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PubMed1988

c-myc protein in normal tissue. Effects of fixation on its apparent subcellular distribution.

The c-myc protein is thought to be a DNA-associated nuclear protein. However, immunohistochemical studies on normal or tumor tissues have shown conflicting findings on its subcellular distribution. By using various fixation procedures on cytospin preparations of HL60 cells, the authors found the subcellular distribution of the c-myc protein to be dependent on the method of fixation. When studying mouse tissues in frozen sections using a biotinylated monoclonal antibody against the c-myc protein, they found the protein to be widely distributed in various normal adult mouse tissues, in most cases localized to the nucleus. However, when these tissues were studied after formalin fixation and paraffin embedding, a loss of nuclear staining was observed concurrent with the appearance of c-myc protein immunoreactivity in the cytoplasm. It is concluded that immunohistochemical studies on the expression of this oncogene should take into consideration the effects of fixation when its subcellular distribution is being examined.

Animals↗

PubMed1985

Adrenochrome uptake and subcellular distribution in the isolated perfused rat heart.

Adrenochrome uptake and its subcellular distribution were examined using isolated perfused rat heart preparation. The heart was perfused for 30 min with a medium containing 1 to 50 mg/l of 14C-adrenochrome and the subcellular fractions were isolated to measure their radioactivities. A decline in contractile force, a rise in resting tension and an increase in adrenochrome uptake by the heart were seen to depend upon the time of perfusion and the concentration of adrenochrome in the medium. The sarcolemmal fraction had the highest uptake of adrenochrome and this was followed by the microsomal fraction; some accumulation of adrenochrome was also observed in the myofibrillar and mitochondrial fractions. Either 10 or 20 min reperfusion of the heart previously exposed to 25 mg/l of adrenochrome, resulted in approximately 50 or 37% of the radioactivity remaining in the heart; this indicates irreversible binding of adrenochrome to the tissue. Reperfusion of the heart showed restoration of the resting tension but the contractile force did not show any recovery. Propranolol and iproniazid, which have been shown to inhibit the adrenochrome induced cardiotoxicity, reduced adrenochrome uptake by the heart, and prevented adrenochrome-induced depression in contractile force and rise in resting tension. These results indicate that adrenochrome is taken up by the heart and induces cardiac disturbances through its action on different subcellular organelles in the myocardium.

Adrenochrome↗

PubMed1985

Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue.

The endogenous concentrations and subcellular distribution of estrone and estradiol were measured in malignant and nonmalignant human breast tissue from pre- and postmenopausal women. The most striking finding was the significantly higher concentration of estradiol per g of tissue in the malignant tissues than in the nonmalignant tissues. The tissue concentrations of estradiol in pre- and postmenopausal women were similar despite the large differences in the peripheral plasma levels. No correlation was found between the estradiol receptor content and endogenous concentration and subcellular distribution of estradiol. No difference in the estrone tissue concentration was found between malignant and nonmalignant tissues. In comparison with human uterine tissues, which we have reported previously, human breast tissue "handles" estrogenic hormones differently from human uterine tissue. At equal concentrations of the estradiol receptor, concentrations and subcellular distribution of the estrogens are different in both tissues. It is concluded that the mechanism of action of estradiol via its receptor, a mechanism mainly based on studies in animal uterine tissue, applies only qualitatively to human breast cancer tissue.

Adolescent↗

PubMed1969

Lipid composition of subcellular particles of human blood platelets.

Human platelets can be fractionated into three main subcellular components: granules, membranes, and a soluble fraction. In this study we determined the phospholipid and neutral lipid content of the granules and membranes. Quantitative relationships between lipids and protein were examined. The fatty acid and aldehyde composition of individual phospholipids and neutral lipids was also determined. Whole platelets had a lower lipid to protein ratio than did the subcellular particles, but the basic lipid composition of the granules, membranes, and platelets was similar. The phospholipid composition of platelets and subcellular fractions was found to differ only in that granules had a lower percentage of lecithin. Each of the phospholipid classes displayed a distinctive fatty acid pattern which was the same in all fractions and in whole platelets. The major neutral lipid was free cholesterol. Cholesteryl esters, triglycerides, and free fatty acids were minor components. The molar ratio of cholesterol to phospholipid in the platelet membranes was lower than that of brain myelin and erythrocyte ghosts. Some differences in fatty acid composition of the neutral lipids of platelet fractions were found. A special lipid composition or constituent that would correlate with platelet function has not been found.

