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PubMed2002

Synthetic neuromelanin is toxic to dopaminergic cell cultures.

In the present study, primary cultures of mesencephalic dopaminergic cells were exposed to synthetic dopamine neuromelanin (NM) for 48 hrs at concentrations of 0, 1, 10, 20, 50 and 100 microg NM/ml medium. Differently prepared synthetic NM with or without incorporated iron and NM oxidatively damaged by hydrogen peroxide were used. All NMs affected cellular structures e.g. as swelling of neural processes, rounding of cells, and occasional inclusion of neuromelanin particles. Cell numbers were uniformly and dose dependently reduced. Exposure to MPP(+) and ferric iron led to cytotoxic changes which could be further aggravated by oxidatively damaged NM, suggesting cytotoxicity of soluble compounds of NM in predamaged neurons.

1-Methyl-4-phenylpyridinium↗

PubMed2003

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers.

Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 microm thick. Immunolabeling also showed that callose (beta-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers.

Cell Wall↗

PubMed2006

Propionic acid induces cytoskeletal alterations in cultured astrocytes from rat cerebral cortex.

Severe neurological symptoms, cerebral edema, and atrophy are common features of the inherited metabolic disorder propionic acidemia. However, the pathomechanisms involved in the neuropathology of this disease are not well established. In this study, we investigate the effects of propionic acid (PA), a metabolite accumulating in this disorder, on cytoskeletal reorganization, on cell viability, and on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes from cerebral cortex of neonatal rats. We observed that the astrocytes changed their usual polygonal morphology when exposed to 5 mM PA for 72 h, leading to the appearance of fusiform or process-bearing cells, without elicit cell death. We also noticed that after 72 h treatment with 5 mM PA cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments and disorganized actin stress fibers, as revealed by immunocytochemistry. In addition, the morphological alterations were accompanied by increased in vitro 32P incorporation into GFAP and vimentin recovered into the high-salt Triton-insoluble cytoskeletal fraction. In conclusion, our results indicate that PA lead to cytoskeletal reorganization and to increased in vitro phosphorylation of Triton-insoluble GFAP and vimentin. On the basis of our results we could suppose that Triton-insoluble GFAP and vimentin hyperphosphorylation could be implicated in the reorganization of cellular structure and these findings could be involved in the brain damage characteristic of propionic acidemia patients.

Actin Cytoskeleton↗

PubMed2004

Constitutive expression of pea Lhcb 1-2 in tobacco affects plant development, morphology and photosynthetic capacity.

Lhcb1-2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1-2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 degrees C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 degrees C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1-2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

Amino Acid Sequence↗

PubMed1998

Acute cytotoxicity testing with cultured human lung and dermal cells.

An extensive in vitro study with cultured cells was conducted to test the basal cytotoxicity theory. This theory suggests that most chemical injury, at least in vitro, is a manifestation of one or more insults to the basic cellular structures and functions common to mammalian cells. This accounts for the similarity of results in multilaboratory studies. Human fetal lung fibroblasts (HFL1), and human skin fibroblasts (WS1, Detroit551) were studied in culture to evaluate their potential to screen for cytotoxicity. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Twenty-nine chemicals were tested with each cell line and the cytotoxicity data compared to rodent and human lethal concentrations. The data suggest that the experimental IC50 values are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD50s. In addition, lung and skin fibroblasts revealed no significant differences among the three cell lines. The results support the conclusion that finite cell lines of human origin have the potential for screening chemicals for human toxicity. In combination with previously published reports, the data suggest that a basal cytotoxic phenomenon may explain the similarity of results among different human cell lines.

Cells, Cultured↗

PubMed1993

Laser-induced stimulation of thromboxane B2 synthesis in human blood platelets: role of superoxide radicals.

Exposure of platelet-rich plasma to laser radiation at 3.5 W for 30 seconds reduced the threshold concentrations of adenosine diphosphate and L-epinephrine needed from complete platelet aggregation by 20% to 60% and by 30% to 50%, respectively. The irradiation of platelet-rich plasma with laser also increased the basal level of thromboxane A2 from < 0.5 pmol/10(8) platelets for each second of exposure. In contrast, the exposure of gel-filtered platelets to laser produced no effect on the prostanoid formation. However, the addition of laser-exposed platelet-free plasma to gel-filtered platelets stimulated the synthesis of thromboxane A2 in these cells. The effect of laser was completely blocked by adding superoxide dismutase or catalase to the platelet-rich plasma, indicating that the radiation-induced stimulation of thromboxane A2 production was mediated through the generation of superoxide radicals. Electron microscopic studies indicated that the laser-induced stimulation of thromboxane A2 production in platelet can occur without any noticeable damage in the cellular structure.

