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Biomedical subjects

R A Farrell

Publications and source records attributed to R A Farrell.

At least 19 recordsLinked to original sources
PubMed1996

Identification of the Zn(II) site in the copper-responsive yeast transcription factor, AMT1: a conserved Zn module.

The N-terminal metal-binding domains of the copper-activated yeast transcription factors, ACE1 and AMT1, bind to specific DNA sequences in a Cu-dependent fashion. Recombinant AMT1 and ACE1 metal-binding domains are isolated as Cu4Zn1-protein complexes. Site-directed mutagenesis of AMT1 was used in this study to map the ligands of the Cu(I) and Zn(II) ions. The results are consistent with the N-terminal halves of AMT1 and ACE1 consisting of two independent submodules, one binding a single Zn(II) ion and the second binding the tetracopper cluster. The basis of this conclusion is, first, that mutations of two cysteinyl codons and a histidyl codon in the first 42 residues of AMT1 do not alter DNA binding. In contrast, serine substitutions at four cysteine positions at codons 43, 61, 90, and 98 abolish DNA binding. We demonstrated previously that population of the Zn(II) site in AMT1 does not alter the ability of the protein to bind DNA but bound Cu(I) ions are essential for DNA binding [Thorvaldsen, J. L., et al. (1994) Biochemistry 33, 9566-9577]. Second, mutations in the N-terminal 42 residue segment reduce the Zn(II) content of purified mutant AMT1 molecules. Third, a synthetic peptide consisting of the N-terminal 42 residues in AMT1 forms a stable Zn(II) complex and substitution with Co(II) reveals an electronic spectrum identical to that of the Co-substituted intact Cu4AMT1 protein. 113Cd(II) NMR studies reveal that the divalent metal site consists of ligands provided by three cysteinyl thiolates and a single histydyl imidazole. The sequence homology between AMT1, ACE1, and MAC1 in the N-terminal 42 residues suggests that ACE1 and MAC1 will, likewise, contain N-terminal Zn modules. A 42-residue ACE1 synthetic peptide gives identical metal binding properties to the corresponding AMT1 synthetic peptide. Thus, AMT1 and likely ACE1 consist of two contiguous modules, residues 1-42 forming an independent Zn(II) module and residues 43-110 enfolding a tetracopper cluster.

Amino Acid Sequence↗
PubMed1996

Spatial mapping of polarized light transmission in the central rabbit cornea.

In this paper polarized light transmission measurements are made under conditions that closely approximate the physiological state in order to probe lamellar structure in the central cornea of New Zealand white rabbits. The results are interpreted with the aid of a newly developed theory (published elsewhere) in which the cornea is modeled as stacked birefringent layers corresponding to the lamellae. The theory enables predictions of the statistical properties of lamellar ordering based on characteristics of the transmission of polarized light. The experimental results are consistent with a structure in which a number of lamellae have a fixed azimuthal orientation (i.e. about an axis normal to the corneal surface), whereas the remainder are essentially randomly oriented. Comparisons with the theoretical predictions are consistent with a model in which the preferred direction in the apical region of the cornea does not vary significantly among rabbits; and the preferred lamellar orientation direction determined for the population measured here is very close to that suggested in previous experiments on a smaller number of rabbits. Mapping experiments using a new goniometric holder showed that the preferential order at the apical region is closely preserved throughout the central approximately 4 mm diameter optical zone in individual corneas.

Animals↗
PubMed1996

A numerical test of the normal incidence uniaxial model of corneal birefringence.

The suggestion that the central cornea can be modeled as a uniaxial birefringent material with its optic axis normal to the surface is explicitly tested by numerical calculations. A theoretical framework is presented to model the corneal stroma as a series of stacked, uniaxial birefringent layers (lamellae). Calculations are then made of the transmission of normally incident, linearly polarized light through model systems having various azimuthal orientations of the layers, motivated by the suggestion of an overall "random" organization of the stromal lamellae. It is concluded that the uniaxial description, and the assumptions upon which that description is based, do not hold for the cornea. In particular, the calculations are in agreement with recent experiments in which one always observes a non-zero cross-polarized transmission (hence birefringence) at normal incidence.

