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At least 37 records · Page 2Linked to original sources
PubMed2025

Measuring Cell Dimensions in Fission Yeast Using Machine Learning.

In fission yeast (Schizosaccharomyces pombe), cell length is a crucial indicator of cell cycle progression. Microscopy screens that examine the effect of agents or genotypes suspected of altering genomic or metabolic stability and thus cell size are crucial for studying disruptions to cell cycle dynamics. This method is based on using an automated cell segmentation algorithm to measure S. pombe cells imaged by brightfield (BF) microscopy methods. PhotoPhenosizer (PP) is a machine learning-based tool designed for automated cell measuring and dimensional analysis of morphology frequency distributions. Integration of this method into large-scale pipelines for tracking cell dimension change streamlines morphological measurements, which facilitates the examination of cellular responses to genomic and metabolic stresses. In this protocol, we use PP to observe the effect of genomic instability on cell size dynamics over a 12-day chronological lifespan assay. Our results show that relative to wild-type cells, a replication stress mutant shows larger cells during chronological aging in excess glucose media. Our results are consistent with activation of checkpoints that regulate cell morphology in response to DNA damage. This method's application highlights the relevance of its incorporation in experimental routines that require large-scale image processing and its adoption by users with routine needs in S. pombe molecular research projects.

Schizosaccharomyces↗
PubMedDate not supplied

Computer tracking of germ cells in the cycle of the seminiferous epithelium and prediction of changes in cycle duration in animals commonly used in reproductive biology and toxicology.

A computer program called Stages was written to aid the tracking of germ cells and stages forward and backward through time in the cycle of the seminiferous epithelium. The program incorporates the basic kinetics of spermatogenesis in the rat, mouse, hamster, guinea pig, dog, rabbit, bull, stallion, ram, boar, quail, monkey, and man. The program is flexible and permits the user to alter the cycle duration time and the frequency of each stage. Compiled for use on personal computers and available on floppy disks free of charge, Stages is menu-driven and requires no knowledge of programming. The program was tested using published data of testicular toxicity and vitamin A synchronization of stages. In general, predicted cell types were similar to those observed; discrepancies between observed and predicted data are discussed. When cycle duration time was changed, predicted data for stage synchronization coincided with the observed data. This program will improve the speed and accuracy of studying factors that affect spermatogenesis. By using Stages, it is possible to predict the target cell types in a toxicity study and to track affected cells over long periods of time. Predicted cell types may also indicate which cells to examine biochemically as well as morphologically in subsequent experiments. The program will also be useful to beginning students learning the complex patterns of cellular associations and the progression of germ cells in the cycle of the seminiferous epithelium.

Animals↗
PubMed1991

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

Animals↗
PubMed1986

Chemokinetic behaviour of insect haemocytes in vitro.

Time-lapse microphotography was used to film the locomotory behaviour of cockroach haemocytes in vitro, and the cell tracks were analysed for speed and persistence; the percentage mobilization and the diffusion rate of the population were calculated. Haemocytes are either fast locomotor or spread moving cells, or non-motile spread or rounded cells; the first three types are plasmatocytes and their behaviour is interchangeable. Approximately 20% of the cells are motile under control conditions and there is no correlation between orthokinesis and klinokinesis. If activated haemocyte lysate supernatant (HLS), a source of components of the prophenoloxidase enzyme sequence, is added to the cell monolayer, up to 80% of the cells switch to fast locomotor behaviour, rounding up and moving faster and for longer in straight lines. Neither heat-inactivated HLS nor zymosan supernatant, used to activate HLS, had any effect. If the chemokinins present in activated HLS are also released in vivo on haemocyte activation or during cuticular wounding, then they and the induced changes in haemocyte adhesion could contribute to haemocyte recruitment to sites of infection.

Animals↗
PubMed1990

All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium.

An analogue of all-trans retinal in which all-trans/13-cis isomerization is blocked by a carbon bridge from C12 to C14 was incorporated into the apoproteins of sensory rhodopsin I (SR-I) and sensory rhodopsin II (SR-II, also called phoborhodopsin) in retinal-deficient Halobacterium halobium membranes. The "all-trans-locked" retinal analogue forms SR-I and SR-II analogue pigments with similar absorption spectra as the native pigments. Blocking isomerization prevents the formation of the long-lived intermediate of the SR-I photocycle (S373) and those of the SR-II photocycle (S-II360 and S-II530). A computerized cell tracking and motion analysis system capable of detecting 2% of native pigment activity was used for assessing motility behavior. Introduction of the locked analogue into SR-I or SR-II apoprotein in vivo did not restore phototactic responses through any of the three known photosensory systems (SR-I attractant, SR-I repellent, or SR-II repellent). We conclude that unlike the phototaxis receptor of Chlamydomonas reinhardtii, which has been reported to mediate physiological responses without specific double-bond isomerization of its retinal chromophore (Foster et al., 1989), all-trans/13-cis isomerization is essential for SR-I and SR-II phototaxis signaling.

