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PubMed1984

The subcellular distribution of beta-carotene in bovine corpus luteum.

The distribution of beta-carotene was determined in various subcellular fractions of bovine corpus luteum. It was found in significant amounts in all subcellular fractions examined including nuclear, mitochondrial, microsomal, cytosolic, and floating lipid. Much of the beta-carotene found in the crude nuclear and mitochondrial fractions was loosely bound and could be removed with repeated washings. In contrast, the microsomal beta-carotene could only be removed by detergent extraction suggesting that it is an integral component of this membrane preparation. In the cytosol fraction beta-carotene was bound to high-molecular-weight protein(s), quite possibly a plasma-derived lipoprotein. The subcellular distribution of beta-carotene in corpus luteum is quite similar to the distribution of its metabolite, retinol, in liver. This finding coupled with other recently published data suggests that beta-carotene could play a distinct role in corpora lutea function.

Animals↗

PubMed2006

Clinical significance of subcellular localization of KL-6 mucin in primary colorectal adenocarcinoma and metastatic tissues.

AIM: To assess subcellular localization of KL-6 mucin and its clinicopathological significance in colorectal carcinoma as well as metastatic lymph node and liver tissues. METHODS: Colorectal carcinoma tissues as well as metastatic lymph node and liver tissues were collected from 82 patients who underwent colorectomy or hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody. RESULTS: Of the 82 colorectal carcinoma patients, 6 showed no staining, 29 showed positive staining only in the apical membrane, and 47 showed positive staining in the circumferential membrane and/or cytoplasm. Positive staining was not observed in non-cancerous colorectal epithelial cells surrounding the tumor tissues. The five-year survival rate was significantly lower in cases showing positive staining in the circumferential membrane and/or cytoplasm (63.0%) than those showing positive staining only in the apical membrane (85.7%) and those showing no staining (100%). Statistical analysis between clinicopathological factors and subcellular localization of KL-6 mucin showed that KL-6 localization in the circumferential membrane and/or cytoplasm was significantly associated with the presence of venous invasion (P = 0.0003), lymphatic invasion (P<0.0001), lymph node metastasis (P<0.0001), liver metastasis (P = 0.058), and advanced histological stage (P<0.0001). Positive staining was observed in all metastatic lesions tested as well as in the primary colorectal carcinoma tissues. CONCLUSION: The subcellular staining pattern of KL-6 in colorectal adenocarcinoma may be an important indicator for unfavorable behaviors such as lymph node and liver metastasis, as well as for the prognosis of patients.

Adenocarcinoma↗

PubMedDate not supplied

Subcellular localization of hepatitis C virus structural proteins in the liver of transgenic mice.

Hepatitis C virus (HCV) core and envelope proteins are suggested to be responsible for the pathogenesis of hepatic and extrahepatic manifestations in chronic hepatitis C. Moreover, the core protein is implicated in the regulation of the transcription of cellular genes including c-myc, RB and p53. Determining the subcellular localization of the core and envelope proteins is therefore necessary to elucidate their behaviors, particularly in vivo ones, regarding the interaction with transcriptional regulatory proteins or gene elements. We defined the subcellular localization of HCV envelope and core proteins which were expressed in substantial levels in the liver of transgenic mice. Subcellular fractionation by ultra-centrifugation revealed that the envelope proteins were present principally in the microsomes of the liver, while a small amount of the protein was detected also in the nuclei. Immunohistochemistry confirmed the localization of envelope proteins in the nuclei. In contrast, the core protein was detected principally in the cytoplasmic fraction, where it was closely associated with lipids. A low level of the core protein was detected also in the nuclei and microsomal fraction. These results suggest possible interaction of the HCV structural proteins with some factors in hepatocytes thereby perturbing intracellular circumstances.

Animals↗

PubMed1999

Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

3T3 Cells↗

PubMed1999

Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation.

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.

Amino Acid Sequence↗

PubMed2000

[Altered subcellular distribution of daunorubicin in the non-P-glycoprotein-mediated multidrug-resistant cell line HL-60/ADR].

