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At least 91 records · Page 5Linked to original sources

PubMed1980

Cellular and molecular mechanisms in angiogenesis.

Low concentrations of copper sulphate, Dispirin, or Walker carcinoma extract elicit intraocular vascularization when tested by anterior chamber implants in rats. The response is markedly depressed by pre-treatment of animals with methylprednisolone acetate, suggesting that such induced vascularization is mediated by leucocytes. Since many agents inducing vascularization also induce migration of cultured endothelial cells, it raises the problem of how to isolate and study the action of possible leucocyte-derived angiogenic factors. Regardless of the identity of the natural angiogenic factor or factors, it is proposed that during blood vessel formation specialized endothelial cells migrate in response to an angiogenic signal and deposit fibronectin on which other cells can track and subsequently adhere, forming an endothelium.

Animals↗

PubMed1991

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Ammonium Chloride↗

PubMed1989

Microdosimetry of rat alveolar type II cells irradiated with alpha particles from 239PuO2.

The alveolar type II cell is one of the critical cells for radiation damage in the lungs after inhalation of radioactive aerosols. With the aid of a Quantimet-970 image analyzer and a VAX-11/780 computer, we calculated the radiation dose to rat alveolar type II cells from alpha particles emitted by 239PuO2. A series of dosimetric parameters for type II cells, including track length distribution, linear energy transfer (LET), values of the specific energy for a single hit of a spherical target (z1), cellular dose, hit number, and their spatial distributions were calculated. By comparing the volume density of type II cells and lung tissue with energy deposited in alveolar type II cells, we found that the energy deposited per unit volume of type II cells was larger than that of lung tissue excluding type II cells. The z1 for spherical targets and the LET across type II cells were less than those in lung tissue excluding type II cells. The age of the rat and damage to lung by inhalation may significantly influence some of the parameters. The neoplastic transformation probability for type II cells is also discussed. The results suggest that the type II cell is an important target cell in the rat lung for exposure to inhaled 239PuO2.

Aerosols↗

PubMed1988

Real time computer tracking of free-swimming and tethered rotating cells.

A computerized image processing system has been developed that tracks individual free-swimming cells and rotating bacterial cell bodies tethered by their flagella in real time. Free-swimming bacteria of Rhodobacter sphaeroides, Rhodospirullum rubrum, and Salmonella typhimurium have been tracked swimming at speeds from 0 to over 120 microns s-1. A high level of discrimination is exerted against noncellular objects, allowing analysis of stopped as well as moving cells. This enabled detection of both speed and qualitative change in the swimming patterns of R. sphaeroides WS8 upon tactic stimulation. Comparison with darkfield microscopy indicated that the two techniques were in substantial agreement. The unidirectional rotation of cells of R. sphaeroides WS8 could be detected when the cells were either parallel to the microscope slide or end on. Frequencies of rotation of up to 10 Hz were monitored before image blurring became a problem. True rods would be easier to analyze at higher speeds of rotation. Although developed for photosynthetic bacteria, a wide range of bacteria, eucaryotic organisms, and subcellular organelles could be tracked with this system. Minor modifications to the software allow customization to different types of motility analysis.

Bacterial Physiological Phenomena↗

PubMed1990

Rapid flow of passive neutrophils into a 4 microns pipet and measurement of cytoplasmic viscosity.

Neutrophils from five different individuals are isolated with a density separation technique. A total of 151 unactivated (passive) cells are rapidly aspirated at constant suction pressure and at room temperature into a pipet with a diameter of 4 microns. The suction pressures in excess of an initial yield threshold are 0.5, 1 and 2 kPa and are comparable to those encountered in the microcirculation. These pressures are well in excess of the small suction pressure of approximately 20 Pa that is required to form a static hemispherical bump on the cell. At a given aspiration pressure, the leading edge of an individual cell is "tracked" as it flows into the pipet. A theory based on the flow of a Newtonian liquid from either a hemisphere or a spherical segment into a cylinder is used to model the entry process. Both theory and experiment show that during most of the entry process the leading edge of the cell moves at a nearly constant velocity with a rapid acceleration at the end. For cells from five different individuals at the three different excess aspiration pressures, Newtonian theory gives a cytoplasmic viscosity of 135 +/- 54 Pa.s and overall entry times of 3.3s (0.5 kPa), 1.6s (1 kPa) and 0.82s (2 kPa). These results and those of Evans and Yeung at lower aspiration pressures indicate that the complex cytoplasm inside unactivated neutrophils behaves as a nearly Newtonian fluid with a viscosity on the order of 10(2) Pa.s over almost a two order of magnitude range in aspiration pressure and, thus, rate of deformation.

