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At least 217 records · Page 12Linked to original sources
PubMed2000

Subcellular localization of presenilins: association with a unique membrane pool in cultured cells.

We have investigated the subcellular distribution of presenilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol-based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, Golgi SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast to PS1, PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.

Alzheimer Disease↗
PubMed2001

Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.

Animals↗
PubMed1995

A physiologically based pharmacokinetic model for fluoride uptake by bone.

A sex-specific, physiologically based pharmacokinetic (pbpk) model has been developed to describe the absorption, distribution, and elimination of fluorides in rats and humans. Growth curves generated by plotting mean body weights (kg) against age (weeks or years) are included in the simulation model to allow the integration of chronic fluoride exposure from birth to old age. The model incorporates age and body weight dependence of the physiological processes that control the uptake of fluoride by bone and the elimination of fluoride by the kidneys. Six compartments make up the model. These are lung, liver, kidney, bone, and slowly and rapidly perfused compartments. The model also includes two bone subcompartments: a small, flow-limited, rapidly exchangeable surface bone compartment and a bulk virtually nonexchangeable inner bone compartment. The inner bone compartment contains nearly all of the whole body content of fluoride, which, in the longer time frame, may be mobilized through the process of bone modeling and remodeling. The model has been validated by comparing the model predictions with experimental data gathered in rats and humans after drinking water and dietary ingestion of fluoride. This physiological model description of absorption, distribution, and elimination of fluoride from the body permits the analysis of the combined effect of ingesting and inhaling fluorides on the target organ, bone. Estimates of fluoride concentrations in bone are calculated and related to chronic fluoride toxicity. The model is thus useful for predicting some of the long-term metabolic features and tissue concentrations of fluoride that may be of value in understanding positive or negative effects of fluoride on human health. In addition, the pbpk model provides a basis for across-species extrapolation of the effective fluoride dose at the target tissue, bone, in the assessment of risk from different exposure conditions.

Adult↗
PubMed2000

Protein degradation in mitochondria.

The biogenesis of mitochondria and the maintenance of mitochondrial functions depends on an autonomous proteolytic system in the organelle which is highly conserved throughout evolution. Components of this system include processing peptidases and ATP-dependent proteases, as well as molecular chaperone proteins and protein complexes with apparently regulatory functions. While processing peptidases mediate maturation of nuclear-encoded mitochondrial preproteins, quality control within various subcompartments of mitochondria is ensured by ATP-dependent proteases which selectively remove non-assembled or misfolded polypeptides. Moreover; these proteases appear to control the activity- or steady-state levels of specific regulatory proteins and thereby ensure mitochondrial genome integrity, gene expression and protein assembly.

Adenosine Triphosphate↗
PubMed1995

Postnatal development of cytochrome P4501A1 and 2B1 in rat lung and liver: effect of aged and diluted sidestream cigarette smoke.

Earlier studies have shown that both mainstream and sidestream cigarette smoke increase the activities of cytochrome P4501A1 and 2E1 in the lungs of adult animals; however, little information is available on the influence of ambient levels of sidestream cigarette smoke on cytochrome P450 monooxygenase activity in the developing lung. The present studies were conducted to define the developmental profiles of cytochrome P450 monooxygenases 1A1 and 2B1 in rat lung and liver and to assess the effects of aged and diluted sidestream cigarette smoke (ADSS) on the developmental profile of these two enzymes. Accordingly, pulmonary and hepatic microsomal P4501A1 and 2B1 activities were determined by measuring ethoxy- and pentoxyresorufin-O-delakylase (EROD and PROD, respectively) activity in animals exposed to filtered air or ADSS from birth to 7, 14, 21, 50, and 100 days of age. Pulmonary P4501A1 activity in control rats was not detected until 14 days of age. Activities increased threefold between 14 and 21 days of age and remained unchanged to 100 days of age. In animals exposed to ADSS from birth, pulmonary EROD activities were detected as early as 7 days postnatal and were elevated three- to fourfold above control at all other ages examined. Hepatic EROD activities were unaltered by ADSS exposure. Short-term (4-day) ADSS exposure was as effective in upregulating pulmonary microsomal EROD activities as 100-day exposures. Induction of pulmonary EROD activities and the associated increases in mRNA levels were dependent upon the particulate fraction. Stimulation of EROD activities in major and minor daughter subcompartments was three- to fourfold higher in ADSS-exposed animals compared to controls, while there was no induction in the trachea and less than a twofold increase in the parenchyma. Pulmonary PROD activities developed more slowly than EROD and did not reach adult levels until Day 50. ADSS did not alter pulmonary or hepatic PROD activities. These studies show that P4501A1 and 2B1 develop at different rates in rat lung and liver and that exposure to ADSS markedly increases P4501A1 activities in the lung at all ages examined.

