Intracellular distribution of hexokinase in rabbit brain

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed, that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.     

P-糖蛋白的细胞内转运及表达调控

肿瘤多药耐药(multidrug resistance,MDR)的发生多与P-糖蛋白(P-glycoprotein,P-gp)过度表达相关。作为一种糖蛋白,P-糖蛋白在内质网中合成、折叠,然后转运到高尔基体进行加工、修饰,最终定位于细胞膜,且只有定位于细胞膜的P-糖蛋白才与肿瘤多药耐药的产生相关。P-糖蛋白的表达与多种信号通路如MAPK、Wnt/β-catenin、PKC、NF-κB有关。研究证实,还有多种miRNA与肿瘤多药耐药的发生相关。本文综述了P-糖蛋白的细胞内转运过程及P-糖蛋白表达相关信号通路的研究进展,为以P-糖蛋白为靶标的肿瘤多药耐药逆转剂提供新的研究策略。     

Intracellular distribution of hepatic copper in macular mutant mice

To evaluate the role of lysosomes in copper-mediated hepatocellular injury, copper was administered, sc, to both normal and macular mutant mice at doses of 4.5, 9.0, and 18 mg Cu/kg, and the subcellular distribution of copper has been investigated in the liver of normal and mutant mice 24h after injection. The amount of copper in all fractions of copper-treated mutant mice was markedly lower than those in copper-treated normal mice, with the exception of microsomal fraction. However, there were no distinct differences in the proportion of copper in subcellular fractions between normal and mutant mice.     

Intracellular distribution and origin of pentacyclic triterpenes in Calendula officinalis leaves

In Calendula officinalis leaves the cyclization of squalene to β-amyrin and its further oxidation to oleanolic acid as well as the biosynthesis of all derivatives of oleanolic acid 3-glucoside and some derivatives of oleanolic acid 3-glucuronoside occur in the microsomal fraction. The final metabolites of oleanolic acid 3-glucoside series i.e. pentaglycosides, are translocated from this fraction, one to the cell wall and plasmalemma fraction and the other to the cytosol. The derivatives of oleanolic acid 3-glucoronoside are synthesized partially in other fractions and accumulate in the different membraneous structures of the cell.     

Intracellular ion concentrations in the isolated frog skin epithelium: Evidence for different types of mitochondria-rich cells

Summary Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Under control conditions, principal cells and mitochondria-rich cells (MR cells) had a similar intracellular ion composition, only the Cl concentration in MR cells was significantly lower. Inhibition of transepithelial Na transport by low concentrations of ouabain (2 × 10^–6 m, innerbath) resulted in a Na concentration increase of principal cells from 10.9 to 54.3 mmol/kg wet wt. The increase was completely abolished by simultaneous application of amiloride (10^–4 m, outer bath). Amiloride alone resulted in a significant decrease of the Na concentration to 6.1 mmol/kg. w. w. Among MR cells, two different groups of cells could be distinguished; cells that showed a Na increase after ouabain which was even larger than that in principal cells and cells that did not respond to ouabain. In about half of all ouabain-sensitive MR cells the Na increase could be prevented by amiloride. According to these results, a subpopulation of MR cells displays the transport characteristics expected for a transepithelial Na transport compartment, an apical amiloride-sensitive Na influx and abasal ouabain-inhibitable Na efflux. Given the small number of cells, however, it is unlikely that this subtype of MR cells contributes significantly to the overall rate of transepithelial Na transport.I wish to thank Cathy Langford, Cindy Partain, and Ray Whitfield for their excellent technical assistance. Financial support was provided by NIH grants DK35717 and 1S10-RR0-234501.     

Intracellular distribution and origin of sterols in Calendula officinalis leaves

In Calendula officinalis leaves 66% of all steryl forms are present in the ‘microsomal fraction’ (IV), 24% in the mitochondrial and Golgi membranes (III), 5% in the ‘chloroplast’ (II), 4% in the ‘cell wall and membrane’ (I) fraction and 1%. in the cytosol. Free sterols, their esters, glycosides and acylated glycosides are present in varying proportions in all cellular subtractions. Mevalonate-[2¹⁴C] labelling of sterols derived from various steryl forms showed that free sterols and all their derivatives, i.e. steryl esters and glucosides, are formed in fraction IV and are then translocated to other organelles. Fraction III is the main site of glycosylation of transported sterols as well as of acylation of steryl glycosides.     

Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites

Background information. Spherulites are multi‐lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B‐subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non‐toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. Results. We demonstrate that STxB‐spherulites (ST×B‐functionalized spherulites) are internalized into HeLa cells in a receptor‐dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Conclusions. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB‐spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB‐spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Capacity of Spirulina platensis to accumulate manganese and its distribution in cell

Effects of manganese salt (MnCl₂) on growth of Spirulina platensis and capacity of the cyanobacteria to accumulate the metal in various cell components were studied. S. platensis cells were shown to tolerate high concentrations of manganese and preserve, although strongly suppressed, the capacity to grow in the medium containing 5.1 mM MnCl₂. The concentrations of manganese that did not inhibit growth considerably altered cell ultrastructure and changed the protein profile. The accumulation of manganese in S. platensis cells was proportional to the period of culturing and manganese concentration in the medium, reaching a plateau at about 2.5 mM. A threshold intracellular concentration of this metal is estimated as 28 ± 3 μmol/g dry wt. The fractionation of the manganese-enriched biomass demonstrated that the major portion of intracellular manganese (over 90%) was found in the total protein fraction. The chromatographic separation of the soluble protein fraction showed that manganese was incorporated into proteins with molecular weight of 5 to 15 kD. Dry biomass adsorbed manganese cations; this evidence seems to indicate a considerable contribution of biosorption to manganese accumulation by S. platensis cells.     

High‐Throughput Quantitation of Intracellular Trafficking and Organelle Disruption by Flow Cytometry

Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry‐based method that circumvents these limitations. The method utilizes fluorescent pulse‐width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse‐width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso‐endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane‐to‐endosomes‐to‐Golgi, by decreasing pulse‐width values. A block in transport upon RNAi‐mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse‐width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.      

Intracellular distribution of the steroidal glycoalkaloid α-tomatine in Lycopersicon esculentum fruit

Pericarp tissue from green tomato fruits was homogenized and separated into organelle fractions by differential centrifugation. Tomatine was found mainly in the final (105 000g) supernatant, with small amounts in the microsomes. Expressed sap from intact tissue was also rich in the alkaloid. It is suggested that tomatine accumulates in the vacuoles and/or soluble phase of the cytoplasm and is possibly synthesized in microsomal organelles.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Intracellular distribution of proteasomes in A-431 cells

The intracellular proteasome distribution in A-431 cells was shown using methods of cell fractionation and immunofluorescence. In growing cells the distribution of proteasomes was EGF-dependent. In unstimulated cells and within 30 min of EGF treatment, proteasomes were localized in the cytoplasm and nuclei, but not on the plasma membrane. After 30 min of EGF treatment they were observed on the plasma membrane as well. In A-431 cells cultivated for 24 h in the medium with a lowered serum concentration, proteasomes were detected on the plasma membrane already in unstimulated cells. It is suggested that dephosphorylation of the EGF receptor and signalling proteins in unstimulated cells may depend on the proteolytic activity of proteasomes.     

Intracellular localization and domain organization of human TRIM41proteins

A human gene previously identified as a partial cDNA homologous to the gene of RET finger protein was characterized. Northern hybridization detected three messages of 3.3, 4.2, and 7.5kb. The coding sequences of the more abundant of the three messages, the 4.2 and the 3.3kb, were determined. The former encodes a 630 amino acid protein (TRIM41) and the latter a 518 amino acid protein (TRIM41). Green fluorescent protein (GFP) fusions of full-length TRIM41 and TRIM41 were both observed as speckles in the cytoplasm and the nucleus. The result was corroborated by Western analysis of cellular fractions. Results with GFP fusions of various segments of the TRIM41 proteins indicated that the nuclear transport of the proteins is mediated by an N-terminal segment common to both isoforms, but independent of a classical nuclear localization signal sequence.     

