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Biomedical subjects

G W Fisher

Publications and source records attributed to G W Fisher.

At least 19 recordsLinked to original sources
PubMed1995

Four-dimensional microscopic analysis of the filopodial behavior of primary mesenchyme cells during gastrulation in the sea urchin embryo.

During gastrulation of the sea urchin embryo, primary mesenchyme cells (PMCs) migrate from the vegetal pole to a site below the equator of the embryo where they form a ring-like structure and begin producing the larval skeleton. As these cells migrate, they extend and retract filopodia which appear to interact with the basal lamina and underlying ectoderm. To better characterize this behavior in vivo, we studied PMC migration using differential interference contrast (DIC) microscopy in combination with four-dimensional imaging (x, y, z space and time). We were able to determine the persistence and direction of extension of each filopodium and were also able to observe the dynamic behavior of each using colorized movie loops. This analysis showed that: (1) Most filopodia are quite transient, usually persisting for less than 0.5-6.0 min, during which time they continuously survey their surroundings; (2) PMCs extend an average of 121 filopodia/hr during migration; (3) the initial direction of extension of filopodia from the cell body is random, with just as many filopodia projecting toward as away from the direction of migration; (4) as a consequence of (2) and (3) above, each PMC explores the area surrounding its cell body approximately once every 5 min; (5) PMCs nearer to the target site migrate faster than those located farther away. To further investigate filopodial distribution, confocal microscopy was used to collect z series of PMCs transplanted to different locations in the embryo and fixed during migration. We found that more filopodia tended to be distributed toward the target site as cells approached the ring, suggesting that filopodial distribution may reflect regional differences in directional cues.

Animals↗
PubMed1995

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies.

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, kappa) at ratios of 1.2-35 mol dye/mol antibody and 9.2.27 (IgG2a, kappa) at 0.6-6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.

Animals↗
PubMed1995

Fibronectin induces protease dependent focal matrix depletion and cell overlap in cultured rheumatoid synoviocytes.

OBJECTIVE: To characterize the effect of added fibronectin (Fn) on human rheumatoid synoviocytes (RAS). METHODS: Early passage cultured RAS were studied by fluorescent imaging microscopy with multiple parameter overlaying and confocal laser scanning microscopy. RESULTS: Added Fn induced a localized decrease in matrix Fn at sites of cell overlap. Similar matrix depletion could be induced by collagen VI, cell binding (120 k) and heparin binding (60 k) fragments of Fn, but not by gelatin binding fragment (45 k). CONCLUSION: The decrease in matrix Fn was associated with the induction of a transformation-like phenotype of overlapping cell growth. Both phenomena were inhibited by serine protease inhibitors.

Arthritis, Rheumatoid↗
PubMed1988

Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts.

Wound healing in Swiss 3T3 cultures was investigated with video-enhanced contrast (VEC) microscopy. The formation of protrusions at the leading edge of cells along wound was investigated in detail during the spreading stage, which usually lasted from 1 to 4 hr postwounding. Lamellipodia exhibited a continuous rearward, or centripetal, transport of a variety of cellular constituents at rates of approximately 0.26 microns/sec from the leading edge. The lamellipodia were also the sites of lateral migration as well as extension and retraction of actin microspikes. Actin fibers oriented transversely to the direction of movement were also observed to transport centripetally at similar rates. These fibers may in part give rise to large actin fibers forming at the interface between the base of the lamellipodia and the lamellae. Beads 0.5 microns in diameter attached to the dorsal surfaces of lamellipodia also transported centripetally at rates of approximately 0.21 microns/sec. Thus there is an apparent correlation between transport of a variety of structures within lamellipodia and with surface movements of lamellipodia.

Actins↗
PubMed1988

The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts.

The formation of protrusions at the leading edge of the cell is an essential step in fibroblast locomotion. Using fluorescent analogue cytochemistry, ratio imaging, multiple parameter analysis, and fluorescence photobleaching recovery, the distribution of actin and myosin was examined in the same protrusions at the leading edge of live, locomoting cells during wound-healing in vitro. We have previously defined two temporal stages of the formation of protrusions: (a) initial protrusion and (b) established protrusion (Fisher et al., 1988). Actin was slightly concentrated in initial protrusions, while myosin was either totally absent or present at extremely low levels at the base of the initial protrusions. In contrast, established protrusions contained diffuse actin and actin microspikes, as well as myosin in both diffuse and structured forms. Actin and myosin were also localized along concave transverse fibers near the base of initial and established protrusions. The dynamics of myosin penetration into a relatively stable, established protrusion was demonstrated by recording sequential images over time. Myosin was shown to be absent from an initial protrusion, but diffuse and punctate myosin was detected in the same protrusion within 1-2 min. Fluorescence photobleaching recovery indicated that myosin was 100% immobile in the region behind the leading edge containing transverse fibers, in comparison to the 21% immobile fraction detected in the perinuclear region. Possible explanations of the delayed penetration of myosin into established protrusions and the implications on the mechanism of protrusion are discussed.