Blood Platelets↗

PubMed1984

Quantitative analysis of endothelial mosaic pattern changes in anterior keratoconus.

The corneal endothelia of 21 eyes with anterior keratoconus and 15 eyes of age-matched controls were investigated with a specular microscope. Of the 21 eyes, 15 had definite keratoconus while six apparently normal fellow eyes were designated as latent. The corneal endothelial photographs were subjected to a computerized digitizer analysis of the area and shape of individual cells. The endothelium in keratoconus showed a significant increase in the extent of polymegethism (the coefficient of variation in cell area; 0.36 +/- 0.07) as compared with controls (0.24 +/- 0.03), with the mean cell area staying within normal limits. The relative frequency of hexagonal cells in keratoconus (50.5 +/- 5.7%) was significantly lower than that of controls (70.6 +/- 5.5%). Additionally, an increase of various cell shapes was noted, indicating that there was also a significant increase in cellular pleomorphism. In the latent group, alterations in cellular structure could not be detected by analysis of cell area when compared to controls. However, when these eyes were subjected to cell shape analysis and compared to fellow keratoconic eyes, both eyes showed similar pleomorphic characteristics. These results suggest that in the fellow eye in unilateral keratoconus, even when there are no ocular signs and symptoms, diagnosis can be made early with specular microscopy and computerized cell shape analysis.

Adolescent↗

PubMed1977

Labeling of membranes from erythrocytes and corn with fluorescamine.

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.

Cell Fractionation↗

PubMed1985

A rapid isolation procedure of plasma membranes from human neutrophils using self-generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes.

In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self-generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils.

5'-Nucleotidase↗

PubMed1989

Isolation of myelin bodies from the kidney cortex of gentamicin-treated rats.

Myelin bodies have been isolated from the kidney cortex of gentamicin-treated rats (100 mg gentamicin sulfate/kg body weight i.p. twice daily for 3 days) by a simple procedure involving differential centrifugation followed by equilibrium density centrifugation on a discontinuous sucrose gradient. Electron microscopy and assay of acid phosphatase suggest that the myelin bodies were obtained in virtually quantitative yield, essentially uncontaminated by other cellular structures and relatively intact. The method developed here may also prove applicable for the isolation of myelin bodies arising in connection with other drug treatments and may provide information on a number of toxic side-effects of clinical importance.

Acid Phosphatase↗

PubMed1975

Relationships between non-extractable DNA and the bacterial growth cycle.

Escherichia coli DNA has been fractionated into extractable and non-extractable DNA after deproteinization of detergent-lysed cell preparations with chloroform-isoamyl alcohol. The former was extracted with dilut buffered saline whereas the latter remained in the interphase layer associated with residual cellular debris from which almost 40 percent could be released by incubating with pronase. About 20-25 percent more amino acid residues were bound to the pronase-released DNA than to the extractable DNA, but the relative distribution of the residues in the two DNA samples was virtually identical. The specific activities and the relative amounts of denser (1.709g-cm-3) and lighter (on the surface of CsCl gradients) DNA fractions from E. coli, grown in the presence of labeled thymidine, indicated that these two corresponded to extractable and non-extractable DNA, respectively. The relative amounts of the two fractions varied with the growth phase primarily as a function of the growth rate. Age and metabolic state of cells in the culture or those used as inocula could modify this relative distribution. When growth rate was maximal, the ratio of the two remained at about 1. During lag phase when no appreciable net synthesis of DNA could be detected, there was a rapid and preferential incorporation of labeled thymidine into non-extractable DNA. A disproportionate increase in the fraction of the total DNA which was extractable, was also abserved but only when stationary phase cultures were used as inocula. Complete equilibration of the label in the two DNA fractions was attained only after cultures had reached mid-log phase of growth. Similar results were obtained when prelabeled cells were used. These data have been interpreted as suggesting that the rate of cell growth and DNA synthesis are related to the number or size of sites of attachment of DNA to some cellular structure. Newly synthesized DNA would be attached to different sites on this structure and initiation of DNA replication in lag phase would require reorientation of the two kinds of DNA. Small peptides which are firmly bound to the DNA and which vary quantitatively with the rate of DNA synthesis could perhaps be involved in the attachment to the sites.

