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Biomedical subjects

D Axelrod

Publications and source records attributed to D Axelrod.

At least 37 records · Page 2Linked to original sources
PubMed1997

Is axillary dissection always indicated in invasive breast cancer?

In light of the changing trends in the diagnosis and management of invasive breast cancer, the practice of routine axillary dissection should be reevaluated. A growing number of patients with breast cancer are diagnosed as having small tumors with an associated low risk of lymph node metastases. The pathologic features of the primary tumor are increasingly being used as a prognostic guide for recommendations about adjuvant systemic therapy, and there are recent reports suggesting a superior prognostic value for tumor cells detected in bone marrow, as compared to axillary lymph node metastases. Consequently, axillary lymph node status is no longer the single prognostic guide for recommendations about adjuvant systemic therapy. For treatment of the axilla, there is evidence that, in clinical N0 patients, radiation therapy to the axilla is an effective alternative to axillary dissection. Finally, there are cost and morbidity considerations for patients undergoing axillary dissection in whom the indications of the procedure are equivocal. In the management of invasive breast cancer, a selective policy toward axillary lymph node dissection should be considered. This review discusses the nonsurgical management of the axilla; ie, radiation therapy to the axilla and observation of the axilla as an alternative to axillary dissection.

Axilla↗
PubMed1996

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.

Calcium↗
PubMed1996

Random sequence phosphorothioate oligonucleotides evoke dramatic phenotypic alterations in cardiac myocyte cultures.

Cardiac myocytes displayed modest and uncoordinated contractile activity regardless of whether they are cultured in the presence or absence of unmodified random sequence oligonucleotides (oligos), as expected. Much to our surprise, however, when cardiac myocytes were cultured in the presence of random sequence phosphorothioate (PS) oligos, they reorganized into cablelike aggregates and displayed surging and coordinated contractile activity. Consistent with these observations, photobleaching experiments revealed that gap junction conductivity between affected cardiac myocytes was enhanced fourfold relative to control cultures. Furthermore, whereas atrial natriuretic factor (ANF) gene expression was induced in control cultures relative to intact hearts, this aberrant expression was selectively repressed in response to PS oligos. As PS oligos appear to mitigate deleterious effects that result from the proteolytic dispersal or culturing of cardiac myocytes or both, we suggest that these may useful cell culture reagents. It is interesting to contemplate whether cardiac myocytes might also be responsive to PS oligos within intact hearts, as this issue has potential clinical significance.

Animals↗
PubMed1995

Subnanosecond polarized fluorescence photobleaching: rotational diffusion of acetylcholine receptors on developing muscle cells.

Polarized fluorescence recovery after photobleaching (PFRAP) is a technique for measuring the rate of rotational motion of biomolecules on living, nondeoxygenated cells with characteristic times previously ranging from milliseconds to many seconds. Although very broad, that time range excludes the possibility of quantitatively observing freely rotating membrane protein monomers that typically should have a characteristic decay time of only several microseconds. This report describes an extension of the PFRAP technique to a much shorter time scale. With this new system, PFRAP experiments can be conducted with sample time as short as 0.4 microseconds and detection of possible characteristic times of less than 2 microseconds. The system is tested on rhodamine-alpha-bungarotoxin-labeled acetylcholine receptors (AChRs) on myotubes grown in primary cultures of embryonic rat muscle, in both endogenously clustered and nonclustered regions of AChR distribution. It is found that approximately 40% of the AChRs in nonclustered regions undergoes rotational diffusion fast enough to possibly arise from unrestricted monomer Brownian motion. The AChRs in clusters, on the other hand, are almost immobile. The effects of rat embryonic brain extract (which contains AChR aggregating factors) on the myotube AChR were also examined by the fast PFRAP system. Brain extract is known to abolish the presence of endogenous clusters and to induce the formation of new clusters. It is found here that rotational diffusion of AChR in the extract-induced clusters is as slow as that in endogenous clusters on untreated cells but that rotational diffusion in the nonclustered regions of extract-treated myotubes remains rapid.

