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Biomedical subjects

D E Wolf

Publications and source records attributed to D E Wolf.

At least 19 recordsLinked to original sources
PubMed1998

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells.

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 x 10(-7) cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (</=1 x 10(-7) cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 x 10(-7) cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Animals↗
PubMed1998

Mobility of TrkA is regulated by phosphorylation and interactions with the low-affinity NGF receptor.

The nerve growth factor (NGF) receptor is a complex of two proteins, gp75 and the tyrosine kinase TrkA. Using fluorescence recovery after photobleaching, we have studied the diffusion properties of the TrkA receptor. For PC12 cells that express both gp75 and TrkA, TrkA was relatively immobile with only 28 +/- 1% of receptor molecules free to diffuse with D = (3.64 +/- 0.23) x 10(-9) cm2/s. Addition of NGF decreased the mobile fraction to 21 +/- 1% with D = (4.11 +/- 0.18) x 10(-9) cm2/s. Using the Sf9 baculovirus expression system, we were able to study TrkA in the absence and presence of gp75. On Sf9 cells, TrkA showed a mobile fraction of 46 +/- 2% with D = (2.64 +/- 0.21) x 10(-9) cm2/s in the absence of gp75 and 43 +/- 2% with D = (2.31 +/- 0.25) x 10(-9) cm2/s in its presence. Thus, gp75 did not alter TrkA mobility. Addition of NGF to the medium approximately halved the mobile fraction for TrkA in both the absence and presence of gp75. However, using a kinase-deficient mutant of TrkA, we found that ligand-induced immobilization requires an active kinase in the absence of gp75 but not in its presence. In addition, using point mutations at specific TrkA autophosphorylation sites, we determined that mobility is controlled by multiple phosphorylation sites, but the SHC binding site at Y490 may be particularly important for ligand-induced immobilization of TrkA. Therefore, two mechanisms lead to NGF-induced immobilization of TrkA--the first resulting from autophosphorylation of TrkA and the second occurring through TrkA's association with gp75.

Animals↗
PubMed1998

Changes in lipid diffusibility in the hamster sperm head plasma membrane during capacitation in vivo and in vitro.

The technique of fluorescence recovery after photobleaching (FRAP) was employed on spermatozoa labeled with the fluorescent lipid analogue C14dil to provide two measures of lateral diffusion in the plane of the sperm plasma membrane during capacitation in vivo and in vitro: the diffusion coefficient (D) for C14dil and the fraction of C14dil that is free to diffuse (%R) within the domain. To evaluate changes in lipid diffusibility during capacitation in vivo, spermatozoa were recovered from the uterus within 30 min after ejaculation or from the oviduct at 2, 4, 6 and 8 hr after mating. To compare the changes which occur in vivo with those which occur during capacitation in vitro, caudal epididymal spermatozoa were incubated under capacitating or non-capacitating (control) conditions for 4 hr. Although transient changes in D occurred during the course of capacitation, there was no net change in D for either anterior (AH) or posterior head (PH) domains following capacitation in vitro or in vivo. Significant differences in the lipid diffusion coefficient between the two head domains were observed during the course of capacitation. A transient decrease in %R was observed for the AH domain during capacitation in vitro and incubation under control conditions, but no significant change in %R was observed in the AH domain during capacitation in vivo. A significant decline in %R of the PH domain was observed for spermatozoa during capacitation in vivo, in vitro and following incubation under non-capacitating conditions. These data indicate that the changes in the lipid diffusibility of the AH and PH domains which occur during capacitation in vivo exhibit both similarities and differences to those which occur during capacitation in vitro.

Animals↗
PubMed1998

Quantitative video microscopy.

Obviously there are many variations and embellishments on the topic of quantitating your image. I have tried here to offer you a very practical guide which highlights some of the critical issues paying particular attention to problems which can prevent or compromise your success. These include: errors due to aspect ratio, use of automatic camera settings, improper setting of dynamic range, and use of integer arithmetic. Further information can be found in several other chapters in this volume as well as in the references below. Additionally you shouldn't overlook the technical manuals which come with your video systems. The field of video microscopy has evolved as a cooperative effort between academia and industry. As a result you will find that many of the technical support personnel from microscope, video, and image processing companies are well versed on the issue of video imaging in biology and are more than willing to assist you.

Image Enhancement↗
PubMed1996

Fluorescence resonance energy transfer analysis of ribozyme kinetics reveals the mode of action of a facilitator oligonucleotide.

