Griffith, O. Hayes H.

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Open Access
Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform
(Proceedings of the National Academy of Sciences, 1995-11) Kaneko, K.; Peretz, David; Pan, K. K.; Blochberger, Thomas C.; Wille, H.; Gabizon, R.; Griffith, O. H.; Cohen, F. E.; Baldwin, Michael A.; Prusiner, Stanley B.
Conversion of the cellular isoform of prion protein (PrPc) into the scrapie isoform (PrPSc) involves an increase in the /3-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPc and PrPSc form a complex during PrPScformation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPc to determine whether its properties were altered. Peptides encompassing two a-helical domains of PrP when mixed with PrPc produced a complex that displayed many properties of PrPSc. The PrPcpeptide complex formed fibrous aggregates and up to 65% of complexed PrPc sedimented at 100,000 x g for 1 h, whereas PrPc alone did not. These complexes were resistant to pro teolytic digestion and displayed a high /3-sheet content. Un expectedly, the peptide in a /3-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPc sensitive to protease digestion. While the pathogenic AllTV mutation increased the efficacy of complex formation, anti-PrP mono clonal antibody prevented interaction between PrPc and pep tides. Our findings in concert with transgenetic investigations argue that PrPc interacts with PrPSc through a domain that contains the first two putative a-helices. Whether PrPc-peptide complexes possess prion infectivity as determined by bioassays remains to be established.
Open Access
Electron optical benches for in-line and branched systems. A new bench designed for mirror-based aberration correction and low energy electron microscopy
(Review of Scientific Instruments, 1994-10) Skoczylas, W. P.; Rempfer, G. F.; Griffith, O. H.
A review of electron optical bench literature is presented, and the designs of two optical benches used by the authors are described. One bench was designed for testing individual electrostatic electron lenses and in-line optical systems, for example, emission electron microscopes and transmission electron microscopes. It has been in operation for many years. The second electron optical bench is new. It is a branched system designed for several purposes: to study correction of spherical and chromatic aberration with an electron mirror, and to gain experience with low energy electron microscopy (LEEM) optics. The alignment of the electron optical support structure is independent of the vacuum housing, and the bench is designed to be operated either horizontally or vertically. As a demonstration of the performance of the new bench in the horizontal mode, a test pattern on a silicon surface was imaged with LEEM optics.
Open Access
Phorbol ester-induced actin cytoskeletal reorganization requires a heavy metal ion, probably Zn2+.
(Cell Regulation, 1991-12) Hedberg, K. K.; Birrell, G. B.; Griffith, O. H.
The cell-permeant heavy metal chelator N,N,N',Ntetrakis( 2-pyridylmethyl)ethylenediamine (TPEN) was found to counteract phorbol ester-induced actin reorganization in PTK2 and Swiss 3T3 cells. By us ing fluorescence and the higher resolution tech nique of photoelectron microscopy to monitor actin patterns, 15-min pretreatment with 25-50 pM TPEN was found to dramatically reduce actin alterations resulting from subsequent phorbol ester treatment in PTK2 cells. Similar results were obtained with Swiss 3T3 cells using 50 mM TPEN for 1.5 h. Phorbol ester-induced actin alterations are thought to de pend on activation of protein kinase C (PKC). In contrast to the phorbol ester effect, the PKC-independent actin cytoskeletal disruption caused by staurosporine and cytochalasin B was unaffected by TPEN pretreatment. TPEN did not block phorbol ester-induced activation of PKC in Swiss 3T3 cells, as observed by the phosphorylation of the 80K PKC substrate protein (MARCKS protein). TPEN also did not inhibit partially purified PKC from Swiss 3T3 cells in an in vitro PKC-specific commercial assay. To establish that the effect of TPEN is the removal of metal ions and not some other nonspecific effect of TPEN, a series of transition metal ions was added at the end of the TPEN pretreatment. The results indicate that the transient but dramatic phorbol ester-induced reorganization of the actin cytoskeleton in cultured cells depends on an inter action of PKC with a heavy metal, probably zinc.