Animals↗
PubMed1995

Spontaneous recovery of fluorescence by photobleached surface-adsorbed proteins.

Fluorescence photobleaching of a carboxyfluorescein-labeled protein (erythrocyte cytoskeletal protein 4.1) immobilized on bare glass is found to be spontaneously reversible, provided that the sample is deoxygenated. After a short (hundredths of seconds) photobleaching laser flash, the subsequent fluorescence excited by a dim probe beam partly recovers on a long (tenths of second) time scale, even in the absence of chemical exchange or diffusion processes. Neither the fraction of the fluorescence that bleaches reversibly nor its recovery rate is a strong function of fluorophore surface concentration. At a fixed surface concentration, the reversibly photobleached fraction and its recovery rate decreases with increasing duration or intensity of the bleaching flash. On the other hand, nondeoxygenated air-equilibrated samples exhibit almost total irreversible bleaching on this time scale. Quantitative fluorescence microscopy experiments occasionally require deoxygenation to avoid photochemical crosslinking or photobleaching or to enhance the triplet state population. The observation presented here indicate that fluorescence recovery after photobleaching (FRAP) experiments performed under deoxygenated conditions for measuring diffusion or chemical kinetics should be interpreted with caution: fluorescence recoveries may be due to intrinsic photochemical processes rather than fluorophore mobility. The recovery effect appears too slow to be ascribed simply to a relaxation of a triplet state; other possible explanations are offered.

Adsorption↗
PubMed1994

Time-lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes.

To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time-lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine alpha-bungarotoxin (R-BT) continuously present in the medium. Since TIRF illuminates only cell-substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced relative to standard epi- (or trans-) illumination. During the experiment, cells were kept alive on the microscope stage at 37 degrees C in an atmosphere of 10% CO2. Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week-long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest-lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell-cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour.

Animals↗
PubMed1994

Lateral mobility of tetramethylrhodamine (TMR) labelled G protein alpha and beta gamma subunits in NG 108-15 cells.

Multi-step signal transducing events, such as those mediated by G proteins, have been difficult to study in intact cells. We prepared fluorescently labelled G protein subunits, tetramethylrhodamine-alpha o (TMR-alpha o) and TMR-beta gamma, in order to study their subcellular distribution and lateral mobility. Heterotrimeric G proteins labelled in the alpha (TMR-alpha o/beta gamma) or beta (TMR-beta gamma/alpha o) subunit were reconstituted into lipid vesicles and fused to NG-108-15 cells using polyethylene glycol (PEG). Vesicles fused completely to the cells as determined by dequenching of a fluorescent lipid probe, octadecyl rhodamine B. The orientation of G protein beta gamma subunits after fusion followed the expected random distribution; the quenching of surface fluorescence with anti-fluorescein antibodies showed that about 50% of the label was accessible extracellularly. G proteins incorporated by the fusion method were able to couple to endogenous alpha 2 adrenergic receptors based on the restoration of high affinity agonist binding to pertussis toxin-treated cells. The subcellular localization of TMR-alpha o and TMR-beta gamma determined by differential centrifugation and confocal microscopy indicated that TMR-alpha o was present in the plasma membrane and in intracellular membranes, whereas TMR-beta gamma was mainly localized in the plasma membrane. The lateral mobility of TMR-alpha o and TMR-beta gamma measured using fluorescence recovery after photobleaching (FRAP) demonstrated low mobile fractions of 0.34 +/- 0.03 and 0.16 +/- 0.03, respectively. The translational diffusion coefficients of the mobile components were similar, 4.0 x 10(-9) and 2.0 x 10(-9) cm2/s, for alpha and beta gamma respectively. Neither activation of Gi-linked receptors nor cytoskeletal disruption with nocodozole or cytochalasin D changed the mobile fraction or diffusion coefficient of the alpha or beta gamma subunits. The FRAP data combined with the localization of fluorescent subunits by confocal microscopy suggest that the beta gamma subunits are highly constrained to localized regions of the plasma membrane while the alpha subunit may diffuse in intracellular regions to transmit signals from receptors to effector proteins.

