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PubMedDate not supplied

Subcellular localization of immunoreactive alpha-melanocyte stimulating hormone in human brain.

The regional and subcellular distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in the post mortem adult human brain was investigated. alpha-MSHi was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of alpha-MSHi were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of alpha-MSHi in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, alpha-MSHi was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of alpha-MSHi were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that alpha-MSH is a neuronal peptide in the human brain.

Brain↗

PubMedDate not supplied

Regional and subcellular localization of luteinizing hormone releasing hormone in the adult human brain.

The localization of luteinizing hormone releasing hormone (LHRH) in the post mortem adult human brain was investigated. LHRH was highly concentrated in medial basal hypothalamic tissue (1.14 ng/mg protein); lower levels of LHRH were present in tissue from the optic chiasm (0.05 ng/mg protein) and mammillary bodies (0.07 ng/mg protein). The concentrations of LHRH in hypothalamic tissue of men and women were similar. LHRH was undetectable (< 0.001 ng/mg protein) in the frontal cerebral cortex and cerebellum. When homogenates of the medial basal hypothalamus were fractionated on continuous or discontinuous sucrose density gradients, LHRH was found to be associated with subcellular particles. Upon examination by transmission electron microscopy, we found that these subcellular particles resembled isolated neuron terminals, i.e., synaptosomes. Low to undetectable amounts of LHRH were found in the cytosol or the myelin + microsome fraction of the gradients. The results of these studies are supportive of the view that LHRH is highly concentrated in neuron terminals of the adult human brain and may, therefore, be a central neurotransmitter or neuromodulator in the human.

Adolescent↗

PubMed1987

Subcellular, regional and immunohistochemical localization of adenosine deaminase in various species.

Immunohistochemical and subcellular fractionation techniques were employed to compare the cellular and subcellular localization of adenosine deaminase (ADA) in various brain regions of several mammalian species. A relatively restricted distribution of ADA-immunoreactive neurons in rat brain was previously reported. Mouse brain exhibited a pattern similar in many respects to rat and, in addition, contained intensely immunostained neurons in lateral habenula and hippocampus. Glial immunostaining was absent or very light in rat but evident in mouse. Prominent immunoreactive fibers and neurons were observed in hamster spinal cord and anterior hypothalamus, respectively. ADA-immunostaining in guinea-pig was localized to presumptive fibers in the superficial layers of spinal cord dorsal horn and to glial cells throughout the brain. Demonstration of specific immunostaining in rabbit was not possible. ADA activity was far more heterogeneously distributed in rat and most brain areas in guinea-pig and rabbit contained up to 5-fold and 10-fold higher levels of activity, respectively, compared with rat. Crude synaptosomal (P2) fractions of rat cortex contained a greater proportion of ADA activity than those of rabbit cortex. Within rat, relatively high activity was found in P2 fractions of whole hypothalamus, cerebellum, and hippocampus. ADA activity was greater in P2 fractions of rat anterior compared with whole hypothalamus and the greatest proportion of the enzyme in this fraction was localized to purified synaptosomes. The large variations in the activity and cellular location of ADA in the animals examined suggest species differences in mechanisms governing adenosine metabolism in brain and possible differences in the relationships between cellular metabolism, ADA and the neuroregulatory role of adenosine in the CNS.

Adenosine Deaminase↗

PubMed1991

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes of Trypanosoma cruzi.

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of L-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biochem. 192 (1990) 363-368).

Alanine Transaminase↗

PubMed1992

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi.

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.

Animals↗

PubMed1993

Subcellular and gel chromatographic distribution of manganese in the mouse brain: relation to the chemical form of chronically-ingested manganese.

The subcellular distribution of manganese and the binding characteristics of manganese to protein in the mouse brain were examined on G-75 Sephadex gel columns. Four manganese compounds were included at 2 g/kg in each food eaten by ddY mice for 12 months. The cerebral cortex manganese concentrations in the virtually insoluble manganese compounds were significantly higher than those in the control group. The brain striatal subcellular distribution and gel chromatographic profiles of manganese were similar among the divalent manganese compounds. On the contrary, the behaviour of MnO2 was little different from the divalent manganese compounds. There was more manganese associated with fast-migrating ligands in the striatal cytosol of the manganese-exposed group than in the control groups.

Acetates↗

PubMed1987

Gel electrophoresis of intact subcellular particles.