Animals↗
PubMed1996

Identification of SLF1 as a new copper homeostasis gene involved in copper sulfide mineralization in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, at least 12 genes are important for cells to propagate in medium containing elevated concentrations of copper salts (J. Welch, S. Fogel, C. Buchman, and M. Karin, EMBO J. 8:255-260, 1989). Complementation studies were carried out on a copper-sensitive mutation (cup14) from this group. A new yeast gene, designated SLF1, was identified as a multicopy suppressor of the cup14 mutation. Slf1 is important for the physiological process of copper sulfide (CuS) mineralization on the surface of cells cultured in medium containing copper salts. CuS mineralization causes the cells to turn brown. Disruption of SLF1, which is located close to the telomere region of chromosome IV, leads to limited copper sensitivity, and the resulting cells lack the normal brownish coloration when grown in CuSO4-containing medium. Overproduction of Slf1 in wild-type cells confers superresistance to CuSO4 and enhances the coloration of cells cultured in the presence of CuSO4. Upon addition of KCN to Cu-grown cells, the brownish coloration was bleached instantly, and copper ions were solubilized. These data are consistent with Slf1-dependent accumulation of CuS complexes on the cell surface. Disruption of SFL1 also results in loss of the ability of yeast cells to deplete Cu but not Cd ions from the growth medium, whereas overexpression enhances Ca depletion ability and the resulting deposition of CuS particles. It is proposed that Slfl participates in a copper homeostasis pathway, distinct from the Cup1 detoxification system, that leads to sulfide generation and CuS biomineralization on the cell surface. This process may coordinate with the Cup1 pathway at different copper concentrations to prevent copper-induced toxicity.

Amino Acid Sequence↗
PubMed1995

Numerical modeling of the cornea's lamellar structure and birefringence properties.

A model of the cornea's lamellar structure is proposed that is capable of explaining experimental results obtained for the transmission of normal-incidence polarized light through rabbit and bovine cornea. The model consists of a large number of planar lamellae, each approximated as a uniaxial birefringent layer, stacked one upon another with various angular orientations. Polarized light transmission through the composite system is modeled theoretically by use of the Jones matrix formalism. The light transmission is calculated numerically for a large number of model lamellae arrangements, each generated from a statistical description, and histograms are constructed of various properties of the light transmission, including the minimum and maximum cross-polarized output intensities. It is demonstrated that various structural and optical parameters of the lamellae arrangements of actual corneas may be estimated by comparison of the calculations with detailed experimental data. Certain characteristics of the histograms are identified that permit a clear distinction between random and partially ordered systems. Comparisons with previously published experimental data provide strong evidence that the lamellae orientations are not entirely random, but rather a significant fraction are oriented in a fixed, preferred direction.

Animals↗
PubMed1995

Ultrastructure in anterior and posterior stroma of perfused human and rabbit corneas. Relation to transparency.

PURPOSE: The authors sought to discover whether there are differences in the degree of spatial order in the fibrillar ultrastructure between anterior and posterior stroma. METHODS: Human corneas were obtained from eye bank eyes. Although they had been classified as normal, some swelling remained after 3 hours of deturgescence. Freshly excised, unswollen rabbit corneas also were used. Image analysis methods were applied to transmission electron micrographs of the anterior, middle, and posterior stroma of these corneas to determine the positions and radii of fibrils, the fraction of total area occupied by fibrils, and the fibril number density. Results were used to calculate the interference factor that appears in the direct summation of the fields for light scattering theory and to estimate the total scattering cross-section per fibril. The interference factor is a measure of the spatial order in the positions and sizes of the fibrils. RESULTS: Electron micrographs showed anterior-posterior variations in size and number density of fibrils. The interference factor at wavelengths of visible light was lower in posterior stroma than in anterior stroma for humans and rabbits. In some instances in humans, the anterior interference factor was characteristic of mildly swollen cornea. When averaged for the electron micrographs analyzed, the anterior stroma was predicted to scatter approximately twice as much light per unit depth as the posterior stroma in humans (at any given wavelength) and approximately three times as much in rabbits. CONCLUSIONS: Calculations of the interference factor showed that there were differences in the anterior-posterior spatial ordering of fibrils. In human corneas, the differences could have been caused by intrinsic in vivo differences between anterior and posterior stroma; however, possible anterior-posterior variations in swelling between the two regions in vitro also could have affected the results.