Open Access
Phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis
(Methods In Enzymology, 1991) Griffith, O. H.; Volwerk, J. J.; Kuppe, A.
Open Access
Biological Photocathodes
(Proceedings of the National Academy of Sciences, 1989-03) Griffith, O. H.; Habliston, D. L.; Birrell, G. B.; Skoczylas, W. P.; Hedberg, K. K.
Biological surfaces emit electrons when subjected to UV light. This emission is increased greatly after exposure to cesium vapor. Increases from 2 to 3 orders of magnitude are observed, depending on the biochemicals present. Heme and chlorophyll exhibit unusually high photoemission currents, which are increased further after cesiation. Photoemission from proteins and lipids is much less but also is increased by exposure to cesium. The formation of photocathodes with cesium greatly increases the practical magnifications attainable in photoelectron microscopy of organic and biological specimens. Photoelectron micrographs taken at magnifications ;;;: x 100,000 of chlorophyll-rich thylakoid membranes and of colloidal gold-labeled cytoskeleton preparations of cultured epithelial cells demonstrate the improvement in magnification. The selectivity and stability of the photocathodes suggest the possibility of detecting chromophore binding proteins in membranes and the design of photoelectron labels for tagging specific sites on biological surfaces.
Open Access
Migration in polyacrylamide gels of the 80K protein substrate for protein kinase C in mouse fibroblasts is dependent on the choice of crosslinker
(Biotechniques, 1989) Hedberg, K. K.; Birrell, G. B.; Griffith, O. H.
Open Access
Electron spin resonance of biological membranes ‑ Spin‑labeled lipids and proteins
(Magnetic Resonance Review, 1988) Volwerk, J. J.; Griffith, O. H.
Open Access
Immunophotoelectron Microscopy: The Electron Optical Analog of Immunofluorescence Microscopy
(Proceedings of the National Academy of Sciences, 1985-01) Birrell, G. B.; Habliston, D. L.; Nadakavukaren, K. K.; Griffith, O. H.
The electron optical analog of immunofluo rescence microscopy combines three developments: (i) photoelectron microscopy to produce a high-resolution image of ex posed components of the cell, (jQ site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, pro vides a way to extend the useful immunofluorescence micros copy technique to problems requiring much higher resolution. The resolution limit of fluorescence microscopy is limited to about 200 nm by the wavelength of the light used to form the image, whereas in photoelectron microscopy the image is formed by electrons (current resolution: 10-20 nm; theoretical limit: 5 nm or better depending on the electron optics). As a test system, cytoskeletons of CV-1 epithelial cells were pre pared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immu nofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing en hanced photoemission from labeled cellular components so that they stand out against the darker background of the re maining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemissionof the colloidal gold mark ers is sufficiently high that the microtubules are easily recog nized without reference to the immunofluorescence micro graphs, indicating that this approach can be used, in combina tion with antibodies, to correlate structure and function in cell biological studies.
Open Access
Photoelectron imaging of guinea pig, hamster, and human spermatozoa
(Journal of Reproductive Fertility, 1985) Mrsny, R.J.; Griffith, O. H.
Photoelectron images of mammalian spermatozoa were obtained by subjecting the specimens to u.v.-irradiation and focussing the emitted electrons by electron optics (photoelectron microscopy). Guinea-pig, hamster and human sperm atozoa were fixed in glutaraldehyde, deposited on conductive glass discs, and dehydrated. Sufficient quantities of photoelectrons were released from the surface of spermatozoa to produce images without staining, coating or metal shadowing. The large planar heads of guinea-pig spermatozoa were easily resolved with good delineation of acrosomal and postacrosomal regions. Residual vesicles could be visualized on the surface of the inner acrosomal membrane of spermatozoa that had undergone the acrosome reaction. Also detectable in these photoelectron images were finer membrane surface details, periodicities in the midpiece region of the tail which coincided with the distribution of mitochondria, and periodicities in the principal piece which appeared to be related to fibrous sheath components. Hamster spermatozoa were similarly well resolved but human spermatozoa were more difficult to image because of their increased surface curvature. The mechanism responsible for detection of these surface details is primarily topographical contrast rather than material contrast, since spermatozoa coated with a thin layer of gold or platinum exhibited similar features, althoughat reduced resolution, as the uncoated specimens.
