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T Frey

Publications and source records attributed to T Frey.

At least 37 records · Page 2Linked to original sources
PubMed1992

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.

Affinity Labels↗
PubMed1992

Office probing of congenital nasolacrimal duct obstruction.

We report the results of office nasolacrimal duct probing under topical anesthesia in 2369 eyes with symptomatic nasolacrimal duct obstruction. The overall success rate for cure by initial probing was 92%. In patients less than 9 months old, a 94% cure rate was achieved with one probing; in patients 9 months and older, the rate decreased to 84%. The decrease in success rate after 9 months was statistically significant (P less than .00001 by chi-square analysis). A majority of 180 parents of these patients surveyed said they were happy with the office procedure and preferred having the probing performed to resolve symptoms, even though they realized that the symptoms most probably would resolve by 1 year of age without it. We conclude that office probing for congenital nasolacrimal duct obstruction is a safe and effective method for treatment of symptomatic nasolacrimal duct obstruction, allowing the cost and risk of general anesthesia as well as the cost and inconvenience of prolonged medical management to be avoided.

Age Factors↗
PubMed1992

Acanthoma supratrochantericum.

We report the cases of five patients with lesions of benign reactive hyperplasia of the skin in the hip joint region. In view of the distinct clinical features, and in analogy with previously reported cases of acanthoma fissuratum, we termed this condition acanthoma supratrochantericum.

Aged↗
PubMed1990

High-affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes.

Soybean membranes possess high-affinity binding sites for fungal beta-glucans that elicit phytoalexin synthesis. The ability of 1,3-1,6-beta-glucans, released by acid hydrolysis from mycelial walls of Phytophthora megasperma f.sp. glycinea, to compete for the putative phytoalexin elicitor receptors increases with their average degree of polymerization (DP). The results suggest a function where the probability for glucan fragments of containing a structural determinant that is optimal for binding approaches 1 as the DP tends to infinity. Ligand displacement data obtained against a 125I-labeled glucan elicitor (average DP = 18) provided a theoretical minimum IC50 (50% inhibitory concentration) for 1,3-1,6-beta-glucans of 3 nM. The IC50 value obtained for a synthetic hepta-beta-glucoside having a known elicitor-active structure was 8 nM, remarkably close to the predicted value. Displacement of the 125I-glucan of large DP was uniform and complete showing that the heptaglucoside had access, with similar affinity, to all sites available to the radioligand. Further analysis using a 125I-labeled aminophenethylamine derivative of the heptaglucoside suggested that the putative glucan-elicitor receptors bind a basic structural determinant present in all elicitor-active glucans from the soybean pathogen P. megasperma.

Binding, Competitive↗
PubMed1990

Solubilization of soybean membrane binding sites for fungal beta-glucans that elicit phytoalexin accumulation.

Soybean membranes contain high-affinity binding sites for fungal beta-glucans. These sites may play a role in the recognition by soybean tissues of fungal phytoalexin elicitors. We have solubilized beta-glucan-binding activity from microsomal membranes using two C12-alkyl zwitterionic detergents, Zwittergent 3-12 (ZW 3-12) and the lysolecithin analog 1-dodecanoyl propanediol-3-phosphorylcholine [corrected] (ES12H). The solubilized binding sites displayed identical affinity for beta-glucans as that found in membranes (KD = 11-34 nM). Detergent-protein micelles with glucan binding activity eluted with approximate Mr values of 300,000 in ZW 3-12 and 380,000 in ES12H in gel permeation chromatography. Maximal binding activity eluted from a chromatofocusing column in the pH range between 6.2 and 6.6 with both ES12H and ZW 3-12, suggesting an apparent pI close to neutral.

Binding Sites↗
PubMed1988

Line-shape analysis of NMR difference spectra of an anti-spin-label antibody.