Archaeal Proteins↗
PubMed1988

Maintenance of peritoneal macrophages in the steady state.

Resident peritoneal macrophages (M phi) were labeled in situ by intraperitoneal (i.p.) injection of the green fluorescent cell tracking dye PKH-1. After immunofluorescence staining with M phi specific monoclonal antibodies (Mabs) and phycoerythrin (PE) second antibody, the resident M phi were labeled with both the green dye and red Mab label, while recruited M phi were labeled only with the red Mab tag. These populations were distinguished by two-color flow cytometry. PKH-1 labeled resident peritoneal M phi were followed for 1-49 days in mice that received no further treatment (steady state). Dye labeled M phi were still detectable after 49 days in vivo, although their green fluorescence intensity had decreased steadily over time. The decrease in dye intensity was limited to M phi, as the fluorescence intensity of PKH-1 labeled peritoneal lymphocytes did not change. Resident M phi populations were clearly separated from recruited M phi by the intensity of their staining with PKH-1 for up to 28 days. No decrease in the number of resident (dye labeled) peritoneal M phi was observed over 1-28 days. These data indicate that resident peritoneal M phi were not replaced by recruited blood monocytes in the steady state.

Animals↗
PubMed1991

Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin.

The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).

Animals↗
PubMed1988

Microorganism track reconstruction: an image processing approach.

This paper presents an automatic system for the analysis of microorganism behaviour. The movements of free swimming microorganisms are videotaped by means of a television camera mounted on a microscope. The analysis is performed off-line by digitizing the video signal through the use of an image processor unit. Microorganism tracks are reconstructed spatially and chronologically by means of image processing techniques. From these tracks cell movement parameters are obtained. The results of our experiment in testing photoinduced movements follow.

Algorithms↗
PubMed1991

A visual assay for quantitating neutrophil chemotaxis in a collagen gel matrix. A novel chemotactic chamber.

The chemotactic behavior of polymorphonuclear leukocytes (PMNs) suspended in a three-dimensional gel of native collagen fibers was analyzed using a new visual assay aided by computer assisted tracking. Cell behavior was observed in a 7 microliters chamber closed at either end with capillary tubes tipped with dialysis membrane. The chemoattractant, LTB4, was placed in one capillary tube and the control substance in the opposite tube. Under microscopic observation neutrophils were videotaped, their images digitized, and the x and y coordinates of the cell centroids captured at 30 s intervals for 15 min and subsequently analyzed. The data generate a global perspective of neutrophil behavior in a medium simulating a collagenous tissue. The results show that when leukotriene B4 was substituted for HBSS the PMN population underwent chemotactic displacement. PMN chemotaxis was increased greatly when the concentration of LTB4 was increased from 10 nM to 1 microM in separate experiments. This result was partly achieved by movement of an increasing percentage of the PMN population, less frequent stops, and longer durations of motility for individual cells. The most dramatic effect of LTB4 on neutrophil behavior was a large increase in directional movement toward the chemotactic source. The effects of LTB4 fell dramatically when the gradient source concentration was increased to 10 microM. The visual assay described here provides clear evidence that LTB4 induces true neutrophil chemotaxis in a collagenous matrix.

Chemotaxis, Leukocyte↗
PubMed1978

An economical, semi-automatic system for measuring cellular tree structures in three dimensions, with special emphasis on Golgi-impregnated neurons.

The hardware and software of a semi-automatic system are described for the measurement of cellular tree structures in thick microscopical sections such as the process of Golgi-impregnated neurons. With this system, the morphologist--viewing the neuron directly through the microscope--measures the tree structures using three cartesian coordinates of characteristic points, e.g. centre of cell, origin of processes, bifurcation points, end points, deflection points, etc. The system requires an on-line computer, but does not demand a dedicated computer since a joy-stick generator usually drives the microscopical stage during cell tracking. A digital computer is utilized for acquisition of data, and, when needed, to return the stage to a required position. The microscope stage is coupled to three stepping motors with 0.5 micron as the smallest step. The stepping motor for movement of the stage parallel to the optical axis (z-direction) is coupled directly to a spindle mechanism, replacing the original cog wheel mechanism. This construction shows no measurable backlash. The practical advantages of this new measuring system in relation to the existing digital systems currently in use, its accuracy and its costs, are discussed.

Animals↗
PubMed1989

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation.

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.

Cell Survival↗
PubMed1988

Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks.