The review describes the application of gel electrophoresis to the characterization and separation of viruses, ribosomes, vesicles and other subcellular particles. The preparation of the sample, the choice of the buffer, the gel medium, the apparatus and the detection of the particle (staining and scanning) as well as the necessary theory are discussed. This includes the mathematical evaluation of experimental data on the basis of Ferguson plots using the extended Ogston theory. Simple methods and sophisticated computer simulation techniques are described and exemplified in application to the determination of particle size and charge, the pore size of the gel (unpublished data) and the two-dimensional agarose electrophoresis (unpublished). It is shown that the nature of the particle (e.g. spherical or rod-shaped, pliable or rigid texture) determines the shape of the non-linear Ferguson plot. In addition, the review gives a number of practical applications of gel electrophoresis, isoelectric focusing, titration curves and immuno-electrophoresis to subcellular particles. Pros and cons are evaluated. A comparison with other analytical procedures is made. The review is concluded by a futuristic outlook.

Animals↗

PubMedDate not supplied

Human liver aldehyde dehydrogenase: subcellular distribution in alcoholics and nonalcoholics.

Activity assay and isoelectric focusing analysis of human biopsy and autopsy liver specimens showed the existence of two major aldehyde dehydrogenases (ALDH I, ALDH II). Subcellular distribution of these isozymes was determined in autopsy livers from alcoholics and nonalcoholics. Nearly 70% of the total ALDH activity was recovered in the cytosol which contained both the major isozymes. Densitometric evaluation of isozyme bands showed that about 65% of the cytosolic enzyme activity was due to ALDH II and the rest due to ALDH I isozyme. Only about 5% of the total ALDH activity was found in the mitochondrial fraction (70% ALDH I and 30% ALDH II). Significantly reduced total and specific ALDH activities were noted in all the subcellular fractions from cirrhotic liver specimens. Apparently, ALDH I isozyme from cytosol and mitochondria is primarily responsible for the oxidation of small amounts of acetaldehyde normally found in the blood of nonalcoholics after drinking moderate amounts of alcohol. However, in alcoholics who exhibit higher blood acetaldehyde concentrations after drinking alcohol, ALDH II isozyme may be of greater physiological significance.

Alcohol Dehydrogenase↗

PubMed1992

Seasonal changes in subcellular distribution of zinc, copper, cadmium and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible essential role of cadmium and metallothionein in the hepatic metabolism of copper.

1. An increase in total Zn concentration in the liver of sexually mature bank voles in spring and summer was primarily accompanied by an increase in Zn levels in the nuclear and mitochondrial-lysosomal fractions, while in immature voles in an analogous situation, Zn tended to concentrate mainly in the post-mitochondrial fraction. 2. Seasonal changes in Cu concentration in all subcellular fractions followed closely those of total hepatic Cu; however, the mitochondrial-lysosomal Cu underwent the most dramatic changes. 3. Multiple regression analysis between the concentration of metallothionein (MT) in the postmitochondrial fraction and the levels of Zn, Cu and Cd in the particular subcellular fractions showed that MT was principally induced by small amounts of Cd concentration of which in the liver ranged from 0.019 microgram/g in winter to 1.1 micrograms/g wet wt in spring. 4. The analysis revealed further that the level of MT in the post-mitochondrial fraction concurrently decreased as total hepatic Cu, as well as mitochondrial-lysosomal Cu, increased. 5. The data indicate that MT, first induced by small amounts of Cd, is involved in the hepatic metabolism of Cu in bank voles; most probably the protein sequesters free Cu ions in cytoplasm and then transfer them into the mitochondrial-lysosomal fraction.

Animals↗

PubMed1990

Haloperidol-sensitive (+)[3H]SKF-10,047 binding sites (sigma sites) exhibit a unique distribution in rat brain subcellular fractions.

The distribution of haloperidol-sensitive (+)[3H]N-allylnormetazocine ((+)[3H]SKF-10,047) binding sites (sigma sites) in subcellular fractions of rat brain homogenates was extensively characterized. In synaptosomal fractions, enriched in choline acetyltransferase activity, sigma sites were present in lower concentrations than in whole brain homogenates. On the other hand, microsomal and myelin fractions were found to be enriched in sigma sites. A similar pattern of enrichment was seen for 5'-nucleotidase activity, a general plasma membrane marker. However, subsequent experiments in which microsomes were subfractionated on linear sucrose gradients led to the recovery of sigma sites over a significantly lower density range than 5'-nucleotidase activity or ATP-stimulated [3H]ouabain binding, an additional plasma membrane marker. In addition, previously reported distributions of a number of other subcellular markers, including those for endoplasmic reticulum, were found to contrast with the observed distribution of sigma sites. It is concluded that rat brain sigma sites are not concentrated at synaptic regions of plasma membrane. However, the possibility that sigma sites are localized to specialized areas of nonsynaptic plasma membrane cannot be excluded.

5'-Nucleotidase↗

PubMed1993

Subcellular fractionation of cultured normal human melanocytes: new insights into the relationship of melanosomes with lysosomes and peroxisomes.