Cytoplasm↗

PubMed2025

Multicolor Cell Lineage Tracing Using MAGIC Markers Strategies.

Multicolor MAGIC Markers strategies are useful lineage tracing tools to study brain development at a multicellular scale. In this chapter, we describe an in utero electroporation method to simultaneously label multiple neighboring progenitors and their respective progeny using these multicolor reporters. In utero electroporation enables the introduction of any gene of interest into embryonic neural progenitors lining the brain ventricles through a simple pipeline consisting of a micro-injection followed by the application of electrical pulses. Successful in utero electroporation requires a concise yet complete understanding of each step of the surgical protocol, spanning from the preoperative preparation to the postoperative care, as well as the MAGIC Markers tool outlined in this study. Besides a detailed protocol, we present non-integrative and integrative approaches to demonstrate the range of cell and lineage tracking possibilities of multicolored progenitors and their descent over time.

Cell Lineage↗

PubMed2024

Transient Zn2+ deficiency induces replication stress and compromises daughter cell proliferation.

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn2+) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown. Using fluorescent reporters to track single cells via long-term live-cell imaging, we find that Zn2+ is required at the G1/S transition and during S phase for timely completion of S phase. A short pulse of Zn2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn2+. Finally, we find that if Zn2+ is chelated during the mother cell's S phase, daughter cells enter a transient quiescent state, maintained by sustained expression of p21, which disappears upon reentry into the cell cycle. In summary, short pulses of mild Zn2+ deficiency in S phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells.

Zinc↗

PubMed2023

Transient Zn2+ deficiency induces replication stress and compromises daughter cell proliferation.

Cells must replicate their genome quickly and accurately, and they require metabolites and cofactors to do so. Ionic zinc (Zn2+) is an essential micronutrient that is required for hundreds of cellular processes, including DNA synthesis and adequate proliferation. Deficiency in this micronutrient impairs DNA synthesis and inhibits proliferation, but the mechanism is unknown. Using fluorescent reporters to track single cells via long-term live-cell imaging, we find that Zn2+ is required at the G1/S transition and during S-phase for timely completion of S-phase. A short pulse of Zn2+ deficiency impairs DNA synthesis and increases markers of replication stress. These markers of replication stress are reversed upon resupply of Zn2+. Finally, we find that if Zn2+ is removed during the mother cell's S-phase, daughter cells enter a transient quiescent state, maintained by sustained expression of p21, which disappears upon reentry into the cell cycle. In summary, short pulses of mild Zn2+ deficiency in S-phase specifically induce replication stress, which causes downstream proliferation impairments in daughter cells.

Biological sciences↗

PubMed1985

Possible mechanisms by which repeated clozapine administration differentially affects the activity of two subpopulations of midbrain dopamine neurons.

Extracellular single-cell recording techniques were employed to study the mechanism of action of repeated oral clozapine administration on the in vivo spontaneous activity of substantia nigra (A9) and ventral tegmental area (A10) dopamine (DA)-containing neurons in the rat. Clozapine was observed to affect DA neurons differentially within these two regions when compared to haloperidol. Acute treatment (1 hr) with both drugs increased the number of spontaneously firing neurons in both A9 and A10. Chronic (21 day) treatment with haloperidol decreased the number of cells encountered in both regions, whereas repeated treatment with clozapine reduced the number of DA cells per track only in A10. In all cases, the silent DA neurons were inferred to be in a state of depolarization inactivation since they could be induced to discharge normally by the microiontophoretic application of the inhibitory neurotransmitter gamma-aminobutyric acid. These effects were not due to an effect of chloral hydrate anesthesia since they were also observed in gallamine-paralyzed, artificially respired animals. Chronic co-administration with haloperidol of either an anticholinergic (trihexyphenidyl) or the alpha 1-norepinephrine (NE) receptor antagonist, prazosin, but not an alpha 2-NE antagonist, RX781094, resulted in a differential effect on A9 and A10 DA neurons identical to that observed with repeated clozapine administration alone. Thus, chronic treatment with these combinations of drugs resulted in the depolarization inactivation of only A10 cells. These data suggest that anticholinergic and/or alpha 1-NE-blocking properties of clozapine may, in part, mediate its differential effects on A9 and A10 midbrain DA neurons.

Anesthetics↗

PubMed1990

The specification of neuronal identity in the mammalian cerebral cortex.

The determination of neuronal fate in the developing cerebral cortex has been studied by tracking normal cell lineages in the cortex, and by testing the commitment of young cortical neurons to their normal fates. These studies together suggest that neuronal progenitors are multipotent during development and have the potential to produce neurons destined for many or all of the cortical layers. However, the laminar identity of an individual neuron appears to be specified through environmental interactions at the time of the cell's terminal mitotic division, prior to its migration into the cortical plate.

