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Biomedical subjects

A W Parker

Publications and source records attributed to A W Parker.

At least 19 recordsLinked to original sources
PubMed2005

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan.

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.

5-Hydroxytryptophan↗
PubMed2005

Depth profiling in diffusely scattering media using Raman spectroscopy and picosecond Kerr gating.

We demonstrate how pulsed laser Raman excitation (approximately 1 ps) followed by fast optical Kerr gating (approximately 4 ps) can be used to effectively separate Raman signals originating from different depths in heterogeneous diffusely scattering media. The diffuse scattering slows down photon propagation through turbid samples enabling higher depth resolution than would be obtained for a given instrumental time resolution in an optically transparent medium. Two types of experiments on two-layer systems demonstrate the ability to differentiate between surface and sub-surface Raman signals. A Raman spectrum was obtained of stilbene powder buried beneath a 1 mm over-layer of PMMA (poly(methyl methacrylate)) powder. The signal contrasts of the lower stilbene layer and upper PMMA layer were improved by factors >or=5 and >or=180, respectively, by rejecting the Raman component of the counterpart layer. The ability to select the Raman signal of a thin top surface layer in preference to those from an underlying diffusely scattering substrate was demonstrated using a 100 mum thick optically transparent film of PET (poly(ethylene terephthalate)) on top of stilbene powder. The gating resulted in the suppression of the underlying stilbene Raman signal by a factor of 1200. The experiments were performed in back-scattering geometry using 400 nm excitation wavelength. The experimental technique should be well suited to biomedical applications such as disease diagnosis.

Complex Mixtures↗
PubMed2005

Subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy.

We describe a simple methodology for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The technique is based on the collection of Raman scattered light from surface regions that are laterally offset away from the excitation laser spot on the sample. The Raman spectra obtained in this way exhibit a variation in relative spectral intensities of the surface and subsurface layers of the sample being investigated. The data set is processed using a multivariate data analysis to yield pure Raman spectra of the individual sample layers, providing a method for the effective elimination of surface Raman scatter. The methodology is applicable to the retrieval of pure Raman spectra from depths well in excess of those accessible with conventional confocal microscopy. In this first feasibility study we have differentiated between surface and subsurface Raman signals within a diffusely scattering sample composed of two layers: trans-stilbene powder beneath a 1 mm thick over-layer of PMMA (poly(methyl methacrylate)) powder. The improvement in contrast of the subsurface trans-stilbene layer without numerical processing was 19 times. The potential applications include biomedical subsurface probing of specific tissues through different overlying tissues such as assessment of bone quality through skin, providing an effective noninvasive means of screening for bone degeneration, other skeletal disease diagnosis, and dermatology studies, as well as materials and catalyst research.

Feasibility Studies↗
PubMed2005

Numerical simulations of subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy.

We present the first elementary model predicting how Raman intensities vary for a range of experimental variables for spatially offset Raman spectroscopy (SORS), a recently proposed technique for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The model was able to reproduce the key observations made from the first SORS experiments, namely the dependence of Raman signal intensities on the spatial offset between the illumination and collection points and the relative contributions to the overall spectrum from the top layer and sub-layer. The application of the SORS concept to a three-layer system is also discussed. The model also clearly indicates that an annular geometry, rather than a point-collection geometry, which was used in the earlier experiments, would yield much improved data.

Algorithms↗
PubMed2003

Spectroscopy of photoinduced charge-transfer reactions between tetrasulfonated aluminium phthalocyanine and methyl viologen.

Interactions between tetrasulfonated aluminium phthalocyanine (AlPcTS4-) and methyl viologen (Mv2+) have been studied in water and ethanol solutions using several experimental techniques. UV-visible absorption and fluorescence spectroscopies show that ion-pair complexes occur in ethanol, disappearing in more polar environments such as water. Time-resolved fluorescence spectroscopy (picosecond timescale) reveals the existence of several emissive species in ethanol solutions, of which one of the components is attributed to the charge-transfer complex (AlPcTS4-)(Mv2+)2, another to higher-order aggregates and yet another to the isolated AlPcTS4- molecule. The AlPcTS4- emission is quenched by Mv2+, leading to transient diffusion in the fluorescence decay kinetics. On the other hand, the emissive complex has an exponential decay with a relatively long lifetime (above 1 ns). Time-resolved absorption measurements did not reveal the existence of radicals in aqueous solution, even on the picosecond timescale. The spectra reveal the presence of excited singlet state AlPcTS4-, which decays via the triplet excited state back to the ground state.