Aged↗
PubMed1994

Validity of pulsatile ocular blood flow measurements.

The determination of average net pulsatile ocular blood flow form measurements of the intraocular pressure (IOP) pulse is based upon 1) an accurate measurement of IOP and its time variation; 2) a knowledge of the relation between the volume of the living eye and its IOP; 3) a physical model for the flow of blood through the eye: and 4) a concept of steady venous outflow from the eye. Each of these premises needs to be examined. The present analysis assesses the validity of the pneumatic tonometer for measuring the pressure in a flowing column of gas that is directed toward a thin membrane that is in contact with the surface of the cornea. The pressure of this stream of gas exerts a force against the cornea that depresses the corneal surface against the opposing force of the IOP. The balance of these forces and the resultant effect on the tonometer pressure sensor is described by the theories of elasticity, thermodynamics and fluid mechanics. In this treatment, differential equations are solved for the elastic deflections of the cornea subject to the opposing intraocular and tonometer pressures. This permits the pressure in the chamber of the pneumatic tonometer to be related through first principles to the IOP. The response of the tonometer pressure sensor to a range of IOPs (5-60 mmHg) is obtained, as well as the dynamical response of the tonometer to IOP oscillations. The conclusion is that the pneumatic tonometer provides a high fidelity, noninvasive measure of the IOP and its time variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Flow Velocity↗
PubMed1993

Effects of metallothionein on the observed copper distribution in cell extracts.

Systematic studies have been undertaken to compare the effects of cell lysis and chromatography conditions on the observed distribution of Cu amongst Cu-binding proteins in cultured cells. The variables included rate of centrifugation, presence or absence of non-ionic detergent, and presence or absence of dithiothreitol. The application of an improved FPLC gel filtration system has permitted us to examine the effects of the addition of exogenous metallothionein (MT) to cell extracts. When the cell extract contains low levels of endogenous MT, the addition of MT in the presence of dithiothreitol causes a shift of copper to the MT peak. High levels of MT can therefore remove copper from other Cu-binding ligands during cell homogenization, hence producing artifactual Cu distribution results. The use of an anaerobic buffer system has greatly reduced the observed level of Cu exchange, and has allowed comparison of Cu distribution in normal cells and cells from patients with Menkes' disease.

Animals↗
PubMed1993

Image processing of electron micrographs for light scattering calculations.

Theoretical light scattering calculations, when applied to models based on structures seen in electron micrographs of corneal stroma, require knowledge of the constituent fibril positions and radii. Obtaining this information manually is a difficult and time-consuming task. In order to facilitate this problem, we present a simple and flexible computer algorithm that allows the process to be automated using a Macintosh computer. The accuracy of the results is checked by comparing light scattering calculations using fibril positions and radii found manually with those found by the computer. The results show that the computer algorithm is a viable and accurate means of obtaining the needed data.

Algorithms↗
PubMed1992

Cornea epithelial damage thresholds in rabbits exposed to Tm:YAG laser radiation at 2.02 microns.

We have determined exposure conditions for minimal damage to the corneal epithelium in rabbit using a continuous wave Tm: YAG laser operating in the TEM00 mode at incident powers between 80 and 450 mW. The 1/e beam diameter was 0.94 mm and exposure durations for threshold damage ranged from 4.3 to 0.08 sec. Calculated temperature increases on the beam axis 10 microns beneath the surface at the measured thresholds were essentially constant and averaged 44 degrees C. This is basically the same temperature increase found for threshold CO2 laser damage and suggests that the critical temperature damage model, which correlates CO2 laser damage, can predict damage thresholds for mid-infrared laser radiation. We also showed that reliable thresholds can be determined in freshly enucleated eyes, thus opening up the possibility of using available laser sources in laboratories not equipped and approved for animal experiments to determine damage thresholds.