Specifically deuteriated Fab fragments of the anti-spin-label antibody AN02 were prepared. NMR difference spectra were obtained, in which the spectrum of Fab with some fraction of the binding sites occupied with spin-label hapten was subtracted from the spectrum of Fab with no spin-label. The peak heights were analyzed as a function of the fractional occupation of the binding site, using a computer program that calculates a best fit to the observed spectra. This method treats all of the peaks in the spectra simultaneously. Analyzing all peaks at once allows for the interdependencies in the spectra arising from overlap of positive and negative signals from different peaks. The fitting program calculates line widths for the peaks arising from protons in the binding site region. Almost all of the line widths calculated for the spectrum of the Fab complex with diamagnetic hapten dinitrophenyldiglycine were found to be narrower than the line widths of the corresponding resonances in the spectrum of Fab with an empty binding site. The distances of the binding site region protons from the unpaired electron of the hapten were also obtained from this calculation. Two tyrosine protons were found to be close (less than A) to this electron. These line-width and distance results are discussed with respect to the structure and dynamics of the antibody binding site.

Immunoglobulin Fab Fragments↗
PubMed1987

Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody.

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase.

A new substrate for ubiquitin carboxyl-terminal hydrolase, the carboxyl-terminal ethyl ester of ubiquitin, has been synthesized by a trypsin-catalyzed transpeptidation. In the presence of 1.6 M glycylglycine ethyl ester, trypsin removes the carboxyl-terminal glycylglycine of ubiquitin and replaces it with the dipeptide ester. The equilibrium mixture under these conditions contains 30% ubiquitin ethyl ester and 70% hydrolysis product, the 74-residue fragment of ubiquitin. Ubiquitin ethyl ester can be purified by gel filtration and ion-exchange chromatography. The structure of this product has been verified by identification of the products of base hydrolysis, tryptic cleavage in aqueous solution, and peptide mapping. When ubiquitin ethyl ester is incubated with purified ubiquitin carboxyl-terminal hydrolase, specific cleavage of the ester linkage is observed. A rapid, sensitive assay is described utilizing high-performance liquid chromatography. By use of this assay, it has been shown that ubiquitin carboxyl-terminal hydrolase is inactivated in the absence of thiols. Optimal protective effects are seen with 10 mM dithiothreitol. The rate of catalysis is maximal at pH 8.5, with evidence for catalytically important groups with pK values of 5.2, 7.6, and 9.5. These findings are consistent with the participation of a thiol group in the active site. Native ubiquitin is a competitive inhibitor of ubiquitin ethyl ester hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocytes↗
PubMed1984

Nonaromatic amino acids in the combining site region of a monoclonal anti-spin-label antibody.

The nuclear magnetic resonance spectra of monoclonal Fab antibody fragments have been recorded in the absence and presence of the specific spin-label dinitrophenyl hapten. The difference spectra reveal the presence of about 50 amino acids in the region of the combining site. By selective deuteration and by use of double difference spectra, all the resonances in the spectral region -1 to 1.5 ppm have been identified. We have found that in the combining site region there are four or five valines, certainly three and possibly five threonines, three or four leucines, two or three isoleucines, and six or seven alanines. Selective deuteration of methionine and lysine reveals one methionine and two lysines in the difference spectra. All of these amino acids are estimated to be within 17 A of the paramagnetic hapten. By using difference spectra involving low fractional occupancy of the combining site with the spin-label hapten, it is established that one threonine and one valine are very close to the paramagnetic hapten.

Amino Acids↗
PubMed1984

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody.

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.

Antibodies, Monoclonal↗
PubMed1984

Activatable esterase activity of murine natural killer cell--YAC tumour cell conjugates.

Natural killer (NK) cells have been obtained from mouse spleens and grown in vitro. These cells: retained cytotoxicity against YAC targets; and were homogeneous as judged by morphology and surface markers. The alpha-naphthol acetate esterases (ANAE) and diisopropyl-fluorophosphate (DFP)-binding proteins of NK cells, YAC cells and NK-YAC conjugates have been examined. Ultrastructural cytochemistry indicates that NK cells have two types of ANAE: an enzyme primarily associated with the granule externum and an activatable ANAE associated with the granule internum. Both of these activities are inhibited by DFP. The activatable ANAE appears during incubation of NK cells with YAC cells. DFP-binding proteins were examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autofluorography. NK cells and YAC cells have major DFP-binding proteins of 35 X 10(3) and 20 X 10(3) Mr, respectively. NK-YAC conjugates had a new band at 55 X 10(3) Mr. This new protein may be identical to the activatable ANAE described above. Our studies provide evidence for an NK cell esterase that becomes activated when incubated in the presence of tumour cells. This esterase may be related to the cytolytic mechanism.