Aldehydes↗

PubMed1966

The intracellular distribution of sterols in Eurycotis floridana and its possible relation to subcellular membrane structures.

The roach Eurycotis floridana was reared on a semisynthetic diet containing minimal cholesterol-4-(14)C (0.005%) supplemented with cholestanol-7alpha-(3)H (0.1%), and various tissues of the insect were separated into "nuclear," "mitochondrial," and "microsomal" fractions by differential centrifugation. Muscle and salivary gland were satisfactorily fractionated and results from these are reported together with some, as yet, incomplete results for nerve. The major concentrations of unesterified sterol and lipid phosphorus were in the mitochondrial and microsomal fractions. The unesterified sterols of the subcellular fractions were separated by alumina and gas-liquid chromatography. The emergent peaks corresponding to cholesterol, cholestanol, and Delta(7)-cholestenol (a metabolite of cholestanol) were assayed for (3)H and (14)C. No sterol was confined to any one subcellular fraction. The proportions of the three sterols were closely similar in all membranous components of the same tissue, but were different in different tissues. It is suggested that the different subcellular membranes of a given tissue contain similar repeating structural units in which the individual sterols occupy sterically characteristic spaces.

Animals↗

PubMedDate not supplied

[Distribution of gamma-hexachlorcyclohexane in subcellular fractions of the liver and brain].

The character of the gamma-HCCH distribution in the subcellular fractions of the liver and brain of albino rats with peroral threefold introduction of the compound in doses of 34 mg/kg (1/5 LD50) and in a dose of 1.7 mg/kg (1/100 LD50) for a space of 1, 3 and 6 months was investigated. It was found that gamma-HCCH penetrates the cells of the liver and brain and is non-uniformly distributed among subcellular fractions, viz. nuclear, mitochondrial and supernatant. Both in acute and chronic experiments the greatest amounts of the preparation were found in the nuclear fraction of the cell. The established features distinguishing the accumulation and distribution of the preparation in the subcellular fractions of the liver and brain explain the previously elicited biochemical and physiological shifts occurring in the organism after entrance of gamma-HCCH thereinto.

Animals↗

PubMed1983

Styrene oxide metabolism in rhesus monkey liver: enzyme activities in subcellular fractions and in isolated hepatocytes.

The activity and the kinetic behavior of expoxide hydrolase were studied in various subcellular fractions of rhesus monkey liver isolated by differential centrifugation. The purity of the fractions was estimated by morphometric electron microscopy. Hydrolase activity was measured by a specific radiometric assay with [7-3H]styrene oxide as substrate. All isolated subcellular fractions catalyzed the hydration of styrene oxide at a significant rate. With a saturating concentration of substrate (1 mM), the enzymatic activity (nanomole of product per minute per milligram of protein; mean +/- S.E.) turned out to be 1.51 +/- 0.45 (nuclear fraction), 3.50 +/- 1.11 (mitochondrial fraction), 14.8 +/- 2.26 (microsomal fraction) and 1.69 +/- 0.37 (soluble fraction). The hydrolase obeyed Michaelis-Menten kinetics in each fraction. Vmax (nanomole per minute per milligram; mean +/- S.E.) was 1.64 +/- 0.65 (nuclear fraction) 3.87 +/- 1.71 (mitochondrial fraction), 19.8 +/- 5.4 (microsomal fraction) and 2.72 +/- 1.36 (soluble fraction). The Km (millimole; mean +/- S.E.) values in the fractions were 0.09 +/- 0.02, 0.07 +/- 0.01, 0.23 +/- 0.15 and 0.64 +/- 0.40, respectively. The metabolism of styrene oxide was also studied in isolated hepatocytes from rhesus monkey. These cells hydrated the substrate easily whereas the conjugation of styrene oxide with glutathione was not measurable. Our results show that epoxide hydrolase is present in all subcellular fractions of the rhesus monkey liver. Styrene oxide is preferentially metabolized by hydration to styrene glycol in the isolated hepatocytes of this species and no conjugation with glutathione was found under the incubation conditions used.

Animals↗

PubMedDate not supplied

Subcellular distribution of inflammatory mediators in guinea-pig polymorphonuclear granulocytes (PMNL).

