Kinetics of uptake, transport, and accumulation of liposome-associated fluorescent dolichol esters in human fibroblasts

1994 ◽  
Vol 72 (7-8) ◽  
pp. 283-288 ◽  
Author(s):  
Lianwei Jiang ◽  
Jack W. Rip
Keyword(s):  
Laser Scanning ◽  
Gradient Centrifugation ◽  
Human Fibroblasts ◽  
Laser Scanning Confocal Microscopy ◽  
Fluorescence Labeling ◽  
Half Maximum ◽  
Scanning Confocal Microscopy ◽  
Percoll Density Gradient Centrifugation ◽  
Time Required ◽  
Kinetics Of Uptake

The anthroyl and n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) hexanoyl esters of dolichol were synthesized and incorporated into phospholipid liposomes. Fluorescence spectrometric methods were used to estimate the kinetics and dynamics of the incorporation and turnover of these esters in normal human fibroblasts. For anthroyl dolichol a saturable uptake of 2.5 × 103 pmol/106 fibroblasts was obtained. Half-maximum uptake was seen at a labeling concentration of 19 μM. The time required for half-maximum uptake of fluorescence (t1/2) was about 10 h. Over 50% of the anthroyl dolichol taken up remained in fibroblasts 24 h after the labeling medium was removed. Uptake was higher for esters of 9 isoprenes than for those with 16–21. Dolichol labeled with the NBD fluorophore appeared to enter fibroblasts in higher concentration than the same dolichol labeled with anthracene. Uptake was not influenced by the presence of agents that disrupt lysosome function (leupeptin and chloroquine) prior to or during fluorescence labeling. The amount of fluorescent dolichol in the (i) lysosomes and endosomes and (ii) nuclei of labeled fibroblasts was determined after Percoll density gradient centrifugation and cell lysis in culture, respectively. Most of the fluorescent dolichyl ester (and most of the free alcohol form) taken up by fibroblasts was recovered in lysosomes.Key words: anthroyl dolichol, n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoyl dolichol, lysosome, localization, laser scanning confocal microscopy, turnover.

scholarly journals In situ localization of mitochondrial DNA replication in intact mammalian cells.

1996 ◽  
Vol 135 (4) ◽  
pp. 883-893 ◽  
Author(s):  
A F Davis ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Brdu Incorporation ◽  
Double Labeling ◽  
Scanning Confocal Microscopy ◽  
Mtdna Replication ◽  
Time Required

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1-2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.

scholarly journals An Intralysosomal hsp70 Is Required for a Selective Pathway of Lysosomal Protein Degradation

1997 ◽  
Vol 137 (4) ◽  
pp. 825-834 ◽  
Author(s):  
Fernando A. Agarraberes ◽  
Stanley R. Terlecky ◽  
J. Fred Dice
Keyword(s):  
Heat Shock ◽  
Protein Degradation ◽  
Laser Scanning ◽  
Human Fibroblasts ◽  
Immunoblot Analysis ◽  
Laser Scanning Confocal Microscopy ◽  
Rnase A ◽  
Serum Deprivation ◽  
Scanning Confocal Microscopy ◽  
Cultured Human Fibroblasts

Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

1993 ◽  
Vol 51 ◽  
pp. 562-563
Author(s):  
Hakan Ancin
Keyword(s):  
Laser Scanning ◽  
Population Analysis ◽  
Three Dimensional ◽  
Large Data ◽  
Laser Scanning Confocal Microscopy ◽  
Tissue Slices ◽  
Scanning Confocal Microscopy ◽  
Tissue Sections ◽  
Spatially Adaptive ◽  
Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

scholarly journals Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 861
Author(s):  
Jacopo Cardellini ◽  
Arianna Balestri ◽  
Costanza Montis ◽  
Debora Berti
Keyword(s):  
Drug Delivery ◽  
Fluorescence Microscopy ◽  
Drug Delivery Systems ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Confocal Microscopy ◽  
Delivery Systems ◽  
Scanning Confocal Microscopy ◽  
Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

