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S W Paddock

Publications and source records attributed to S W Paddock.

31 records · Page 2Linked to original sources
PubMed1993

Three-color immunofluorescence imaging of Drosophila embryos by laser scanning confocal microscopy.

We present a simple means for triple-labeling biological specimens by immunofluorescence using a laser scanning confocal microscope for imaging with a krypton/argon laser as a light source. Three separate images of fluorescein-, lissamine rhodamine- and cyanine-5-labeled antibodies are collected and subsequently merged to form the triple-labeled image, which is displayed at full-image resolution (24 bit) on a second image processing system. The technique is illustrated using immunofluorescence localization of three segmentation proteins in Drosophila embryos.

Animals↗
PubMed1992

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.

Aniline Compounds↗
PubMed1991

Human immunodeficiency virus (HIV) transcripts identified in HIV-related psoriasis and Kaposi's sarcoma lesions.

Persons with HIV infection sometimes develop aggressive psoriasis or Kaposi's sarcoma (KS) not usually seen in other immunosuppressed patients. However, a specific and direct pathophysiological role for HIV-1 in these AIDS-associated disorders remains unclear since HIV has not been easily detected in these skin lesions. By combining in situ hybridization with the sensitive detection technique of confocal laser scanning microscopy, we have demonstrated HIV RNA transcripts in 5 of 15 lesional skin biopsies from HIV-infected psoriasis patients, and in 3 of 8 Kaposi's sarcoma biopsies from HIV-infected patients. HIV transcripts were not detected in normal appearing skin from HIV-infected patients or in psoriatic and normal skin biopsies from uninfected individuals (P = 0.006). Although previous attempts to demonstrate viral sequences in psoriasis and KS lesions have been unsuccessful, in situ hybridization with confocal microscopy has shown the presence of HIV RNA transcripts predominantly within CD4+, Factor XIIIa positive dermal dendrocytes. HIV or cytokines produced by infected cells in skin lesions may therefore play a direct role in the pathogenesis of HIV-associated psoriasis and KS.

Adult↗
PubMed1990

Confocal microscopic detection of human immunodeficiency virus RNA-producing cells.

A central anomaly in the pathogenesis of AIDS is that few actively infected CD4+ cells (1 in 10(4)-10(5) have been detected in the peripheral blood, even though dramatic depletion (often greater than 90%) of CD4+ cells is the hallmark of disease progression. A sensitive, 35S-based human immunodeficiency virus (HIV) RNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. In 35 symptomatic HIV-infected individuals (AIDS and AIDS related complex), an average of 1 in 350 mononuclear cells produced HIV RNA. In contrast, in an asymptomatic group of 30 individuals, an average of 1 in 2000 mononuclear cells produced HIV RNA. These data, obtained using this improved detection method, suggest there are more HIV RNA-producing cells in HIV-infected individuals than previously reported. In addition, increased numbers of HIV transcribing cells were found to correlate with declining clinical condition as assessed by Karnofsky performance score. These data suggest that viremia per se may account for the pathologic consequences in HIV infection.

AIDS-Related Complex↗
PubMed1989

Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells.

This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because of the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laser-based confocal microscope, the actual colour of the specimen is viewed directly with TSM in almost real-time.

Amido Black↗
PubMed1988

Rigidity of the nucleus during nuclear rotation in 3T3 cells.

Using near infrared microscopy and ultraviolet fluorescence microscopy of living 3T3 cells stained with the fluorochrome Hoechst 33342, we have demonstrated that the nucleoli and Hoechst 33342-stained chromocenters in the nucleus maintain a fixed pattern during nuclear rotation. We conclude that the term "nuclear rotation" refers to rotation of the entire nucleus in the cytoplasm of interphase cells, and that nuclear rotation is not an expression of karyoplasmic streaming. In conjunction with earlier results on nuclear rotation the data imply that the interface of nuclear rotation is located either between the two nuclear membranes or in the adjacent cytoplasm.

Benzimidazoles↗
PubMed1986

Distribution of microfilament bundles during rotation of the nucleus in 3T3 cells treated with monensin.

