SEARCH · PubMed Health

Results for “subcellular”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources

PubMed2004

A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules.

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.

Actins↗

PubMed2004

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences.

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.

3T3 Cells↗

PubMed2004

Nuclear shuttling and TRAF2-mediated retention in the cytoplasm regulate the subcellular localization of cIAP1 and cIAP2.

Dynamic subcellular localization is an important regulatory mechanism for many proteins. cIAP1 and cIAP2 are two closely related members of inhibitor of apoptosis (IAP) family that play a role both as caspase inhibitors and as mediators of tumor necrosis factor (TNF) receptor signaling. Here, we report that cIAP1 and cIAP2 are nuclear shuttling proteins, whose subcellular localization is mediated by the CRM1-dependent nuclear export pathway. Blocking export with leptomycin B induces accumulation of both endogenous cIAP1 and epitope-tagged cIAP1 and cIAP2 in the nucleus of human cancer cells. We have identified a new CRM1-dependent leucine-rich nuclear export signal (NES) in the linker region between cIAP1 BIR2 and BIR3 repeats. Mutational inactivation of the NES, which is not conserved in cIAP2, reduces cIAP1 nuclear export. Forced relocation of cIAP1 to the nucleus did not significantly alter its ability to prevent apoptosis. Interestingly, co-expression experiments showed that the cIAP1 and cIAP2-interacting protein TNF receptor-associated factor 2 (TRAF2) plays an important role as regulator of IAP nucleocytoplasmic localization, by preventing nuclear translocation of cIAP1 and cIAP2. TRAF2-mediated cytoplasmic retention of cIAP1 was reduced upon TNFalpha treatment. Our results identify molecular mechanisms that contribute to regulate the subcellular localization of cIAP1 and cIAP2. Translocation between different cell compartments may add a further level of control for cIAP1 and cIAP2 activity.

Active Transport, Cell Nucleus↗

PubMed2003

Characterization of proteins localized to a subcellular compartment associated with an alternate secretory pathway of the malaria parasite.

Monoclonal antibodies recognizing proteins localized to a unique subcellular compartment within the malaria parasite are described in this report. These monoclonal antibodies recognize Plasmodium falciparum proteins of 68, 45 and 22 kDa proteins which are also conserved in rodent Plasmodium species. Co-localization studies indicate that these proteins are located in a brefeldin A-induced compartment which was previously proposed to be an early step in the export of proteins from the parasite into the infected erythrocyte. COPII coat proteins, Sar1p and Sec31p, and the endoplasmic reticulum-associated chaperone, BiP, all partially co-localize with the 68 and 22 kDa proteins, thus suggesting that this subcellular compartment has some similarities to the endoplasmic reticulum or that this compartment represents a domain of the endoplasmic reticulum. The 68 and 22 kDa proteins are highly soluble in non-ionic detergent and are likely to be located within the lumen of a membrane-bound compartment. These proteins found within this subcellular compartment are present throughout the blood stage from very early rings to segmenters. The results of this study further substantiate the existence of an alternate secretory pathway in the malaria parasite which plays a role in the export of proteins into the host erythrocyte.

Animals↗

PubMed1986

Postobstructive subcellular organelle and biliary lipid composition in the rat. A selective increase in biliary lecithin output is not reflected by changes in organelle composition.

Rats infused intravenously with taurocholate shortly after relief of 48 h of biliary obstruction excrete in bile twice as much phospholipid in relation to cholesterol and bile acid as controls. In an effort to identify a subcellular compartment as the source of this biliary lipid, we examined several hepatic subcellular organelles for an increase in phospholipid to cholesterol ratio or an increase in biliary-type lecithins (16:0-18:2; 16:0-20:4) either absolute or relative to non-biliary-type lecithins (18:0-20:4; 18:0-22:6) as analyzed by high-performance liquid chromatography. Subcellular fractions studied were: three microsomal subfractions, Golgi heavy and intermediate fractions, and plasma membrane light (canaliculus-enriched) fraction. No organelle fraction from rats with biliary obstruction displayed either a significant increase in phospholipid to cholesterol ratio or a relative or absolute increase in biliary type lecithin. This suggests that biliary lipid changes are not attributable to measurable alterations in lipid composition of any anatomical compartment in the liver cells; changes are probably more related to changes in lipid turnover than to organelle total lipid pool sizes.

Animals↗

PubMed1997

Influence of dietary fat on the activities of subcellular membrane-bound enzymes from different regions of rat brain.