OBJECTIVE: To investigate DNR subcellular distribution in the non-P-glycoprotein-mediated multidrug-resistant cell line HL-60/ADR and its relation to multidrug resistance. METHODS: DNR subcellular disposition was studied by confocal scanning laser microscopy, fluorescent methods, MTT and RT-PCR. The effects of verapamil, brefeldin A, chloroquine were also examined. RESULTS: In the drug-sensitive cell line HL-60 DNR fluorescence distributed evenly in the nucleus and cytoplasm, while in the resistant cell line DNR distributed in a punctate pattern in the cytoplasm and was reduced in the nucleus. Verapamil, brefeldin A, but not chloroquine could recover the intracellular distribution of DNR from punctate to even in the resistant cell line. CONCLUSION: Altered subcellular disposition of DNR in resistant cell line was involved in the mechanism of multidrug resistance.

Antibiotics, Antineoplastic↗

PubMed2002

Differential long-term subcellular responses in heart and liver to adriamycin stress. Exogenous L-carnitine cardiac and hepatic protection.

In order to evaluate the heart and liver responses after adriamycin (ADR) toxic aggression, with and without exogenous L-carnitine (CAR) protection, female Sprague-Dawley rats, body weight 40-60 g, were randomized into four groups: CON, ADR, CAR and CAR-ADR. ADR was injected i.v. at a dose of 15-18 mg/kg body wt (0.1 ml). CAR was administered i.v. at a dose of 20 mg (0.1 ml) before each subdose of ADR, and then orally at 180 mg/kg body wt daily for 12 weeks. Long-term cardiac and hepatic subcellular damage were determined by transmission electron microscopic analysis of ultrathin sections. The ADR-induced long-term cardiac subcellular pathology included loss, disruption and disassembly of myofibrils, and mitochondrial swelling and condensation. On the other hand, the ADR-induced subcellular hepatic alterations consisted of polymorphic mitochondria, cytoplasmic vacuolization and accumulation of lipid droplets. Apparently, cardiac tissue was more affected by ADR toxic aggression than hepatic tissue. However, these alterations were of less severity in protected groups, in both heart and liver, suggesting CAR as a possible hepatoprotector agent against ADR toxicity. Because of the liver-L-carnitine-heart relationship, studying ADR-hepatotoxicity could be helpful in the further understanding of severe ADR-cardiotoxicity.

Administration, Oral↗

PubMed2004

[Expression of CTLA4-EGFP fusion protein in K562 cells and its subcellular localization].

AIM: To construct a eukaryotic expression vector for cytotoxic T lymphocyte antigen-4 (CTLA4) gene fused with enhanced green fluorescent protein(EGFP) gene and to analyze the expression and subcellular localization of the fusion protein in K562 cells. METHODS: The human CTLA4 gene was cloned by RT-PCR and was then inserted into plasmid pEGFP-N1 to construct the expression vector for CTLA4-EGFP fusion gene. The expression and subcellular localization of the fusion protein in transfected K562 cells were analyzed by flow cytometry and confocal microscopy, respectively. RESULTS: The cDNA of CTLA4 gene was cloned from human peripheral blood cells. The expression vector for CTLA4-EGFP fusion gene was constructed by PCR through introducing a Kozak sequence before the initiation site and deleting the stop codon. Flow cytometry analysis revealed that the proportion of both CTLA4+ and EGFP+ K562 cells was 16% at 24 h after the transfection. Confocal microscopy observation showed that the expressed fusion protein was mainly distributed in the intracellular compartments and had small part on the cell membrane. In contrast, only EGFP expression could be detected in K562 cells transfected with empty vector with diffuse distribution in the cells. Moreover, the expression level of the fusion protein on the cells increased to 29% after stimulation with phorbol ester and ionomycin. In addition, the fusion protein inside the cells tended to move towards and fused with the cell membrane. CONCLUSION: The expression vector for CTLA4-EGFP fusion gene has been constructed successfully and expressed in K562 cells. The expressed fusion protein has similar subcellular localization and transport characteristics to native CTLA4 in activated T cells.

Abatacept↗

PubMed2004

[Restorative effect of quercetin on subcellular distribution of daunorubicin in multidrug resistant leukemia cell lines K562/ADM and HL-60/ADM].