The experiments were performed for localizing the inflammation-inducing factors in the subcellular fractions of human and guinea pig PMNL. The magnitude of the induced inflammation tested on rats and guinea-pigs represented combined effects of all proteins extracted from PMNL. The isolation of PMNL subcellular components did not elevate the potency of the inflammatory response. The inflammation was completely abolished with SH-dependent protease inhibitor. The inflammation induced by PMNL factors is a complex phenomenon which could not be attributed to special subcellular components.

Animals↗

PubMed1982

Regional and subcellular distribution of biopterin in the rat.

Autoradiography: Intravenous (2--14C)-Tetrahydrobiopterin (BH4; 5 muCi, spec. act. 50 Ci/mol) is incorporated into liver and kidney of the rat. The gastrointestinal tract is labelled possibly due to biliary excretion of BH4 and its metabolites. BH4 and its metabolites obviously do not pass the blood brain barrier. Estimation of Crithidia active material shows a regional and subcellular distribution of the coenzyme. The highest levels were found in the liver (4.6 mumol/kg), and in spleen (2.3 mumol/kg). In the brain, highest coenzyme concentration is in the hypothalamic region with 0.49 mumol/kg as compared with 0.25 mumol/kg for total brain value. After subcellular fractionation, biopterins (expressed as Crithidia activity) are located mainly in the soluble fraction of liver (61.3 +/- 6.7) and kidney (89.1 +/- 14.0), whereas subcellular fractions of brain show additional higher percentage distribution in lysosomal (23.4 +/- 3.3) and microsomal (24.9 +/- 5.2) fractions due to their synaptosomal content as shown by density gradient centrifugation (values in brackets: percentage +/- SD).

Animals↗

PubMed1980

Subcellular fractionation of human intestinal mucosa by large-scale zonal centrifugation. Separation of the organelles in diseased ileum of a patient with Crohn's disease.

The subcellular organelles in diseased ileal mucosa of a patient with Crohn's disease have been separated by rate zonal density gradient centrifugation. Mucosal scrapings were obtained from a surgical specimen of diseased intestine and the cells disrupted by extrusion under controlled pressure. The homogenized cells were then centrifuged to prepare a cell extract and the extract fractionated in a single-step procedure by zonal centrifugation on a continuous sucrose gradient. The subcellular organelles (brush borders, basal-lateral membranes, lysosomes, mitochondria and endoplasmic reticulum) were located in the density gradient by assay of marker enzymes and their overall distribution compared with the distribution of organelles from a normal tissue. The centrifugal properties of individual organelles in the diseased tissue were largely unaffected but the lysosomes were more fragile than in the normal tissue. Since the fractionation technique used is on a large scale, each fraction of interest can be further analyzed for a variety of compounds. Therefore, the subcellular pathology and pathophysiology in this and other cases of Crohn's disease may eventually be studied and discussed in other than morphological and descriptive terms.

Aged↗

PubMed1995

Altered subcellular location of an activated and tumour-associated epidermal growth factor receptor.

The epidermal growth factor (EGF) receptor is a membrane bound tyrosine kinase whose activity is initiated by ligand binding. The malignant brain tumour glioblastoma frequently shows amplification and rearrangements of the EGF receptor gene that are associated with the synthesis of a constitutively activated tyrosine kinase, lacking amino acids 6-273 near the protein's N-terminus. When expressed in Chinese hamster ovary (CHO) cells, this mutant receptor (p140EGFR) displays ligand-independent tyrosine kinase activity, stimulates DNA synthesis, and promotes cell proliferation. Here, we investigate the subcellular location of p140EGFR in CHO cell transfectants as well as in human glioblastoma tumours. p140EGFR had an intracellular location that contrasted sharply with the plasma membrane location of the wild-type EGF receptor. Endoglycosidase H sensitivity analysis and the pattern of p140EGFR immunoreactivity suggested that the aberrant tyrosine kinase resided primarily in the endoplasmic reticulum. The half-life of p140EGFR in the endoplasmic reticulum was extended several-fold over that of the ligand-activated wild-type receptor. The altered subcellular location of p140EGFR in combination with its prolonged half-life suggest that this activated tyrosine kinase may escape the regulatory mechanisms utilized for the attenuation of wild-type receptor signaling. Therefore, the previously reported growth stimulatory property of the ligand-independent p140EGFR may be attributed to a sustained tyrosine kinase activity resulting from an altered subcellular location.

Animals↗

PubMed1994

Subcellular localization of p-boronophenylalanine-delivered boron-10 in the rat 9L gliosarcoma: cryogenic preparation in vitro and in vivo.