Cell Membrane↗
PubMed1994

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes.

Ankyrins↗
PubMed1994

Reduction-of-dimensionality kinetics at reaction-limited cell surface receptors.

It has been suggested for several years that reactions between ligands and cell surface receptors can be speeded up by nonspecific adsorption of the ligand to the cell surface followed by two-dimensional surface diffusion to the receptor, a mechanism referred to as "reduction-of-dimensionality" (RD) rate enhancement. Most of the theoretical treatments of this and related problems have assumed that the receptor is an irreversibly absorbing perfect sink. Such receptors induce a depletion zone of ligand probability density around themselves. The reaction rate in this case (called "diffusion-limited") is limited only by the time required for ligands to diffuse through this depletion zone. In some cases, however, the receptor may be far from "perfect" such that a collision with a ligand only rarely leads to binding. Receptors then do not create significant local depletion zones of ligand probability density, and the reaction rate becomes strongly affected by the (small) probability of reaction success per diffusive encounter (the "reaction-limited" case). This article presents a simple theory of RD rate enhancement for reaction-limited receptors that are either reversible or irreversible binders. In contrast to the diffusion-limited theories, the reaction-limited theory presented here: (a) differs quantitatively from diffusion-limited models; (b) is simple and algebraic in closed form; (c) exhibits significant rate enhancement in some realistic cases; (d) depends strongly on the actual Brownian rather than pure diffusive nature of the ligand's motion; (e) depends (for irreversibly binding receptors only) on the kinetic rates (not just equilibria) of reversible adsorption to nontarget regions, in contrast to some previous approximate theories of reduction of dimensionality; and (f) is applicable to actual ligand/receptor systems with binding success probabilities at the opposite extreme from the perfect sink/diffusion-limited models.

Adsorption↗
PubMed1993

Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeast Saccharomyces cerevisiae.

We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (diI) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma membranes of Saccharomyces cerevisiae ino1 and opi3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobility was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe diI, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized spheroplasts, mobility was still somewhat lower than the mobility observed in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.

Carbocyanines↗
PubMed1992

Reaction rate enhancement by surface diffusion of adsorbates.

Ligands can be captured by a surface target through either direct bulk diffusion or surface diffusion following reversible adsorption to the surface. We have solved a steady state boundary value problem for a perfect sink disk target in the surface, taking into account bulk and surface diffusion coefficients D and Ds and adsorption/desorption kinetic rate constants ka and kd at non-target regions. Solutions have been successfully found by numerical computation. The results show that the rate of capture from the surface depends non-linearly on Ds, D, ka, kd and geometrical dimensions. In particular, we demonstrate that not only is the non-target region equilibrium constant Keq (= ka/kd) important in determining the rate of capture from the surface, but so are the kinetic rate constants ka and kd separately. In all cases, the surface adsorption/diffusion combination enhances the total rate of capture. The results should be useful for predicting reaction rates of biological membrane bound receptor clusters and substrate-immobilized enzymes.

Adsorption↗
PubMed1991

Mutational analysis of the receptor-activating region of human parathyroid hormone.

The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.

Amino Acid Sequence↗
PubMed1990

A polarized photobleaching study of chromatin reorientation in intact nuclei.

