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Biomedical subjects

L Powers

Publications and source records attributed to L Powers.

72 records · Page 4Linked to original sources
PubMed1980

Screening and replica plating of anti-hapten hybridomas with a transfer template hemolytic spot assay.

A localized hemolysis in gel assay is described for screening microcultures of hybridomas for the production of anti-hapten antibody. The keys to the rapid screening assay reported here are a special fenestrated transfer template and an improved hapten conjugated target cell. The transfer template is a 96-well plate with a calibrated hole in the bottom center of each well. To assay for anti-hapten antibodies, the transfer template is positioned over a 96-well microculture plate containing the growing hybridomas. After making contact with the tissue culture supernatant each orifice of the transfer template retains approximately 2 microliter of tissue culture supernatant. The transfer template is then placed onto an assay slide containing a thin layer of hapten conjugated target erythrocytes incorporated into agarose. After incubation with an anti-immunoglobulin and complement, areas of localized hemolysis in the gel indicate hybridomas which are secreting anti-hapten antibodies. The assay detects as little as 10 pg of antibody. Since the transfer template can be used as a replica plate, one can repeatedly transfer samples to various slides which contain either different hapten target cells or different hapten analog inhibitors in the agarose layer. Therefore, in addition to rapidly screening microcultures for positive hybridomas this procedure permits the characterization of each monoclonal antibody's fine specificity.

Animals↗
PubMed1979

The nature of the copper atoms of cytochrome c oxidase as studied by optical and x-ray absorption edge spectroscopy.

X-ray absorption edge spectroscopy has been used to study the copper of 1--2 mM cytochrome c oxidase in the resting oxidized, mixed-valence, and fully reduced states. A comparison was made of this protein with copper complexes and with natural and artificial copper proteins. Spectra were obtained with synchrotron radiation from the SPEAR storage ring using highly sensitive fluorescence detectors. Temperatures of -80 to -120 degrees C were employed further to improve the stability of the samples and to avoid the possibility of either auto- or photon-induced reduction of the materials, which might have occurred in previous studies. In order to characterize the valence states of the Cu and Fe components, the samples were monitored by infrared and visible spectroscopy before and after irradiation by the X-ray beam. The combination of the optical and X-ray absorption techniques has afforded a deconvolution of the four species of copper in the various states of cytochrome c oxidase and the tentative assignment of Cu alpha, the copper redox coupled to the heme alpha of cytochrome alpha, as a highly covalent type of copper and Cu alpha 3, the copper of cytochrome alpha 3, as a more ionic 'blue' type I copper. The implications of these findings upon the mechanism of action of cytochrome oxidase are briefly outlined.

Absorptiometry, Photon↗
PubMed1979

Optical studies of the phase behavior of monodomain samples of dipalmitoyl phosphatidylcholine containing calcium chloride.

Optical birefringence of the phases exhibited by monodomain samples of dipalmitoyl phosphatidylcholine containing 0.1 M-6 mM calcium chloride is measured over a range of temperature and water content. Little change was observed in the birefringence for this calcium chloride content range, and a phase diagram is constructed from these data and compared with that of monodomain samples of dipalmitoyl phosphatidylcholine and water. Effects of the presence of calcium chloride are most pronounced at low temperature and water content but the interaction with phosphatidylcholine cannot account for the effects observed with calcium on intermembrane interactions.

Birefringence↗
PubMed1979

Direct observation of the hydrocarbon chain tilt angle in phospholipid bilayers.

By means of X-ray diffraction we have observed the tilting of hydrocarbon chains within a dipalmitoyl phosphatidylcholine multilayer. The tilting produces a distribution of intensity in the (4.2 A)-1 region that provides a quantitative measure of the angle of tilt. Detailed diffraction investigation of multilayers prepared by a recently developed method and mounted to form a "freestanding" sample has made possible the accurate measurement of intensity in this region. We find the intensity distribution to be different from that previously reported and proceed to develop a new model for tilted chains. The model permits calculation of the intensity distribution as a function of tilt angle which is then fit to the data.

Fourier Analysis↗
PubMed1978

Head-group conformation in phospholipids: a phosphorus-31 nuclear magnetic resonance study of oriented monodomain dipalmitoylphosphatidylcholine bilayers.

