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PubMed1995

Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in P-glycoprotein-expressing MCF7 human breast adenocarcinoma cells.

A triazinoaminopiperidine derivative synthesized as a modulator of multidrug resistance, S9788, was investigated in the human breast adenocarcinoma MCF7DXR cell line expressing P-glycoprotein. In addition to being less sensitive to daunorubicin, the resistant cell line showed dramatic alterations in the subcellular distribution of daunorubicin, as observed via fluorescence microscopy and quantified via tritiated daunorubicin nuclear distribution analysis. Compared to verapamil and cyclosporin A at 2 and 5 mumol/liter, S9788 proved to be more potent in restoring the cellular accumulation and the subcellular distribution of daunorubicin in the resistant cells. Significant activity of S9788 was observed at 2 mumol/liter, which is clinically achievable, and S9788 restored the nuclear distribution of the drug to the level observed in the parental sensitive cell line. Consequently, the restoration of the cytotoxicity of daunorubicin by S9788 was nearly complete (> 90%) at 2 mumol/liter, wheras cyclosporin A reached this level of activity at 5 mumol/liter, and verapamil was always less active at both concentrations. These results suggest that the modulation of multidrug resistance by S9788 is not only related to the enhancement of the cellular accumulation but also especially by the restoration of the subcellular distribution of the drugs to their nuclear sites of action.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Subcellular localization of (latent) transforming growth factor beta and the latent TGF-beta binding protein in rat hepatocytes and hepatic stellate cells.

Recently, the existence of the large latent transforming growth factor beta (TGF-beta) complex, consisting of TGF-beta, the N-terminal part of its precursor (latency-associated peptide [LAP]), and the latent TGF-beta binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF-beta complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF-beta1,-2,-3, beta1-LAP, and LTBP-1 after cultivation using double-immunofluorescent staining, followed by high-resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF-beta1 enzyme-linked immunosorbent assay (ELISA) and Western blotting for beta1-LAP and LTBP-1. By confocal microscopy, a diffuse distribution of TGF-beta and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF-beta, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF-beta, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle--actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent TGF-beta complex in PC and HSC, respectively. Baseline information is provided from which new hypotheses regarding intracellular functions of TGF-beta, LAP, and LTBP in liver parenchymal and stellate cells can be concluded.

Animals↗

PubMed1985

Subcellular localization of histone messenger RNAs on cytoskeleton-associated free polysomes in HeLa S3 cells.

We have examined the subcellular distribution of histone mRNA-containing polysomes in HeLa S3 cells to assess the possible relationship between localization of histone mRNAs and the regulation of cellular histone mRNA levels. The distribution of histone mRNAs on free and membrane bound polysomes was examined as well as the association of histone mRNA-containing polysomes with the cytoskeleton. The subcellular localization of histone mRNAs was compared with that of HLA-B7 mRNAs which encode a cell surface antigen. Histone mRNAs were localized predominantly on the free polysomes, whereas the HLA-B7 mRNA was found almost exclusively on membrane bound polysomes. However, both species of mRNA were found associated with the cytoskeleton. Interruption of DNA synthesis by hydroxyurea treatment resulted in a rapid and selective destabilization of histone mRNAs in each subcellular fraction; in contrast, the stability of HLA-B7 mRNA appeared unaffected. The results presented confirm that histone mRNAs are predominantly located on non-membrane bound polysomes and suggest that these polysomes are associated with the cytoskeletal framework.

Cycloheximide↗

PubMed1984

A comparative study of L[3H]-glutamate and L[3H]-cysteine sulfinate binding sites in subcellular fractions of rat brain.

A comparative study of the binding of L-cysteine sulfinic acid (CSA) and L-glutamic acid (GLU) to various subcellular fractions of membranes from rat brain was made. Kinetic parameters were determined in all fractions for both types of binding. The effects of membrane preincubation, freezing, and thawing were also examined. The GLU and CSA specific binding levels increased in medium-density (C) and high-density (D) synaptic membranes as compared to the crude mitochondrial/synaptosomal membranes (wP2). Freezing and thawing reduced CSA binding in all tested subcellular fractions. GLU binding is reduced in wP2, C, and D. Binding to the "light" synaptic membranes (B) was not significantly affected, suggesting the presence of two GLU sites. Kinetics of the GLU binding indicated that the temperature-sensitive and -insensitive sites have Kd of 600 nM and 1,100/nM, respectively. Preincubation of fresh membranes conversely affected CSA and GLU binding to the various subcellular fractions, increasing CSA binding in wP2, B, C and decreasing it in D suggesting the existence of distinct sites for GLU and CSA. Preincubation of previously frozen membranes similarly modified CSA and GLU binding except in B fractions. CSA and GLU binding exhibited different pH sensitivities in both fresh and frozen membranes. These results indicate that multiple acid amino acid binding sites exist in membranes and that they can be differentiated according to their sensitivity to temperature. They also suggest the existence of distinct sites for CSA and GLU in fresh membranes, giving further support to the hypothesis that CSA may also serve a neurotransmitter role in the rat central nervous system.