A defining characteristic of catalysts is the rate at which they can process multiple copies of substrate. In the case of synthetic hammerhead ribozymes that cleave an RNA sequence, binding of the ribozyme to the substrate and products is through base-paired duplexes. The kinetics of formation and dissociation of these duplexes can determine the turnover of the ribozyme. We have followed these processes in real time by using fluorescent labels that can interact through fluorescence resonance energy transfer (FRET). This approach has been used to identify the rate-limiting steps for a particular ribozyme and to reveal how turnover was improved by a facilitator oligonucleotide. It was found that dissociation of the ribozyme-substrate complex is faster than cleavage to products. Hence, to undergo cleavage, most substrate molecules must interact with a ribozyme more than once. In the presence of a facilitator oligonucleotide, the complex is stabilized so that cleavage is faster than dissociation. Under these circumstances, cleavage of the substrate becomes the most likely outcome following binding to the ribozyme.

Base Sequence↗
PubMed1996

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA.

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.

Animals↗
PubMed1995

Distribution and dynamics of mouse sperm surface galactosyltransferase: implications for mammalian fertilization.

It has been proposed that a mouse sperm surface beta-1,4-galactosyltransferase functions as a receptor for the zona pellucida during fertilization. In this paper we used two monovalent fluorescent probes specific for galactosyltransferase: a trinitrophenylated derivative of UDP-galactose and rhodaminated alpha-lactalbumin. We found that galactosyltransferase was initially present over the posterior head of acrosome-intact sperm but became progressively localized to the plasma membrane overlying the acrosomal region after it was cross-linked with an anti-galactosyltransferase polyclonal antibody. Labeled mouse sperm that were treated with the calcium ionophore A23187 revealed that galactosyltransferase remained on the posterior head after acrosomal exocytosis. However, if galactosyltransferase was first cross-linked and redistributed with antibody and then acrosome reacted with A23187, all head fluorescence was lost. In addition, although anti-galactosyltransferase antibody induced a surface redistribution, it did not, by itself, lead to the release of acrosin, the endpoint of the acrosome reaction. Finally, using the technique of fluorescence recovery after photobleaching, we found that, in the absence of bivalent antibody, mouse sperm surface galactosyltransferase exhibited 40-50% recovery with a high diffusion coefficient on the anterior head (5-8 x 10(-9) cm2/s) approximately 2 times greater than on the posterior head (2-4 x 10(-9) cm2/s). When galactosyltransferase was cross-linked and redistributed to the anterior head using the bivalent antibody, the mobile fraction decreased to 20-30% with no significant change in the diffusion coefficient.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Interaction with TrkA immobilizes gp75 in the high affinity nerve growth factor receptor complex.

It has been proposed that the high affinity nerve growth factor (NGF) receptor required for NGF response is a complex of two receptor proteins, gp75 and the tyrosine kinase TrkA, but direct biochemical or biophysical evidence has been lacking. We have previously shown using fluorescence recovery after photobleaching that gp75 is highly mobile on NGF-nonresponsive cells, but relatively immobile on NGF-responsive cells. In this report, we show that a physical interaction with TrkA causes gp75 immobilization. We found that gp75 is relatively mobile on TrkA negative nnr5 cells, a PC12 variant which is nonresponsive to NGF. In contrast, on T14 nnr5 cells (which bear a TrkA expression vector) gp75 is relatively immobile. Similarly, using baculoviruses to express gp75 and TrkA on Sf9 insect cells, we found that TrkA immobilizes gp75 molecules. The related receptor, TrkB, caused a more modest immobilization of gp75. Immobilization was found to require intact TrkA kinase and gp75 cytoplasmic domains, paralleling the requirements of high affinity binding of NGF. Analysis of gp75 diffusion coefficients indicates that mutated gp75 and TrkA molecules may form a complex, even in the absence of the ability to bind NGF with high affinity.

Animals↗
PubMed1995

Differential biological effects of K252 kinase inhibitors are related to membrane solubility but not to permeability.

K252a and K252b are related protein kinase inhibitors that, dependent on conditions, can either inhibit or potentiate the effects of neurotrophic factors. K252a, an ester, is more potent and more cytotoxic on intact cells than K252b, a carboxylic acid. To understand better why these drugs elicit different degrees of biological responses, we analyzed their hydrophobicity, cell permeability, and subcellular distribution. As judged by partitioning between organic and aqueous phases, both compounds are hydrophobic. The partition coefficients were 15.6:1 (organic/aqueous phases) for K252a and 4.4:1 for K252b. The ratio of fluorescence excitation at 352 nm to that at 340 nm for the K252 compounds in the organic alcohol 1-decanol versus water provides a simple assay of binding of these compounds to phospholipid membranes. This ratio shifted for K252a, but not K252b, in the presence of phospholipid vesicles, indicating that K252a dissolved in the hydrophobic interior of the membrane. Using quantitative video fluorescence microscopy, we found that K252a strongly labeled both Sf9 insect cells and PC12 rat pheochromocytoma cells, probably staining intracellular membranes. The uptake of K252a was rapid and apparently irreversible. K252b also quickly entered Sf9 and PC12 cells, but staining was much weaker. Hence, K252a and K252b are similar in that they both rapidly enter cells but greatly differ in their membrane solubility.