Aging↗
PubMed1998

Cytochrome P450 2E1 in rat tracheobronchial airways: response to ozone exposure.

The distal trachea and centriacinus of the lung are primary sites of acute injury during short-term ozone exposure; long-term exposure yields cells in these areas that are resistant to high doses of oxidant gases. Epithelial cells located in primary sites for ozone injury are also targets for chemicals that undergo cytochrome P450 (CYP)-dependent activation. These studies were designed to compare the effects of ozone exposure on pulmonary CYP2E1 in susceptible and nonsusceptible sites within the airway tree of lung. CYP2E1 activity was measured in well-defined regions of airways using p-nitrophenol, a CYP2E1-selective substrate, with HPLC/ electrochemical detection of the p-nitrocatechol. Alterations in distribution of CYP2E1 were evaluated by immunohistochemistry. CYP2E1 activities were highest in the distal bronchioles and minor daughter airways but were much lower in the lobar bronchi/ major daughter airways and trachea. Immediately after short-term ozone exposures (8 h, 1 ppm), CYP2E1 activities were elevated only in the lobar bronchi/major daughter airways. These activities remained above the filtered air control at 1 day but returned to control levels by 2 days. Immunohistochemical assessment of CYP2E1 protein in ozone and filtered air-exposed animals was consistent with the activity measurements. After long-term ozone exposures (90 days, 1 ppm), CYP2E1 activities were decreased in the major and minor daughter airways. These studies indicate that CYP2E1 activities vary substantially by airway level. However, ozone exposure only results in minimal alterations in activity with varying concentration of ozone, length of exposure, and time after exposure in any of the lung subcompartments examined.

Animals↗
PubMed2006

Chaperones in preventing protein denaturation in living cells and protecting against cellular stress.

A variety of cellular internal and external stress conditions can be classified as proteotoxic stresses. Proteotoxic stresses can be defined as stresses that increase the fraction of proteins that are in an unfolded state, thereby enhancing the probability of the formation of intracellular aggregates. These aggregates, if not disposed, can lead to cell death. In response to the appearance of damaged proteins, cells induce the expression of heat shock proteins. These can function as molecular chaperones to prevent protein aggregation and to keep proteins in a state competent for either refolding or degradation. Most knowledge of the function and regulation (by co-factors) of individual heat shock proteins comes from cell free studies on refolding of heat- or chemically denatured, purified proteins. Unlike the experimental situation in a test tube, cells contain multiple chaperones and co-factors often moving in and out different subcompartments that contain a variety of protein substrates at different folding states. Also, within cells folding competes with the degradative machinery. In this chapter, an overview will be provided on how the main cytosolic/nuclear chaperone Hsp70 is regulated, what is known about its interaction with other main cytosolic/nuclear chaperone families (Hsp27, Hsp90, and Hsp110), and how it may function as a molecular chaperone in living mammalian cells to protect against proteotoxic stresses.

Animals↗
PubMed1984

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat. Morphological and cytochemical studies.

The effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 microM, was studied on the Golgi apparatus of absorptive cells in the small intestine of the rat. At 2-3 min after treatment most of the Golgi stacks exhibited dilated cisternae. At 4-5 min stacked cisternae were absent; they were replaced by groups of smooth-surfaced vacuoles. Dilatation and vacuolization occurred in the entire stacks without preferential effect on any particular Golgi subcompartment. Monensin did not influence the cytochemical Golgi reaction of thiamine pyrophosphatase and acid phosphatase. The characteristic staining pattern of these two enzymes in all Golgi cisternae of absorptive cells in the proximal small intestine, and the reactivity restricted to trans cisternae in distal segments of the small intestine, were unchanged after treatment with monensin. In the distal small intestine, the cytochemical pattern allowed the monensin-induced vacuoles to be attributed to the former cis- or trans-Golgi face. Further, the cytochemical results demonstrate that vacuolization is not restricted to the stacked cisternae, but includes the trans-most cisterna. The latter, usually located at some distance from the Golgi stacks, has been defined as belonging to the GERL system in several types of cells. The clear response to monensin, an agent that selectively affects the Golgi apparatus, indicates common properties between trans-most and stacked Golgi cisternae.

Acid Phosphatase↗
PubMed1991

Steroid receptor profile and receptor stability in subfractions of human prostatic tissues. Critical aspects on microassays.

Androgen (AR), progesterone (PR), and estrogen (ER) receptor contents in cytosol and salt-extractable nuclear subcompartments from 6 normal, 39 benign hyperplastic (BPH), and 7 malignant prostatic tissue specimens were analyzed by radioligand-binding assay techniques. In addition, the temperature stability of AR and PR was measured in another three BPH specimens. Five punch-needle biopsy samples from prostate cancers were also analyzed for AR and PR content. All receptor data were calculated from saturation analyses. The highest AR content was found in the cytosol and nucleic from malignant prostatic tissues. The highest PR concentrations were found in BPH cytosol, whereas nuclei of all types of tissues were negative with regard to this receptor. Markedly lower concentrations of ER were found in cytosol and nuclei from BPH as compared with malignant and normal tissues. PR was the most temperature-stable receptor; a marked receptor loss at room temperature was not registered until after 12 h. AR was stable for 4-5 h in cytosol and for 8-9 h in nuclei. Needle-biopsy specimens from prostate cancer showed highly variable and confusing results for AR and PR content, indicating that microassay studies using biochemical techniques on small tissue samples are unreliable and should not be recommended.

Biopsy↗
PubMed1988

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes.

Binding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternae of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.

Animals↗
PubMed1989

Ultrastructural identification of sulphated glycoconjugates in the Golgi apparatus in human colonic absorptive cells.

The subcellular localization of sulphated glycoconjugates was determined at the ultrastructural level by using the high iron diamine (HID) technique for sulphate groups in the absorptive cells of human colonic mucosa. Stained material was observed on the apical plasma membrane, in intracytoplasmic vesicles and in the Golgi complex. In this organelle, the last two or three cisternae of the trans side and the trans-Golgi network (TGN) were labelled, as well as a variable number of coated and noncoated vesicles facing the trans side and surrounding trans-Golgi network. These findings point to the trans side of the Golgi apparatus and trans-Golgi network as the subcompartments functionally involved in the sulphation of glycoconjugates.

Chlorides↗
PubMed1993

Targeting anticancer drugs to the brain: II. Physiological pharmacokinetic model of oxantrazole following intraarterial administration to rat glioma-2 (RG-2) bearing rats.

