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At least 1,243 records · Page 69Linked to original sources
PubMed2003

Subcellular localization and tumor-suppressive functions of 15-lipoxygenase 2 (15-LOX2) and its splice variants.

15-Lipoxygenase 2 (15-LOX2), the most abundant arachidonate (AA)-metabolizing enzyme expressed in adult human prostate, is a negative cell-cycle regulator in normal human prostate epithelial cells. Here we study the subcellular distribution of 15-LOX2 and report its tumor-suppressive functions. Immunocytochemistry and biochemical fractionation reveal that 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 we previously cloned, i.e. 15-LOX2sv-a/b/c, are mostly excluded from the nucleus. A potential bi-partite nuclear localization signal (NLS),203RKGLWRSLNEMKRIFNFRR221, is identified in the N terminus of 15-LOX2, which is retained in all splice variants. Site-directed mutagenesis reveals that this putative NLS is only partially involved in the nuclear import of 15-LOX2. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor AA-metabolizing activity. To our surprise, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. More importantly, when orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Together, these results suggest that both 15-LOX2 and 15-LOX2sv-b suppress prostate tumor development, and the tumor-suppressive functions apparently do not necessarily depend on AA-metabolizing activity and nuclear localization.

Arachidonate 15-Lipoxygenase↗
PubMed2003

Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system.

The acid-sensitive ion channel ASIC1 is a proton-gated ion channel from the mammalian nervous system. Its expression in sensory neurons and activation by low extracellular pH suggest that ASIC is involved in transmitting nociceptive impulses produced by the acidification caused by injury or inflammation. However, ASIC1 expression is not restricted to sensory neurons. To understand the functional role of ASIC1 in the CNS we investigated its expression and subcellular distribution therein. In particular, we examined the presence of ASIC1 in domains where the local pH may drop sufficiently to activate ASIC1 under physiological conditions. Immunostaining with specific antibodies revealed broad expression of ASIC1 in many areas of the adult rat brain including the cerebral cortex, hippocampus and cerebellum. Within cells, ASIC1 was found predominantly throughout the soma and along the branches of axons and dendrites. ASIC1 was not enriched in the microdomains where pH may reach low values, such as in synaptic vesicles or synaptic membranes. Pre- or postsynaptic ASIC1 was not gated by synaptic activity in cultured hippocampal neurons. Blockage or desensitization of ASIC1 with amiloride or pH 6.7, respectively, did not modify postsynaptic currents. Finally, the ontogeny of ASIC1 in mouse brain revealed constant levels of expression of ASIC1 protein from embryonic day 12 to the postnatal period, indicating an early and almost constant level of expression of ASIC1 during brain development.

Acid Sensing Ion Channels↗
PubMed2000

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Aging of human glyceraldehyde-3-phosphate dehydrogenase is dependent on its subcellular localization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.

Aging↗
PubMed1992

Cellular and subcellular localization of enzymes of arginine metabolism in rat kidney.

Rat kidneys extract citrulline derived from the intestinal metabolism of glutamine and convert it stoichiometrically into arginine. This pathway constitutes the major endogenous source of arginine. We investigated the localization of enzymes of arginine synthesis, argininosuccinate synthase and lyase, and of breakdown, arginase and ornithine aminotransferase, in five regions of rat kidney, in cortical tubule fractions and in subcellular fractions of cortex. Argininosuccinate synthase and lyase were found almost exclusively in cortex. Arginase and ornithine aminotransferase were found in inner cortex and outer medulla. Since cortical tissue primarily consists of proximal convoluted and straight tubules, distal tubules and glomeruli, we prepared cortical tubule fragments by collagenase digestion of cortices and fractionated them on a Percoll gradient. Argininosuccinate synthase and lyase were found to be markedly enriched in proximal convoluted tubules, whereas less than 10% of arginase and ornithine aminotransferase, were recovered in this fraction. Arginine production from citrulline was also enriched in proximal convoluted tubules. Subcellular fractionation of kidney cortex revealed that argininosuccinate synthase and lyase are cytosolic. We therefore conclude that arginine synthesis occurs in the cytoplasm of the cells of the proximal convoluted tubule.

Animals↗
PubMed2000

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine.

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like positive terminals were 60-85 nm in diameter. The present data provide immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.

Animals↗
PubMed1986

The effect of sodium thiosulfate on subcellular localization of platinum in rat kidney after treatment with cisplatin.

Female Sprague-Dawley rats were given 10 mg/kg of i.v. cis-diammine-dichloroplatinum(II) (cisplatin) simultaneously with i.v. sodium thiosulfate (STS) at a 200-fold molar excess to cisplatin, then subcellular (nuclei, mitochondria, microsomes, cytosol) distribution of platinum within rat kidney cells was determined 15 min, 1 h, 8 h and 24 h after cisplatin injection. Blood urea nitrogen levels were measured in rats treated in the same manner described above. STS was found to block cisplatin-induced nephrotoxicity. However, differences in platinum concentrations in total homogenate or each subcellular fraction between STS-treated rats and controls were not significant enough to fully account for the drastic protective effect of STS against cisplatin nephrotoxicity.

Animals↗
PubMed1997

Distribution and subcellular localization of motilin binding sites in the rabbit brain.

We previously reported the existence of motilin receptors in the cerebellum of the rabbit. We now explored the existence of motilin receptors in other brain regions and determined their association with neurons by subcellular fractionation studies. Autoradiographic studies with [(125)I]nle13-porcine motilin on rabbit coronal brain sections revealed specific binding sites in the hippocampus, thalamus, hypothalamus and amygdaloid body. Receptor binding studies allowed the identification of two binding sites. In all regions the density of the high-affinity binding site was lower than in the cerebellum, but its affinity was the same, except for the hypothalamus. No differences were found for affinity or density of the low-affinity receptor site. Homogenates of rabbit cerebellum were subjected to differential centrifugation. The highest motilin binding (10-times more than in the postnuclear supernatant) was found in the fraction which also showed maximal enrichment of [11-3H]saxitoxin binding (selective marker for voltage-sensitive Na+ channels), 6.9-fold, and cytochrome c oxidase activity (mitochondrial marker), 2.4-fold. In discontinuous sucrose density gradient centrifugation the motilin and saxitoxin binding both peaked in the 0.85-1 M layer, while cytochrome c oxidase was maximal in the 1.2 M layer. In conclusion, motilin receptors exist in several regions of the rabbit brain and are probably associated with synaptosomes. These findings further support a neurotransmitter role for motilin in the brain.

5'-Nucleotidase↗
PubMed1991

Subcellular localization of enzymes related to PAF metabolism in Krebs-II ascites cells: evidence for the lack of intracellular PAF transfer activity.

We have investigated various steps in the metabolism of Platelet-Activating Factor (PAF-acether or PAF) at the subcellular level in Krebs-II ascites cells. Microsomes contained an active acetyltransferase located on a heavy-rough domain of the endoplasmic reticulum quite rich in ribosomes, as monitored by [3H]uridine labelling, and which displayed a very high density across the Percoll gradient. This membrane domain, which was separated from all other cellular organelles including peroxisomes, also contained a membrane-bound acetylhydrolase with similar activity as the acetyltransferase. However most part of the cellular acetylhydrolase was located in the cytosol, which was actually devoid of PAF transfer activity, normally involved in transport of the mediator within the cell.

Acetyltransferases↗
PubMed2006

ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations.

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.

Adenosine Triphosphate↗
PubMed2003

Subcellular localization of Toll-like receptor 3 in human dendritic cells.

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Animals↗