Plasmi copy number quantification by spectrofluorometry

No data (2012)

The role of ultraviolet-A reflectance and ultraviolet-A induced fluorescence in the appearance of budgerigar plumage: insights from spectrofluorometry and reflectance spectrophotometry

Fluorescence has so far been found in 52 parrot species when illuminated with ultraviolet-A (UVA) 'black' lamps, and two attempts have been made to determine whether such fluorescence plays any role in sexual signalling. However, the contribution of the reflectance versus fluorescence to the total radiance from feathers, even in the most studied species to date (budgerigars), is unclear. Nor has the plumage of this study species been systematically assessed to determine the distribution of fluorescent patches. We therefore used spectrofluorometry to determine which areas of budgerigars fluoresce and the excitation and emission spectra involved; this is the first time that such a technique has been applied to avian plumage. We found that both the yellow crown and (normally hidden) white downy chest feathers exhibit strong UVA-induced fluorescence, with peak emissions at 527 nm and 436 nm, respectively. Conversely, the bright-green chest and dark-blue tail feathers do not fluoresce. When comparing reflectance spectra (400700 nm) from the yellow crown using illuminants with a proportion of UVA comparable to daylight, and illuminants with all UVA removed, no measurable difference resulting from fluorescence was found. This suggests that under normal daylight the contribution of fluorescence to radiance is probably trivial. Furthermore, these spectra revealed that males had fluorescent crowns with substantially higher reflectance than those of females, in both the UV waveband and at longer wavelengths. Reflectance spectrophotometry was also performed on a number of live wild-type male budgerigars to investigate the chromatic contrast between the different plumage areas. This showed that many plumage regions are highly UV-reflective. Overall our results suggest that rapid surveys using UVA black lamps may overestimate the contribution of fluorescence to plumage coloration, and that any signalling role of fluorescence emissions, at least from the yellow crown of budgerigars, may not be as important as previously thought.

Comparative study of dissolved organic matter from groundwater and surface water in the Florida coastal Everglades using multi-dimensional spectrofluorometry combined with multivariate statistics

Dissolved organic matter (DOM) in groundwater and surface water samples from the Florida coastal Everglades were studied using excitation–emission matrix fluorescence modeled through parallel factor analysis (EEM-PARAFAC). DOM in both surface and groundwater from the eastern Everglades S332 basin reflected a terrestrial-derived fingerprint through dominantly higher abundances of humic-like PARAFAC components. In contrast, surface water DOM from northeastern Florida Bay featured a microbial-derived DOM signature based on the higher abundance of microbial humic-like and protein-like components consistent with its marine source. Surprisingly, groundwater DOM from northeastern Florida Bay reflected a terrestrial-derived source except for samples from central Florida Bay well, which mirrored a combination of terrestrial and marine end-member origin. Furthermore, surface water and groundwater displayed effects of different degradation pathways such as photodegradation and biodegradation as exemplified by two PARAFAC components seemingly indicative of such degradation processes. Finally, Principal Component Analysis of the EEM-PARAFAC data was able to distinguish and classify most of the samples according to DOM origins and degradation processes experienced, except for a small overlap of S332 surface water and groundwater, implying rather active surface-to-ground water interaction in some sites particularly during the rainy season. This study highlights that EEM-PARAFAC could be used successfully to trace and differentiate DOM from diverse sources across both horizontal and vertical flow profiles, and as such could be a convenient and useful tool for the better understanding of hydrological interactions and carbon biogeochemical cycling.