The disposition of the anticancer drug oxantrazole (OX) was characterized in rats bearing the rat glioma-2 (RG-2) brain tumor. Following intraarterial administration of 3 mg/kg of OX, serial sacrifices were completed from 5 min to 5 hr after administration. Blood and tissue samples collected at the time of sacrifice were processed and measured for OX concentrations by HPLC. The kidney had the greatest affinity for OX with the Cmax being 40.6 micrograms/ml at 15 min after administration. OX concentrations in brain tumor were higher than in normal right and left brain hemispheres, and consistent with enhanced drug blood-tumor barrier (BTB) permeability seen in experimental models for brain tumors. Observed heart, liver, lung, and spleen OX concentrations were similar, ranging from approximately 2 micrograms/ml to 20 micrograms/ml. A unique technique was used to develop a global physiological pharmacokinetic model for OX. A hybrid or forcing function method was used to estimate individual tissue compartment biochemical parameters (i.e., partition and mass transfer coefficients). A log likelihood optimization scheme was used to determine the best model structure and parameter sets for each tissue. Most tissues required a 3-subcompartment structure to adequately describe the observed data. The global model was then reconstructed with an arterial blood and rest of body compartments that provided predicted OX concentrations in agreement with the data. The model development strategy provides a systematic approach to physiological pharmacokinetic model development.

Animals↗
PubMed1983

Glial membrane specializations and the compartmentalization of the lamina ganglionaris of the housefly compound eye.

Membrane specializations in the lamina ganglionaris of the housefly are investigated using conventional thin-section EM, freeze-fracture replication and the diffusion of colloidal lanthanum. All glial cells in the lamina are coupled by gap junctions. Desmosomes also link all glia except the epithelial glia. Extensive glia-glial and glia-neuronal septate junctions are present in the pseudocartridge zone and nuclear layer. Septate junctions in the nuclear layer intermingle with bands of interglial and glia-neuronal tight junctions. Tight junctions are also found between satellite and epithelial glia at the border of the nuclear and plexiform layers, between adjacent epithelial glial cells in the plexiform layer, between epithelial and marginal glia at the proximal boundary of the optic neuropil, between marginal glial cells, and between marginal glia and axons. Colloidal lanthanum, introduced through an incision in the cornea, penetrates the retina but is occluded from the neuropil by septate junctions in the pseudocartridge zone. The disposition of tight and septate junctions is described in relation to the compartmentalization of the lamina. Two major compartments are delineated. The first represents the nuclear layer and contains the cell bodies of second-order visual neurons (monopolar neurons). The second compartment constitutes the plexiform layer of the lamina. Within the plexiform layer, each optic cartridge is partitioned into a separate subcompartment. Also, tracheoles and axons of long visual fibres are isolated from the optic cartridges by glial tight junctions. Morphological evidence for compartmentalization is correlated with previously established electrical properties of the insect lamina ganglionaris.

Animals↗
PubMed1985

Energy metabolism and transduction in smooth muscle.

Early investigations into the nature of the coupling between energy transduction and metabolism in smooth muscle, particularly from the laboratories of Bülbring and Lundholm, suggested that specific metabolic pathways could independently supply energy for ion transport and actin-myosin interactions. Subsequent work has solidified the concept that oxidative phosphorylation is specifically coupled to tension generation and maintenance, whereas, aerobic glycolysis is not only a vital characteristic of smooth muscle metabolism, but also is likely to be independently coupled to Na-K transport at the plasmalemma. The independence of oxidative and glycolytic metabolism is reflected as a compartmentation of carbohydrate metabolism in the porcine carotid artery. The coupling of these independent metabolic pathways with specific energy utilizing processes, indicates a means by which energy production and transduction can be closely and efficiently regulated. The coupling of glycogenolysis to mitochondrial respiration may have evolved as a direct response to the energetic needs of VSM. That is, the large glycogenolytic response in the initial minutes of stimulation may be necessary to maximize the cellular production of ATP during the presteady state. Likewise, the coupling between aerobic glycolysis and Na-K transport indicates a sensitive and efficient means of coordinating energy metabolism with ion transport at the membrane level. Additionally, the regulation of substrate supply, i.e. glucose transport, also may be closely coordinated with changes in ion transport. One may speculate that alterations in the microenvironment of each compartment can independently regulate intermediary metabolism and therefore allow the cell to quickly and efficiently respond to localized stimuli. Thus, stimulation of Na-K transport could effectively regulate energy production at the membrane level without mobilizing or competing with the energy transduction of other cellular processes. This compartmentation of energy utilization may be highly advantageous, since oxidative metabolism is closely coordinated with mechanical activity and therefore regulation of blood flow. Future investigations will attempt to elucidate which intracellular signals which are responsible for the regulation of these functionally independent compartments of energy metabolism and transduction in VSM. In more general terms, our findings provide a basis from which future questions concerning the regulation of cellular metabolism must be directed. The cellular cytoplasm can no longer be envisioned as a homogeneous compartment, but rather a complex array of functional subcompartments which may be individual

