Subcellular localization and internalization of the four human leptin receptor isoforms.

There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.     

Nuclear inclusions in the human adenohypophysis. A fine-structural study of nontumorous and adenomatous pituitaries.

Electron microscopy revealed the presence of nonviral nuclear inclusions in human nontumorous as well as adenomatous adenohypophysiocytes, regardless of cell type. Based on ultrastructural appearances, the inclusions have been classified as simple bodies, complex bodies and filamentous aggregates. Many transitional forms were noted between the simple and complex bodies, however, no relationship between the nuclear bodies and filamentous aggregates was found. It can be concluded that the three types of inclusions are normal nuclear constituents. Since no accumulation of these structures was observed in cells associated with enhanced secretion it appears that they are not related to hormonal hyperactivity in the human adenohypophysis.     

Double-labeling immunogold electron-microscopic study of hormonal colocalization in nontumorous and adenomatous rat pituitaries

According to the one cell, one hormone theory, the pituitary gland is composed of 5 cell types which secrete 6 hormones. Recent investigations indicate that this theory must be modified, as there are some bihormonal cells containing 2 hormones, i.e., mammosomatotrophs prolactin-growth hormone (PRL-GH). This study was undertaken in order to elucidate whether other adenohypophysial cells are capable of producing 2 hormones and to demonstrate the presence of cells coexpressing PRL-GH, PRL-thyrotropin (TSH), or TSH-GH. Sixteen nontumorous and 16 adenomatous male and female Sprague-Dawley and Long Evans rat pituitaries were removed immediately after the animals were killed and processed for transmission electron microscopy and the immunogold double-labeling technique. Coexpression of PRL-GH, PRL-TSH, and TSH-GH was found in both nontumorous and adenomatous pituitaries. Double labeling was present not only in the same cell cytoplasm but also in the same secretory granules. The question of whether these double-labeled cells represent different cell populations, transitional cell types, or precursor cells requires further investigation.     

Comparative immunoenzymatic localization of prolactin and growth hormone in human and rat pituitaries.

Twelve human and twelve rat pituitaries were stained by an immunohistochemical method using a rabbit anti-ovine prolactin serum, a rabbit anti-human growth hormone serum and a sheep anti-rabbit immunoglobulin serum conjugated with horseradish peroxidase. On the same pituitary section, growth hormone cells were stained brown by using 3-3'-diaminobenzidine as peroxidase substrate, and prolactin cells were stained purplish blue by using 4-chloro-1-naphtol. Growth hormone cells outnumbered prolactin cells, especially in human pituitaries where the proportion is at least 10:1. No cells containing both brown granules stained for growth hormone and blue granules stained for prolactin were found in any of the sections examined. In the fetal pituitaries, there was no apparent hypertrophy of the prolactin cells, although the circulating levels of the hromone are known to be as high in the fetus at term as in the mother and much higher than in nonpregnant women.     

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.     

Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.     

Subcellular localization of Gi alpha in human neutrophils

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.     

Subcellular localization of acetaldehyde dehydrogenase in human liver

The subcellular distribution of aldehyde dehydrogenase activity was determined in human liver biopsies by analytical sucrose density-gradient centrifugation. There was bimodal distribution of activity corresponding to mitochondrial and cytosolic localizations. At pH 9.6 cytosolic aldehyde dehydrogenase had a lower apparent Kappm for NAD (0.03 mmol l-1), than the mitochondrial enzyme (Kappm NAD = 1.1 mmol l-1). Also, the pH optimum for cytosolic aldehyde dehydrogenase activity (pH 7.5) was lower than that for the mitochondrial enzyme activity (pH 9.0), and the cytosolic enzyme activity was more sensitive to inhibition by disulfiram in vitro. Disulfiram (40 mumol l-1) caused a 70% reduction in cytosolic aldehyde dehydrogenase activity, but only a 30% reduction in mitochondrial enzyme activity after 10 min incubation. The liver cytosol may therefore be the major site of acetaldehyde oxidation in vivo in man.     