Polarized fluorescence recovery after photobleaching (pFRAP) was used to monitor the effects that condensation, i.e. compaction and aggregation, have on the (microseconds and ms) internal dynamics of chromatin in intact nuclei. When divalent cations were present with physiological (approximately 90 mM) monovalent salt the chromatin was found to exist in a compact and aggregated state which was characterized by rotational immobilization over timescales that range from 10 microseconds to 40 milliseconds. This immobilization is attributed to suppression of internal dynamics by intermolecular interactions. When the divalent cations were removed, the compact fibers no longer aggregated and were free to reorient with a characteristic decay time of about 1.2 milliseconds. It is shown that this millisecond relaxation could represent rigid rotation of topologically independent structural domains. Dilution of the monovalent salt induced a gradual change in the structural state of the chromatin that was manifest as a dramatic increase in internal flexibility. At the lowest salt concentration studied (11 mM-monovalent salt) the chromatin reorients in fewer than ten microseconds. These changes in flexibility are continuous with salt concentration, indicating that there are no well-defined endpoints to structural transitions and that the microsecond-millisecond internal dynamics of chromatin are a sensitive measure of structure. Measurements made on nuclei from cells that are either transcriptionally quiescent or active indicate that the dynamics mirrors biological activity.

Animals↗
PubMed1990

A polarized photobleaching study of DNA reorientation in agarose gels.

Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions with gel matrices apparently do perturb the larger amplitude, more slowly (microseconds to milliseconds) relaxing internal motions of large DNAs. The relationship between this work and recent studies which indicate that internal motions of DNA play an important role in the separation achieved with pulsed-field gel electrophoresis techniques is discussed. The polarized photobleaching technique is also analyzed in some detail. In particular, it is shown that "reversible" photobleaching phenomena are probably related to depletion of the ground state by intersystem crossing to the triplet state.

DNA↗
PubMed1990

Rotational diffusion of acetylcholine receptors on cultured rat myotubes.

The rotational mobility of acetylcholine receptors (AChR) in the plasma membrane of living rat myotubes in culture is measured in this study by polarized fluorescence recovery after photobleaching (PFRAP). These AChR are known to exist in two distinct classes, evident by labeling with rhodamine alpha-bungarotoxin; clustered AChR that are aggregated in a pattern of highly concentrated speckles and streaks, with each cluster occupying an area of approximately 1,000 microns 2; and nonclustered AChR that appear as diffuse labeling. PFRAP results reported here show that: (a) most clustered AChR (approximately 86%) are rotationally immobile within a time scale of at least several seconds; and (b) most nonclustered AChR (approximately 76%) are rotationally mobile with characteristic times ranging from less than 50 ms to 0.1 s. External cross-linking with the tetravalent lectin concanavalin A immobilizes many nonclustered AChR. PFRAP experiments in the presence of carbachol or cytochalasin D show that the restraints to rotational motion in clusters are remarkably immune to treatments that disperse clusters or disrupt cytoplasmic actin. The experiments also demonstrate the feasibility of using PFRAP to measure rotational diffusion on selected microscopic areas of living nondeoxygenated cells labeled with standard fluorescence probes over a very wide range of time scales, and they also indicate what technical improvements would make PFRAP even more practicable.

Animals↗
PubMed1990

Effect of habitual knuckle cracking on hand function.

The relation of habitual knuckle cracking to osteoarthrosis with functional impairment of the hand has long been considered an old wives' tale without experimental support. The mechanical sequelae of knuckle cracking have been shown to produce the rapid release of energy in the form of sudden vibratory energy, much like the forces responsible for the destruction of hydraulic blades and ship propellers. To investigate the relation of habitual knuckle cracking to hand function 300 consecutive patients aged 45 years or above and without evidence of neuromuscular, inflammatory, or malignant disease were evaluated for the presence of habitual knuckle cracking and hand arthritis/dysfunction. The age and sex distribution of the patients (74 habitual knuckle crackers, 226 non-knuckle crackers) was similar. There was no increased preponderance of arthritis of the hand in either group; however, habitual knuckle crackers were more likely to have hand swelling and lower grip strength. Habitual knuckle cracking was associated with manual labour, biting of the nails, smoking, and drinking alcohol. It is concluded that habitual knuckle cracking results in functional hand impairment.

Aged↗
PubMed1989

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters.

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to alpha-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotube-substrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.

Actin Cytoskeleton↗