Animals↗
PubMed1990

Biogenesis of mitochondrial porin: the import pathway.

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavable 'signal' sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein which is characterized by its extreme protease resistance.

Animals↗
PubMed1996

Protein glycosylation in the endoplasmic reticulum and the Golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein A-gold and lectin-gold techniques.

High resolution immunolabeling applying the protein A-gold technique and carbohydrate cytochemistry using lectin-gold labeling on Lowicryl K4M and thawed-frozen thin sections are most useful approaches for the detection of protein antigens and lectin binding sites in intracellular organelles and the plasma membrane. They provided the basis for modern electron microscopic studies on protein glycosylation reactions and the identification of their subcellular localization as reviewed here. These studies have demonstrated organelle subcompartments and the cell type-specific compartmentation of endoplasmic reticulum and Golgi apparatus-associated glycosylation reactions. The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.

Animals↗
PubMed1986

The effects of ACTH on acid phosphatase activity in endosomes, GERL and lysosomes of cultured adrenal tumor cells.

Cultured cells derived from a mouse adrenocortical tumor transplant are unspecialized in appearance, but produce basal levels of steroids and demonstrate a near-immediate steroidogenic response to ACTH. There is biochemical evidence that ACTH induces increases in the uptake of serum lipoproteins by these cells and that this material is hydrolyzed in lysosomes to free cholesterol, a precursor for steroid end products. To investigate morphologically the role of lysosomes in the steroidogenic activity of these cells, cultures were incubated for 4 h with and without ACTH, then processed for the ultrastructural localization of acid phosphatase (ACPase), a marker enzyme for lysosomes, and for GERL, the lysosome-forming subcompartment of the Golgi, and examined by TEM and HVEM. Steroid output was determined by a fluorometric technique. Unstimulated cells secreted basal levels of steroids. By TEM, large endosomes, some containing semi-compact material and ACPase reaction product, were occasionally seen at the cell periphery and in the Golgi region. The Golgi and GERL were poorly developed. Residual bodies, a few of them ACPase+, appeared in the Golgi region and in microtubule-associated clusters near the cell membrane. ACTH-stimulated cells secreted steroids at 8-10 fold basal values. In TEM records, they displayed numerous ACPase+ endosomes between the cell periphery and the Golgi. The Golgi and GERL regions appeared to be hypertrophied and many large, inclusion-containing, strongly ACPase+ residual bodies appeared here and in elongated microtubule-containing cell processes. HVEM micrographs showed more definitively that ACTH produced distinct increases in the size of GERL and in the number of ACPase+ organelles. Our results suggest that in unstimulated cells, endosomes, presumably containing media-derived material, gain lysosomal enzymes in or near GERL, are transformed to residual bodies as their contents are hydrolyzed, and are subsequently translocated by microtubules to the cell periphery for exocytosis. ACTH appears to intensify all of these effects. The "giant" lysosomes seen in stimulated cells may result from a fusion of smaller lysosomes. Their amorphous contents may reflect an inefficient hydrolysis of LDL to free cholesterol.

Acid Phosphatase↗
PubMed1999

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae.

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.

ATP-Dependent Proteases↗