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PubMed1998

Role of endothelins and nitric oxide in hepatic reperfusion injury in the rat.

We determined the functional role of nitric oxide (NO) and endothelins (ET), two potent vasoactive mediator systems in the liver, for the pathogenesis of sinusoidal perfusion failure and lethal hepatocyte injury after low-flow ischemia/reperfusion in the isolated perfused rat liver. NO synthase blockade with Nomega-nitro-L-arginine methyl ester (L-NAME) (10[-3] mol/L) before reperfusion prevented increased N02-/NO3- the final products of NO oxidation, which could be observed in the vehicle group. Epifluorescence microscopy revealed that the decrease in functional sinusoid density during reperfusion was much more profound compared with vehicle. This was associated with a lower surface PO2, a substantially higher number of nonviable hepatocytes, as assessed by in situ propidium iodide staining, and enhanced enzyme release into the perfusate compared with vehicle. In contrast, reperfusion in the presence of the endothelinA+B receptor antagonist bosentan (2 x 10(-4) mol/L) restored functional sinusoid density and surface PO2 to baseline values, resulted in a small reduction in the number of propidium iodide-positive hepatocytes, and caused similar increases in enzyme release as compared with vehicle. This indicates that hepatic generation of NO attenuates sinusoidal perfusion failure and improves liver tissue oxygenation, thus limiting hepatocyte injury during early reperfusion after hepatic low-flow ischemia. In contrast, endothelins counteract the microcirculatory effects of NO, i.e., mediate the no-reflow in hepatic sinusoids; however, the restoration of functional sinusoid density with bosentan resulted only in a small reduction in tissue damage, suggesting that additional components, which are independent of microcirculatory failure, contribute to hepatic reperfusion injury under these conditions.

Animals↗
PubMed1990

Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenyl-aziridinium.

Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N-dimethyl-2-phenyl-aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270-278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be prevented by edrophonium; the sequence of one of the peptides assumed to be part of the anionic site in the catalytic centre was found to be DLFR (positions 217-220). Decamethonium efficiently blocked alkylation by DPA in all three investigated peptides.

Acetylcholinesterase↗
PubMed1993

The absence of supercoiling in kinetoplast DNA minicircles.

Crithidia fasciculata kinetoplast DNA is a mitochondrial DNA composed of 5000 minicircles and approximately 25 maxicircles, all catenated into a giant network. By comparing the linking number of minicircles released from the network by limited sonication with that of control minicircles, we demonstrate that not only does the elaborate catenation of the network not cause supercoiling, but that there is no minicircle supercoiling at all. The absence of catenation-induced supercoiling is explained by our finding [using electron microscopy (EM) and gel electrophoresis] that network minicircles are joined by only one interlock; single interlocking can be accommodated without helix distortion. EM revealed that propidium diiodide supertwists all the network minicircles and thereby condenses the network into a much smaller size while maintaining its planarity. At high dye concentration the network is condensed to a size comparable to that found in vivo. Nevertheless, network minicircles bind less propidium than free minicircles, indicating that catenation into a network restricts the supercoiling of individual rings. These studies show that the mitochondrion of trypanosomatids may be a unique niche in nature where a covalently-closed circular DNA is not supercoiled. This absence of supercoiling may be a major factor in promoting the formation of the network.

Animals↗
PubMed1986

Cellular effects of a brain extract factor or factors inhibiting neuroblastoma cell growth in vitro.

The mechanism of inhibition of neuroblastoma cell proliferation in vitro by extract prepared from the brains of newborn mice was partially elucidated. Flow cytometric analysis of propidium iodide-stained cells showed that a significant increase occurred in the pre-G0/G1 cell population after exposure to the extract for 24 hr. Fluorescence microscopy indicated that the pre-G0/G1 cells had small "micronuclei" in association with their nucleus, whereas post-G2/M cells had enlarged propidium iodide-staining nuclei. 3H-thymidine incorporation was unchanged from that of control cells, in cells exposed to the extract for either 6 or 24 hr. No effect was seen on choline acetyl transferase activity or on morphology. These results suggest that the inhibitor blocks neuroblastoma cell proliferation either by inducing an uneven distribution of DNA at the time of cell division or by altering its packaging within the nucleus.

Animals↗
PubMed1993

Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations.

Boar spermatozoa loaded with the Ca2+ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged ("dead") cells. If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonate-mediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it. The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times.

Animals↗
PubMed1993

Induction of programmed cell death in a dorsal root ganglia X neuroblastoma cell line.

Growth factor-dependent neurons die when they are deprived of their specific growth factor. This "programmed" cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD.

