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1.
Lysosomal acid phosphatase is transported to lysosomes via the cell surface. 总被引:31,自引:9,他引:22
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Lysosomal acid phosphatase (LAP) is transported as a transmembrane protein to dense lysosomes. The pathway of LAP to lysosomes includes the passage through the plasma membrane. LAP is transported from the trans-Golgi to the cell surface with a half-time of less than 10 min. Cell surface LAP is rapidly internalized. Most of the internalized LAP is transported back to the cell surface. On average, each LAP molecule cycles greater than 15 times between the cell surface and the endosomes before it is transferred to dense lysosomes. At equilibrium approximately 4 times more LAP precursor is present in endosomes than at the cell surface. Exposing cells to reduced temperature or weak bases such as NH4Cl, chloroquine and primaquine decreases the steady-state concentration of LAP at the cell surface. The recycling pathway is operative at greater than or equal to 20 degrees C and does not include passage of the Golgi/trans-Golgi network. LAP is transferred with a half-time of 5-6 h from the plasma membrane/endosome pool to dense lysosomes, from where it does not recycle to the endosome/plasma membrane pool at a measurable rate. 相似文献
2.
Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box 总被引:7,自引:1,他引:6
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The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA. 相似文献
3.
A major mechanism of human immunodeficiency virus-induced cell killing does not involve cell fusion. 总被引:33,自引:24,他引:9
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In vitro studies indicate that human immunodeficiency virus (HIV) infections are cytopathic for T4+ peripheral blood lymphocytes and for most continuous lines of T4+ lymphocytes. These cytopathic effects have been largely attributed to the formation of syncytia by HIV-infected cells. We report that HIV infections killed cultured peripheral blood lymphocytes and a line of T4+-lymphoid cells (CEM cells) without causing cell fusion. We also report that the occurrence of syncytia is an early and transitory phenomenon following infection of a fusion-susceptible line of T4+-cells (H9 cells). Mixing experiments and flow cytometry have been used to demonstrate that susceptibility to HIV-induced fusion is not determined by differences in presentation of viral envelope antigens or the surface levels of T4 receptor antigens on fusion-susceptible and -resistant cells. We conclude that a major mechanism of HIV-induced cell killing does not involve cell fusion and that HIV-induced cell fusion, when it does occur, requires factors in addition to viral envelope antigens and host T4 receptors. 相似文献
4.
Y Tanaka S Yano K Okada T Ishikawa M Himeno K Kato 《Biochemical and biophysical research communications》1990,166(3):1176-1182
Involvement of endosomes in transport of newly synthesized acid phosphatase to lysosomes was investigated using the Golgi fraction (GF1 + 2), enriched in endosomes. The Golgi fraction (GF1 + 2) was prepared from the livers of rats given [35S]methionine and asialofetuin conjugated-horseradish peroxidase (HRP). Newly synthesized acid phosphatase in the endosomes containing internalized asialofetuin-HRP was measured as a loss of the detectable labeled enzyme after 3,3'-diaminobenzidine (DAB) and H2O2 reaction, due to formation of insoluble polymers which reduce protein antigenicity. With this procedure, acid phosphatase was all but undetectable in the Golgi fraction. Thus, newly synthesized acid phosphatase is apparently transported to lysosomes by endosomes. 相似文献
5.
Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane 总被引:28,自引:12,他引:16
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We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail. 相似文献
6.
Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation
Chandler MP Kerner J Huang H Vazquez E Reszko A Martini WZ Hoppel CL Imai M Rastogi S Sabbah HN Stanley WC 《American journal of physiology. Heart and circulatory physiology》2004,287(4):H1538-H1543
Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon. 相似文献
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Hepatic lysosomes have been fractionated by rate sedimentation and by isopycnic banding. In all experiments, the distribution of acid phosphatase differed from that of the other lysosomal enzymes. Evidence is presented that this difference is due not to the separation of lysosomes from different cell types, but simply reflects the membrane location of a part of the acid phosphatase. 相似文献
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A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 μM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 μM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5–40 μM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 μM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential. 相似文献
12.