Amino Acids↗

PubMed1987

Membrane transport of mitoxantrone by L1210 leukemia cells.

Transport of radiolabeled mitoxantrone, a new antineoplastic agent, was studied using cultured mouse L1210 leukemia cells. The initial velocity of influx remained linear for about 90 sec and was 110 pmoles/10(6) cells measured at 60 sec. The steady-state accumulation of about 480 pmoles/10(6) cells was not reached until 30 min. The unidirectional drug influx was linear from 0 to 1000 microM extracellular drug concentration. The initial uptake was relatively temperature independent between 37 degrees and 27 degrees, but accumulation at steady state was 17% lower at 27 degrees. None of six metabolic inhibitors had an appreciable effect on initial uptake. Efflux was initially exponential with a half-life of 2.8 min; this efflux and the residual drug concentration plateau were not affected by KCN or verapamil. Under steady-state conditions, about 86% of the cell-associated label was contained in parent drug and the remainder in an unidentified metabolite. These studies indicate that the mechanism of mitoxantrone uptake is passive diffusion. The efflux is not energy requiring, but there is considerable tight binding of the drug to cellular structures.

Animals↗

PubMed1990

Lipid composition and fluidity of liver mitochondria, microsomes and plasma membrane of rats with chronic dietary iron overload.

The effect of chronic dietary iron overload on the lipid composition and physical state of rat liver mitochondria, microsomes and plasma membranes was investigated. After 9 weeks of iron treatment, a significant decrease of polyunsaturated and a parallel increase of saturated fatty acids was observed in mitochondrial and plasma membrane phospholipids. By contrast, no appreciable modification of the fatty acid composition of microsomal membranes was detected. The cholesterol/phospholipid molar ratio as well as the lipid/protein ratio, did not reveal any significant difference in any of the fractions studies. Finally, no change in the molecular order of the various membranes, as assessed by electron spin resonance spectrometry, was observed following iron intoxication. These data indicate that, although in vivo chronic hepatic iron overload induces a modification of fatty acid profile in cellular structures consistent with the in vivo occurrence of lipid peroxidation, these changes do not bring about appreciable modifications of other physico-chemical parameters relevant to membrane integrity and cell viability.

Animals↗

PubMed1993

Unidirectional membrane uptake of the ether lipid antineoplastic agent edelfosine by L1210 cells.

We have studied the cellular uptake of edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH3), a membrane active anticancer drug of the ether lipid family, by L1210 murine leukemia cells. Initial unidirectional linear uptake velocity was 1.1 nmol/min per 2 x 10(6) cells; at about 30 min it reached a steady-state phase of accumulation of approximately 5 nmol/2 x 10(6) cells. Concentration studies indicated no saturation kinetics from 0 to 40 microM. Studies with metabolic inhibitors displayed no energy dependence. There was no effect of chloroquine, monensin or cytochalasin B, which are known inhibitors of endocytosis. The inhibitory effect of lower temperature on uptake was moderate in extent and compatible with passive diffusion. There was no efflux of drug from preloaded cells which indicates intense binding of incorporated drug to cells. In human serum, edelfosine bound to several protein components, primarily high density lipoprotein and albumin, and this may explain why cellular uptake was slowed considerably by the presence of serum or albumin in the incubation medium. We conclude that the lipophilic ether lipid derivative edelfosine is taken up by passive diffusion by the L1210 cell. It is tightly bound to cellular structures, probably by insertion into the membrane lipid bilayer.

Animals↗

PubMed1993

Effects of chronic ethanol administration on acetylcholinesterase activity in the somatosensory cortex and basal forebrain of the rat.