Open Access
Photoelectron Imaging in Cell Biology
(Annual Review of Biophysics and Biophysical Chemistry, 1985) Griffith, O. H.; Rempfer, G. F.
Open Access
The potential role of photoelectron microscopy in the analysis of biological surfaces
(Scanning Electron Microscopy, 1984-05-07) Griffith, O. H.; Nadakavukaren, K. K.; Jost, P. C.
The photoelectric effect provides the basis for an imaging technique useful for the study of biological surfaces. The photoelectron microscope (PEM) employs a UV lamp to photoeject electrons from the specimen surface. The electrons are then accelerated and imaged using electron optics. Photoelectron micrographs often resemble scanning electron micrographs, but the origin of contrast is different and these two techniques are complementary. Scanning Electron Microscopy (SEM) is unsurpassed in applications where specimens have pronounced relief or where elemental analysis is required. The advantages of PEM are a new origin of contrast, high sensitivity to fine topographical detail, short depth of information, and low specimen conductivity requirements. Photoelectron Images of model systems, cell surfaces and cytoskeletal elements have been obtained.
Open Access
Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells
(Proceedings of the National Academy of Sciences, 1983-07) Nadakavukaren, K. K.; Chen, L. B.; Habliston, D. L.; Griffith, O. H.
Pt K2 rat kangaroo epithelial cells and Rat-I fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin , and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actincontaining stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.
Open Access
Strategies in applying spin labeling to the structure and dynamics of biological membranes
(Kemia-Kemi, 1982) Griffith, O. H.; Jost, P. C.
Open Access
A high vacuum photoelectron microscope for the study of biological specimens
(Scanning Electron Microscopy, 1981) Griffith, O. H.; Rempfer, G. F.; Lesch, G. H.
A photoelectron microscope (photoemission electron microscope) has been designed and built for the study of organic and biological samples. The microscope is an oil-free stainless steel high vacuum instrument pumped by a titanium sublimation pump, an ion pump, and molecular sieve roughing pumps. The electron lenses are of the electrostatic unipotential type. The microscope is equipped with a dewar for sample cooling, an internal cryogenic camera, TV-image intensifier, and vibration isolation support. Applications include studies of biological cell surfaces, photosynthetic membranes and aromatic chemical carcinogens. A representative micrograph of mouse 3T3 cells is included. In some respects, photoelectron micrographs resemble scanning electron micrographs, but the basis for contrast is different in these two techniques.
Open Access
Photoelectric properties and detection of the aromatic carcinogens benzo(a)pyrene and dimethylbenzanthracene
(Proceedings of the National Academy of Sciences, 1979-09) Houle, W. A.; Brown, H. M.; Griffith, O. H.
The absolute fhotoelectron quantum yield spectra for benzo[ a ]pyrene an dimethylbenzanthracene are present~d in th~ wavelength range 180-230 nm. These polycyclic aromatic carcmogens have photoelectron quantum yields of approximately 2 X 10-3 electrons per incident photon at 180 nm. The quantum yields fall off quickly and monotonically at wavelengths longer than 210 nm (5.9 eV). Threshold values for benzo[ a ]pyrene and dimethylbenzanthracene are 5.25 ± 0.06 eV and 5.27 ± 0.04 eV, respectively. The photoelectron quantum yields of benzo[ a ]pyrene and dimethylbenzanthracene are several orders of magnitude greater than typical components of biological membranes (amino acids, phospholipids, and polysaccharides). Preliminary micrographs of benzo[a]pyrene and dimethylbenzanthracene sublimed onto poly(L-lysine) and onto dimyristoyl phosphatidylcholine demonstrate the high contrast of small crystallites of carcinogens against a background of membrane components. These results and calculations involving relative contrast factors suggest that the distribution of these carcinogens in biological membranes can be determined by using photoelectron microscopy.