BACKGROUND & OBJECTIVE: Quercetin, a widely distributed natural flavonoid with a variety of biological functions, can reverse multidrug resistance (MDR) in leukemia according to recent researches. This study was to investigate the mechanism of quercetin restoring subcellular distribution of daunorubicin (DNR) in multidrug resistant leukemia cell lines, K562/ADM and HL-60/ADM, and reversing their MDR. METHODS: MTT cell viability assay was used to verify the sensitization of DNR by quercetin in K562/ADM and HL-60/ADM cells,and determine the reverse concentration extent,confocal laser scanning microscope was used to observe the subcellular distribution of DNR in K562/ADM and HL-60/ADM cells,and relevant sensitive cell lines, K562/S and HL-60/S,before and after quercetin exposion. RESULTS: Compared with K562/S and HL-60/S cells,20-40 micromol/L of quercetin in vitro remarkably enhanced the sensitivity of K562/ADM and HL-60/ADM cells to DNR, restore the subcellular distribution of DNR, so as to reverse MDR. CONCLUSION: quercetin could be a candidate of effective multidrug resistance-reversing agent in leukemia chemotherapy.

Antibiotics, Antineoplastic↗

PubMed2005

Expression pattern and subcellular localization of five splice isoforms of human PXK.

Phox homology (PX) domains specifically bind to phosphoinositides, and this interaction is crucial for their cellular function. A full-length cDNA of human PX domain containing serine/threonine kinase gene (PXK) that we termed PXK_v1 had previously been cloned. PXK_v1 consists of a S_TKc domain (serine/threonine kinases, catalytic domain), but lacks several residues that are indispensable for intrinsic catalytic activity. Evidence obtained in the present study demonstrated the existence of four other splice isoforms of human PXK in fetal brain, designated as PXK_v2, PXK_v3, PXK_v4 and PXK_v5. The results of RT-PCR indicated that human PXK_v1, PXK_v2 and PXK_v4 transcripts were widely expressed in human adult tissue, except heart tissue. In contrast, PXK_v3 transcripts were only expressed in peripheral blood leukocytes at a low level and PXK_v5 transcripts were not detectable in any of the tissue analyzed. Subcellular localization analysis of EGFP-PXK fusion proteins in COS7 cells indicated that EGFP-PXK_v3 had a different subcellular localization compared to other EGFP-PXK fusion proteins. Mutation analysis of EGFP-PXK_v1 showed that PXK_v1-Tyr56 and Arg92 are essential for subcellular localization of the protein in the cytoplasm.

Alternative Splicing↗

PubMed1991

A morphometric comparison of subcellular structures between primary breast tumors and their nodal metastases.

A morphometric study of subcellular structures in human primary breast tumors (of the ductal infiltrating type) and their synchronous lymph node metastases was carried out using planimetry and stereology. Each primary neoplasm was compared separately with its axillary metastatic growth in order to detect differences in the quantitative estimates of their subcellular components. A set of 24 morphometric parameters was used to quantitatively describe each neoplastic cell profile. Although some subcellular parameters showed significant differences between the cells of a primary tumor and its metastases, no parameter was found to be consistently altered in all cases. No consistent increase or decrease in the values of the affected parameters was observed in the metastases. More than being due to the expression of a biologically variant (more-or-less differentiated) metastatic phenotype, the differences in stereologic parameters detected in metastatic cells appear to reflect the existence of different functional states of the same cell type.

Breast Neoplasms↗

PubMed2006

[Comparative proteome analysis of hepatoma cells at subcellular level].

The subcellular proteome strategy can complement the separation power of two-dimensional electrophoresis, a step for subcellular fractionation is introduced before electrophoresis is conducted, and more proteins will be displayed in two-dimensional gels. A comparative analysis of proteomic profiling of mitochondria and nuclei was conducted between hepatoma cell and hepatocyte, in order to find more information involved in cancer development. The cultured hepatoma cell line QGY-7703 and hepatocyte line LO2 were used as research models in this work. Subcellular fractionation for mitochondrion and nucleus were done by ultracentrifugation, then two-dimensional electrophoresis was applied to the separation of mitochondrial and nuclear proteins, imaging analysis for the selection of differentially-expressed protein spots, and MALDI-TOF-MS for the identification of proteins. 54 spots from electrophoresis gels were selected as differentially-expressed protein for MS analysis which resulted in identification for 22 proteins. Among the 22 differentially-expressed proteins, 17 show a up-regulated expression and 5 show a down-regulated expression. These differentially-expressed proteins found in this work have a wide coverage of functions which are related to energy metabolism, cytosheleton, protein biosynthesis, pre-mRNA splicing and processing, apoptosis regulation. These results imply that cancer cell has experienced a fundamental change in structure and metabolism pattern.