Angular-dependent 31P NMR spectra of oriented biaxial monodomain DPPC.H2O multilayers are employed to study head-group conformation in this phospholipid. The results indicate that the O-P-O plane of the phosphate, where the O's are the nonesterified oxygens of the phosphodiester, is tilted at 47 +/- 5 degrees with respect to the bilayer normal. This PO4 orientation could result in the choline moiety being extended parallel to the bilayer plane, and it will explain the breadth of the axially symmetric 31P powder spectrum observed for DPPC in excess water. This work is the first direct observation of this conformation for lecithins and it illustrates the utility of high-resolution solid-state NMR in structural studies of disordered systems.

Magnetic Resonance Spectroscopy↗
PubMed1978

Brillouin light scattering measurement of the elastic properties of aligned multilamella lipid samples.

Brillouin measurements of the elastic properties are presented for aligned multilamella samples of both pure dipalmitoyl phosphatidylcholine at various temperatures and water concentrations and for the same compound containing 33 mol% cholesterol at various temperatures and two water concentrations. For pure dipalmitoyl phosphatidylcholine the elastic moduli change significantly at the gel transition and the modulus for area compression of individual bilayers is inferred to be an order of magnitude larger below the gel transformation than above. The presence of cholesterol is shown to influence the elastic behavior of dipalmitoyl phosphatidylcholine.

Journal Article↗
PubMed1978

Characteristics of the 'life spanning' phenomenon in Amoeba proteus. Independent nuclear and cytoplasmic ability to impose finite 'life span'.

Amoeba proteus given adequate food grows exponentially and clones of amoebae are normally immortal. After periods of food supply restriction to that necessary for maintenance, or food intake restriction by agitation, cells subsequently given a normal growth diet produce clones of finite life span. Reciprocal nuclear transfer between maintained and normal cells demonstrated that the nucleus and cytoplasm of maintained cells have acquired the independent ability to impose a finite life span on clones developing from cells whose other components are normal. In clones developing from maintained cells, inviable cell production is enhanced and inter-division times are prolonged. Inter-division times and clones mean doubling times do not show normal distribution.

Amoeba↗
PubMed1977

Monodomain samples of dipalmitoyl phosphatidylcholine with varying concentrations of water and other ingredients.

Methods are presented for the preparation of large monodomain phospholipid bilayer arrays containing variable amounts of water approaching the two-phase limit. The optical birefringence of these lamellar phases of dipalmitoyl phosphatidylcholine (DDPC) is measured over a range of temperature and water content, and phase transitions are observed. The techniques employed for pure DPPC and water are extended in order to produce macroscopically aligned samples containing varying concentrations of cholesterol, inorganic salts, antibiotics, and chlorophyll a. Polarization studies of the 670-nm band of chlorophyll a indicate macroscopic orientational order in the chromophore under the same conditions.

Anti-Bacterial Agents↗
PubMedDate not supplied

Multiple structures and functions of cytochrome oxidase.

X-ray absorption studies have been used to investigate the structure of the four redox centers (2Fe, 2Cu) of the terminal enzyme in the respiratory chain, cytochrome c oxidase in the resting oxidized form as well as in the functional intermediates that are freeze-trapped. Methods of x-ray fluorescence detection for these low-concentration samples together with low-temperature cryostats and simultaneous optical monitoring were developed to ensure good signal-to-noise data and sample integrity. The resting oxidized form contains a sulfur bridge between the copper and iron of the active site which are separated by approximately 3.8 A. This separation of the active site metal atoms was uniquely identified by comparison of both the iron and copper EXAFS data and iron EXAFS of the copper-depleted enzyme. In the reduced state, the CO or O2 is bound to the active site iron having a structure identical to CO or oxy hemoglobin while the sulfur remains with the active site copper. Little change in structure is observed for the other iron and copper. It is the sulfur bridged active site form that is isolated by the Yonetani and Caughy methods with greater than or equal to 85% homogeneity but not the Hartzell-Beinert or similar methods. Another form observed in the redox cycle is also fully oxidized but lacks the sulfur bridged active site with the iron of the active site having a structure identical to that of the peroxidases. This form exhibits peroxidase as well as oxidase activity, and a stable intermediate is formed with hydrogen and ethylhydrogen peroxide in which the iron of the active site is structurally similar to that of the peroxidase intermediate. The active site copper, however, does not participate in the peroxidatic role and the structures of the other iron and copper are identical to those of the sulfur bridged resting oxidized form. Thus this unique enzyme has peroxidase activity which may serve to safeguard its main oxidase function.

Animals↗