A well-characterized in vitro cryogenic preparation for ion microscopic isotope imaging, which minimizes redistribution of diffusible species, was used to determine the distribution of boron in GS-9L gliosarcoma cells incubated with the boron neutron capture therapy agent, p-boronophenylalanine (BPA). At the subcellular level, boron from BPA distributes relatively homogeneously within the glioma cell. Boron from BPA was eliminated rapidly, indicating that most is unbound. Thus a large pool of boron is susceptible to diffusion artifact. Removal of this artifact increases the degree of confidence in microdosimetric results inferred from the homogeneous subcellular distribution. The ion microscopic imaging of boron in subcutaneous tumors cryofixed in situ was achieved in rats treated with BPA. Boron signals from BPA were adequate to image microdistributions at the 1-micron resolution level. As in the in vitro case, boron did not localize discretely at the subcellular level. However, boron heterogeneity was seen at the tissue level. Physiologically valid cellular potassium and sodium levels were seen, which demonstrates minimized redistribution artifact. Future tissue studies designed to correlate ion microscopic boron images to microscopic structure are feasible using cryogenic sample preparation and ion microscopy.

Animals↗

PubMed1994

Subcellular localization of peripheral benzodiazepine receptors on human leukocytes.

BACKGROUND: The peripheral-type benzodiazepine receptor (PBR) was initially identified in many peripheral tissues and in some blood cells. Drugs that bind with high affinity to PBRs have previously been described as having immunomodulating properties. The number of PBRs varies according to the cell population considered. The aim of this study was to study the localization of PBRs in two human leukocyte populations, T4-lymphocytes, and monocytes. EXPERIMENTAL DESIGN: Both cell populations were purified by negative immunoselection in order to keep only the physiologically accessible sites on the viable cells. Mitochondria were quantified by electron microscopy and flow cytometric analysis. Subcellular localization was then studied after PBR photoaffinity labeling using electron microscopic ultrastructural autoradiography. RESULTS: We have shown that monocytes contain twice as many mitochondria as lymphocytes. We have also shown that the global labeling of monocytes by ultrastructural autoradiography is actually higher than that of lymphocytes and the labeling of monocyte mitochondria is higher than that of lymphocyte mitochondria. In addition, the distribution of subcellular labeling indicates that there are different populations of mitochondria in one cell, i.e., labeled and unlabeled, and that the percentage of labeled mitochondria is greater in monocytes. These results are consistent with those obtained in previous binding studies. Finally, over 50% of receptors are localized in cell compartments devoid of visible mitochondria. CONCLUSIONS: The subcellular distribution of the PBR shows that this receptor could have other physiologic functions towards immune cells than a function associated with mitochondria.

Autoradiography↗

PubMed1993

High level O-acetylation of sialic acids on N-linked oligosaccharides of rat liver membranes. Differential subcellular distribution of 7- and 9-O-acetyl groups and of enzymes involved in their regulation.

O-Acetylation of sialic acids has previously been considered an uncommon modification found on certain salivary mucins and neural gangliosides. We show here that glycosidically bound sialic acids from total membranes of rat liver have surprisingly high levels (approximately 20%) of O-acetylation at the 7- or 9-position. This O-acetylation is further enriched in N-linked oligosaccharides but is barely detectable in ganglioside fractions from the same tissue. The position of O-acetylation on the sialic acid side chain varies between different subcellular fractions. In particular, 7-O-acetylation was enriched in lysosomal membranes and 9-O-acetylation in plasma membranes, whereas Golgi membranes contained both types. This distribution fits with the ability of the rat liver sialate: O-acetyltransferase(s) to synthesize both 7- and 9-O-acetyl esters (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426) and the fact that 7-O-acetyl esters can migrate to the 9-position at physiological temperature but only under neutral or mildly alkaline conditions. Subcellular fractionation shows that sialate:O-acetyltransferase activity directed toward endogenous acceptors is enriched in Golgi fractions, whereas an intralumenal sialic acid-specific O-acetylesterase activity is not. The O-acetyltransferase is labile and difficult to solubilize in the intact state and cannot be assayed with exogenous acceptors. However, a prelabeled [3H]acetyl intermediate can be solubilized from Golgi membranes with Triton X-100 and is stable for a prolonged time in the cold. In contrast to the transferase, the lumenal esterase is easily released in a stable and water-soluble form from membrane fractions by saponin permeabilization or repeated freeze-thaw. In keeping with this finding, differential subcellular fractionation and continuous sucrose gradients indicate that this enzyme is enriched in lysosomal fractions (see also the accompanying paper (Butor, C., Higa, H. H., and Varki, A. (1993) J. Biol. Chem. 268, 10207-10213). Based upon findings reported in this and previous studies, a model is proposed for the biosynthesis, maturation, and turnover of 7- and 9-O-acetyl esters on the sialic acids of N-linked oligosaccharides that are attached to membrane-bound proteins in the rat liver.