Open Access
Photoelectron microscopy of organic surfaces: The effect of substrate reflectivity
(Journal of Applied Physics, 1976-03) Dam, R. J.; Griffith, O. H.; Rempfer, G. F.
Photoelectron measurements of thin organic films deposited on a metal substrate may contain information from deep within the sample, derived from reflected ultraviolet light. This effect depends on the reflectivity of the substrate, the sample thickness and optical absorption coefficient, and the photoelectron escape depth. Calculations are given for phthalocyanine as a specific example. Contrast reversal and apparent seethrough effects resulting from reflection are predicted in overlapping thin films. Photoelectron micrographs of thin films and grid patterns of phthalocyanine show that the reflection model is essentially correct. This effect can be substantially reduced by using a nitrocellulose-coated carbon substrate.
Open Access
Photoelectron microscopy and quantum yields of membrane phospholipids
(Proceedings of the Electron Microscopy Society of America, 1976) Griffith, O. H.; Dam, R. J.
Open Access
Photoelectron microscopy of biological surfaces. Excitation source brightness requirements
(Society of Photo-Optical Instrumentation Engineers, 1976) Dam, R. J.; Griffith, O. H.
Photoelectron microscopy is a surface technique which provides topographical infor mation using the photoelectric effect as a basis for contrast. Progress in the biolo gical applications of this technique is briefly reviewed. Due to relatively low quantum yields, photoemission from biological samples is weak and an image intensifier is used in order to visualize and record the photoelectron image. Currently the limiting magnification is determined by UV power incident on the sample. Power requirements for high-magnification imaging are calculated in terms of microscope, sample, and image intensifier parameters. To approach 40 A resolution, an instrument magnification of 12,000-50,000 is required along with a UV intensity of 0.01 to 10 Watts/cm2 depending on the wavelength and sample. For a tightly focused laser source the total power re quirement is 1 mWatt or less.
Open Access
Photoelectron microscopy of biomembranes: Observation of external photomission from spinach chloroplasts
(Annual Proceedings of the Electron Microscopy Society of America, 1975) Dam, R. J.; Lesch, G. H.; Deamer, D. W.; Griffith, O. H.
Open Access
Lipid binding to the amphipathic membrane protein cytochrome b5
(Proceedings of the National Academy of Sciences, 1974-06) Dehlinger, P. J.; Jost, P. C.; Griffith, O. H.
ABSTRACT The lipid hinding properties of the membrane protein cytochrome b:. (detergent-extracted from calf liver microsomal preparations) were characterized by studying the interaction of spin-labeled lipid,- (5-, 12-, and 16-doxylstcaric acid and 5- and 16-cloxylphosphatidylcholine, where cloxyl refers to the nitroxide moiety) with cytochrome b :, using electron "pin resonance spectroscopy. The intact cytochrome b, n1olec11lc immobilizes all of the lipid spin label,;, while the segment of cytochrome b, released by trypsin doei. not afTcct lipid mobility. The immobilization of lipid spin label,-; on the h) ·drophohic surface of intact cytochrome b:, is not appreciably altered by associating the protein with liposomc" · DHTcrcnccs in polarity of the lipid binding site" between c, ·tochromc b, and phospholipid vc,.icles were also ob,-cr\'ed. The lipid binding sites on cytochrome b.; arc hydrophobic by conventional criteria, but arc more polar than the interior of fluid phospholipid bilayers.