Animals↗

PubMed1986

The beta subunit of the guanine nucleotide regulatory proteins: identification of its location on two-dimensional gels of brain tissue and its regional and subcellular distribution in brain.

The beta subunit of the guanine nucleotide regulatory proteins (also termed G proteins) has been examined in both rat and human brain. Proteins contained within samples of fresh rat and human brain tissue were separated by two-dimensional gel electrophoresis and either stained with silver or reacted with various antisera raised against the G proteins. In both rat and human brain, a single protein of molecular weight 36,000 daltons and pI 5.8 reacted the antisera. This protein also comigrated with one of the proteins present in a purified preparation of bovine brain G proteins. Based upon molecular weight, pI, and reaction with specific antisera, it was concluded that this protein is the beta subunit of the G proteins in brain. Using this information, the regional and subcellular distribution of the G protein beta subunit was studied in rat brain. Of 25 distinct neuroanatomical areas examined, cortical regions were generally found to contain the largest amount of this protein. The subcellular distribution of the G protein beta subunit revealed that large amounts are present in the synaptic membrane, crude synaptic vesicles, and microsomes. These studies serve to identify another protein visible on silver-stained two-dimensional electrophoretograms of rat and human brain. The regional and subcellular distribution of the G protein beta subunit correlate well with the proposed physiological function of this protein.

Animals↗

PubMed1998

Using discriminant function for prediction of subcellular location of prokaryotic proteins.

The discriminant function algorithm was introduced to predict the subcellular location of proteins in prokaryotic organisms from their amino-acid composition. The rate of correct prediction for the three possible subcellular locations of prokaryotic proteins studied by Reinhardt and Hubbard (Nucleic Acid Research, 1998, 26:2230-2236) was 90% by the self-consistency test, and 87% by the jackknife test. These rates are considerably higher than the results recently reported by them using the neural network method. Furthermore, the test procedure adopted here is also more rigorous. The core of the current algorithm is the covariance matrix, through which the collective interactions among different amino-acid components of a protein can be reflected. It is anticipated that, owing to the intimate correlation of the function of a protein with its subcellular location, the current algorithm will become a useful tool for the systematic analysis of genome data.

Algorithms↗

PubMed2000

Subcellular trafficking of the nuclear receptor COUP-TF in the early embryonic cell cycle.

The nuclear receptor SpCOUP-TF is the highly conserved sea urchin homologue of the COUP family of transcription factors. Previous results from our laboratory demonstrated that SpCOUP-TF transcripts are localized in the egg and asymmetrically distributed in the early embryonic blastomeres (A. Vlahou et al., 1996, Development 122, 521-526). To examine the subcellular localization of SpCOUP-TF protein, polyclonal antibodies were separately raised against the divergent N-terminus as well as the conserved DNA-binding and ligand-binding domains. Immunohistochemical analyses suggest that SpCOUP-TF is a maternal protein residing in the cytoplasm of the unfertilized egg. After fertilization, and as soon as the two-cell-stage embryo, most of the receptor translocates from the cytoplasm to the cell nuclei. During the rapid embryonic cell division, SpCOUP-TF was found to shuttle from the interphase nuclear periphery to the condensed chromosomes in mitosis, in a cell-cycle-dependent manner. In an attempt to confirm these observations, the subcellular localization of myc-tagged human COUP-TF I introduced into the sea urchin embryo by RNA injection of fertilized eggs was examined. The pattern of human COUP-TF I subcellular localization, detected with a monoclonal myc antibody, recapitulated the essential features described for the endogenous SpCOUP-TF trafficking. Replacement of the N-terminus of the human receptor with the unique sea urchin N-terminus enhanced its localization to the nuclear rim during interphase. Deletion of the DNA-binding domain of human COUP-TF I resulted in loss of all aspects of nuclear periphery and chromosomal localization. Taken together these data suggest that SpCOUP-TF transcriptional activity is keyed on a cell-cycle-dependent mechanism that regulates chromosomal protein traffic.