Animals↗
PubMed1990

A variational principle for the scattered wave.

A Schwinger-type variational principle is presented for the scattered field in the case of scalar wave scattering with an arbitrary field incident on an object of arbitrary shape with homogeneous Dirichlet boundary conditions. The result is variationally invariant at field points ranging from the surface of the scatterer to the farfield and is an important extension of the usual Schwinger variational principle for the scattering amplitude, which is a farfield quantity. Also, a generic procedure, physically motivated by the general principles of boundary conditions and shadowing, is presented for constructing simple trial functions to approximate the fields. The variational principle and the trial function design are tested for the special case of a spherical scatterer and accurate answers are found over the entire frequency range.

Acoustics↗
PubMed1989

Epithelial damage in rabbit corneas exposed to CO2 laser radiation.

Corneal injury thresholds are determined for conditions not previously explored for CO2 laser radiation, including multiple-pulse exposures and a systematic investigation of the effect of beam diameter on single-pulse damage thresholds. Multiple-pulse exposures from pulse trains up to 999 pulses, having pulse repetition frequencies between 1 and 100 Hz and individual pulse durations between 10(-3) and 0.5 s, were explored. Damage thresholds are discussed in terms of an approximate critical temperature model, the damage integral model and other empirical correlations. Single-pulse exposures are accurately correlated by an empirical critical temperature model in which the critical temperatures have a weak dependence on exposure duration. However, certain aspects of the single-pulse damage data led us to propose a new thermal damage model that incorporates an endothermic phase transition as the damage mechanism. This physical model accurately correlates single-pulse damage for exposures between 10(-3) and approximately 10 s.

Animals↗
PubMed1989

Estimation of pulsatile ocular blood flow from intraocular pressure.

A relationship has been derived between intraocular pressure and pulsatile blood flow in the eye. Measurements of intraocular pressure show a time variation that is associated with the pulsatile component of arterial pressure. Experimental results provide a means of transforming intraocular pressure changes into ocular volume changes. The eye is represented by a chamber with elastic walls, a pulsatile incoming flow of incompressible fluid (blood), and a steady outgoing flow of blood. Under these conditions, the rate of pulsatile blood flow through the eye can be approximated from the instantaneous intraocular pressure measurements. Data from a healthy human eye are used to illustrate the analysis.

Blood Flow Velocity↗
PubMed1986

Effects of fibril orientations on light scattering in the cornea.

It is generally assumed that the collagen fibrils in the stroma are the primary scatters of light in the nearly transparent cornea of the eye. We derive a scaling relationship between scattering angle and light wavelength that should hold if this hypothesis is valid. The derivation accounts for the cornea's layered nature and the azimuthal orientations of the fibrils in the different layers. The fibrils are treated as finite-length cylinders, and the scaling relation is obtained in both the far- and the intermediate-field zones. The predicted relationship is verified experimentally for normal-thickness rabbit corneas.

Animals↗
PubMed1983

Stromal damage in rabbit corneas exposed to CO2 laser radiation.

Threshold damage to the cornea from CO2 lasers is confined to the epithelium. Exposures well above the threshold for epithelial damage produce bowl-shaped stromal wounds. Light and electron microscopy and slit-lamp photographs all show a sharp demarcation between the damaged and undamaged regions 48 hr after exposure. The micrographs also show that the damaged region is accellular. Calculations of the expected temperature increases combined with analyses of slit lamp photographs show that the wound boundary corresponds to a surface of equal peak temperature increase. Comparisons with epithelial and endothelial damage conditions suggest that stromal, endothelial and epithelial cells have essentially the same thermal damage mechanism.

Animals↗