Animals↗
PubMed1989

Cell cycle alterations and cell death in cyclophosphamide teratogenesis.

Litters of pregnant mice treated with cyclophosphamide (CP) exhibit malformations of the limbs ranging from oligodactyly to amelia. Previous studies have indicated that cell death occurs in limb buds shortly after maternal exposure. We have investigated the relationship of cell death, cell cycle perturbation, and embryo/fetal toxicity in the mouse using vital staining and flow cytometry (FCM). CP (20, 30, and 40 mg/kg) was investigated via intraperitoneal administration to Swiss-Webster mice on day 10 of gestation. At 4, 8, or 28 hours later, embryos were removed. Cell death was identified with Nile blue sulphate (NBS). Two embryos per litter were stained with NBS, and the remaining embryos were frozen at -70 degrees C prior to FCM analysis. After thawing, the forelimb buds were removed for the isolation of nuclei. Tissues were dissociated through a wire mesh followed by cytolysis with 0.1% nonidet P-40 in PBS with 0.5 mg/ml RNase. Nuclei were stained with the fluorescent nucleic acid probe propidium iodide and analyzed (10,000 nuclei per sample) for propidium iodide fluorescence by FCM. NBS revealed a dose-related increase in cell death by 8 hours after dosing. CP-induced cell death was greatest in areas of rapid cell proliferation (DNA synthesis). FCM analysis revealed retardation of progression through the S-phase of the cell cycle by 4 hours post-exposure at all doses. This retardation occurred earlier in S-phase with increasing dose and persisted through 8 hours. At 28 hours, cell cycle histograms were normal in the low-dose embryos, but remained perturbed in the intermediate- and high-dose embryos. On day 17 of gestation, the last group of dams was killed. A high incidence of fetal malformations, including limb defects, occurred at the 20 mg/kg dose, and fetal mortality was observed at 30 and 40 mg/kg. The pattern and magnitude of cell death correlated with cell cycle perturbation and fetal toxicity at term, suggesting a relationship between cell cycle perturbation, cell death, and malformations produced by CP.

Animals↗
PubMed1994

A cytotoxicity assay for tumor necrosis factor employing a multiwell fluorescence scanner.

Tumor necrosis factor (TNF) is an important mediator of many biological processes. This study sought to develop a sensitive bioassay allowing quantitative determination of TNF. The assay is based on uptake of the membrane impermeant dye, propidium iodide, by L929 cells when their viability is lost. L929 cells were plated on 96-well plates and cultured overnight in Eagle's minimum essential medium containing 10% horse serum. Propidium iodide (30 microM) was added, and fluorescence was monitored with a multiwell fluorescence scanner using 560-nm excitation and 645-nm emission filters. Human recombinant TNF caused a dose- and time-dependent increase in fluorescence, indicating onset of cell death. Cycloheximide (50 micrograms/ml) and actinomycin D (5 micrograms/ml) sensitized L929 cells to TNF-induced killing. By measuring cell killing early (12 h) and late (30 h), responses proportional to TNF concentration were obtained over a dynamic range of 1 to 5000 pg/ml. These findings serve as the basis for a simple TNF assay that is easy to perform and sensitive over a very broad range of TNF concentrations.

Animals↗
PubMed1998

Regional vulnerability to endogenous and exogenous oxidative stress in organotypic hippocampal culture.

Organotypic cultures of the brain provide a unique opportunity to directly examine the regional vulnerability of specific brain regions like the hippocampus. Two well-characterized models of oxidative stress were used to examine the regional vulnerability of the hippocampus. Endogenous oxidative stress was induced by blocking synthesis of the endogenous antioxidant, glutathione with buthionine sulfoximine (BSO). Exogenous oxidative stress was induced with paraquat, an intracellular generator of superoxide. Injury was measured by quantitative fluorescence microscopy using the vital dye propidium iodide. BSO caused dose- and time-dependent injury that took at more than 24 h to develop. Injury began in discrete patches in the culture. In any given culture, each patch increased in size and intensity as incubation continued. The pattern was not clearly correlated with neuronal anatomy and may demonstrate glial vulnerability. Injury caused by BSO could be prevented with the antioxidants trolox or the 21-aminosteroid U-83836E, both of which are vitamin E derivatives. Paraquat also caused dose- and time-dependent injury, but the CA1 region of the hippocampus was most vulnerable. The same pattern of selective CA1 injury was caused by brief exposures to high concentrations and by prolonged exposures to much lower concentrations. Under some conditions, paraquat injury was prevented by iron chelation with deferoxamine or by blockade of either NMDA or AMPA/ kainate glutamate receptors. During paraquat exposure, glutathione concentration in the cultures was reduced prior to onset of propidium staining. The observation that the hippocampus has a similar selective regional pattern of vulnerability to paraquat and ischemia suggests that their mechanisms of injury may be related.

