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Biomedical subjects

K Moffat

Publications and source records attributed to K Moffat.

At least 19 recordsLinked to original sources
PubMed1995

Optical studies of a bacterial photoreceptor protein, photoactive yellow protein, in single crystals.

Photoactive yellow protein (PYP), isolated from Ectothiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYP crystal has a bright yellow color and displays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73 degrees (or 107 degrees) with respect to the six-fold crystallographic symmetry axis. The crystal absorbance can be bleached reversibly as indicated by absorption changes. A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degrees C; a slower component of 1.4 +/- 0.2 s-1 accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s-1 for the other 40%. As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies. The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 kcal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our results show that the solution kinetic model is not directly applicable to crystals. A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.

Bacterial Proteins↗
PubMed1994

Photolysis-induced structural changes in single crystals of carbonmonoxy myoglobin at 40 K.

Myoglobin's reversible binding of oxygen is a model for studies of protein control of ligand binding and discrimination. Protein relaxation and geminate ligand rebinding subsequent to ligand photodissociation have been studied extensively by a variety of techniques. The ps to ns time scales for these processes are still much shorter than the ms time resolution of X-ray diffraction experiments, but it may be possible to trap these intermediates at low temperatures. We report here an X-ray diffraction investigation of structural changes induced by photolysis of carbonmonoxy myoglobin crystals at 40 K. Our results provide a structural basis for the interpretation of ambient and low temperature spectroscopic observations and molecular dynamics simulations of the ligand photodissociation and binding processes in haem proteins.

Animals↗
PubMed1992

Dye yellow vs. argon green laser in panretinal photocoagulation for proliferative diabetic retinopathy: a comparison of minimum power requirements.

We compared the power required to achieve a retinal burn with the dye yellow laser (wavelength 577 nm) and the argon green laser (wavelength 514 nm) in 49 eyes of 38 patients with proliferative diabetic retinopathy who underwent panretinal photocoagulation. All eyes were treated with both lasers. The dye laser required on average 35.7% less power to achieve a retinal burn than the argon laser (p = 0.0001). With both lasers more power was needed in patients older than 55 years than in those aged 55 years or less and in patients with light skin versus those with dark skin (p less than 0.02). The lower power required with the dye yellow laser allows facilitated photocoagulation in patients in whom treatment with the argon green laser would be problematic.

Adult↗
PubMed1992

Effect of dietary phosphoric acid supplementation on acid-base balance and mineral and bone metabolism in adult cats.

Experimental evidence indicates that maintenance of urinary pH < or = 6.4 is the single most effective means of preventing feline struvite crystalluria or urolithiasis of noninfectious causes. This may be accomplished by dietary acidification, but must be moderated to avoid potential adverse effects of excessive acidification, including bone demineralization, negative calcium balance, potassium depletion, and renal disease. Effects of chronic dietary phosphoric acid supplementation on acid-base balance and on mineral and bone metabolism were investigated in adult, domestic cats. One group of 6 cats was fed a basal, naturally acidifying diet without added acidifiers, and another group of 6 cats was fed 1.7% dietary phosphoric acid. Changes observed during 12 months of study included development of noncompensated metabolic acidosis, increased urinary calcium excretion, and lower but positive calcium balance in cats of both groups. Urinary pH decreased in cats of both groups, but was significantly (P < 0.05) and consistently maintained < or = 6.4 in cats given dietary phosphoric acid. Urinary phosphorus excretion increased in cats of both groups, but was significantly (P < 0.05) greater in phosphoric acid-supplemented cats, leading to lower overall phosphorus balance as well. Potassium balance decreased in cats of both groups, but was only transiently negative in the phosphoric acid-supplemented cats midway through the study, and normalized at positive values thereafter. Plasma taurine concentration was not affected by dietary acidification, and remained well within the acceptable reference range for taurine metabolism. Double labeling of bone in vivo with fluorescent markers was followed by bone biopsy and histomorphometric measurement of several static and dynamic variables of bone formation. Overall indices of bone formation decreased in cats of both groups with age and confinement, but were not affected by dietary phosphoric acid supplementation. Dietary supplementation with phosphoric acid used as the principal inorganic P source to achieve moderate and stable degree of urinary acidification, did not appear over the course of 1 year, to have induced adverse effects on mineral, bone, or taurine balance in these adult domestic cats.

Acid-Base Equilibrium↗
PubMed1991

Effect of vitrectomy on the ocular distribution of gentamicin in the rabbit.

To determine whether vitrectomy affects the ocular distribution of intravitreally injected gentamicin sulfate, 25 pigmented rabbits received an intravitreal injection of gentamicin (control group), and 25 other pigmented rabbits underwent pars plana vitrectomy followed by an intravitreal injection of gentamicin (experimental group). The animals were killed in groups of five 1, 3, 6, 12 and 24 hours after the injection. The mean retinal gentamicin levels 1 and 3 hours after the injection were significantly higher in the experimental group than in the control group (p less than 0.01). The mean gentamicin level in the aqueous humour was significantly lower in the experimental group than in the control group 1 hour (p less than 0.01), 6 hours (p less than 0.01) and 24 hours (p less than 0.001) after the injection. The mean corneal gentamicin level 24 hours after the injection was significantly lower in the experimental group than in the control group (p less than 0.05). There was no significant difference in mean choroidal gentamicin level between the two groups. We conclude that vitrectomy significantly affects the ocular distribution of gentamicin in the rabbit.

Animals↗
PubMed1986

The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins.