Observation of Fine Structure in Bicontinuous Phase-Separated Domains of a Polymer Blend by Laser Scanning Confocal Microscopy

2001 ◽  
Vol 34 (15) ◽  
pp. 5186-5191 ◽  
Author(s):  
Hiroshi Jinnai ◽  
Hiroshi Yoshida ◽  
Kohtaro Kimishima ◽  
Yoshinori Funaki ◽  
Yoshitsugu Hirokawa ◽  
...  
Keyword(s):  
Fine Structure ◽  
Confocal Microscopy ◽  
Polymer Blend ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

scholarly journals The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

1994 ◽  
Vol 42 (11) ◽  
pp. 1413-1416 ◽  
Author(s):  
S L Erlandsen ◽  
E M Rasch
Keyword(s):  
Confocal Microscopy ◽  
Genome Size ◽  
Dna Content ◽  
Giardia Lamblia ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Evolutionary Development ◽  
Scanning Confocal Microscopy ◽  
Dividing Cells ◽  
C Value

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

The Effect of Chloroquine on the Apoptosis Induced by Cisplatin in Human Gastric Cancer BGC823 Cells

2014 ◽  
Vol 926-930 ◽  
pp. 1124-1127
Author(s):  
Zhen Xun Jin ◽  
Li Li Zhang ◽  
Yan Wang ◽  
Lin Chuan Zeng ◽  
Yang Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Combination Group ◽  
Human Gastric Cancer ◽  
Apoptotic Cells ◽  
Toxic Dose ◽  
Scanning Confocal Microscopy ◽  
Mtt Tests

The aim of this study is to investigate the effects and mechanism of chloroquine (CQ) on the apoptosis induced by cisplatin in human gastric cancer BGC823 cells. MTT assay was used to detect the state of cell growth. The appearances of cellular apoptosis were detected by laser scanning confocal microscopy and light microscopy. The expressions of LC3 and p62 were detected by laser scanning confocal microscopy. MTT tests showed that the non-toxic dose of CQ could increase the inhibition rate of BGC823 cells induced by cisplatin. Under the light microscope, the ratio of apoptotic cells in the group treated with non-toxic dose of CQ combined with cisplatin was higher than that in the group treated with cisplatin alone. Hoechst33342 staining showed that the ratio of apoptotic cells in the combination group was higher than that in the cisplatin group. The expression and colocalization of LC3 and p62 proteins were significantly increased in the combination group. These results indicate that CQ can enhance the cell apoptosis induced by cisplatin in BGC823 cells, which is through the inhibition of autophagy.

In-situ Observation of Surface Relief Formation and Disappearance during Order-Disorder Transition of Equi-atomic CuAu alloy using Laser Scanning Confocal Microscopy

2004 ◽  
Vol 842 ◽  
Author(s):  
Seiji Miura ◽  
Hiroyuki Okuno ◽  
Kenji Ohkubo ◽  
Tetsuo Mohri
Keyword(s):  
Surface Relief ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Strain Effect ◽  
In Situ Observation ◽  
Stress Effect ◽  
Scanning Confocal Microscopy ◽  
Crystallographic Orientation Relationship

ABSTRACTIn-situ observation of the formation and disappearance of the surface relief associated with the twinning during the order-disorder transitions among CuAu-I (L10), CuAu-II (PAP) and disordered fcc phases was conducted using Confocal Scanning Laser Microscopy equipped with a gold image furnace. The Retro effect was confirmed in poly-crystal samples, however no evidence was found in single-crystal samples. Also observed in poly-crystal samples are that the disordering temperature detected by the disappearing of relieves is different from grain to grain, and that grain boundary cracking alleviates the Retro effect. The observed phenomena were explained based on the crystallographic orientation relationship among grains investigated by FESEM/EBSD in terms of the elastic strain effect around grain boundaries induced by transition. It was confirmed that in each grain the surface relieves correspond to a set of two {011} planes having a <100> axis perpendicular to both planes in common. It was also found that the larger the average strain of two neighboring grains is, the lower the transition temperature. This observation was explained by the stress effect on the stability of a phase.

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