Animals↗
PubMed1999

Connexin alpha1 and cell proliferation in the developing chick retina.

During the formation of the eye, high levels of connexin alpha1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin alpha1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2-4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin alpha1, to one eye of early chick embryos results in a rapid and significant reduction of alpha1 protein which lasts for 24-48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin alpha1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 +/- 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin alpha1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.

Animals↗
PubMed1994

The trolox analog, U-78517F, attenuates hypoxic injury in isolated cardiac myocytes.

Neonatal rat cardiac myocytes were subjected to 5 h of hypoxia (PO2 < 5 mmHg) in glucose-free, modified Tyrodes solution. Prior to hypoxic exposure, cells were pretreated for 90 min with media alone or containing the trolox analog, U-78517F, at 0.3, 1, or 3 microM. Hypoxic injury was characterized by a significant loss of sarcolemmal integrity [76 +/- 12% of the total cell fluorescence (mean +/- S.E., n = 14 cultures)] quantified by nuclear staining with the membrane-impermeant fluorophore propidium iodide. In addition, hypoxia also induced significant decreases in the intracellular content of creatine kinase (43 +/- 7%, n = 12), lactate dehydrogenase (25 +/- 5%, n = 8), adenosine 5'-triphosphate (49 +/- 12%, n = 6), and sulfhydryls (34 +/- 4%, n = 6). Sarcolemmal integrity was significantly preserved by pretreatment with 1 microM and 3 microM U-78517F which reduced propidium iodide staining below the hypoxic control value by 51 +/- 9% (n = 12) and 75 +/- 4% (n = 14, P < 0.01 v hypoxic control), respectively. Similar results were obtained in cells pretreated with alpha-tocopherol suggesting that these two compounds are equipotent at preserving the sarcolemmal integrity of cardiac myocytes exposed to hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

The mechanism for hexachlorocyclohexane-induced cytotoxicity and changes in intracellular Ca2+ homeostasis in cultured cerebellar granule neurons is different for the gamma- and delta-isomers.

The cytotoxic action of the delta- and gamma-isomers of hexachlorocyclohexane (HCH) as well as their ability to induce changes in intracellular Ca2+ homeostasis was studied in cultured rat cerebellar granule neurons. Changes in the free intracellular Ca2+ concentration ([Ca2+]i) related to Ca2+ influx and release from intracellular stores were investigated using, in addition to the physiological incubation medium, media without added Ca2+ (nominally Ca2+ free) or containing verapamil or dantrolene, drugs which block influx through voltage-gated Ca2+ channels and release from intracellular stores, respectively. Cytotoxicity was monitored using leakage of lactate dehydrogenase (LDH) and staining of damaged cells with either trypan blue or propidium iodide. In the latter case, when fluorescence microscopy was employed, undamaged cells were visualized using the vital stain calcein acetoxymethyl ester (calcein-AM). The delta-isomer of HCH was found to be more potent and active as a cytotoxic agent than the gamma-isomer (lindane) and it was demonstrated that the moderate cytotoxic action of lindane could be ameliorated by dantrolene which on the other hand had no effect on cytotoxicity induced by delta-HCH. It was noticed that pronounced (50% increase in fluorescence) staining with propidium iodide was associated with only a marginal (20%) LDH leakage and staining of only a few (<25%) cells with trypan blue. Both delta- and gamma-HCH induced an increase in [Ca2+]i which was most pronounced in case of delta-HCH. Analysis of the different mechanisms governing the increase in [Ca2+li using dantrolene, verapamil, and Ca2+-free medium revealed distinct differences between the two isomers with regard to the Ca2+ pools affected. Both isomers stimulated Ca2+ influx through voltage-gated Ca2+ channels but only the gamma-isomer affected a Ca2+-dependent, dantrolene-sensitive pool. On the other hand, delta-HCH affected mainly a Ca2+-independent dantrolene-insensitive pool. The finding that delta-HCH was more toxic than lindane may be correlated to the differences between the isomers with regard to the action on the different Ca2+ pools. Thus, delta-HCH affected primarily the dantrolene-insensitive Ca2+ pools which partly reflect influx. On the contrary, lindane had little effect on these Ca2+ pools but affected primarily dantrolene-sensitive intracellular Ca2+ stores. This may suggest that delta-HCH may exert its cytotoxic action by stimulating a large influx of Ca2+ possibly leading to release of Ca2+ from dantrolene-insensitive stores. In contrast, the toxic action of lindane may be primarily related to release of Ca2+ from the dantrolene-sensitive stores.