Recycling of the hepatocyte asialoglycoprotein receptor does not require delivery of ligand to lysosomes 总被引:10,自引:0,他引:10
The hepatocyte asialoglycoprotein receptor is able to mediate the internalization of ligand in buffer devoid of Na+ but containing 0.15 M K+. Under these conditions, degradation of internalized ligand does not occur due to an inability to deliver the ligand to lysosomes. Instead, the ligand becomes localized in a vesicle with the same density as plasma membrane on Percoll gradients. This vesicle may be the functional equivalent of the uncoated vesicles observed by electron microscopy. Internalization of more than 20 glycoprotein molecules/high affinity surface receptor was observed under these conditions, indicating that delivery of ligand to lysosomes is not necessary for receptor reutilization. 相似文献
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14.
Moshe Wolman 《Cell and tissue research》1964,65(1):1-9
Summary Treatment with M/2 CaCl2 or fresh frozen sections of various organs of mice inhibits the subsequent histochemical demonstration of acid phosphatase by various procedures. This inhibition can be reversed by treatment with M/I NaCl. Fixation in cold formalin solution, or treatment with fat solvents weakens or abolishes this effect with the result that phosphatase staining is not markedly influenced by pretreatment with salts. The effect of surfactants in inhibiting acid phosphatase staining is diminished by fixation in formalin. Treatment with M/2 CaCl2 of fresh frozen sections of mice organs partly inhibited the histochemical staining of phospholipids.The findings indicate that acid phosphatase molecules are bound to polar lipids and that under the influence of excess of Ca ions the lipoproteic molecules of acid phosphatase form droplets bounded by hydrophobic groups of the lipids. In such droplets the enzyme molecules are not available to act on substrates dissolved in the medium, and the droplets may be regarded as lysosomes. Under the influence of excess Na ions in the milieu the lipoprotein layer breaks into droplets bounded on the outside by the enzyme protein and the hydrophilic groups of the phospholipids. Such particles probably represent microsomes. The implications of these conclusions are discussed.A preliminary communication dealing with the present study has been published (in Hebrew) in the Harefuah (Wolman, 1964). 相似文献
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Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation. 相似文献
17.
Epoxidation in vivo of hyoscyamine to scopolamine does not involve a dehydration step 总被引:1,自引:0,他引:1
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Hyoscyamine is epoxidized to scopolamine via 6β-hydroxyhyoscyamine in several solanaceous plants. 6,7-Dehydrohyoscyamine has been proposed to be an intermediate in the conversion of 6β-hydroxyhyoscyamine to scopolamine on the basis of the observation that this unsaturated alkaloid is converted to scopolamine when fed to a Datura scion. To determine whether a dehydration step is involved in scopolamine biosynthesis, [6-18O]6β-hydroxyhyoscyamine was prepared from l-hyoscyamine and 18O2 using hyoscyamine 6β-hydroxylase obtained from root cultures of Hyoscyamus niger L. When [6-18O]6β-hydroxyhyoscyamine was fed to shoot cultures of Duboisia myoporoides R. BR., the labeled alkaloid was converted to scopolamine which retained 18O in the epoxide oxygen. It is concluded that 6β-hydroxyhyoscyamine is converted in vivo to scopolamine without a dehydration step. 相似文献
18.
Summary A quantitative X-ray microanalytical study of 80 spleen lysosomes after histochemical reaction for acid phosphatase was carried out. Lysosomal Fe concentrations showed a skewed distribution, with most lysosomes having relatively low concentrations. The concentration of Pb in the lysosomes (indicative of acid phosphatase activity) showed a close to normal distribution. Although the plot of lysosomal Pb concentrations against Fe concentrations showed a marked scattering of the points, there is a statistically highly significant correlation between accumulated metal and acid phosphatase activity as determined from the Pb concentrations. The relation between concentrations of P and those of Pb in the lysosomes points to the possibility that Pb(H2PO4)2 is the main reaction product of the histochemical acid phosphatase reaction. 相似文献
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Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle. 相似文献