Animals↗
PubMed1983

Pediatric eye trauma.

The pediatrician, faced with a child with an injured eye, faces a large responsibility. The key to proper disposition is an orderly approach with a good history and with a methodical examination and the use of ophthalmologic aids, such as topical anaesthetics, good light and magnification. The pediatrician can handle many problems in his office, such as tarsal foreign bodies and corneal abrasions, but he should be reassured that he has in the wings an ally, the ophthalmologist, standing to help anywhere along the line.

Child↗
PubMed1983

Applications of energy filtered imaging in biology.

We describe the use of a magnetic sector spectrometer positioned below the projection chamber of an electron microscope for energy filtered transmission imaging. The spectrometer used has circular pole face edges and is corrected for second order aberrations. A round EM lens is placed after the sector to form a real image of the virtual achromatic image produced by the spectrometer. A slit placed in the dispersion plane allows the passage of electrons in a selected energy range. The filtered image is projected onto a transmission phosphor and acquired with a silicon intensified TV camera and stored in digital form on computer disk. Filtered images are taken at two energies, one immediately preceding (pre-edge) and one on the characteristic energy loss (edge). To obtain images showing the distribution of elements, background subtraction is performed by either subtraction or division of edge and pre-edge images. The optical properties of the imaging system are described and the results are illustrated by energy filtered images of single ferritin molecules (Fe M2,3 and C k), the phosphorus distribution in ribosomes (PL2,3) and the localization of calcium in muscle (Ca L2, 3). The major advantage of the system, compared to other energy filtered imaging methods, is that it can be readily adapted to existing high vacuum microscopes without the necessity of modifying the column to insert a spectrometer.

Animals↗
PubMed1982

Electron microscopic study of natural killer cell-tumor cell conjugates.

Natural killer (NK) cells were obtained from C3H mouse spleens according to a modified version of the method of Kuribayashi et al. [Kuribayashi, K., Gillis, S., Dern, D. E. & Henney, C. S. (1981) J. Immunol. 126, 2321-2327]. These cells retain in vitro cytotoxicity against certain model tumor cell targets and appear homogeneous by morphological criteria. NK cells, YAC (tumor) cells, and NK cell-YAC cell conjugates have been examined with scanning (SEM) and transmission (TEM) electron microscopy. SEM experiments have shown that: (i) NK cells are large and possess various shapes in contrast to the YAC target cells which are smaller and round, (ii) YAC cells have uniformly distributed microvilli whereas the NK cell microvilli are most prominent in the area of effector-to-target contact, and (iii) in the absence of target cells, NK cell microvilli are found in a small number (usually 1-3) of cell surface locations. The region of NK cell-tumor cell contact has also been examined with TEM. The cells were stained with ruthenium red/OsO(4). The electron-dense ruthenium red/OsO(4) reaction product was consistently found in regions of close cell-cell contact, suggesting that carbohydrates were not completely cleared from areas of contact and that target and effector membranes do not fuse extensively. TEM observations indicate that NK cells have structurally unique granules. The granules are composed of at least two distinct compartments. The outer compartment contains the lysosome-associated enzymes acid phosphatase and inorganic trimetaphosphatase. No enzymatic activities have been found associated with the inner compartment. NK cells appear to degranulate when incubated with YAC cells. Under those circumstances, limited areas of the NK cytoplasm contain vacuole-like areas possessing granules and apparent granular debris. Degranulation appears to be involved in the cytotoxic function of NK cells.

Animals↗
PubMed1982

Isolated paresis of the inferior oblique.

Four cases of isolated superior oblique paresis are presented. The usual course of events was that of a gradually increasing diplopia and head tilt. All patients fulfilled the three-step test criteria, with a hypertropia that increased on side gaze to the ipsilateral side and on head tilt to the side opposite that of the paretic muscle. All patients were treated with a superior rectus recession. Three patients had their hypertropia reduced to between zero and 2 diopters. One patient needed, in addition to his superior rectus recession, an inferior rectus resection to eliminate the hypertropia. Vertical rectus surgery presents an alternative to superior oblique muscle tenotomy in treating inferior oblique paresis.

Adult↗
PubMed1980

Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins.

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.

Animals↗