Journal Article↗
PubMed2002

Measurement of long-range repulsive forces between charged particles at an oil-water interface.

Using a laser tweezers method, we have determined the long-range repulsive force as a function of separation between two charged, spherical polystyrene particles (2.7 microm diameter) present at a nonpolar oil-water interface. At large separations (6 to 12 microm between particle centers) the force is found to decay with distance to the power -4 and is insensitive to the ionic strength of the aqueous phase. The results are consistent with a model in which the repulsion arises primarily from the presence of a very small residual electric charge at the particle-oil interface. This charge corresponds to a fractional dissociation of the total ionizable (sulfate) groups present at the particle-oil surface of approximately 3 x 10(-4).

Journal Article↗
PubMed2001

Photochemistry of the pi-extended 9,10-bis(1,3-dithiol-2-ylidene)-9,10-dihydroanthracene system: generation and characterisation of the radical cation, dication, and derived products.

Flash photolysis of bis[4.5-di(methylsulfanyl) 1,3-dithiol-2-ylidene]-9,10(-dihydroanthracene (1) in chloroform leads to formation of the transient radical cation species 1.+ which has a diagnostic broad absorption band at lambdamax approximately 650 nm. This band decays to half its original intensity over a period of about 80 micros. Species 1.+ has also been characterised by resonance Raman spectroscopy. In degassed solution 1.+ disproportionates to give the dication 1(2+), whereas in aerated solutions the photodegradation product is the 10-[4,5-di(methylsulfanyl) 1,3-dithiol-2-ylidene]anthracene-9(10 H)one (2). The dication 1(2+) has been characterised by a spectroelectrochemical study [lambdamax (CH2Cl2) = 377, 392, 419, 479 nm] and by an X-ray crystal structure of the salt 1(2-) (ClO4)2, which was obtained by electrocrystallisation. The planar anthracene and 1,3-dithiolium rings in the dication form a dihedral angle of 77.2 degrees; this conformation is strikingly different from the saddle-shaped structure of neutral 1 reported previously.

Journal Article↗
PubMed2001

Structure of the radical from one-electron oxidation of 4-hydroxycinnamate.

Radicals from one-electron oxidation of 4-hydroxycinnamate, ferulate and 3,4-dihydroxycinnamate have been formed by reaction with the oxidising triplet state of duroquinone. All three compounds react with triplet duroquinone with second order rate constants close to the diffusion-controlled limit. The identity of the resulting radicals is confirmed by observation of their characteristic visible absorption spectra. Time-resolved resonance Raman (TR3) spectra of the radical from 4-hydroxycinnamate were measured using a probe laser wavelength of 600 nm, to be in resonance with the long wavelength absorption band of the radical. The TR3 spectra contain prominent bands ascribed to the C-O and ring C-C stretching vibrations. The spectra are interpreted as indicating strong delocalisation of the radical site to the double bond in conjugation with the aromatic ring in 4-hydroxycinnamate. This contributes to the low reduction potential of the radical and the antioxidant properties of hydroxycinnamates.

Benzoquinones↗
PubMed2000

Luminescence imaging microscopy and lifetime mapping using kinetically stable lanthanide(III) complexes.

The sensitised luminescence from stable lanthanide complexes (1 and 2) bearing a phenanthridine antenna has been used to generate time-resolved images of silica particles. The millisecond order luminescent lifetime of these complexes is utilised to demonstrate time-gated imaging of the sample from a fluorescent background and to facilitate lifetime mapping over the area of the sample.

Europium↗
PubMed1999

An in vivo analysis of the effectiveness of the osteoarthritic knee brace during heel-strike of gait.

The objective of this study was to analyze the effects of off-loading knee braces in patients diagnosed with symptomatic unicompartmental osteoarthritis. Under fluoroscopic surveillance, 15 patients were asked to perform normal gait on a treadmill. Each patient was asked initially to walk without using a knee brace and then to walk while wearing a brace. The fluoroscopic images of the patients at heel-strike were downloaded to a workstation computer. Condylar separation angle of the knee joint and the distances from the medial and lateral femoral condyles to the tibial plateau (condylar separation) were measured. Twelve of 15 patients (80%) reported relief of pain and demonstrated condylar separation of the degenerative compartment with the use of the off-loading brace. The 3 patients who did not demonstrate condylar separation were obese, making accurate brace fitting difficult. The average change in condylar separation and condylar separation angle was 1.2 mm (range, 0.0-4.5 mm) and 2.2 degrees (range, 0.0 degrees-7.8 degrees). This study demonstrated that condylar separation of a degenerative knee compartment can be achieved with off-loading braces with subsequent subjective relief of knee pain.