A PAL1 gene promoter-green fluorescent protein reporter system to analyse defence responses in live cells of Arabidopsis thaliana

Arabidopsis thaliana ecotype Columbia-0 was transformed with a green fluorescent protein (GFP) gene under control of a phenylalanine ammonia-lyase (PAL) promoter. PAL is a key enzyme of the phenylpropanoid pathway and is induced to high levels during plant stress. Constitutive expression of PAL1 promoter-controlled GFP occurred in vascular tissues within stems, leaves and roots and in developing flowers. PAL1 promoter–GFP expression was examined in leaves of transgenic plants subjected to an abiotic elicitor, mechanical wounding or to inoculation with the pathogens Pseudomonas syringae pv. tomato or Peronospora parasitica. Wounding of leaves and treatment with an abiotic elicitor and compatible interactions produced low to moderate levels of GFP. However, in incompatible interactions there were high levels of GFP produced. In incompatible interactions, the intensity of GFP fluorescence was similar to that produced in transgenic plants expressing GFP driven by the CaMV promoter. The bright green fluorescence produced in live cells and tissues was readily visualised using conventional fluorescence microscopy and was quantified using spectroflourometry. This is the first report of the use of GFP as a reporter of defence gene activation against pathogens. It has several advantages over other reporter genes including real time analysis of gene expression and visualisation of defence gene activation in a non-invasive manner.

The platelet intracellular calcium response to serotonin and thrombin in patients with panic disorder.

Serotonin is implicated in both the biology of depression and anxiety. The aim of this study was to examine the platelet intracellular calcium response to serotonin and thrombin using spectrofluorometry in 14 patients with DSM-4 panic disorder compared to 14 matched controls. Patients did not show significantly higher baseline platelet intracellular calcium levels and serotonin stimulated levels of intracellular calcium than control subjects. There was a much smaller standard deviation in the control subjects than in the panic patients. The intracellular calcium response to thrombin activation was however greater in panic patients than in control subjects (P<0.001). The failure of this study to find enhanced sensitivity of 5-HT2 receptors in panic disorder is compatible with the findings of previous challenge studies that found no consistent dysregulation of serotonin in panic disorder. The enhanced thrombin sensitivity, nevertheless suggests some receptor mediated second messenger changes independent of serotonin in the disorder.

The specificity of platelet glutamate receptor supersensitivity in psychotic disorders.

Hypoglutamatergic function is implicated in the pathogenesis of schizophrenia, and supersensitivity of platelet NMDA receptors has been reported in schizophrenia. The aim of this study was to examine the platelet glutamate receptor sensitivity in patients with schizophrenia (n=12), mania with psychotic features (n=10) and depression with psychotic features (n=10) and matched controls (n=12) in order to assess if this is a marker of schizophrenia or occurs in other psychotic conditions. Glutamate receptor sensitivity was assessed using the intracellular calcium response to glutamate measured with spectrofluorometry. The percentage response of the schizophrenic and depressed psychotic subjects to glutamate stimulation was significantly greater than control subjects (p<0.005). The mania with psychotic features group was not significantly different to controls. This data suggests that platelet glutamate receptors may be supersensitive in schizophrenia and depression with psychotic features. Furthermore, the platelet may be a possible peripheral marker of glutamate function in schizophrenia and depression with psychotic features.

Blunted adenosine A2a receptor function in platelets in patients with major depression.

There is provisional evidence of involvement of adenosine in depression. In this study, the second messenger intracellular calcium response in platelets was measured in patients with major depression and controls using spectrofluorometry. The primary result of this study was a statistically significantly blunted second messenger response to agonist stimulation in the depressed group compared to the control group at the 50 and 100 nM and 1 microM dosage levels. This suggests that dysregulation of the adenosine A2a receptor may be present in depression.

Changes in the platelet intracellular calcium response to serotonin in patients with major depression treated with electroconvulsive therapy: state or trait marker status.

