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^–6m, 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^–4m, 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.     

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.     

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.     

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.     

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 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.     

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 distribution and chemical forms of arsenic in rabbits exposed to arsenate

Inhibition of the methylation of arsenic in rabbits by ip injection of periodate-oxidized adenosine (PAD) prior to an iv injection of⁷⁴As-arsenate (AsV; 0.4 mg As/kg body wt) caused a marked increase in the retention of⁷⁴As in both the cellular organelles and the soluble fractions of liver and kidney. One day after exposure, almost 30% of the arsenic in the liver and about 40% of the arsenic in the kidney was recovered in the nuclear fraction. In the liver nuclei, the inhibition of the methylation increased the⁷⁴As content of the insoluble fraction and most of this arsenic was protein-bound. The major part of the soluble intranuclear⁷⁴As was in the form of AsIII, formed by reduction of the administered AsV. In the liver, PAD also caused a pronounced increase in the⁷⁴As content of the microsomal fraction. In the kidneys, where most of the arsenic was present as AsV, there was a marked accumulation of arsenic in the mitochondria.     

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.     

Intracellular Na and K distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.     

Intracellular Localization of the HCS2 Gene Products in Identified Snail Neurons In Vivo and In Vitro

SUMMARY 1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca²⁺ concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy.2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system.3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons.4. The immunogold particles were practically absent in the bodies of cultured neurons.5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.     

The Golgi apparatus in parasitic protists

This review summarizes the current reports on the Golgi apparatus of parasitic protists. Numerous recent publications have demonstrated that studies on intracellular traffic in parasites essentially advanced our knowledge on the Golgi structure and function, which has been traditionally based on research on yeast and mammalian cultured cells. It has been reported that the parasitic lifestyle determines the functional and structural peculiarities of the secretory systems in unrelated groups of unicellular parasites that make them different from those in mammalian and yeast cells. This review covers the best-studied protists, predominantly those of high medical importance, belonging to the following taxa: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma, Plasmodium), and Kinetoplastida (Trypanosoma, Leishmania). The morphology of the Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of the six highest taxa of Eukaryota (Adl et al., 2005) a minimum of eight groups are represented by species lacking Golgi dictiosomes. However, biochemical and/or molecular (genomic) evidence indicate that an organelle with the functions of the Golgi was present in every lineage of eukaryotes studied thus far. Loss of the Golgi organelle is a secondary event as proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. The loss might have occurred independently several times in evolution. Neither the number of stacks, nor the size of the organelle correlates with the intensity of secretion or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Intracellular alkalinization in dexamethasone-induced thymocyte apoptosis

Glucocorticoid can induce apoptosis of thymocytes, but its mechanism is not clear yet. In this study, we reported that dexamethasone-induced apoptosis was associated with intracellular alkalinization. Dexamethasone induced a higher percentage of apoptosis in 138 mM than in 50 mM NaCl, total abrogation of apoptosis was noted in NaCl-depleted culture medium. Highest apoptotic rate was observed in medium with pH 7.2, whereas it was partially and completely inhibited at pH 6.5 and pH 6.0, respectively. Intracellular pH was higher in pre-apoptotic thymocytes than non-apoptotic ones. The Na+ /H+ antiporter inhibitor of 5-(N,N'-dimethyl)-amiloride inhibited the dexamethasone-induced increase in pHi and apoptosis of thymocytes. Glucocorticoid antagonist RU486 also blocked the dexamethasone-induced effect. Furthermore, the apoptosis and increase in intracellular pH induced by dexamethasone were inhibited by cycloheximide, actinomycin D. It seems that intracellular pH is increased during the development of thymocyte apoptosis and inhibiting its increment would retard the rate of progression to cell death.     

Intracellular trafficking and secretion of cerebral dopamine neurotrophic factor in neurosecretory cells

Cerebral dopamine neurotrophic factor (CDNF) is a novel evolutionary conserved protein which can protect and restore the function of dopaminergic neurons in the rat model of Parkinson's disease, suggesting that CDNF might be beneficial for the treatment of Parkinson's disease. CDNF is widely expressed in neurons in several brain regions including cerebral cortex, hippocampus, substantia nigra, striatum and cerebellum. Human CDNF is glycosylated and secreted from transiently transfected cells; however, the mechanism underlying CDNF secretion is currently unclear. In this study, we found that CDNF could be secreted primarily via the regulated secretion pathway in PC12 cells. The glycosylation of CDNF is not required for its secretion. Moreover, we identified two key subdomains in CDNF which are important for its intracellular localization and secretion. Disrupting helix-1 of CDNF significantly reduces its constitutive and regulated secretion and the helix-1 mutant is retained in the endoplasmic reticulum. Although helix-7 mutation only decreases CDNF regulated secretion and has no effect on its constitutive secretion, which is further supported by the reduction in co-localization of helix-7 mutant with secretory granules. In all, these findings will advance our understanding of the molecular mechanism of CDNF trafficking and secretion.     

Intracellular pH based controlled cultivation of yeast cells: I. Measurement methodology

A method has been developed to continuously measure the intracellular pH (pH(i)) of cells cultivated in a bioreactor in an on-line fashion over extend time periods. The methods is attractive in its simplicity and involves the use of a fluorescent pH(i) indicator 9-aminoacridine (9A A) which is a week base. An expression has been derived to calculate changes in pH(i) from measured 9AA-fluorescence changes. The indicator 9AA was found t be nontoxic to yeast cells at concentrations used to measure pH(i) (7 muM). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA-fluorescence. The pH(i) change in yeast cell following the addition of a proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) measured by 9AA compared favorably with that measured by the well-established pH(i), indicator (which is however unsuitable for on-line applications in a bioreactor) bis-carboxyethyl carboxy fluorescein (BCECF). The pH(i) of yeast under substrate starved conditions was 6.4 units. The responses of pH(i) of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pH(i) increased by 0.12 unit following a 100-ppm glucose pulse addition and by 0.25 unit following a 300-ppm ethanol pulse addition. Under anaerobic condition, pH(i) increased by 0.1 unit following a 500-ppm glucose pulse addition. Comparison of pH(i) with other indicators of cellular metabolic state suggests that pH(i) is a cellular metabolic state indicator. (c) 1993 John Wiley & Sons, Inc.     

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 2-p-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.