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D Axelrod

Publications and source records attributed to D Axelrod.

At least 19 recordsLinked to original sources
PubMed2003

Influence of postmenopausal hormone replacement therapy on an estrogen metabolite biomarker of risk for breast cancer.

Whether postmenopausal hormone-replacement therapy (HRT) increases the risk of breast cancer remains controversial, despite numerous epidemiological studies. We approached the question from a biochemical rather than an epidemiological direction - we hypothesized that if estrogen administration increases the risk of breast cancer, it should also alter a known estrogen biomarker of risk towards what has been observed in patients who already have breast cancer. The specific biomarker we studied was the ratio of the urinary excretion of two principal estradiol metabolites, 2-hydroxyestrone and 16 alpha-hydroxyestrone, which is markedly decreased in women with breast cancer and women with familial risk for breast cancer. We studied 34 healthy postmenopausal women not on HRT and 19 women on HRT (Premarin 0.625 mg daily plus Provera, 2.5 mg daily, in women with a uterus and Premarin alone in women without a uterus); treatment duration ranged from 3 months to 15 years. We also studied four women with recently diagnosed, untreated breast cancer. The women with breast cancer showed a significantly lower 2-hydroxyestrone to 16 alpha-hydroxyestrone ratio than control women on HRT (1.35 +/- 0.13 vs. 2.71 +/- 0.84; p < 0.0001). There was no significant difference in the metabolite ratio between healthy women on HRT and women not on HRT (2.82 +/- 0.92 vs. 2.71 +/- 0.84). There was no significant difference between women receiving Premarin alone and women receiving Premarin plus Provera (2.46 +/- 0.84 vs. 3.13 +/- 0.90), and neither differed significantly from women not on HRT (2.71 +/- 0.84). The finding that the ratio of women on HRT was not decreased to or toward the ratio in women with breast cancer can be interpreted, we believe, as a suggestive item of biochemical evidence that HRT is not a risk for breast cancer.

Biomarkers, Tumor↗
PubMed2001

Restriction of secretory granule motion near the plasma membrane of chromaffin cells.

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

Actins↗
PubMed2001

Mammotome core biopsy for mammary microcalcification: analysis of 160 biopsies from 142 women with surgical and radiologic followup.

BACKGROUND: Although stereotaxic fine-needle aspiration biopsy or core biopsy (14-gauge) have proven to be accurate techniques for the evaluation of mammographically detected microcalcification, the development of the Mammotome Biopsy System (Biopsys Medical, Inc., Irvine, CA) has led many medical centers to use this vacuum-assisted device for the sampling of microcalcification. METHODS: One hundred forty-two women underwent 160 stereotaxic Mammotome core biopsies of mammographic calcification over a 1-year period. The stereotaxic procedure was performed by radiologists using the Mammotome Biopsy System. Microcalcification was evident on specimen radiographs and microscopic slides in 99% of the cases. Excisional biopsy was recommended for diagnoses of atypia or carcinoma. Patients with benign diagnoses underwent mammographic followup. RESULTS: One hundred thirty-two benign, 12 atypical, and 15 adenocarcinoma diagnoses (comprising 1 lobular adenocarcinoma in situ [LCIS], 1 invasive ductal adenocarcinoma [IDC], and 13 intraductal adenocarcinomas [DCIS]: 10 comedo, 1 cribriform, 2 mixed cribriform and micropapillary) were rendered. Surgical excision in eight patients with atypia on Mammotome biopsy (two refused surgery, two were lost to followup) showed ductal hyperplasia in three, atypical ductal hyperplasia (ADH) in three and DCIS (low grade, solid) in two patients. Surgical excisions in 14 patients diagnosed with carcinoma (1 patient lost to followup) showed ADH in 3, ADH and LCIS in 1, residual DCIS in 8, IDC in 1, and microinvasive carcinoma in 1 patient. CONCLUSIONS: A diagnosis of atypia on Mammotome biopsy warranted excision of the atypical area, yet the underestimation rate for the presence of carcinoma remained low. The likelihood of an invasive component at excision was low for microcalcification diagnosed as DCIS on Mammotome biopsy. Mammotome biopsy proved to be an accurate technique for the sampling and diagnosis of mammary microcalcification.

Adenocarcinoma↗
PubMed2001

Total internal reflection fluorescence microscopy in cell biology.

Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.

Cytoplasmic Granules↗
PubMed2001

Selective imaging of surface fluorescence with very high aperture microscope objectives.

Three approaches to selective surface fluorescence detection are described. All three of them depend on the use of extremely high numerical aperture (NA) objectives now commercially available (1.45 NA from Zeiss and Olympus and 1.65 NA from Olympus). The first two approaches are elaborations of "prismless" total internal reflection fluorescence (TIRF), one approach with a laser illumination and the second with arc lamp illumination. The new higher NA objectives are much more suitable for TIRF work on biological cells in culture than are 1.4 NA objectives previously described for prismless TIRF. The third approach is not TIRF at all. It uses the high aperture objective to selectively gather the emission of fluorophores located close enough to the substrate for their near-field energy to be captured by the substrate. Schematic diagrams, experimental demonstrations, and practical suggestions for all these techniques are provided.

Animals↗
PubMed2000

Imaging constitutive exocytosis with total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

Animals↗
PubMed2000

The incidence of positive margins with breast conserving therapy following mammotome biopsy for microcalcification.