In order to obtain information on the disputed nature of melanosomes a comparison was made between the localization of melanosomal markers with those of other well-defined subcellular organelles such as lysosomes and peroxisomes. The distribution of marker enzymes was studied using two different density gradient systems, i.e., Percoll and Nycodenz. Furthermore, the subcellular localization of various types of antigens was analyzed using indirect immunofluorescence and immuno-electron microscopy. All methods revealed the existence of partial co-localization of melanosomal and lysosomal proteins and different localization of peroxisomal markers. The results suggest that melanosomes may share a common origin with lysosomal structures.

Cell Fractionation↗

PubMed1994

The subcellular distribution of glucocorticoid-receptor complexes as studied by chemical crosslinking of intact HTC cells.

Treatment of intact HTC cells with glutaraldehyde results in redistribution of glucocorticoid binding sites between cytosolic and nuclear fractions. The decrease in cytosolic receptors and their accumulation at the nuclear level were found to be directly related to the glutaraldehyde concentrations employed in our procedure and inversely related to the cell density of samples. When the data from eleven separate experiments were combined, and analyzed by linear regression of cytosolic and nuclear levels of receptor complexes vs the ratios between the DNA and glutaraldehyde concentration of our samples, two lines were obtained whose intercepts on the ordinate yielded values of cytosolic and nuclear receptors corresponding to 37.5 and 62.5% of the total cellular pool, respectively. When we compared the subcellular redistribution of glucocorticoid receptor to that of the cytosolic enzyme lactate dehydrogenase upon HTC cell crosslinking with glutaraldehyde, we found that the cytosolic and nuclear levels of the enzyme were 53.2 and 46.8% of the total content, respectively. If the subcellular distribution of glucocorticoid receptor is corrected for the artefactual redistribution induced by crosslinking, using the values obtained for lactate dehydrogenase, it can be concluded that glucocorticoid receptors in HTC cells are distributed between cytosol and nuclei in a ratio which is about 2:1. Our findings lend further support to the conclusion that only a portion of glucocorticoid receptor is cytosolic in intact cells.

Cell Line↗

PubMed2003

A new hybrid approach to predict subcellular localization of proteins by incorporating gene ontology.

Based on the recent development in the gene ontology and functional domain databases, a new hybridization approach is developed for predicting protein subcellular location by combining the gene product, functional domain, and quasi-sequence-order effects. As a showcase, the same prokaryotic and eukaryotic datasets, which were studied by many previous investigators, are used for demonstration. The overall success rate by the jackknife test for the prokaryotic set is 94.7% and that for the eukaryotic set 92.9%. These are so far the highest success rates achieved for the two datasets by following a rigorous cross-validation test procedure, suggesting that such a hybrid approach may become a very useful high-throughput tool in the area of bioinformatics, proteomics, as well as molecular cell biology. The very high success rates also reflect the fact that the subcellular localization of a protein is closely correlated with: (1). the biological objective to which the gene or gene product contributes, (2). the biochemical activity of a gene product, and (3). the place in the cell where a gene product is active.

Algorithms↗

PubMed2004

Prediction of protein subcellular locations by GO-FunD-PseAA predictor.

The localization of a protein in a cell is closely correlated with its biological function. With the explosion of protein sequences entering into DataBanks, it is highly desired to develop an automated method that can fast identify their subcellular location. This will expedite the annotation process, providing timely useful information for both basic research and industrial application. In view of this, a powerful predictor has been developed by hybridizing the gene ontology approach [Nat. Genet. 25 (2000) 25], functional domain composition approach [J. Biol. Chem. 277 (2002) 45765], and the pseudo-amino acid composition approach [Proteins Struct. Funct. Genet. 43 (2001) 246; Erratum: ibid. 44 (2001) 60]. As a showcase, the recently constructed dataset [Bioinformatics 19 (2003) 1656] was used for demonstration. The dataset contains 7589 proteins classified into 12 subcellular locations: chloroplast, cytoplasmic, cytoskeleton, endoplasmic reticulum, extracellular, Golgi apparatus, lysosomal, mitochondrial, nuclear, peroxisomal, plasma membrane, and vacuolar. The overall success rate of prediction obtained by the jackknife cross-validation was 92%. This is so far the highest success rate performed on this dataset by following an objective and rigorous cross-validation procedure.

Algorithms↗

PubMed2004

Subcellular localization of the camptothecin analogues, topotecan and gimatecan.