Animals↗
PubMed1994

Feedback control of the metaphase-anaphase transition in sea urchin zygotes: role of maloriented chromosomes.

To help ensure the fidelity of chromosome transmission during mitosis, sea urchin zygotes have feedback control mechanisms for the metaphase-anaphase transition that monitor the assembly of spindle microtubules and the complete absence of proper chromosome attachment to the spindle. The way in which these feedback controls work has not been known. In this study we directly test the proposal that these controls operate by maloriented chromosomes producing a diffusible inhibitor of the metaphase-anaphase transition. We show that zygotes having 50% of their chromosomes (approximately 20) unattached or monoriented initiate anaphase at the same time as the controls, a time that is well within the maximum period these zygotes will spend in mitosis. In vivo observations of the unattached maternal chromosomes indicate that they are functionally within the sphere of influence of the molecular events that cause chromosome disjunction in the spindle. Although the unattached chromosomes disjoin (anaphase onset without chromosome movement) several minutes after spindle anaphase onset, their disjunction is correlated with the time of spindle anaphase onset, not the time their nucleus breaks down. This suggests that the molecular events that trigger chromosome disjunction originate in the central spindle and propagate outward. Our results show that the mechanisms for the feedback control of the metaphase-anaphase transition in sea urchin zygotes do not involve a diffusible inhibitor produced by maloriented chromosomes. Even though the feedback controls for the metaphase-anaphase transition may detect the complete absence of properly attached chromosomes, they are insensitive to unattached or mono-oriented chromosomes as long as some chromosomes are properly attached to the spindle.

Anaphase↗
PubMed1993

Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152.

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm.

Animals↗
PubMed1992

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.

Acrosome↗
PubMed1992

Theory of fluorescence recovery after photobleaching measurements on cylindrical surfaces.

The theory of fluorescence recovery after photobleaching measurements of isotropic diffusion on a cylindrical surface is developed for Gaussian beam illumination centered perpendicular to an infinitely-long cylinder. A general analytical solution is obtained which is a function of the ratio of the cylindrical radius (r) to the beam exp [-2] radius omega. Numerical analysis of this solution demonstrates that significant deviations from one dimensional recovery are observed for omega less than 3r and from two-dimensional recovery for omega greater than or equal to 0.5r. Numerical data and an algorithm for analysis of recovery data where 0.5r less than or equal to omega less than or equal to 3r is presented.

Biophysical Phenomena↗
PubMed1991

Nerve growth factor receptors are preaggregated and immobile on responsive cells.

It has been hypothesized that signal transduction occurs by ligand-induced receptor clustering and immobilization. For many peptide receptors, cross-linking by anti-receptor antibodies is sufficient for receptor activation. This is not, however, the case for nerve growth factor receptor (NGFR). Using fluorescence microscopy and fluorescence recovery after photobleaching (FRAP), we have analyzed the distribution and diffusibility of NGFR on a series of cell lines. We have found the following: (1) Cells expressing high-affinity responsive NGFR's display clustered NGFR's even in the absence of ligand. In contrast, NGFR's in nonresponsive cell lines are diffusely distributed. (2) Receptors on responsive cell lines are largely nondiffusing while most receptors on nonresponsive cell lines are relatively free to diffuse. (3) NGF does not greatly alter the distribution or diffusion properties of the NGFR on either nonresponsive or responsive cell lines. Thus, NGFR is preclustered and immobile on responsive cells, which suggests that immobilization of NGFR prior to ligand binding is required for signal transduction.

Animals↗
PubMed1991

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction.

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.

Acrosome↗
PubMed1990

Synthesis, purification, and characterization of 2,4,6-trinitrophenyl-UDP-galactose: a fluorescent substrate for galactosyltransferase.

Glycosyltransferases enzymatically transfer monosaccharides from sugar-nucleotides to complex oligosaccharide chains and, as cell surface molecules, exhibit receptor-like activity. We have modified the substate UDP-galactose to produce a compound that has useful absorbance and fluorescence properties upon binding to galactosyltransferase (GalTase). Using strategies similar to those for preparing fluorescent ATP analogs, we were able to synthesize 2,4,6-trinitrophenyl-5'-UDP-galactose (TUG). In solution, the absorbance properties of TUG are pH dependent, with absorbance maxima at 260, 408, and 453 nm and an isobestic point at 353 nm. In the presence of soluble GalTase extracted from bovine milk, TUG exhibited an excitation maximum at 368 nm with emission maxima at 436 and 533 nm; in the absence of GalTase only the 533-nm peak was present. Under enzymatic conditions, TUG acted as a competitive substrate of UDP-galactose with GalTase. Under noncatalytic conditions, the fluorescence emission of TUG at 436 nm increased monotonically with Gal-Tase concentration, with a half-maximal response at approximately 4 microM. This compound may be useful for quantifying GalTase function as both an enzyme and a cell adhesion molecule.

Binding, Competitive↗