Modeling Fusion/Fission‐Dependent Intracellular Transport of Fluid Phase Markers

A fundamental feature of eukaryotic cells is the presence of distinct membrane‐bound compartments having unique protein and lipid composition. These compartments are interconnected by active trafficking mechanisms that must direct macromolecules to defined locations, and at the same time maintain the protein and lipid composition of each organelle. It is well accepted that Rab proteins play a central role in intracellular transport regulating the recognition, fusion and fission of organelles. However, how the transport is achieved is not completely understood. We propose a model whereby a soluble component in the luminal compartment is transported along different Rab‐containing organelles that interact according to the following simple principles: (i) only organelles with the same or compatible Rab membrane domains can fuse; (ii) after fusion, an asymmetric fission occurs producing a tubule and a round‐shaped vesicle; and (iii) Rab membrane domains distribute asymmetrically between the two resulting organelles. When this model was tested in a simulation, efficient unidirectional transport was observed, while the compartment identity was preserved. All three principles were absolutely necessary for transport. The model is compatible with Rab association/dissociation dynamics and with Rab conversion. In simulations mimicking a simplified endocytic pathway, soluble and membrane‐associated markers were efficiently transported preserving the identity of the interacting compartments.     

Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells

Although multidrug resistance (mdr) may arise through a variety of mechanisms, the most widely studied and accepted form is associated with an increased concentration of P-glycoprotein (P-gp), a 170kd protein found in the membrane fraction of a number of mammalian cells. Since mdr seems to be related to the ability of resistant cells to extrude drugs and the circumvention of mdr is supposed to be due to the restored ability to accumulate drugs, membrane has been regarded as the crucial site for such a regulation and an important role for membrane ion exchangers has been suggested. The aim of this work was to elucidate whether the Na⁺/H⁺ antiporter is involved in the mechanism of regulation and circumvention of mdr and if 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a selective inhibitor of the Na⁺/H⁺ exchanger, can modulate the functional expression of the mdr phenotype. The effect of EIPA on doxorubicin (DX) resistant cells (LoVo/DX) obtained from a human colon adenocarcinoma cell line (LoVo) was studied. EIPA at concentrations ranging from 10 to 50 μM was able to increase the antibiotic cytotoxicity in the resistant Lovo/DX cells. The reversal of DX resistance paralleled an increase of the ability of the cells to accumulate the drug. Both drug loading and sensitivity to the inhibitory effect of DX on cell proliferation were restored by EIPA in a dose-dependent way. These results suggest a new mechanism of mdr reversal and indicate that amiloride and its derivatives may be useful in reversing DX resistance and in enhancing the clinical effectiveness of chemotherapeutics.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and $\text{2-p-}\text{toluidinyl-6-naphthalene}$ sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Intracellular transport of sulfated macromolecules in parotid acinar cells

Intracellular transport of sulfated macromolecules in parotid acinar cells was investigated by electron microscopic radioautography after injection of 35S-sulfate. Ten minutes after injection radiosulfate was concentrated in the Golgi region. By 1 hr, much of the radioactive material had been transported to condensing vacuoles. These vacuoles were subsequently transformed into zymogen granules which contained almost 70% of the radioactivity 4 hrs after injection. These results indicate that, in addition to its packaging function, the Golgi apparatus in parotid acinar cells is capable of utilizing inorganic sulfate for the production of sulfated macromolecules. These molecules, following an intracellular route similar to that taken by digestive enzymes, become an integral component of zymogen granules. The possibility that sulfated macromolecules play a role in exocrine secretion by aiding in the packaging of exportable proteins is discussed.     

ABCA1的胞内运输及功能研究新进展

三磷酸腺苷结合盒转运体A1(ABCA1)具有介导细胞内脂质流出,维持细胞脂质稳态的功能．新生的ABCA1必须经过胞内运输和各种化学修饰等过程,最终成为具有功能的成熟转运体,才能行使其转运脂质的功能,因此,ABCA1在胞内的运输过程和正确质膜定位对其介导胆固醇流出的功能至关重要．目前ABCA1相关研究主要集中于脂质转运方面,并提出各种胆固醇流出机制的模型,如通道转运模型、蘑菇状突起模型和胞吞-胞吐转运模型等．最近研究显示,ABCA1还具有调节质膜脂筏结构、参与免疫和炎症调节等新功能．本文主要针对ABCA1的胞内运输过程以及各种功能做一综述,以期为动脉粥样硬化相关疾病提供新的治疗靶点和途径．     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.