Lipophilicity of camptothecins derivatives has been reported to improve the stability of the lactone ring and to favor rapid uptake and intracellular accumulation. Recently, a novel series of lipophilic camptothecins substituted at position 7 was developed, and gimatecan (ST1481) was selected for clinical development on the basis of some favorable features, including potent cytotoxicity and the unique feature of the lack of recognition by breast cancer resistance-associated protein (BCRP). In this work the intrinsic fluorescence properties of this compound were exploited to investigate its intracellular disposition in comparison with the water-soluble camptothecin, topotecan (TPT), in HT-29 colon carcinoma cells and in a subline, HT-29/Mit, selected for resistance to mitoxantrone and overexpressing BCRP. The study was performed at single-cell level by means of microspectrofluorometry and fluorescence image analysis. The results indicated a quite different subcellular localization of TPT ST1481, since TPT localized mainly in mitochondria, whereas gimatecan exhibited a lysosomal localization. An increased persistence of DNA damage in gimatecan-treated cells was consistent with the interpretation that lysosomes represent a store of active drug. In contrast to gimatecan, which showed a similar localization in HT-29 cells and in the mitoxantrone-resistant subline, the cellular pharmacokinetic of TPT was markedly influenced by overexpression of BCRP protein in the resistant subline. In conclusion, the present results indicating a quite different behavior of the two camptothecins suggest that, apart from intracellular accumulation, subcellular distribution plays a role in their cytotoxic potency and contributes to their pharmacological features.

Antineoplastic Agents↗

PubMed2004

Dynamics of interactions of photosensitizers with lipoproteins and membrane-models: correlation with cellular incorporation and subcellular distribution.

The incorporation and subcellular localization of photosensitizers are critical determinants of their efficiency. Here, we correlate these properties with the interactions of photosensitizers with membrane-models and low density lipoproteins (LDL) in acellular systems. Focus was given on dynamics aspects. Two amphiphilic photosensitizers, deuteroporphyrin (DP) and aluminum phthalocyanine sulfonated on two adjacent isoindole units (AlPcS2a) were selected. The phthalocyanine was bound to LDL with an overall association constant around 5 x 10(7)M(-1). Biphasic association kinetics was indicative of two types of sites. The release of the phthalocyanine into the bulk aqueous medium occurred within less than a second. A similar behavior was found previously for deuteroporphyrin although its affinity was somewhat higher (5.5 x 10(8)M(-1)). Both compounds were previously characterized by high affinity for membrane-models and quick exchange with the bulk solution. However, they strongly differed by their rate of transfer through the lipid bilayer, in the range of seconds for the porphyrin, several hours for the phthalocyanine. In the case of the porphyrin, fluorescence microscopy on human fibroblasts showed diffuse labeling with no significant modification of the distribution upon vectorization by LDL. In contrast, the phthalocyanine was localized in intracellular vesicles. Vectorization by LDL favored lysosomal localization although little effect was found on the overall uptake as shown by extraction experiments. The role of lipoproteins in the cellular localization of photosensitizers is significantly more important for photosensitizers not freely diffusing through bilayers. The dynamics of the interactions of photosensitizers with membranes appears as an important determinant of their subcellular localization.

Animals↗

PubMed2005

Bcl-2 mediated inhibition of erucylphosphocholine-induced apoptosis depends on its subcellular localisation.

The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2. Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood. To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/DeltaTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria. The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.

Apoptosis↗

PubMed2004

5-HT3A receptor subunits in the rat medial nucleus of the solitary tract: subcellular distribution and relation to the serotonin transporter.

The 5-hydroxytryptamine 3 (5HT3) receptor is a serotonin-gated ion channel implicated in reflex regulation of autonomic functions within the nucleus of the solitary tract (NTS). To determine the relevant sites for 5-HT3 receptor mediated transmission in this region, we used electron microscopic immunocytochemistry to examine the subcellular distribution of the 5HT3 receptor subunit A (5HT3A) in relation to the serotonin transporter (SERT) in the intermediate medial NTS (mNTS) of rat brain. The 5HT3A immunolabeling was detected in many axonal as well as somatodendritic and glial profiles. The axonal profiles included small axons and axon terminals in which the 5HT3A immunoreactivity was localized to membranes of synaptic vesicles and extrasynaptic plasma membranes. In dendrites and glia, the 5HT3A immunoreactivity was located on the plasma membranes or in association with membranous cytoplasmic organelles. The dendritic plasmalemmal 5HT3A labeling was prominent within and near excitatory-type synapses from terminals including those that resemble vagal afferents. The 5HT3A-labeled glial processes apposed 5HT3A-immunoreactive axonal and dendritic profiles, some of which also contained SERT. Terminals containing 5-HT3A and/or SERT were among those providing synaptic input to 5HT3A-labeled dendrites. Thus, 5HT3A has a subcellular distribution consistent with the involvement of 5-HT3 receptors in modulation of both presynaptic release and postsynaptic responses of mNTS neurons, some of which are serotonergic. The results further suggest that the neuronal as well as glial 5HT3 receptors can be activated by release of serotonin from presynaptic terminals or by diffusion facilitated by SERT distribution at a distant from the synapse.

Animals↗