The effect of different dietary fats with varying degrees of unsaturation and essential fatty acid composition, which are commonly consumed in India, on the activity of some important membrane-bound enzymes was assessed in different brain regions of rat. Four groups of male CFY weanling rats were fed nutritionally adequate diets containing groundnut, coconut, safflower or mustard oil as fat source at 20% level for 16 weeks. The synaptosomal, microsomal and myelin membranes were prepared from three brain regions, viz., cerebrum, cerebellum and brain stem from each group. The activities of Na+, K(+)-ATPase, Mg(2+)-ATPase and acetylcholinesterase were assayed and the fatty acid composition was determined in these subcellular membrane fractions. The safflower oil-fed group showed higher Na+, K(+)-ATPase activity in most membrane fractions than the coconut or mustard oil-fed groups. The Mg(2+)-ATPase activity was found to be similar amongst all groups in all the brain regions. The synaptosomal acetylcholinesterase activity was distinctly higher in coconut and groundnut oil-fed groups when compared to safflower or mustard oil consuming groups. Alterations in the activities of these subcellular membrane-bound enzymes are expected to exert a significant impact on the electrophysiological and metabolic functions of brain. Results of the present study show that depending on the nature of dietary fat the fatty acid composition of subcellular membranes is altered, which in turn could regulate the activity of membrane-bound enzymes that are vital for brain function.

Acetylcholinesterase↗

PubMed1998

Independent assembly and subcellular targeting of GABA(A)-receptor subtypes demonstrated in mouse hippocampal and olfactory neurons in vivo.

The ability of neurons to display more than a single GABA(A)-receptor subtype per cell requires intricate targeting mechanisms. Analysis by confocal laser scanning microscopy revealed that the alpha2- and alpha5-subunits differed strikingly in their subcellular distribution in hippocampal pyramidal cells and olfactory bulb granule cells, while the distribution of the gamma2-subunit was rather uniform. In mutant mice lacking the alpha5-subunit gene due to a chromosomal deletion, the absence of the alpha5-subunit was accompanied by a corresponding decrease of the gamma2-subunit immunoreactivity. In striking contrast, the subcellular distribution of the alpha2-subunit was unchanged in these mutant mice. These findings indicate that the assembly of distinct GABA(A)-receptor subtypes in the same neuron is regulated independently. Furthermore, the alpha-subunit is a prime candidate for providing domains which direct subcellular targeting.

Animals↗

PubMed1999

Subcellular fractionation, electromigration analysis and mapping of organelles.

Subcellular fractionation has provided the means required to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation has helped to define membrane boundaries and became necessary for the development of cell-free assays that reconstitute complicated cellular processes. Although cell fractionation techniques have improved over the last decades the purification of organelles to homogeneity is still a barely accessible goal in cell biology. In this article, we will first briefly review the basic principles of subcellular fractionation, and the establishment of different organelle fractions by density centrifugation, using tissue culture cells as a paradigm. Then we will discuss some of the intrinsic problems and will compare gradient purification of cellular extracts with electromigration analysis. Finally, we will describe alternative approaches, such as immunoisolation and flow cytometry to purify organelles from tissue culture cells.

Cell Fractionation↗

PubMed2003

Analysis of subcellular calcium signals in T-lymphocytes.

Subcellular Ca(2+) signals were analysed in Jurkat and peripheral human T-lymphocytes by confocal Ca(2+) imaging employing an off-line deconvolution method. Stimulation of the TCR/CD3 complex in T-lymphocytes resulted in a series of subcellular pacemaker Ca(2+) signals preceding the first global Ca(2+) signal. The pacemaker signals occurred in a cytosolic "trigger" zone, which is localised close to the plasma membrane. The pacemaker signals were almost independent of extracellular Ca(2+) as shown by measurements in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel blocker SK-F 96365. Analysis of the confocal Ca(2+) images revealed characteristic amplitudes of 82 +/- 30 to 109 +/- 21 nM, signal diameters between 2.5 +/- 0.9 and 3.5 +/- 1.5 microm and frequencies between 0.235 and 0.677 s(-1). Taken together, our data constitute the first analysis of subcellular Ca(2+) signals in T cells and indicate that the pacemaker Ca(2+) release events, which are necessary for the development of the global Ca(2+) signal, are composed of Ca(2+) release both from inositol 1,4,5-trisphosphate- and ryanodine receptors.