Biomechanical Phenomena↗
PubMed1998

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes.

Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to 'target' damage at guanine sites.

Base Sequence↗
PubMed1998

Antioxidant reactions of dihydrolipoic acid and lipoamide with triplet duroquinone.

The oxidation of the antioxidants dihydrolipoate and lipoamide by triplet duroquinone (3DQ) has been studied by laser flash photolysis and time-resolved resonance Raman (TR3) spectroscopy. Reaction of 3DQ with lipoamide by electron transfer [k(H2O)/k(D2O approximately 1] was more rapid than with dihydrolipoate, in which a proton is also involved [k(H2O)/k(D2O approximately 2]. For dihydrolipoate at neutral pH the undeprotonated form was the major reactive species with k approximately 10(9) dm3 mol-1 s-1. At higher pH values the reaction of ionised (thiolate) forms was observed with k > or = 4 x 10(9) dm3 mol-1 s-1. The electron transfer mechanism of reaction between 3DQ and lipoamide was confirmed by TR3 spectra in which formation of the durosemiquinone radical anion and lipoamide disulfide radical cation (RSS+.) was observed.

Antioxidants↗
PubMed1998

Laser line-scanning confocal fluorescence imaging of the photodynamic action of aluminum and zinc phthalocyanines in V79-4 Chinese hamster fibroblasts.

Confocal fluorescence microscopy, using a newly constructed laser line-scanning confocal microscope, was applied to an investigation of the early stages of photoinduced destruction of V79-4 Chinese hamster fibroblasts using aluminum and zinc phthalocyanines as photosensitizers. Results obtained in this work show that aluminum and zinc phthalocyanines, once internalized, localize in perinuclear sites that are disrupted upon light exposure resulting in fluorescence redistribution. The combination of laser-line scanning with charge-coupled device detection used in the confocal microscope developed in this work can enable rapid high-resolution sequential imaging, which is ideal for studying photoinduced intracellular fluorescence dynamics.

Animals↗
PubMed1997

Excited triplet state photophysics of the sulphonated aluminium phthalocyanines bound to human serum albumin.

The binding of the sulphonated aluminum phthalocyanines to human serum albumin (HSA) in aqueous phosphate buffer solution at 25 degrees C has been studied by measuring the properties of the triplet excited states of these dyes. The triplet lifetimes were measured by triplet-triplet absorption flash photolysis. The triplet lifetime of the disulphonated AlS2Pc (2.5 microM) varies from 500 +/- 30 microseconds in the absence of protein to 1.100 microseconds and longer with HSA concentrations above 100 microM. Under identical conditions, the maximum triplet lifetimes of the mono-, tri- and tetrasulphonated compounds bound to HSA are shorter than those for the disulphonated species. The increase in the triplet state lifetimes is attributed to the ability of the bulk aqueous phase to interact with the sensitizer at the site of binding; the site of binding being dependent on the degree of sulphonation. For AlS2Pc and AlS3Pc at all HSA concentrations, and regardless of the degree of sulphonation, all the triplet state decay profiles follow simple pseudo-first-order kinetics. The exponential decay of the triplet phthalocyanine at all HSA concentrations is ascribed to the rapid association and dissociation of the phthalocyanine-HSA complex on the time-scales of the triplet state lifetimes. A simplified one-step binding model is utilized to describe the results. The association of AlS1Pc with HSA results in substantial quenching of the triplet state quantum yield, and a more complex model is required to analyze the results. The tetrasulphonated compound (AlS4Pc) binds to the protein at a site where it experiences some protection from the aqueous phase.

Aluminum↗
PubMed1996

Resonance Raman spectroscopy of 4-thiothymidine and oligodeoxynucleotides containing this base both free in solution and bound to the restriction endonuclease EcoRV.

