is introduced and the theoretical basis of such reflectance measurements are discussed. A new method is introduced for extraction of subcomponent proportions from reflectance of a mixture sample, e.g. for retrieving proportion of lingonberry's reflectance in observation of lingonberry-lichen sample. This method was tested by conducting a series of measurements on reflectance properties of artificial samples. The component separation method yielded sound results and brought up interesting aspects in targets' reflectances. The method and the results still need to be verified with further studies, but the preliminary results imply that this method could be a valuable tool in analysis of such mixture samples.

Novel fluorinated surfactants tentatively identified in firefighters using liquid chromatography quadrupole time-of flight tandem mass spectrometry and a case-control approach

Fluorinated surfactant-based aqueous film-forming foams (AFFFs) are made up of per- and polyfluorinated alkyl substances (PFAS) and are used to extinguish fires involving highly flammable liquids. The use of perfluorooctanesulfonic acid (PFOS) and other perfluoroalkyl acids (PFAAs) in some AFFF formulations has been linked to substantial environmental contamination. Recent studies have identified a large number of novel and infrequently reported fluorinated surfactants in different AFFF formulations. In this study, a strategy based on a case-control approach using quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) and advanced statistical methods has been used to extract and identify known and unknown PFAS in human serum associated with AFFF-exposed firefighters. Two target sulfonic acids [PFOS and perfluorohexanesulfonic acid (PFHxS)], three non-target acids [perfluoropentanesulfonic acid (PFPeS), perfluoroheptanesulfonic acid (PFHpS), and perfluorononanesulfonic acid (PFNS)], and four unknown sulfonic acids (Cl-PFOS, ketone-PFOS, ether-PFHxS, and Cl-PFHxS) were exclusively or significantly more frequently detected at higher levels in firefighters compared to controls. The application of this strategy has allowed for identification of previously unreported fluorinated chemicals in a timely and cost-efficient way.