Different biochemical and cytochemical techniques were applied to characterize the sites of localization of thrombospondin in cultured endothelial cells. The results obtained by [35S]methionine labeling, immunoblotting, immunoprecipitation, fluorescence microscopy, ultracytochemistry, immunogold labeling, and silver enhancement experiments revealed that thrombospondin secreted by endothelial cells is structurally organized together with proteoheparan sulfate in spherical granules at the cell surface. These granules are about 100 to 300 nm in size. Heparin or enzymatic degradation with heparitinase, but not with ABC lyase, release thrombospondin from the cell surface. Fibronectin is expressed in the extracellular matrix of endothelial cells in a fibrillar organization, clearly distinct from the punctate pattern of thrombospondin on the cell surface. Furthermore, secreted thrombospondin is highly enriched together with fibronectin and proteoheparan sulfate in cell attachment sites and in cell migration tracks. In cell migration tracks proteoheparan sulfate more clearly resembles the fibrillar distribution pattern of fibronectin, whereas thrombospondin reveals a rather monodisperse pattern. The obtained data suggest preferential sites of interaction between thrombospondin and heparan sulfate proteoglycans on the cell surface and a participation of thrombospondin in cell adhesion and cell migration.

Animals↗
PubMed1989

Microcomputer-based interactive tracking of blood cells at biomaterial surfaces.

A microcomputer-based system for analyzing the motion of human platelets and leukocytes at synthetic surfaces from a sequence of video frames on tape is described. The software is designed to provide convenient interaction with an operator to reduce the burden of manual analysis. In addition, the system computes and stores the cell movement data on disk for subsequent statistical analysis. Measurement include the number and nature of cell-to-surface collisions, residence times, and distances traveled.

Biocompatible Materials↗
PubMedDate not supplied

Evaluation of methods for estimating renal medullary blood flow.

Although only 10-15% of renal blood flow enters the medulla, renal medullary hemodynamics have been implicated as a key factor for the ability of the kidney to excrete concentrated or diluted urine and to be involved in sodium homeostasis. Due to the obvious importance of medullary hemodynamics, several methods have been developed or modified for estimating regional renal blood flow. The aim of the present paper is to evaluate critically the most commonly used methods available today. Those which are considered in this paper are: (1) albumin accumulation (Lilienfield), (2) rubidium-86 uptake, (3) transit time-indicator dilution, (4) red cell velocity tracking, (5) red cell flux (fluorescently labeled red cells) and (6) laser-Doppler flowmetry. Depending on the method of choice, the reported control values for renal medullary blood flow differ up to tenfold. Overall, it appears that the discrepancies in values obtained from the different methods will not be resolved until the methods are compared directly in the same animal and during different experimental conditions. Furthermore, one needs to understand and describe accurately the involved errors so that proper corrections can be made. It seems likely, however, that the majority of the techniques can be used to study relative changes, but attempts should not be made to draw conclusions about the absolute flow value. In this regard, the laser-Doppler method seems to be superior in its simplicity of use and its large advantage of continuous and relatively noninvasive measurement.

Animals↗
PubMed1992

Circadian variations in cell cycle phase progression of mouse epidermal cells measured directly by bivariate BrdUrd/DNA flow cytometry.

Circadian stage-dependent variations in cell cycle traverse of mouse epidermal cells in vivo were investigated. The fate of cohorts of basal cells pulse-labelled with bromodeoxyuridine (BrdUrd) at different times of the day were studied by bivariate BrdUrd/DNA flow cytometry of isolated epidermal basal cells. Basal cells were tracked through the cell cycle up to 96 h after intraperitoneal injection of BrdUrd at 0800 and 2000, or followed for 6 h after BrdUrd injection at 0400, 1200, 1600 and 2400. The results confirmed our previous assumption that the cell cycle progression through S phase and G2 phase is considerably delayed at night, i.e. from 1600 to 0400, compared with daytime. The results indicate variations in G1 phase as well. The data strongly support the hypothesis that the main parameters responsible for circadian fluctuations in mitotic activity are variations in the S and G2 phase durations. The data are also consistent with the notion of proliferative heterogeneity among basal cells as described by a hierarchical proliferation model.

Animals↗
PubMed1990

Track structure, lesion development, and cell survival.

A stochastic track-structure-dependent model is presented based on DNA double-strand breaks (DSBs) interacting in a time- and distance-dependent manner, and in competition with DSB repair, to form exchange-type chromosomal aberrations. Many models of cell survival involve estimation of mean numbers of lesions per cell, which is then related to cell survival. Unless this relationship is linear, this implies that a cell responds not to the number of lesions produced in it, but to the mean number of lesions in all the exposed cells; this is clearly unrealistic, particularly for phenomena such as saturation. In contrast to such deterministic approaches, we describe a stochastic model, in which individual cells are considered and exposed to Monte Carlo-generated tracks of various radiations. The elementary sublesions produced (DSBs) diffuse, repair, or interact, forming lesions (chromosomal exchange-type aberrations) in a time- and distance-dependent manner. Results agree well with experiments for survival of synchronous Chinese hamster V-79 cells exposed to X rays and radiations with LETs from 20 to 170 keV/microns. Thus the main features of survival for low-, medium-, and high-LET radiation are understandable in terms of a single approach, the relative responses to different radiations being determined by their different energy deposition patterns.

Cell Survival↗