Acetyl Coenzyme A↗

PubMed1996

Subcellular localization of the Arg protein tyrosine kinase.

Arg and c-Abl are ubiquitously expressed proteins which represent the mammalian members of the Abelson family of nonreceptor protein tyrosine kinases (PTKs). In contrast to most nonreceptor PTKs, c-Abl is located in the nucleus and cytoplasm. In this study the subcellular localization of Arg was examined to gain insight into its possible functions. Using indirect immunofluorescence, Arg was detected in the cytoplasm but not the nucleus of both transiently transfected COS cells and stable NIH3T3 transfectants. Arg is expressed as IA and IB isoforms which bear alternative amino termini, with the IB isoform containing a potential myristic acid acceptor sequence. Modification of ArgIB by myristic acid was confirmed by the detection of [3H]myristic acid labeling and the observation that labeling was abolished by mutating Gly-2. These results indicate that the subcellular localization of Arg is distinct from that of c-Abl and thus suggest that the two proteins have different functions in intracellular signaling. In addition, Arg, like c-Abl, is expressed as myristoylated and nonmyristoylated isoforms, suggesting that it may have dual cytoplasmic subcellular localizations, and possibly participate in diverse signaling pathways.

3T3 Cells↗

PubMed1996

Immunohistochemical distribution and electron microscopic subcellular localization of the proteasome in the rat CNS.

The proteasome multicatalytic proteinase (MCP) is a 20S complex that plays a major role in nonlysosomal pathways of intracellular protein degradation. A polyclonal antibody against rat liver MCP was used to investigate the distribution of MCP in the CNS of the rat and its subcellular localization within the neurons. As expected, MCP immunoreactivity (MCP-IR) was distributed ubiquitously in the rat CNS but not homogeneously. The most intensely stained neurons were the pyramidal cortical neurons of layer 5 and the motor neurons of the ventral horn in the spinal cord, which show an intense nuclear and cytoplasmatic MCP-IR and clearly stained processes. Additionally, some populations of large neurons in the mesencephalon and brainstem also displayed a moderate MCP-IR in their perikarya. The vast majority of neurons in the remaining structures did not show a strong cytoplasmatic MCP-IR, but their nuclei displayed an intense MCP-IR. The subcellular localization also was studied by immunoelectron microscopy. MCP-IR was intense in the neuronal nuclei, and significant staining also was found in the cytoplasm, dendritic, and axonic processes (including some myelinated axons) and in synaptic boutons, as illustrated in the cerebellar cortex. The distribution of MCP in the rat CNS and its subcellular localization are discussed in relation to (1) the distribution of calpain, the other major nonlysosomal cellular protease, and (2) the possible role of MCP in the degradation of regulatory proteins and key transcription factors that are essential in many neuronal responses.

Animals↗

PubMed1996

[Protective effect of fructus gardeniae on pancreatic subcellular organs of the acute pancreatitis in rats].

The acute pancreatitis (AP) was induced with sodium deoxy-cholate in SD rats. Succinic dehydrogenase (SDH), acid phosphatase (ACP) and cytochrome P450 were taken as marked enzyme in estimation of mitochondria, lysosome and microsome to observe the functional and structural changes of the pancreatic subcellular organs and the protective effects of Fructus Gardeniae (FG) in acute pancreatitis in rats. The histological change was observed simultaneously. The results showed that pancreatic cellular SDH reduced significantly and the releasing rate of ACP raised obviously in AP, in FG treated group, SDH was nearly the same as in normal, the releasing rate of ACP decreased and the content of P450 increased in the same time. Histologic observation showed that there was significant inflammatory change in pancreas and abnormal manifestation in structure of subcellular organs in AP, FG Could alleviate the lesion. These results suggested the FG has a remarkable protective effect on the function and structure of the pancreatic subcellular organs in AP.

Acute Disease↗

PubMed1997

High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos.

We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 microns thick. Such a flattened whole mount allows imaging with a spatial resolution of approximately 500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10,000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.

Animals↗