The structure of bovine intestinal calcium-binding protein (ICaBP) has been determined crystallographically at a resolution of 2.3 A and refined by a least squares technique to an R factor of 17.8%. The refined structure includes all 600 non-hydrogen protein atoms, two bound calcium ions, and solvent consisting of one sulfate ion and 36 water molecules. The molecule consists of two helix-loop-helix calcium-binding domains known as EF hands, connected by a linker containing a single turn of helix. Helix-helix interactions are primarily hydrophobic, but also include a few strategic hydrogen bonds. Most of the hydrogen bonds, however, are found in the calcium-binding loops, where they occur both within a single loop and between the two. Examination of the hydrogen bonding patterns in the calcium-binding loops of ICaBP and the related protein, parvalbumin, reveals several conserved hydrogen bonds which are evidently important for loop stabilization. The primary and tertiary structural features which promote the formation of an EF hand were originally identified from the structure of parvalbumin. They are modified in light of the ICaBP structure and considered as they apply to other calcium-binding proteins. The C-terminal domain of ICaBP is a normal EF hand, with ion binding properties similar to those of the calmodulin hands, but the N-terminal domain is a variant hand whose calcium ligands are mostly peptide carbonyls. Relative to a normal EF hand, this domain exhibits a similar KD for calcium binding but a greatly reduced affinity for calcium analogs such as cadmium and the lanthanide series. Lanthanides in particular may be inappropriate models for calcium in this system.

Amino Acid Sequence↗
PubMed1986

Extended X-ray absorption fine structure studies of Zn2Fe2 hybrid hemoglobins: absence of heme bond length changes in half-ligated species.

Metal hybrid hemoglobins, in which Zn(II) replaces Fe(II), have been structurally characterized by extended X-ray absorption structure (EXAFS) studies. Since Zn and Fe have very different K absorption edge energies, the structures of the ligated (Fe) and unligated (Zn) sites could be examined independently within a single molecule that mimics an intermediate ligation state. The observed EXAFS spectra and associated structural parameters are compared among the ligand free (alpha Zn)2(beta Zn)2, half-ligated (alpha FeCO)2(beta Zn)2 and (alpha Zn)2(beta FeCO)2, and fully ligated (alpha FeCO)2(beta FeCO)2 systems.

Heme↗
PubMed1985

Crystallization and preliminary x-ray characterization of bovine growth hormone. Purification of bovine prolactin and growth hormone.

A new purification scheme for both prolactin and growth hormone from bovine pituitaries has been developed which avoids the use of potentially damaging solution conditions. Both hormones were greater than 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had specific activities similar to or greater than standard samples of the same hormone as judged by several bioassays. Small single crystals of bovine growth hormone were obtained by vapor diffusion techniques. Examination of these crystals by x-ray diffraction, using the Cornell High Energy Synchrotron Source, showed that they were well ordered, and exhibited diffraction to 2.8-A resolution on still photographs. Precession and oscillation photographs showed that they belonged to the orthorhombic space group P2(1)2(1)2(1) (or P2(1)2(1)2) with unit cell dimensions a = 219 A, b = 51.9 A, c = 68.9 A. The density of the crystals was 1.19 +/- 0.02 g/ml from which the presence of eight 45,000-dalton dimers/unit cell was deduced. The protein content of the crystals was shown by isoelectric focusing to be identical to that of purified growth hormone in solution. These crystals appear suitable for use in the x-ray structure determination of bovine growth hormone to at least 3.2-A resolution.

Animals↗
PubMed1984

The lens capsule and rubeosis iridis: an angiographic study.

In 15 pigmented rabbits a pars plana lensectomy, vitrectomy and giant retinal tear with bullous retinal detachment were produced with maintenance of an intact anterior lens capsule. The incidence of subsequent rubeosis iridis, as detected angiographically, was compared with that in a control group of animals in whom the anterior lens capsule was not preserved but whose management was identical in all other respects. The rubeosis in the rabbits was angiographically identical to that seen in humans following vitrectomy, with immediate formation of a mossy hyperfluorescent surface membrane that leaked fluorescein extensively into the anterior chamber in the late phase of the angiography. It was possible to distinguish true neovascularization from breakdown of the blood-ocular barrier by the time and extent of the fluorescein leakage. Preservation of an intact anterior lens capsule reduced the incidence of unequivocal (florid) rubeosis from 73% to 33% (p less than 0.05).

Animals↗
PubMed1983

Cilioretinal artery obstruction.

The cases of 23 patients with untreated cilioretinal artery obstruction were reviewed. Three distinct groups were found: (1) isolated cilioretinal artery obstruction, (2) cilioretinal artery obstruction associated with central retinal venous obstruction, and (3) cilioretinal artery obstruction in conjunction with ischemic optic neuropathy. In the first group 90% of eyes achieved 6/12 or better vision and there was a high incidence of associated atherosclerotic carotid disease. In the second group, 70% of eyes improved to 6/12 or better, while in the last group no eye reached better than 6/120 vision.

Adolescent↗
PubMed1982

Extended X-ray absorption fine structure studies of cytochromes c: structural aspects of oxidation-reduction.

EXAFS fluorescence spectra were recorded for high-potential c-type cytochromes which range in oxidation-reduction potential from +145 to +365 mV. No average Fe-ligand bond length differences greater than 0.03 A were observed, for any cytochrome source of oxidation state. Least-squares analysis in combination with model calculations allowed limits to be set on the average Fe-N bond length (1.97-1.99 A) and the Fe-S bond length (2.29-2.32 A). A change of 0.05 A in either the average Fe-N or the Fe-S bond length is readily detectable with the range and quality of the data presented here. Two major conclusions are drawn from this study: In octahedrally coordinated iron porphyrin systems, Fe-N and Fe-S bond lengths are independent of oxidation-reduction potential, and they are also independent of oxidation state. A model for the regulation of oxidation-reduction potential in cytochrome c is proposed.

Animals↗