Animals↗

PubMed1989

A new calculational method to assess the therapeutic potential of Auger electron emission.

This paper discusses a new computer code to estimate the efficacy of Auger electron sources in cancer therapy. Auger electron emission accompanies the decay of many radionuclides already commonly used in nuclear medicine, for example; 99mTc and 201Tl. The range of these electrons is in general sub-cellular, therefore, the toxicity of the source depends on the site of decay relative to the genetic material of the cell. Electron track structure methods have been used which enable the study of energy deposition from Auger sources down to the Angstrom level. A figure for the minimum energy required per single strand break is obtained by fitting our energy deposition calculations for 125I decays in a model of the DNA to experimental data on break lengths from 125I labeled plasmid fragments. This method is used to investigate the efficiency of double strand break production by other Auger sources which have potential value for therapy. The high RBE of Auger sources depends critically on the distance between the source and target material. The application of Auger emitters for therapy may necessitate a carrier molecule that can append the source to the DNA. Many DNA localizing agents are known in the field of chemotherapy, some of which could be carrier molecules for Auger sources; the halogenated thymidine precursors are under scrutiny in this field. The activation of Auger cascades in situ by high energy, collimated X ray and neutron beams is also assessed.

DNA, Neoplasm↗

PubMed2025

Tracking HIV persistence across T cell lineages during early ART-treated HIV-1-infection using a reservoir-marking humanized mouse model.

Human immunodeficiency virus (HIV) infection depletes CD4 T-cells, and long-term persistence of latent virus prevents full clearance of HIV even in the presence of effective antiretroviral therapy (ART), Here we present the HIV-1-induced lineage tracing (HILT) system, a model that irreversibly marks infected cells within a humanized mouse model, which detects rare latently infected cells. Immunodeficient mice transplanted with genetically modified hematopoietic stem cells develop a human immune system, in which CD4 T-cells contain a genetic switch that permanently labels cells infected by HIV-1 expressing cre-recombinase. Through single-cell RNA sequencing of HILT-marked cells during acute infection and post-ART treatment, we identify distinct CD4+ T-cell transcriptional lineages enriched in either active or latent infections. Comparative gene expression analysis highlights common pathways modulated in both states, including EIF2, Sirtuin, and protein ubiquitination. Critical regulators of these pathways, including JUN, BCL2, and MDM2, change to opposite directions in the two states, highlighting gene expression programs that may support HIV persistence across T-cell lineages and states.

Animals↗

PubMed1987

A computer-assisted image-analysis system for analyzing polymorphonuclear leukocyte chemotaxis in patients with diabetes mellitus.

A computer-assisted image-analysis system that precisely tracks the cell movements of up to 50 polymorphonuclear leukocytes (PMNLs) was developed and used to quantitatively measure cellular chemokinesis and chemotaxis in normal individuals and in diabetic patients with hyperglycemia. The PMNLs were tested in Zigmond chambers with or without a gradient of 10(-7) M n-formylmethionylleucylphenylalanine (f-Met-Leu-Phe). Cellular movement was recorded on videotape by using a videocamera mounted on the microscope. The videotapes were analyzed by computer programs to calculate the speed and direction of each PMNL at 10-sec intervals. Average rates of chemokinesis were 19.6 microns/min without and 25.3 microns/min with f-Met-Leu-Phe. McCutcheon indices, which measure chemotaxis, were 0.01 without and 0.48 with f-Met-Leu-Phe. Similar values were observed in diabetic patients after fasting (average glucose, 217 mg/100 ml) and 2 hr after glucose challenge (average glucose, 309 mg/100 ml). These values demonstrate that PMNLs from diabetic patients with hyperglycemia move at normal rates and respond appropriately to f-Met-Leu-Phe.

Adolescent↗

PubMed1988

Spatial and temporal distribution of energy.

Studies of the spatial and temporal distribution of microscopic radiation doses lead to potentially important questions regarding conventional approaches to radiation protection. The short ranges of alpha-particle and Auger-electron emissions from radionuclides lead to uncertainties in assessing their hazards. The conventional extrapolations from intermediate doses to low doses and dose rates are questioned by observed dose-rate effects in the so-called "initial slope," by the total lack of data for single tracks in cells and by the possibility of multiple-cell effects. At all subcellular levels, even down to DNA, high linear-energy-transfer (LET) radiations can produce unique initial damage, different from that possible with low-LET radiations, and therefore may even, in principle, produce unique final biological effects. This questions simple extrapolations from low- to high-LET radiations and the application of universal quality factors to diverse effects. Further understanding of these questions could lead, in future, to substantial increases or decreases in estimations of risk.