Cytoskeletal aspects of monensin-treated 3T3 cells with rotating nuclei were studied by immunofluorescence. The pattern of intermediate filaments and microtubules appeared unchanged when compared with control cells having a stationary nucleus. In contrast, the actin microfilament bundles appeared to have a consistent distribution in cells with rotating nuclei. Typically, we did not find long microfilament bundles that traverse the length of the cytoplasm of cells that were fixed at the time of nuclear rotation. Instead, there was a local distribution of short microfilament bundles situated ventrally to the nucleus and oriented at various angles to one another and to the predominant distribution of microfilament bundles in the cell. The observations suggest that the actin cytoskeleton is reorganized locally before or during rotation of the nucleus.

Actin Cytoskeleton↗
PubMed1986

The degree of coupling of nuclear rotation in binucleate 3T3 cells.

In order to test the existence of mechanical coupling between the rotational movements of two adjacent nuclei, we prepared binucleate 3T3 cells and observed their nuclear movements by near infrared microscopy and recorded them with time-lapse video techniques. We found that 49 out of 110 (44%) of the selected binucleate cells expressed nuclear rotation. Rotation could occur in just one of the nuclei while the second nucleus remained stationary (31/110) or in both nuclei simultaneously (18/110). In almost all cases where both nuclei rotated simultaneously (15/110) they did so at different speeds and in opposite directions. The nuclei were observed to rotate in the same direction in only three of the examples. The results are consistent with a weak mechanical interaction between a rotating nucleus and its neighbor. Consistent with our previous observations in mononucleate cells, we did not find a characteristic position of the centrosphere or a special distribution of the microtubules or the intermediate filaments in binucleate cells with rotating nuclei. There was an absence of long, well-formed microfilament bundles beneath the nuclei during rotation, even in the local region beneath the rotating nucleus in those cells with one rotating and one stationary nucleus. Also consistent with observations of mononucleate cells, nuclear rotation was inhibited by treatment with colcemid, although the ability of the nuclei to rotate was eventually restored when the colcemid-containing medium was replaced with normal medium.

Actin Cytoskeleton↗
PubMed1986

Analysing collisions between fibroblasts and fibrosarcoma cells: fibrosarcoma cells show an active invasionary response.

We describe a direct way of measuring contact inhibition of locomotion by analysing the changes in motion of pairs of colliding cells. This allows values to be assigned to each type of cell in mixed collisions and will enable certain hypotheses about the relationship between contact inhibition and invasion in culture to be tested critically. We find that fibrosarcoma (FS9) cells, on colliding with chick heart fibroblasts, show a reversed contact-inhibition response that we call contact promotion of locomotion. We also describe a measure of the lateral changes in motion that result from collisions between cells and show that this is dependent on the type of colliding cell but, unlike contact inhibition, it does not appear to be dependent on the type of cell with which it collides for the types studied here. Finally, we analyse how the total response is dependent on the dispositions and motions of the cells before collision and we find that FS9 cells, on colliding with fibroblasts, tend to turn towards the point of initial marginal contact. We conclude that the FS9 cells show a pronounced response on colliding with the fibroblasts, which is in contrast to the subjective impression that the FS9 cells do not respond much. These findings support the thesis of Abercrombie and colleagues, that the infiltration of a population of normal cells by a population of invasive cells in culture is dependent on the nature of the response of each cell type to collision with the other and that the invasive cells fail to show contact inhibition in these heterotypic collisions; but the findings further suggest that these invasive cells show an active invasionary response as opposed to merely failing to show contact inhibition.

Animals↗
PubMed1982

Analysing the motile behaviour of cells: a general approach with special reference to pairs of cells in collision.

As a measure of the change in motion of locomoting cells, acceleration has several advantages over more conventional measures such as rate of change in speed and rate of change in direction. In particular the instantaneous acceleration vector associated with a specific event, such as the collision of the cell with another, may be correlated with directional information contained in the structure of the cell and in the geometrical configuration of the event. It is hoped that this will prove to be a generally useful method for examining the relation between cell structure and motion during both normal and aberrant behaviour. Preliminary results obtained with this method to examine the changes in motion of a normal cell and a malignant cell on colliding with each other are already of interest for elucidating the mechanism of contact inhibition of locomotion and its failure during malignant invasion.

Animals↗