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Freeze-etching and x-ray diffraction of the isolated double-track layer from the cell wall of a gram-negative marine pseudomonad.

The isolated double-track layer of the cell wall of the gram-negative marine pseudomonad studied here contains a cleavage plane. This finding localizes the single cleavage plane of the cell wall and shows that the molecular architecture of this layer provides the lipid-enriched layer which cleaves preferentially in the frozen cell. The observation that the isolated double-track layer of the cell wall is sufficiently ordered at the molecular level to yield a well-defined X-ray diffraction pattern with a d-spacing of 0.44 nm shows that its molecular architecture is very similar to that of true membranes. This specific d-spacing is produced by the highly ordered packing of the hydrophobic portions of phospholipid molecules. Therefore, the double-track layer of the cell wall has been shown, by these two biophysical means, to have a molecular architecture which would allow it to function as the membrane-like "molecular sieve" layer, whose presence has been deduced from physiological data. This layer is important in the retention of cell wall-associated enzymes and in the control of the movement of large molecules through the cell wall.

Bacteria

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

Tracking of mouse cell lineage using microinjected DNA sequences: analyses using genomic Southern blotting and tissue-section in situ hybridizations.

We examined the feasibility of applying DNA microinjection to label cells for lineage studies of mouse embryos. Tissues from three transgenic mice mosaic due to the insertion of an exogenously introduced mouse beta-major globin gene were analyzed by genomic Southern-blotting and in situ hybridization. These studies allowed the direct quantification and localization of lineage descendants derived from the marked or transformed founder cells. The results of these studies suggested an early segregation of cells in the somatic vs. germ-cell lineages. The in situ hybridization data further demonstrated that cells of the transformed lineages were usually finely dispersed, indicative of extensive cell-cell mixing during mouse development. However, a notable exception to this was the patchy distribution of cells in the kidney (corresponding to individual nephrons), the clustering of transformed cells in individual villi of the small intestine, and the segregation of positive and negative seminiferous tubules in the testis. These data suggest a clonal basis for the organization of development in organs like the kidney, intestine, and testis.

Animals

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

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

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

Do cells show an inverse locomotory response to fibronectin and laminin substrates?

Sixteen cell types from a variety of tissues and from primary and secondary cell cultures and established cell lines were tested for their ability to distinguish between fibronectin and laminin substrates during locomotion in vitro. Laminin and fibronectin were presented to the cells as directly adjacent tracks. Most cells, regardless of origin, showed no preference for one substrate over the other. Only two of the cell types tested showed a strong preference for one or other other substrate molecule. Cells were responding to the local substrate, since antibodies directed against one substrate molecule only interfered with locomotion on tracks coated with that molecule. We conclude that many cells simultaneously express functionally active receptors for fibronectin and laminin, and that differential locomotory response to these two molecules cannot be assumed without experimental confirmation.

Animals

Complex-spike activity of cerebellar Purkinje cells related to wrist tracking movement in monkey.