BACKGROUND AND OBJECTIVES: The ability to achieve clean margins with breast conserving therapy varies greatly even when the diagnosis of carcinoma is known beforehand. Although several reports reveal that the incidence of positive margins decreases after stereotaxic core biopsy of nonpalpable lesions and fine-needle aspiration biopsy of palpable lesions, the data on the results following mammotome biopsy (mmbx) is scanty. METHODS: Two hundred and ninety-eight biopsy specimens for mammographically indeterminate microcalcification from 1/97 through 3/30/98 were reviewed. Biopsies were performed using the biopsys method utilizing an 11-gauge multidirectional, vacuum-directed device. RESULTS: Ten percent (n = 31) of the mammotome biopsies were atypical and 9% (n = 27) were malignant. These 58 cases (19%) were recommended for surgical excision. The incidence of positive margins in this subset was determined. Of patients who underwent lumpectomy as their initial surgical procedure 69% had negative surgical margins. Seventy-seven percent of patients with carcinoma diagnosed by mammotome biopsy had definitive initial surgery with a single surgical procedure. CONCLUSIONS: Mmbx facilitates fewer surgical procedures to achieve negative margins, and thus provides a better cosmetic result.

Adult↗
PubMed2000

Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics.

Actins↗
PubMed1999

Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles.

Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

Animals↗
PubMed1999

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.

Adsorption↗
PubMed1998

Rethinking breast cancer risk and the environment: the case for the precautionary principle.

The World Health Organization recently reported that breast cancer has become the most common cancer in women throughout the world. Known risk factors account for less than half of all cases of breast cancer, and inherited germ line mutations occur in at most only 10% of all cases. Cumulative exposure to estradiol and other hormones links many of the established risk factors for breast cancer. This paper reviews epidemiologic and toxicologic evidence on breast cancer risks and presents a comprehensive construct of risk factors intended to focus on the identification of those factors that can be controlled or modified. We attempt to provide a framework for interpreting the etiologic interplay of endogenous metabolic changes and environmental changes in the etiology of breast cancer. The construct we develop distinguishes between those risk factors that are directly causal, such as ionizing radiation and inherited germ cell defects, those vulnerability factors that extend the time period during which the breast undergoes development, and those contributing factors that increase total hormonal stimulation of the breast. Some hormonally active compounds, such as those in soy and broccoli and other phytoestrogen-containing foods, can be protective against breast cancer, while others, such as some environmental contaminants, appear to increase the risk of the disease by increasing levels of harmful hormones. Efforts to explain patterns of breast cancer should distinguish between these different risk factors. Identification of vulnerability and contributing risk factors can foster the development of public policy to reduce the burden of this prevalent cancer. Prudent precautionary principles suggest that reducing exposure to avoidable or modifiable risk factors should receive high priority from the public and private sectors.

Breast Neoplasms↗
PubMed1997

Chromatin dynamics in interphase nuclei and its implications for nuclear structure.

Translational dynamics of chromatin in interphase nuclei of living Swiss 3T3 and HeLa cells was studied using fluorescence microscopy and fluorescence recovery after photobleaching. Chromatin was fluorescently labeled using dihydroethidium, a membrane-permeant derivative of ethidium bromide. After labeling, a laser was used to bleach small (approximately 0.4 microm radius) spots in the heterochromatin and euchromatin of cells of both types. These spots were observed to persist for >1 h, implying that interphase chromatin is immobile over distance scales >/=0.4 microm. Over very short times (<1 s), a partial fluorescence recovery within the spots was observed. This partial recovery is attributed to independent dye motion, based on comparison with results obtained using ethidium homodimer-1, which binds essentially irreversibly to nucleic acids. The immobility observed here is consistent with chromosome confinement to domains in interphase nuclei. This immobility may reflect motion-impeding steric interactions that arise in the highly concentrated nuclear milieu or outright attachment of the chromatin to underlying nuclear substructures, such as nucleoli, the nuclear lamina, or the nuclear matrix.

3T3 Cells↗
PubMed1997

Fatty acid alteration and the lateral diffusion of lipids in the plasma membrane of keratinocytes.

The fluorescent probe diI was used to study the lateral mobility of lipids in in vitro strains of living adult human keratinocytes grown in four different media. One medium was essential fatty acid deficient (EFAD) and low in calcium ion, a medium known to yield cells that proliferate rapidly and contain lipid with extremely low levels of essential fatty acids. Two other media were supplemented with essential fatty acids (FAS), media that are known to result in cells that grow more slowly and have normalized fatty acid proportions. A fourth medium consisted of 1 microM all-trans-retinoic acid added to the fatty acid-supplemented medium (FAS-RA), a medium known to produce cells that are highly proliferative, with a growth rate greater than that of the FAS strains and similar to that of the EFAD strains. The keratinocytes grown in these four media were studied using the fluorescence recovery after photobleaching (FRAP) technique to determine the lateral diffusion rate of diI in the plasma membranes. Our results showed a positive correlation between growth rate and diffusion coefficient (D): the diffusion coefficient of diI was higher in the EFAD or FAS-RA cells than in the FAS cells. The measurement of D among the FAS cells fell into two groups. One group was similar to the single group seen in the EFAD cells, but the other group was composed of much lower D values. The other FRAP parameters (mobile fraction and bleach depth) were larger in the "slow" group than in the "fast" group. This trend of negative correlation between these parameters and D was also found within the fast group. These results are interpreted in terms of possible changes in membrane structure or morphology that might be indirectly associated with the fatty acid alterations, including the possible presence of areas in senescing keratinocytes where plasma membranes collapse to form an interacting system of lipid bilayers.

Affinity Labels↗
PubMed1997

Cytoskeletal protein binding kinetics at planar phospholipid membranes.

It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an electrostatic attraction and is far from saturation at the concentrations employed. These results and the techniques implemented carry general implications for understanding the functional role of nonspecific protein binding to cellular lipid membranes.

Biophysical Phenomena↗