Carcinoma, Hepatocellular↗

PubMed1977

Subcellular distribution and properties of alkaline inorganic pyrophosphatase of maize leaves.

1. Studies on the distribution of alkaline inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) in the subcellular fractions of maize leaves showed that the enzyme is present in cytoplasm, chloroplasts and mitochondria. The activity observed in nuclei and microsomes may result from contamination with the mitochondrial fraction. 2. Alkaline pyrophosphatases from three subcellular fractions were purified by fractionation with (NH4)2SO4, followed by ion-exchange and gel-filtration chromatography, and by isoelectric focusing. Highly purified enzyme preparations, with specific activities ranging from 55 to 188 micronmoles/min/mg protein, were obtained. 3. All the enzymes exhibited the maximum activity at pH 8.5 and the Mg2+/PPi ratio of 5. They differed in electrophoretic mobility, pI, and susceptibility to urea and thermal denaturation. This indicates that they represent isoenzymes compartmentized in particular subcellular fractions.

Cell Fractionation↗

PubMed1976

Catabolism of canine apolipoprotein A-I: purification, catabolic rate, organs of catabolism, and the liver subcellular catabolic site.

The catabolic site(s) of plasma lipoproteins and apolipoproteins has not been established. We have purified one of the major apolipoproteins and explored its catabolic rate and its site of catabolism. Apolipoprotein (Apo) A-1 was purified by column chromatography of canine high-density lipoprotein (HDL) subfraction, HDL3, which was totally delipidated; the purified Apo A-1, molecular weight 28,000, had only one precipitin line to either anti-Apo A-1, or anti-Apo HDL3 serum. Apo A-I was then iodinated, its catabolic rate measured, and the various organs and the subcellular sites of liver involved in the catabolism were investigated. The T 1/2 of 125I-Apo A-I in HDL3 was 3.33 +/- 0.08 days and in plasma 3.52 +/- 0.17 days. The disappearance curve of radioactivity recovered in density fractions d less than 1.110 and d greater than 1.210 Gm. per milliliter was the same as that in HDL3, suggesting identical Apo A-I catabolism in each density class. The liver and kidney absorbed more radioactivity than did the spleen, heart, lung, and intestine. In subcellular fractions of liver, radioactivity was predominantly recovered in the light mitochondrial fraction. There was a statistically significant correlation between the acid phosphatase relative specific activity and the relative specific radioactivity in each subcellular fraction examined at several time intervals after the injection of 125I-Apo A-I. Direct organelle isolation and demonstration of labeled apoplipoprotein uptake indicate that liver lysosomes may play an important role in the catabolism of Apo A-I, as may those of the kidney.

Amino Acids↗

PubMed1979

Iron and the liver: subcellular distribution of iron and decreased microsomal cytochrome P-450 in livers of iron-loaded rats.

To understand better the intracellular iron distribution and metabolic consequences of chronic hepatic iron overload, rats were given large doses of iron dextran or ferric citrate intraperitoneally. They accumulated large quantities of iron within Kupffer cells and hepatocytes. The relative subcellular iron distributions were similar in controls and iron-loaded rats, despite a ten- to 20-fold difference in hepatic iron concentration. Electron microscopy of whole liver and subcellular particulate fractions suggested that iron was present in highest concentration in lysosomes, which were rendered more labile by its presence. Nevertheless, quantitative iron determinations on all subcellular fractions, obtained by two preparative methods, showed that most of the iron was present in the "soluble" fraction. The amount of iron in the "microsomal" fraction varied, depending on the techniques used for preparation of this fraction. Cytochrome P-450 and total heme concentrations were decreased 40% to 50% in microsomes isolated from iron-loaded livers.

Animals↗

PubMed1989

[Organization of enzymes on subcellular structures: relay at the surface].

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".

Cytoplasm↗

PubMed1986

Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing.

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.

Bone Marrow↗

PubMed1986

Action of insulin on the subcellular metabolism of polyphosphoinositides in isolated rat hepatocytes.

The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32Pi incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 32Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma-32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP.

Adenosine Triphosphate↗