Animals↗
PubMed1984

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry.

A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%-30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.

Animals↗
PubMed1987

Antibody labelling and flow cytometric analysis of metaphase chromosomes reveals two discrete structural forms.

Metaphase chromosomes from cultured Chinese Hamster Ovary cells were labelled in suspension with a monoclonal antibody to histone 2B, counterstained with propidium iodide (PI) and analysed by flow cytometry. Contour plots of antibody binding (FITC fluorescence) against DNA content (PI fluorescence) revealed two discrete forms of each individual chromosome, showing high and low levels of antibody binding respectively. The two types of chromosome were easily distinguishable by immunofluorescence microscopy. The distribution of individual chromosomes between the two populations was related to chromosome size, with larger chromosomes predominating in the low-labelling population and vice versa. Variations in ionic strength, pH, divalent cation concentration or preparation procedure influenced the absolute level of antibody binding but did not eliminate the two populations. In contrast, preincubation with intercalating dyes, such as ethidium and propidium, inhibited antibody binding and generated a single, low-labelling population. Preliminary evidence suggests that in vivo changes in chromosome structure can affect the distribution of chromosomes between the two populations. Prolonged exposure of cells to Colcemid prior to chromosome isolation, a procedure known to increase chromosome condensation, resulted in a progressive shift into the low-labelling population. Our results suggest that chromosomes undergo a relatively rapid transition from the high-labelling to the low-labelling form during the prometaphase-metaphase stage of mitosis. The timing of this transition appears to be size dependent, with the larger chromosomes preceding the smaller. The transition may represent a change in chromosome condensation.

Animals↗
PubMed1977

Extended application of flow microfluorometry by means of dual laser excitation.

A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region, DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specificity combined with a great stability under UV illumination. By separating two focussed laser beams at their intersecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.

DNA↗
PubMed1981

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization.

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.

Animals↗
PubMed1992

Division and migration of satellite glia in the embryonic rat superior cervical ganglion.

While distinct precursors committed to a neuronal or glial cell fate are generated from neural crest cells early in peripheral gangliogenesis, little is known about the subsequent generation and maturation of young satellite glia from restricted glial precursor cells. To examine the division and migration of glial precursor cells and their satellite cell progeny, morphological, immunocytochemical and culture techniques were applied to the developing rat superior cervical ganglion. At embryonic day (E)18.5, numerous clusters of nonneuronal cells appeared transiently in the ganglion. Individual cells with a similar morphology were present in E16.5 ganglia, and are likely to represent the precursor cells which generate these clusters. The clustered cells were distinguishable from neighbouring neurons as well as from endothelial cells and fibroblasts. Morphologically similar cells were present in nerve bundles at E18.5 and surrounding principal neurons and nerve bundles in the adult ganglion. Double-label studies of the E18.5 ganglion with tyrosine hydroxylase to identify noradrenergic neurons and propidium iodide counterstaining to visualize all cell nuclei revealed that the cells in clusters stained with propidium iodide but lacked tyrosine hydroxylase immunoreactivity. To determine if cell clusters arose from division, bromodeoxy-uridine, a thymidine analogue, was administered to pregnant mothers between E16.5-E18.5, and ganglionic cells examined at E18.5 both in vivo and in vitro. Numerous non-neuronal cells divided during this period in situ and composed portions of clusters. When dissociated, superior cervical ganglion satellite glia reacted with an NGF-receptor antibody (MAb 217c) and possessed a flattened shape, in contrast to bipolar Schwann cells. Over half of the 217c-immunoreactive glia at E18.5 had incorporated bromodeoxyuridine during E16.5-18.5 in vivo. At birth, non-neuronal cells were no longer grouped in clusters, but were associated with neuronal cell bodies and processes. These findings suggest that, between E16.5-E18.5, glial precursors divide rapidly to form clusters, and that, after the peak of neurogenesis, daughter cells migrate within the ganglion to associate with nerve cell bodies and processes where proliferation continues at a slower rate. Distinct cellular and molecular interactions are likely to trigger the initial rapid division of glial precursors, initiate their migration and association with neuron cell bodies, and control their subsequent slower division.

Animals↗
PubMed1995

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells.

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to investigate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidium-iodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.

Apoptosis↗