A chronic diet of ethanol has detrimental effects on the cholinergic system in adult humans and rats. This study examined the effects of chronic exposure to dietary ethanol on the anatomical organization of true acetylcholinesterase (AChE) active elements in rat cerebral cortex. We focused on the somatosensory cortex because of its highly organized chemical and cellular structure. Following 42 days of exposure to an ethanol diet (6.7% v/v), there were marked changes in the cortical plexus of AChE-positive fibers. The AChE-positive plexus in ethanol-treated rats was reduced in all cortical layers, in comparison to age-matched pair-fed control and chow-fed rats. The most marked reduction was evident in layers II/III, IV, and VIa. Moreover, the density of AChE-positive cell bodies was significantly reduced in the cortices of ethanol-fed rats, particularly in the deep laminae. These alterations in the chemoarchitecture of somatosensory cortex occurred in the absence of changes in the cytoarchitectonic organization of neocortex. There was no detectable ethanol-induced change in the density of Cresyl violet-stained neurons either in the horizontal limb of the diagonal band of Broca or in the nucleus basalis. The density of AChE-positive neurons in the nucleus basalis, however, was significantly lower in ethanol-fed rats than in controls. Thus, it appears that a mere 6 weeks of ethanol exposure is sufficient to alter the cholinergic innervation of the cerebral cortex. These cortical alterations occur despite the lack of an ethanol-induced death of neurons in the basal forebrain. Such changes may contribute to the memory loss associated with alcohol dementia.

Acetylcholinesterase↗

PubMed1983

Bone marrow cell chromosomal aberrations and styrene biotransformation in mice given styrene on a repeated oral schedule.

Styrene's capacity to induce chromosomal aberrations was studied in bone marrow cells of CD1 male mice. No mutagenic effect could be detected after either a 4-day treatment course with daily oral doses of 500 mg/kg or a 70-day course with daily oral doses of 200 mg/kg. Urinary elimination of styrene metabolites related to styrene-7,8-oxide formation (i.e. phenylethylene glycol, mandelic acid, benzoic acid, phenylglyoxylic acid and total mercapturic acids) was quantitatively evaluated in the group of mice given the 200 mg/kg dose. In parallel, kinetic studies were made on styrene and styrene-7,8-oxide blood concentrations in the same group of animals. These determinations were carried out on days 1 and 70 of treatment by spectrophotometric, gas chromatographic and mass fragmentographic procedures. Not even nanograms of styrene-7,8-oxide were found in the blood of styrene-treated mice. This suggests that the metabolite does not migrate from the cellular compartment where it is formed being immediately metabolized or irreversibly bound to cellular structures. This observation could well explain the lack of mutagenic effects observed.

Administration, Oral↗

PubMed1985

A time-lapse, image digitization videomicroscope system based on a mini computer with large peripheral memory.

We describe a time-lapse image digitization videomicroscope system that uses a mini computer as the main processing unit in conjunction with a large peripheral memory for storing multiple digitized images. The advantage of the system lies in its ability to facilitate acquisition and analysis of data relative to size, location and optical density of cells and cellular structure using elementary image processing techniques. This system can also potentially control microscope stage movement, enabling acquisition of larger amounts of data per time-lapse experiment.

Animals↗

PubMed1988

Formation of the transverse nerve in moth embryos. I. A scaffold of nonneuronal cells prefigures the nerve.

We have studied the embryonic development of the transverse nerve (TN), an unpaired segmental nerve of the moth Manduca sexta. Two identified motor neurons and 16 identified neuroendocrine neurons project axons within the larval TN; therefore, the TN is both a peripheral nerve and a neurohaemal organ. At 33% of embryogenesis, and prior to the arrival of any neuronal growth cones, the position, shape, and trajectory of the TN are anticipated by two groups of nonneuronal cells that we call the strap and the bridge. At this time the strap and the bridge together consist of approximately 100 cells, all of which express a cell surface epitope recognized by the monoclonal antibody TN-1. As development proceeds, both the number of nonneuronal cells within the strap and the bridge and the fraction that expresses the TN-1 antigen(s) decrease. Moreover, individual cells within the strap become morphologically identifiable before the arrival of the neuronal growth cones. Most of the axons that project to the TN also express the TN-1 antigen(s) during their period of outgrowth. The two motor neuron growth cones are the first to reach the environment of the strap and the bridge, doing so at approximately 37%; having encountered these cellular structures, the growth cones restrict their navigation to this preexisting scaffolding, until they reach their muscle target. The neuroendocrine growth cones arrive later and also grow within the confines of the strap and the bridge (J.N. Carr and P.H. Taghert, 1988, Dev. Biol, 130, 500-512). In this first paper we describe the development of the strap and the bridge, and the interactions of the motor neuron growth cones with these structures. The observations are novel in documenting the extent and precision to which a peripheral nerve pathway is prefigured by a contiguous assemblage of nonneuronal cells.

Animals↗