Four rhesus monkeys were trained to perform visually guided wrist tracking movements (50). While they performed tasks by wrist flexion or extension from a neutral position, simple-spike (SS) and complex-spike (CS) discharges of a single Purkinje cell (P-cell) were recorded from intermediate and lateral parts of cerebellar hemispheres (lobules IV to VI) ipsilateral to the task-performing wrist. Of approximately 400 P-cells observed, 215 (54%) significantly increased or decreased their SS discharge rate during task performance (task-related P-cells). Of these, 161 were selected for analysis of CS activity; in these P-cells, we could reliably discriminate between CS and background SS by a spike discriminator. The 161 P-cells were further classified into response locked (n = 65) and poorly locked (n = 96) cells according to temporal coupling of the SS frequency modulation to the onset of wrist movements. About 60% of the response-locked P-cells showed a phasic increase (statistical significance level: P less than 0.01) of CS firing rate at the onset of wrist tracking movement. In a few P-cells, a phasic decrease (statistically insignificant) of CS firing rate was observed with the wrist movement. In most P-cells, an increase of CS firing rate was observed with both rapid- and slow-tracking wrist movements. The increase was larger with faster step-tracking movement than with slower ramp-tracking movement. In most P-cells, the CS activity increased with both wrist flexion and extension; in some cells, however, it increased only with either flexion or extension. In most of the response-locked P-cells, the increase of CS firing rate occurred during motor time, i.e., after the onset of the EMG change in prime movers and before the beginning of wrist tracking movement. The increase occurred phasically at the onset and/or at the recovery phase of SS frequency modulation. At neutral wrist position, the maintained frequency of the CS was 0.72 +/- 0.29 CS/s (mean and SD for 161 task-related P-cells). Compared with the frequency at neutral position, the CS frequency did not change tonically during maintained flexed or extended wrist position in any response-locked P-cells. There was no increase of CS firing rate when the monkey returned the handle to center position after completing the tracking task, even in P-cells that had shown a significant increase of CS activity during tracking.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials

Closed loop television tracking of beating heart cells in vitro.

The motion of beating heart cells in vitro has been used as a sensitive indicator of the presence of drugs. The combination of closed loop television video tracking and latex microspheres placed on the cells as markers results in an improved measurement of cell motion. Latex microspheres, 5.2 micrometers, are allowed to settle onto beating myocardial heart cells in vitro, where they move in concert with the cell motion. These microspheres, when viewed by televised transmission microscopy with normal optics, present high contrast circular targets that are excellent for closed loop television video tracking techniques. With video tracking, an analog voltage is generated proportional to the horizontal center of intensity of the target being tracked. This signal represents the location of a video demodulator gate that is maintained centered over the video target by feedback control.

Animals

Micropatterned substratum adhesiveness: a model for morphogenetic cues controlling cell behavior.

It is generally considered that tracks of cell adhesiveness are important in controlling cell migration during the development and regeneration of many tissues. In order to investigate this experimentally, a number of techniques have in the past been employed to make patterns of differential adhesiveness for in vitro studies. However, practical limitations on patterning resolution and the introduction of residual topography to the experimental substrata have restricted their usefulness. Here we describe a simplified photolithographic technique for patterning cell adhesiveness which allows a high degree of flexibility and precision. We have quantified, using adhesion and spreading characteristics of BHK cells, the differential adhesiveness that can be created on patterned surfaces, how this alters with the duration of exposure to serum proteins, and how this, in turn, relates to the persistence of cell patterning despite increases in cell density. We believe that this technique will prove extremely useful for the detailed in vitro examination of the mechanisms controlling cell behavior as it offers a degree of precision and ease of fabrication that has previously been unavailable.

Adhesiveness

Computer-assisted analysis of the vascular endothelial cell motile response to injury.

We have developed an automated, user-friendly method to track vascular endothelial cell migration in vitro using an IBM PC/XT with MS DOS. Analog phase-contrast images of the bovine aortic endothelial cells are converted into digital images (8 bit, 250 x 240 pixel resolution) using a Tecmar Video VanGogh A/D board. Digitized images are stored at selected time points following mechanical injury in vitro. FORTRAN and assembly language subroutines have been implemented to automatically detect the wound edge and the edge of each cell nucleus in the phase-contrast, light-microscope field. Detection of the wound edge is accomplished by intensity thresholding following noise reduction in the image and subsequent sampling of the wound. After the range of wound intensities is determined, the entire image is sampled and a histogram of intensities is formed. The histogram peak corresponding to the wound intensities is subtracted, leaving a histogram peak that gives the range of intensities corresponding to the cell nuclei. Rates of cell migration, as well as cellular trajectories and cell surface areas, can be automatically quantitated and analyzed. This inexpensive, automated cell-tracking system should be widely applicable in a variety of cell biologic applications.