Actins↗
PubMed1987

Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH.

Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.

Animals↗
PubMed1985

Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis.

A burst of endocytosis accompanying microvillar elongation follows cortical granule exocytosis in normal sea urchin development. By 5 min postfertilization the burst is over and a lower level of endocytosis ensues (constitutive phase). To determine whether microvillar elongation and initiation of endocytosis are necessary concommitants of cortical granule exocytosis we utilized Chase's (1967, Ph.D. thesis, University of Washington, Seattle) high-hydrostatic pressure technique to block the latter and then examined developing eggs for endocytosis and microvillar elongation. To accomplish this, eggs were fertilized, after which hydrostatic pressure was quickly raised to 6000-7000 psi at the start of cortical granule exocytosis and maintained for 5 min. Only the cortical granules immediately surrounding the sperm penetration site were secreted (about 3% or less of the egg's total number of cortical granules). Blockage of major cortical granule exocytosis had the following consequences on surface events during first division: (1) The endocytosis burst normally associated with cortical granule exocytosis was effectively eliminated as was early microvillar elongation and elevation. Both occurred to a limited extent around the sperm penetration site which resulted in a highly localized surface transformation. (2) By 20 min after fertilization endocytosis began over the rest of the egg surface in the absence of any further cortical granule exocytosis. (3) Subsequently, during a 30-min period starting midway between fertilization and first cleavage microvilli more than doubled in length and endocytosis levels increased severalfold. These events brought about a complete surface transformation similar to that which normally occurs in early development but in the absence of cortical granule exocytosis. By first cleavage surfaces and cortices of high-pressure-treated and control eggs were nearly indistinguishable except for the presence of cortical granules in cortices of the former. Pressure-treated eggs cleaved normally and developed to larval forms overnight. The period of late surface transformation in high-pressure-treated Strongylocentrotus purpuratus eggs corresponds in timing and some of its characteristics to second phase microvillar elongation observed in normal development in this species and also in S. droebachiensis development. These observations suggest, therefore, that microvillar elongation and endocytosis are necessary membrane remodelling events which must occur for normal development even in the absence of membrane addition from the cortical granules.

Animals↗
PubMed1983

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles.

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This "burst phase" lasts about 3-5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol. 40, 245-255; D. Epel, 1978, Curr. Top. Dev. Biol. 12, 185-246) that initiation of endocytosis is most likely a Ca2+-dependent event.

Animals↗
PubMed1980

A theoretical and experimental study of light absorption and scattering by in vivo skin.

A theoretical treatment has been developed for the optical properties of a layered structure which absorbs and scatters light. This theory predicts that the logarithm of the inverse of reflectance (LIR) of the surface should be a useful parameter for the examination of that structure. This approach has been applied to a study of skin in vivo. An instrument was constructed for use in clinical situations to measure the LIR spectrum of skin over the visible region of the spectrum (450-760 nm). The contributions to the observed spectra made by pigments and the skin structure were deduced by reference to the theoretical model. Numerical indices were used to quantify the changes in skin haemoglobin content following the application of vasoconstricting preparations. The indices also provided a means of measuring erythema and melanin pigmentation induced in the skin by exposure to ultraviolet radiation. The assessments made using this instrument were more reproducible and sensitive than judgments made by eye.

Connective Tissue↗
PubMed1976

[Vinylogous acyl compounds. XV. Thin-layer chromatographic detection of 2-halovinyl ketones with toxicological relevance (author's transl)].

The basic ring-opening of 1-(2-acylvinyl)-pyridinium salts yielding azaoxonol dyes and the spontaneous dehydrohalogenation of 1-(2-acylvinyl)-4-(4-nitrobenzyl)-pyridinium salts to deeply coloured anhydrous bases underlie two sensitive thin-layer chromatographic detection procedures for 2-halovinyl ketones (R-CO-CH=CH-X). The detection limits are 0.06 mug on successive use of methanolic solutions of pyridine and potassium hydroxide as spray reagents and 0.02 mug on spraying with an acetonic solution of 4-(4-nitrobenzyl)-pyridine. RF values of twenty-five trans- and twelve cis-configurated 1-halovinyl ketones for three solvent systems are given.

Chromatography, Thin Layer↗