Subcellular localization of enterokinase in human small intestine

Enterokinase (enteropeptidase, EC 3.4.4.8) was found to be purified to the same extent as sucrase and alkaline phophatase when human intestinal brush border membrane was isolated. It is concluded that, in man as in other mammals, enterokinase activity occurs in close association with the brush border membrane.However, a second localization was also found. A fraction of the mucosal homogenate containing only small amounts of brush border but large amounts of endoplasmic reticulum, basolateral membranes and mitochondria (Fraction P₁) contained a disproportionately high amount of enterokinase. The enzyme in this particulate fraction occurred in a not fully active form.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

Subcellular localization and role of lipin-1 in human macrophages

The lipins have been described as metabolic enzymes that regulate lipid biosynthesis and also signaling processes by controlling the cellular concentration of bioactive lipids, phosphatidic acid, and diacylgycerol. In the present work we have studied the subcellular localization and role of lipin-1 in human monocyte-derived macrophages. Human macrophages express lipin-1 isoforms α and β. A transfected lipin-1α-enhanced GFP construct associates with membranes of cellular organelles that can be stained with Nile Red. Colocalization experiments with lipid droplet (LD)-specific proteins such as adipophilin/adipose differentiation-related protein/perilipin 2 or TIP47/perilipin 3 show that both proteins colocalize with lipin-1α in the same cellular structures. Reduction of the expression levels of lipin-1 by small interfering RNA technology does not impair triacylglycerol biosynthesis but reduces the size of LDs formed in response to oleic acid. In agreement with these data, peritoneal macrophages from animals that carry a mutation in the Lpin-1 gene (fld animals) also produce less and smaller LDs in response to oleic acid. Mass spectrometry determinations demonstrate that the fatty acid composition of triacylglycerol in isolated LDs from lipin-1-deficient cells differs from that of control cells. Moreover, activation of cytosolic group IVA phospholipase A(2)α, a proinflammatory enzyme that is also involved in LD biogenesis, is also compromised in lipin-1-deficient cells. Collectively, these data suggest that lipin-1 associates with LDs and regulates the activation of cytosolic group IVA phospholipase A(2)α in human monocyte-derived macrophages.     

Subcellular localization of hexadecanedioic acid activation in human liver

The activation of hexadecanedioic acid has been studied in subcellular fractions of human liver. The activation capacity in a total homogenate of human liver was found to be 0.5 micro mole/min/g wet wt of tissue, about 10% of that for palmitic acid. Hexadecanedioic acid was activated by the mitochondrial and microsomal fractions. The mitochondrial enzyme is probably localized outside the inner mitochondrial compartment. The subcellular distribution of the hexadecanedioic acid activation was almost identical with the distribution of palmitic acid activation. Hexadecanedioic and palmitic acids seemed to compete for the same enzyme.     

Subcellular localization of leukotriene receptors in human endothelial cells

Leukotriene B₄ (LTB₄), a potent chemotactic and immune-modulating mediator, signals via two receptors, BLT₁ and BLT₂. Recently, we reported that BLT₁ is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC), and that BLT₁ mediated functions are enhanced by LTB₄ and lipopolysaccharide (LPS), but not by TNFα. Here, we demonstrate that BLT₁ is found on the outer cell membrane of HUVECs but also in intracellular granules, co-localized with monocyte chemotactic protein-1 and P-selectin, but not with interleukin-8 and von Willebrand factor. Upon stimulation with LTB₄ or LPS, more BLT₁ protein is found, now evenly distributed over the cytoplasm and in the cell nucleus, but less on the cell surface. An MAP kinase inhibitor prevented this enhancement and translocation, suggesting this signaling pathway to be crucial. Thus, BLT₁, a G-protein-coupled 7-transmembrane receptor, is located in various subcellular compartments in endothelial cells, which may have implications for cellular LT dependent responses and target accessibility for BLT₁ antagonists.     

Subcellular localization of the human proto-oncogene protein DEK

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.     

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.     

Subcellular localization of annexin V in human foreskin fibroblasts: nuclear localization depends on growth state

Annexin V is a major intracellular calcium-binding protein in human foreskin fibroblasts. Immunocytochemistry revealed that annexin V was localized in the nucleus and throughout the cytoplasm in human foreskin fibroblasts. The presence of annexin V in the nucleus was variable depending on the growth state. Nuclear staining was strongest in proliferating cells immediately after sub-culture, and decreased on prolonged culture without changing the culture medium. The cytoplasmic location of annexin V was not greatly affected by the same conditions. Refeeding cells with fresh serum restored annexin V to the nuclei of all cells within in 24 h indicating that nuclear localization of annexin V is dependent on serum factors.     

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.     

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.