Calcium↗

PubMed2000

In vivo subcellular elemental dynamics in liver graft: With special reference to effect of non-selective endothelin receptor antagonist, TAK-044, on the graft injury.

Background: No data are available concerning the in vivo subcellular dynamics of elements in liver grafts and the effect of endothelin receptor antagonist, TAK-044, against graft injury. Methods: Liver transplantation was performed in porcine under active veno-venous bypass. The grafts stored in chilled preservation solution were recirculated following reflush with lactated Ringer's solution with or without TAK-044 (10 mg/kg). Cold and warm ischemic times of the grafts were comparable between the two groups. Elements (Na, K, Cl, Ca, P and S) were measured in three fractions of cytoplasm, mitochondria and nucleus by electron probe X-ray microanalysis for the graft biopsy specimens obtained at various time from donor laparotomy to 1 week after liver grafting. Liver functions also were compared between the two groups. Results: In both groups, concentration of each element changed in parallel among the three subcellular fractions and their changes were less marked in the nucleus. In the control group, there were significant increases in cytoplasmic Na and Cl after portal reperfusion and in cytoplasmic and mitochondrial Ca after hepatic artery reperfusion. These were accompanied by K and mitochondrial S decreases without a statistical significance. In the TAK group, such postreperfusion elemental alterations were significantly suppressed and early deterioration of the liver functions was alleviated, as compared with the control group. Conclusion: A supplemental use of TAK-044 in a rinse solution before reflush contributed to stability of subcellular elements after reperfusion and better preservation of early graft function.

Journal Article↗

PubMed2006

PredSL: a tool for the N-terminal sequence-based prediction of protein subcellular localization.

The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein's function. We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups: chloroplast, thylakoid, mitochondrion, secretory pathway, and "other". When tested in a five-fold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL:http://bioinformatics.biol.uoa.gr/PredSL/.

Algorithms↗

PubMed2006

Predicting the subcellular localization of human proteins using machine learning and exploratory data analysis.

Identifying the subcellular localization of proteins is particularly helpful in the functional annotation of gene products. In this study, we use Machine Learning and Exploratory Data Analysis (EDA) techniques to examine and characterize amino acid sequences of human proteins localized in nine cellular compartments. A dataset of 3,749 protein sequences representing human proteins was extracted from the SWISS-PROT database. Feature vectors were created to capture specific amino acid sequence characteristics. Relative to a Support Vector Machine, a Multi-layer Perceptron, and a Naive Bayes classifier, the C4.5 Decision Tree algorithm was the most consistent performer across all nine compartments in reliably predicting the subcellular localization of proteins based on their amino acid sequences (average Precision=0.88; average Sensitivity=0.86). Furthermore, EDA graphics characterized essential features of proteins in each compartment. As examples, proteins localized on the plasma membrane had higher proportions of hydrophobic amino acids; cytoplasmic proteins had higher proportions of neutral amino acids; and mitochondrial proteins had higher proportions of neutral amino acids and lower proportions of polar amino acids. These data showed that the C4.5 classifier and EDA tools can be effective for characterizing and predicting the subcellular localization of human proteins based on their amino acid sequences.

Algorithms↗

PubMedDate not supplied

System dynamics of subcellular transport.

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Identification of subcellular energy fluxes by P NMR spectroscopy in the perfused heart: contractility induced modifications of energy transfer pathways.

The identification of subcellular fluxes of exchange of ATP, phosphocreatine (PCr) and Pi between mitochondria, cytosol and ATPases and pathways of energy transfer in a whole organ is a challenge specially in the myocardium where 50% of creatine kinases (CK) are found in close vicinity of ATP producing (mito-CK) and utilizing (MM-bound CK) reactions. To dissect their contribution in cardiac energy transfer we recently developed a new experimental 31P NMR spectroscopy approach. This led to identify three kinetically different subcellular CKs and to evidence experimentally the CK shuttle in a rat heart perfused in isovolumy. Here we show that a decreased energy demand alters energetic pathways : two CKs (cytosolic and MM-bound) functioning at equilibrium and a non mitochondrial ATP<-->Pi exchange was sufficient to describe NMR data. Mito-CK fluxes was not detected anymore. This confirms the dependence of energy pathways upon cardiac activity. Indeed the subcellular localization and activity of CKs may have important bioenergetic consequences for the in vivo control of respiration at high work: free ADP estimated from global CK equilibrium might not always adequately reflect its concentration at the ANT.