The resonance Raman spectra of 4-thiothymidine [4ST] have been recorded (a) in the free deoxynucleoside form, (b) when incorporated into the single stranded oligodeoxynucleotide d(AG[4ST]-TC), and (c) within the double-stranded self-complementary dodecamer d(GACGA[4ST]ATCGTC). Vibrational mode assignments of almost all the major Raman bands observed in each spectra have been made, mainly by comparison with the published assignments of related nucleosides and nucleotides. Differences between the spectra were observed, particularly when [4ST] and d(AG[4ST]TC) were compared to d(GACGA[4ST]ATCGTC). This is explained in terms of the variations in structure between single-and double-stranded DNA. Good quality spectra were obtained at nucleotide/oligonucleotide concentrations of between 100 and 500 microM and this coupled with an apparatus that uses small volumes (100 microL) allowed measurement of the spectrum of d(GACGA[4ST]ATCGTC) bound to the EcoRV endonuclease. This well characterised nuclease, for which crystal structures are available, recognizes d(GATAT) sequences. When this is replaced with d(GA[4ST]ATC), a poor substrate results but turnover can be prevented during data accumulation by omission of the essential cation Mg2+. Large shifts in several of the Raman bands were observed, and these have been related to the environment of the [4ST] base in the protein-bound oligonucleotide as deduced from the crystal structure. The wavenumber for the C = S stretch vibration in free d(GACGA[4ST]ATCGTC) has been used to calculate the strength of the Watson-Crick hydrogen bond between the sulphur atom in [4ST] and the 6-NH2 group on its partner dA. On binding to the enzyme, the shift in the wavenumber of the C = S stretch indicates this Watson-Crick hydrogen bond is weakened, in good agreement with X-ray structures. The advantage of using [4ST] as a resonance Raman probe is that it absorbs at 340 nm, a wavelength where other nucleic acid and protein absorbance is minimal. Thus the spectra obtained are very simple and consist of signals that arise predominantly from the thiobase alone, and this facilitates data interpretation.

Base Sequence↗
PubMed1996

The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate.

Resonance Raman spectra were obtained of the free radical site in substrate reduced anaerobic samples of pea seedling copper amine oxidase (PSAO). The spectra differ significantly from those reported previously for E. coli copper amine oxidase [Moenne-Loccoz et al. (1995) Biochemistry 34, 7020]. The spectra were found to be independent of substrate (benzylamine, spermidine or methylamine) used to reduce the TOPA quinone cofactor, however, several of the peaks in the Raman spectrum displayed small shifts on using [15N]benzylamine, proving incorporation of the substrate nitrogen atom onto the cofactor radical. Changes in the spectrum were also observed when measured in D2O solution indicating a strongly bound proton in the radical. The spectra were independent of pH values between 5 and 9 and are interpreted as showing that the radical exists as a semiiminoquinone radical monoanion. Benzylamine and phenethylamine have been caged with 2-nitrobenzaldehyde and shown by laser flash photolysis to uncage on a sub-millisecond timescale. Preliminary experiments have shown the formation of the enzyme radical intermediate on laser flash photolysis of 2-nitrobenzyl-caged benzylamine in the presence of enzyme. This should permit time-resolved resonance Raman spectral investigations of the catalytic cycle of copper amine oxidases.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Quenching of reactive oxidative species by probucol and comparison with other antioxidants.

One-electron oxidation of the antiatherogenic and antiatherosclerotic drug probucol has been studied in relation to its activity as an antioxidant. Oxidation by triplet excited states of duroquinone and benzophenone, and by the inorganic radicals Br2.- and N3., lead to the formation of a transient absorption at 500 nm. This was identified by time-resolved resonance Raman spectroscopy as the phenoxyl radical from probucol, formed by hydrogen atom or electron plus proton loss from one of the phenolic groups of probucol. The reactivity of probucol with triplet duroquinone and triplet benzophenone, and as a quencher of singlet oxygen, was compared with the reactivities of other antioxidants (alpha-tocopherol, palmitoyl ascorbic acid, dihydrolipoic acid and N-acetyl cysteine). In quenching of the triplet states the reactivity of probucol was comparable with that of alpha-tocopherol, whereas as a quencher of singlet oxygen probucol (k < 10(6) M-1 s-1) was less effective than alpha-tocopherol (k = 2.0 x 10(8) M-1 s-1) by more than two orders of magnitude. This difference in reactivity may allow the contribution of singlet oxygen towards oxidative stress to be quantified separately.

Acetylcysteine↗