Platelet serotonin type 2A receptor (5-HT2A) sensitivity changes have previously been documented in depression, although it is unclear if this represents a stable trait marker of the illness, or whether it represents an acute state marker of depression that would change with treatment. Electroconvulsive therapy (ECT) may be a useful intervention to separate out trait and state marker status avoiding the potential confounding effects of pharmacotherapy on receptor function. Using spectrofluorometry, the platelet 5HT2A, receptor sensitivity as reflected by the intracellular calcium response to serotonin stimulation, was measured every week in patients suffering from major depression and undergoing ECT. There was a significant drop in the platelet response to serotonin stimulation over a course of ECT, with an associated progressive decrease in Hamilton Rating Scale of Depression (HAM-D) scores. This may suggest either decreased sensitivity of platelet 5HT2A receptors as a mechanism of action of ECT, or changes in second messengers such as the inositide phospholipid system. This suggests that the enhanced sensitivity of platelet 5HT2A receptors may be a state marker of major depression.

Platelet glutamate receptor supersensitivity in major depressive disorder.

Dysregulation of glutamate has been described in depression, and supersensitivity of platelet glutamate receptors has been found in both psychotic major depression and schizophrenia. The aim of this study was to examine the platelet glutamate receptor sensitivity in patients with nonpsychotic, unipolar major depression to assess whether this is a marker of depression or of psychosis. Glutamate receptor sensitivity was assessed using the platelet intracellular calcium response to glutamate (0-100 micromol) measured by spectrofluorometry. The depression group showed a significantly greater platelet intracellular calcium response to glutamate stimulation than the control group, both in terms of absolute values (p = 0.007) and percentage of response from baseline (p = 0.030). These data suggest that platelet glutamate receptors may be supersensitive in depression and that the platelet may be a possible peripheral marker of glutamate function in depression.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: A solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors. © 2007 The Authors.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

Recent applications of analytical techniques for quantitative pharmaceutical analysis: A review

The intension of this paper was to review and discuss some of the current quantitative analytical procedures which are used for quality control of pharmaceutical products. The selected papers were organized according to the analytical technique employed. Several techniques like ultraviolet/visible spectrophotometry, fluorimetry, titrimetry, electroanalytical techniques, chromatographic methods (thin-layer chromatography, gas chromatography and high-performance liquid chromatography), capillary electrophoresis and vibrational spectroscopies are the main techniques that have been used for the quantitative analysis of pharmaceutical compounds. In conclusion, although simple techniques such as UV/VIS spectrophotometry and TLC are still extensively employed, HPLC is the most popular instrumental technique used for the analysis of pharmaceuticals. Besides, a review of recent works in the area of pharmaceutical analysis showed a trend in the application of techniques increasingly rapid such as ultra performance liquid chromatography and the use of sensitive and specific detectors as mass spectrometers.

Estudo da degradação de resinas compostas pela ação de bebidas alcoólicas: análise de fluorescência e rugosidade superficial

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Charge effect in the interaction of doxorubicin and derivatives with polydeoxynucleotides

The equilibrium interaction of doxorubicin and its N-acetyl derivative with a series of purine-pyrimidine alternating polydeoxynucleotides has been studied through spectrofluorometry to assess the relevance of the electrostatic contribution to DNA intercalation. The results have shown that: (a) the suppression of the positive charge on the aminosugar has: (I) a profound negative effect on the free energy of intercalation, as expected, and (II) a negligible influence on the base specificity, which supports the notion of an essentially electrostatic effect of N-acetylation on intercalation; (b) a reasonably good accord with the demands of a polyelectrolytic model, due to Friedman and Manning, is found.

Respiratory electron transport activity in plankton: determinants and detection

[ES]La presente tesis, se centra en el estudio del Sistema de Transporte de Electrones (ETS) en organismos del plancton marino, los factores que lo influencian la interpretación de estas mediciones y su detección mediante espectrofotometría y espectrofluorometría, en muestras oceánicas naturales y en cultivos de organismos marinos. Se pudo establecer, la biomasa, la respiración (R) y la respiración potencial (ɸ), en tres transectos en los océanos Índico y Atlántico Norte Sur. A su vez, se determino el estado fisiológico, en tres tamaños del zooplancton, midiendo la relación R/ɸ. Se exploró los efectos de la inanición sobre la R y la variación con respecto a la ɸ en el zooplancton