Adenosine Triphosphate↗

PubMed2006

Subcellular organization of GABAergic synapses: role of ankyrins and L1 cell adhesion molecules.

In vertebrate nervous systems, different classes of synaptic inputs are often segregated into restricted compartments of target neurons. For example, distinct types of GABAergic interneurons preferentially innervate subcellular domains and have been implicated in the precise temporal regulation of integration within neurons and activity within networks. Recent studies suggest that the subcellular segregation of different classes of GABAergic synapses is largely genetically determined. The localization and signaling of L1 family immunoglobulin proteins recruited by ankyrin-based membrane adaptors might serve as compartmental labels, which contribute to subcellular synapse organization in cerebellar Purkinje neurons.

Animals↗

PubMed2001

Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells.

The pool of cellular chelatable iron ('free iron', 'low-molecular-weight iron', the 'labile iron pool') is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8+/-2.6 microM within the cytosol and of at least 4.8 microM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6+/-2.9 microM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3+/-2.6 microM; nucleus, 11.8+/-3.9 microM; mitochondria, 9.2+/-2.7 microM); in addition, chelatable iron (approx. 16+/-4 microM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

Animals↗

PubMed2005

Expression and subcellular localization of the AQP8 and AQP1 water channels in the mouse gall-bladder epithelium.

BACKGROUND INFORMATION: Transepithelial transport of water is one of the most distinctive functions by which the gall-bladder rearranges its bile content. Water is reabsorbed from the gall-bladder lumen during fasting, whereas it is secreted into the lumen following meal ingestion. Nevertheless, the molecular mechanism by which water is transported across the gall-bladder epithelium remains mostly unclear. RESULTS: In the present study, we investigate the presence and subcellular localization of AQP (aquaporin) water channels in the mouse gall-bladder epithelium. Considerable AQP8 mRNA was detected in the gall-bladder epithelium of mouse, calf, rabbit, guinea pig and man. Studies of subcellular localization were then addressed to the mouse gall-bladder where the transcript of a second AQP, AQP1, was also detected. Immunoblotting experiments confirmed the presence of AQP8 and AQP1 at a protein level. Immunohistochemistry showed intense expression of AQP8 and AQP1 in the gall-bladder epithelial cells where AQP8 was localized in the apical membrane, whereas AQP1 was seen both in the apical and basolateral membranes, and in vesicles located in the subapical cytoplasm. CONCLUSIONS: The pattern of subcellular distribution of AQP8 and AQP1 strongly corroborates the hypothesis of a transcellular route for the movement of water across the gall-bladder epithelium. Osmotic water would cross the apical membrane through AQP8 and AQP1, although AQP1 would be the facilitated pathway for the movement of water across the basolateral membrane. The presence of two distinct AQPs in the apical membrane is an unusual finding and may relate to the membrane's ability both to absorb and secrete fluid. It is tempting to hypothesize that AQP1 is hormonally translocated to the gall-bladder apical membrane to secrete water as in the bile duct epithelium, a functional homologue of the gall-bladder epithelium, whereas apical AQP8 may account for the absorption of water from gall-bladder bile.

Amino Acid Sequence↗

PubMed1967

The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain.

Protein-bound N-acetylneuraminic acid and hexosamine, including the sialomucopolysaccharides, occur mainly in the least dense particles sedimented in the microsomal fraction from rat whole brain. Particles rich in protein-bound N-acetylneuraminic acid and hexosamine are also found in the subcellular fraction separated as a layer between 0.8m- and 1.2m-sucrose after centrifuging the crude mitochondrial preparation in a density gradient. This distribution is similar to that of the gangliosides and suggests an association of all of these substances in the same subcellular structures. It is postulated that the sialomucopolysaccharides, as well as the gangliosides, are components of cell membranes. Evidence is presented that indicates that there are quantitative differences between distribution of the gangliosides on the one hand, and protein-bound N-acetylneuraminic acid and hexosamine on the other. The ratio of protein-bound N-acetylneuraminic acid (and hexosamine) to gangliosidic N-acetylneuraminic acid (and hexosamine) present in individual subcellular fractions obtained by density-gradient centrifugation tends to increase with increasing particle density. Exposure of the crude mitochondrial fraction to osmotic ;shock' before density-gradient centrifugation causes a shift of the protein-bound N-acetylneuraminic acid and gangliosides to the less dense fractions. In some experiments, a selective shift of the protein-bound N-acetylneuraminic acid was observed.

Animals↗