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Biomedical subjects

J Ramsden

Publications and source records attributed to J Ramsden.

9 recordsLinked to original sources
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↗
PubMed1998

The similarity of action spectra for thymine dimers in human epidermis and erythema suggests that DNA is the chromophore for erythema.

The location of DNA photodamage within the epidermis is crucial as basal layer cells are the most likely to have carcinogenic potential. We have determined the action spectra for DNA photodamage in different human epidermal layers in situ. Previously unexposed buttock skin was irradiated with 0.5, 1, 2, and 3 minimal erythema doses of monochromatic UVR at 280, 290, 300, 310, 320, 340, and 360 nm. Punch biopsies were taken immediately after exposure and paraffin sections were prepared for immunoperoxidase staining with a monoclonal antibody against thymine dimers that were quantitated by image analysis. Dimers were measured at two basal layer regions, the mid and the upper living epidermis. The slopes of dose-response curves were used to generate four action spectra, all of which had maxima at 300 nm. Dimer action spectra between 300 and 360 nm were independent of epidermal layer, indicating comparable epidermal transmission at these wavelengths. Furthermore, we observed 300 nm-induced dimers in dermal nuclei; however, there was a marked effect of epidermal layer between 280 and 300 nm, showing relatively poor transmission of 280 and 290 nm to the basal layer. These data indicate that solar UVB (approximately 295-320 nm) is more damaging to basal cells than predicted from transmission data obtained from human epidermis ex vivo. The epidermal dimer action spectra were compared with erythema action spectra determined from the same volunteers and ultraviolet radiation sources. Overall, these spectral comparisons suggest that DNA is a major chromophore for erythema in the 280-340 nm region.

Adult↗
PubMed1997

Response of human hair cortical cells to fractionated radiotherapy.

Hair cortical cell counting (HCCC) represents a non-invasive, in-vivo measure of cell kill in the human integument. Sixty-six patients undergoing conventionally fractionated, external beam radiotherapy for early stage carcinoma of the prostate had groin hair samples counted. This technique is a sensitive and reproducible measure of radiation effect and may have applicability as an in-vivo prediction tool or in the field of biological dosimetry. A repopulative follicular response occurring at 3-4 weeks may explain flattening of the dose response curve.

Dose-Response Relationship, Radiation↗
PubMed1996

Changes in the cellularity of the cortex of human hairs as an indicator of radiation exposure.

Growing hair follicles with their rapid cell proliferation would be expected to be sensitive organs to cytotoxic agents such as radiation. Various abnormalities in the hair and hair follicles have been reported in the past. Changes in the number of cells in the newly forming hair cortex have been shown in the mouse to be one of the more sensitive assays for radiation effects, and this approach could provide a basis for a biological dosimeter. Here we show for the first time using hair cortex cell counts some preliminary data indicating that the number of cell nuclei in a unit of length (140 microns) of the cortex of human hairs from the chest and scalp of patients undergoing fractionated radiotherapy falls significantly (P = 0.005) by 5%-10% 3 days after the first dose in a fractionated sequence of irradiations. The first dose was delivered on a Friday, and no further exposures were delivered until after the hair sample was taken on the 3rd day (Monday). No significant effect of radiation dose could be detected over the available. limited range of doses studied (5-6.5 Gy with one exit dose sample at 2.6 Gy). Also, the width varies from hair to hair. If the width of the hair is taken into account and the cortical nuclei counts are normalised to the width of each hair, the effects seen at day 3 become slightly more significant (P = 0.002), and those at day 5 also become significant (P = 0.012). Samples taken on the 5th day after the first (Friday) exposure were also 2 days after the second exposure and 1 day after the third exposure. However, little expression of damage attributable to the 2nd and 3rd exposures was anticipated since their effects would take some time to be expressed in the cortical region examined, which is some distance from the proliferative region of the follicle.

Cell Count↗
PubMed1995

The carboxyl terminus of the membrane-binding domain of cytochrome b5 spans the bilayer of the endoplasmic reticulum.

Preliminary studies (Vergères, G., and Waskell, L. (1992) J. Biol. Chem. 267, 12583-12591) have suggested that the carboxyl-terminal membrane-binding domain of cytochrome b5 traverses the membrane and that the carboxyl terminus is in the lumen of the endoplasmic reticulum. In order to confirm and extend these studies, additional experiments were conducted. The gene coding for rat cytochrome b5 was transcribed and the resulting mRNA was translated in vitro in a rabbit reticulocyte lysate in the presence of microsomes. The binding and topology of cytochrome b5 were investigated by treating microsomes containing the newly incorporated cytochrome b5 with carboxypeptidase Y and trypsin. Our studies indicate that cytochrome b5 is inserted both co- and post-translationally into microsomes in a topology in which the membrane-binding domain spans the bilayer with its COOH terminus in the lumen. Cytochrome b5 is also incorporated into microsomes pretreated with trypsin in a topology indistinguishable from the one resulting from the insertion of the protein into untreated microsomes, reconfirming that cytochrome b5 does not use the signal recognition particle-dependent translocation machinery. Our results do not allow a distinction to be made between a spontaneous insertion mode or some other trypsin-resistant receptor-mediated mechanism. A role for Pro115 in the middle of the membrane-binding domain of cytochrome b5 was also examined by mutating it to an alanine and subsequently characterizing the ability of the mutant protein to be incorporated into membranes. The mutant protein inserted more slowly in vitro into microsomes as well as into pure lipid bilayers by a factor of 2 to 3.

Amino Acid Sequence↗
PubMed1993

Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.

Scanning force microscopy allows imaging of biological molecules in their native state in buffer solution. To this end samples have to be fixed to a flat solid support so that they cannot be displaced by the scanning tip. Here we describe a method to achieve the covalent binding of biological samples to glass surfaces. Coverslips were chemically modified with the photoactivatable cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Samples are squeezed between derivatized coverslips and then cross-linked to the glass surface by irradiation with ultraviolet light. Such samples can be imaged repeatedly by the scanning force microscope without loss of image quality, whereas identical but not immobilized samples are pushed away by the stylus.

Azides↗
PubMed1989

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin.

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.

Allosteric Regulation↗
PubMed1982

The effect of local cooling on digital systolic pressure in patients with Raynaud's syndrome.

The effect of local controlled cooling on the digital systolic blood pressure in the hand was studied in 25 healthy volunteers and 25 patients with Raynaud's syndrome. Arterial systolic pressure was indirectly measured using a digital cuff over the proximal phalanx and a photoplethysmographic probe over the digital pulp to detect pulsation. Pressures were measured after occlusion of the circulation (cuff at 180 mmHg) for 5 minutes with water circulating initially at 30 degrees C and then at 10 degrees C. An air filled cuff was used simultaneously on a control finger. The digital systolic pressure at 10 degrees C in the patients was significantly lower (P less than 0.001 Mann-Whitney U test) than in the normal volunteers whilst calculation of the percentage change in pressure induced by cooling the digit to 10 degrees C allowed complete separation between both groups. The range in the controls was +2 per cent to -26 per cent and in the patients, -33 per cent to -100 per cent. The test gives reproducible results (coefficient of variation 5-11 per cent) and offers an objective method of identifying patients with excessive vasospasm due to a cold stimulus. It may prove useful in studying natural history and the effect of drug therapy.

Adult↗