Animals

Target velocity signals of visual tracking in vermal Purkinje cells of the monkey.

Discharges of Purkinje cells were recorded from the vermis, lobules VI and VII, of a monkey trained to track a visual target. When the monkey tracked a sinusoidally oscillating target, cellular activity changes in phase with the velocity signal of the eye movement. When the monkey fixated a stationary point, almost identical modulation in activity occurred, reflecting the velocity signal of the motion of the retinal image of the target. The data suggest that the vermis participates in the control of smooth pursuit eye movements by providing the oculomotor system with the actual target velocity information which is the sum of eye velocity and retinal image velocity signals.

Action Potentials

Seeding of malignant cells into the needle track after lung and pleural biopsy.

Transthoracic needle biopsy is frequently performed for the diagnosis of pulmonary and pleural disease. Seeding of malignant cells is a potential complication but is extremely uncommon. We report the second patient with malignant seeding after fine-needle aspiration biopsy of a bronchogenic carcinoma. We also report a patient with seeding of bronchogenic carcinoma after a pleural biopsy. Because seeding is a rare complication, it does not affect the indications for biopsy.

Adenocarcinoma, Papillary

Time lapse and microscopic examinations of insonated in vitro cells.

A fibroblast cell line (Balb/c 3T3) was sham/exposed to ultrasound (2.2 MHz resonant frequency, 35.4 W/cm2 SPTP, 3 microseconds pulse duration, 200 Hz prf) for 30 min and subsequently scored for cell motility and alterations in cell morphology. Cell motility was assessed by computerized tracking of cells. Cell morphology ("normal" or "abnormal") was determined blindly by three independent scorers. A positive control (1-4 Gy) yielded statistically significant alterations in cell motility and morphology; no such differences were observed for the ultrasound regimen. The results thus fail to confirm an earlier report by Liebeskind et al. (Brit. J. Cancer 45(Suppl. V):176-186; 1982) of ultrasound-induced alterations in cell motility and morphology.

Animals

Cell survival following multiple-track alpha particle irradiation.

In experiments in which mammalian cells were irradiated with 5 . 6 MeV alpha particles from a Tandem Van de Graaff machine, we have confirmed the finding of others that the mean lethal dose (Do) is about 60 rad. However, on measuring the area of the nuclei of the flattened cells as they were irradiated, we found that this mean lethal dose corresponds to the passage of not one or two alpha particles per cell nucleus as expected but to between 10 and 20 particles. This allows for the possibility that the direct action of alpha particles on the nucleus may be the important event in carcinogenesis.

Alpha Particles

Spatiotemporal single-cell profiling reveals T cell clonal dynamics and phenotypic plasticity in human graft-versus-host disease.

Allogeneic hematopoietic cell transplantation cures hematologic diseases but is limited by acute graft‑versus‑host disease. How human T cell clones drive epithelial injury remains poorly mapped. We studied 31 transplant recipients, integrating longitudinal T cell antigen receptor (TCR) profiling with single-cell RNA sequencing/TCR sequencing and spatial transcriptomics to track T cell clonal dynamics. We developed DecompTCR to resolve temporal dynamics and adapted computational tools to map clone phenotypes and niches in tissue. Our analyses revealed that cyclophosphamide selectively depletes alloreactive clones, although insufficient early expansion leads to incomplete depletion and severe disease. Severe graft‑versus‑host disease is marked by persistent expansion of alloreactive clones, rewiring of homeostatic cell types and diversification of donor-derived CD8+ clonotypes that acquire Hobit (ZNF683)+ tissue‑resident memory T (TRM) cell programs during migration to epithelium. Spatial deconvolution identified CD8+ effector/Hobit+ TRM hubs near intestinal stem‑cell-rich crypt bases and crypt‑loss regions. This clonotype‑resolved framework links tissue‑instructed TRM cell remodeling to localized epithelial injury, nominating early-repertoire dynamics and spatial hub burden as biomarkers.

Journal Article