Alpha Particles↗

PubMed1979

A cinematographical study of the development of vitally stained Dictyostelium discoideum.

We report observations from time-lapse films of the development of Dictyostelium discoideum (Dd) stained with the vital dye neutral red. We have used this dye to enable us to track individual cells, as they move through multicellular tissues in later Dd stages. Our observations lead us to new conclusions about the control of cell movement and cell contact during Dd development, including the tentative conclusion that the aggregation control competences regulate cell behaviour throughout the whole of Dd. development. We are led to specific hypotheses for the mechanisms of later morphogenetic movements and pattern formation.

Dictyostelium↗

PubMed1990

Three-dimensional structure of the rat intestinal wall (mucosa and submucosa).

The three-dimensional organization of the mucosa and submucosa of the rat intestine was analysed by scanning electron microscopy (SEM) aided by micro-dissection methods. New functional aspects raised by the SEM observations were examined by transmission electron microscopy (TEM). Morphogenesis of the intestinal villi was also illustrated three dimensionally. Removal of the epithelium by osmic acid maceration revealed the presence of many round fenestrations averaging 3 microns in diameter over the villous basal lamina. TEM confirmed that they were not artifacts but represented passages or tracks of cells of the immune system such as lymphocytes, eosinophils and macrophages. Penetration of the epithelial processes into the lamina propria was also observed. Close contacts between these free cells and the epithelial cells suggest an intercellular communication between these different cell types. The basal lamina is thus appears as a structure that allows dynamic interaction between the epithelial layer and the lamina propria, though it is generally regarded as a rigid structure acting as a barrier. Beneath the basal lamina, a cellular reticulum of fibroblast-like cells overlies the capillary network in the villi. These cells are characterized by bundles of actin filaments and contact with each other by gap junctions. This cellular reticulum probably influences the absorption of nutrients from the villi by its contractile ability in addition to its supportive role. A similar cellular network occurs beneath the epithelium of the intestinal glands. These cells may also mechanically support the glandular organization, maintaining the delicate microvascular bed. The submucosa is considered the skeleton of the intestine. SEM reveals the main framework of the submucosa is as being composed of two sets of collagen fibers running diagonally around the intestinal wall, one set in a clockwise direction, the other, counterclockwise. These fibers--in different arrays--interweave to form a lattice sheet, which presumably provides the tissue with a high resistance to mechanical forces, particularly in respect to radial forces. The diagonal orientation of the collagen fibers is essential for the flexibility of the submucosa in allowing deformation of the intestinal wall during peristalsis. Despite the nonelastic nature of the collagen fibers, the submucosa can adapt to the various shapes of the intestinal lumen by simply changing the angles formed by these fibers. Structures of the villous microcirculation, the muscularis mucosae and of the submucous plexus are also discussed.

Animals↗

PubMed1987

Population response of midbrain dopaminergic neurons to neuroleptics: further studies on time course and nondopaminergic neuronal influences.

In the present study, we examined the effects of the cholecystokinin receptor antagonist, proglumide, on the depolarization-induced inactivation of A9 and A10 dopaminergic neurons produced by repeated administration of a classical antipsychotic drug (dopamine receptor antagonist). In addition, we studied the nature of the effects of acute (1-48 hr) and long-term (7 month) treatment with the butyrophenone neuroleptic haloperidol on both the basal firing rate and population response of dopamine-containing neurons in these 2 regions. Acute oral administration of haloperidol (0.5 mg/kg) results, within 1 hr of administration, in an increase in both the firing rate and number of spontaneously active dopamine neurons encountered in both A9 and A10 regions. These effects of a single treatment persist for a minimum of 6 hr and, with respect to firing rate, are not completely normalized for at least 24 hr. In contrast, 7 month continuous treatment with haloperidol reduces the number of spontaneously active DA neurons encountered in both regions in a manner similar to that observed at 21 d. This effect is inferred to be due to the induction of depolarization-induced inactivation of these neurons, since the acute administration of the normally hyperpolarizing, direct-acting dopamine receptor agonist apomorphine (64 micrograms/kg) immediately reverses this reduced number of cells per track to near control levels. This effect appears to be dependent on the continued presence of haloperidol since, when animals treated for 7 months are sampled 14 d after the cessation of drug administration, spontaneous activity is no different from that observed in age-matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Radioimmunoassay of c-myc protein.

A sensitive radioimmunoassay for human c-myc protein was developed by using an amino-terminal fragment of c-myc protein, c-myc(11-24). Immunoreactive c-myc protein was found to be present in 3 human tumors (HL-60, N231, Lu-65), which are known to have c-myc gene amplification. In contrast, c-myc protein was undetectable in 1 human tumor (H69) without c-myc gene amplification as well as in human heart tissues.

Cell Line↗