Biomedical subjects

P Teesdale-Spittle

Publications and source records attributed to P Teesdale-Spittle.

5 recordsLinked to original sources

PubMed2001

A TSM sensor investigation of low crystallinity cellulose films.

A thickness shear mode (TSM) quartz sensor has been used to characterize the substantivity, viscoelasticity, and mucoadhesive properties of low crystallinity cellulose (LCC) films. LCC is a novel pharmaceutical excipient that has been attributed with mucoadhesive properties. Thin films of LCC were deposited onto TSM sensors by a spin coating technique. The films were treated by passing water or 1.0% w/v mucin solution (pH 3.7 or 7.0) over the surface. Changes in the mass and viscosity of the film were observed by monitoring changes in the impedance spectra of the coated TSM sensors. Scanning electron micrographs (SEMs) of each film were used to assist the interpretation of the TSM sensor data. This study showed that LCC forms highly tenacious and viscoelastic films able to withstand prolonged (approximately 1 h) exposure to both water and mucin solution. Furthermore, these results indicate that the films may have mucoadhesive properties as LCC was found to bind significant (P<0.05) amounts of mucin in comparison with control measurements. Mucin binding to the LCC sensor was greater at pH 3.7 (P<0.05) than at pH 7.0, suggesting that the LCC formulation is mucoadhesive under these conditions.

Cellulose↗

PubMed2000

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy.

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.

Anthraquinones↗

PubMed2000

Characterisation of fatty acid multilayers using a TSM biosensor.

Thickness shear mode (TSM) biosensors have many potential applications within the pharmaceutical sciences as a means of measuring mass changes in the nanogram range, film thickness, viscosity and shear moduli. This study addresses the possible use of the TSM sensor as a biosensor for measuring drug partition coefficients. In order to realise this potential, some fundamental understanding is required of the behaviour of lipid films on the sensor. The present study characterises the behaviour of fatty acid multilayers as a suitable model chemical system. Frequency shifts and impedance spectra are presented for multilayers of three fatty acid films coated on to the sensor using a Langmuir-Blodgett trough. The results indicate that the frequency shift is non-linear at lower numbers of fatty acid layers but the response is Sauerbrey-like at higher numbers of layers. Also at high numbers of layers, changes in the impedance spectra indicate viscoelastic behaviour in thicker membranes. An inverse relationship is observed between chain length and frequency shift, which is attributed to variations in the topography of the sensor surface. This work demonstrates the importance of fully characterising the physical behaviour of the lipid multilayers prior to using these systems for the measurement of drug partition coefficients.

Biosensing Techniques↗

PubMed1997

Structural and functional analysis of a glutathione S-transferase from Ascaris suum.

A recombinant glutathione S-transferase (GST) (EC 2.5.1.18) from the parasitic nematode Ascaris suum (AsGST1) displays specific activity with a variety of model substrates and secondary products of lipid peroxidation. The AsGST1 interacts with a range of model inhibitors, haematin-related compounds, bile acids and anthelminthics. The reported variations in biochemical activity correlate with structural differences observed by homology modelling. Here, differences in the topography of the proposed substrate binding site between the AsGST1 and the host GSTs were identified. A rabbit polyclonal antiserum was raised against the glutathione-binding proteins of A. suum and specific antibodies against AsGST1 were affinity-purified using the recombinant protein. These antibodies were used to localize the AsGST1 in adult worms by immunohistochemical staining. The strongest immunostaining for AsGST1 was localized in the intestine in all worms examined. This suggests that the enzyme may be responsible for the metabolism of materials that are incorporated from the environment, as well as for molecules that are excreted or secreted from the parasite to the environment. It also demonstrates the accessibility of the enzyme to an inhibitor or blocking antibody. In addition, the structure and sequence of the gene encoding AsGST1 have been determined. Southern-blot analyses of the AsGST1 gene suggests that it is a single-copy gene. The nucleotide sequence analysis revealed that the gene is composed of four exons and three introns, and potential regulatory elements were identified in the 5' flanking sequence.

Amino Acid Sequence↗

PubMed1994

Aliphatic amine N-oxides of DNA binding agents as bioreductive drugs.

The DNA binding and cytotoxicity of four intercalating agents, namely bis-alkylamino (-N(CH2)2N(CH3)2) substituted anthraquinone, anthrapyrazole and anthracene, and mono (N(CH2)2N(CH3)2) acridinone, have been compared with their respective aliphatic amine N-oxides -N(CH2)2N+(O-)(CH3)2. The results show that, unlike the intercalators, the N-oxides do not bind to DNA. Molecular modelling illustrates that the delta + nature of the intercalator alkylamino side chains in the protonated form allows for an attractive electrostatic interaction with phosphates of the DNA backbone, whereas the delta- partial charge on the N-oxide makes such an interaction not permissible; indeed, the electrostatic interaction with the DNA phosphates will be repulsive. The N-oxides show little or no cytotoxicity against V79 cells at concentrations equimolar to the IC90 (concentration that inhibits 90% of cell proliferation) of the respective intercalators. However, the cytotoxicity of anthrapyrazole N-oxide against hypoxic V79 cells in the presence of an activating system of S9 liver fraction was enhanced significantly. The results indicate that N-oxides of DNA-affinic agents have potential as bioreductive prodrugs, since they possess low aerobic toxicity but under hypoxic conditions can be metabolised to a potent cytotoxic species presumed to be a DNA-binding tertiary amine.

Amines↗