Animals↗

PubMed2000

Analysis of the subcellular distribution of protein kinase Calpha using PKC-GFP fusion proteins.

One important factor for the determination of the specific functions of protein kinase C (PKC) isoforms is their specific subcellular localization. In NIH 3T3 fibroblasts phorbol esters induce translocation of PKCalpha to the plasma membrane and the nucleus. In order to investigate PKCalpha's subcellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKCalpha and the green fluorescent protein (GFP). Purified GFP-PKCalpha from baculovirus-infected insect cells undergoes nuclear accumulation without any further stimuli in digitonin-permeabilized cells. Interestingly, permeabilization appears to be a trigger for PKCalpha's nuclear translocation, since the fusion protein also translocates to the nucleus in transiently transfected cells following permeabilization. This suggests that PKCalpha has a high nuclear binding capacity even in the case of large protein amounts. In contrast to endogenous PKCalpha, overexpressed GFP-PKCalpha as well as overexpressed PKCalpha itself translocates mainly to the plasma membrane and only to a smaller extent to the nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKCalpha enabled determination of motifs involved PKCalpha's subcellular distribution: A25E and K368R point mutations of PKCalpha showed enhanced affinity for the plasma membrane, whereas sequences within the regulatory domain probably confer PKCalpha's nuclear accumulation.

3T3 Cells↗

PubMed2001

Expression and subcellular localization of the cyclin-dependent kinase inhibitor p27(Kip1) in epithelial ovarian cancer.

OBJECTIVE: To determine if p27(Kip1) expression was altered in epithelial ovarian cancers as compared to normal ovarian surface epithelial (NOSE) cells and to determine if subcellular localization of p27(Kip1) was an important feature. METHODS: Thirteen tumor samples (1 Stage IC [early] and 12 Stage III/IV [advanced]) from patients with epithelial ovarian cancer and five NOSE samples were evaluated. Samples were surgically dissected to obtain an enriched population (90%) of cancer cells. The level of p27(Kip1) protein expression was determined by Western blot analysis. Actin was used as a loading control, and results were quantified by scanning densitometry using the ratio of the p27(Kip1) signal to the actin signal for comparison. To evaluate the subcellular localization of p27(Kip1), immunocytochemical staining was performed. Clinical pathological parameters were correlated to nuclear p27(Kip1) staining to establish if any association existed. RESULTS: When comparing the expression of p27(Kip1) between NOSE and ovarian cancer samples, only 2 of 13 ovarian cancer samples had altered p27(Kip1) expression. No correlation was found between the expression level of p27(Kip1) on Western blot and clinical pathological correlates. While no correlation between expression level of p27(Kip1) and subcellular localization was found, decreased nuclear staining (1+) was associated with shorter survivals using the log-rank test (P < 0.001). More importantly, in all tumor samples examined under the microscope, no nuclear p27(Kip1) staining was noted in cells that were undergoing mitosis. CONCLUSIONS: p27(Kip1) protein degradation may not be modified in ovarian cancer cells undergoing mitosis. Altered expression of p27(Kip1) is not an overwhelming feature in certain epithelial ovarian cancers. Decreased nuclear staining of p27(Kip1) is associated with poor survival in some epithelial ovarian cancers.

Adult↗

PubMed1998

Transport and subcellular distribution of nickel in the olfactory system of pikes and rats.

Occupational exposure to nickel by inhalation may result in impaired olfactory sense. Recent studies have shown that nickel is transported from the olfactory epithelium along the axons of the primary olfactory neurons to the brain. In the present study 63Ni2+ was applied in the olfactory chambers of pikes (Esox lucius) and the rate at which the metal was transported in the primary olfactory neurons was determined by beta-spectrometry. The results showed a wave of 63Ni2+ in the olfactory nerves, which slowly moved toward the olfactory bulbs. The maximal 63Ni2+ transport rate corresponding to the movement of the base of the wave front was found to be about 0.13 mm/h at the experimental temperature (10 degrees C). This rate of 63Ni2+ transport falls into the class of slow axonal transport. Radioluminography of tape sections of a pike given 63Ni2+ in the right olfactory chamber showed a selective labeling of the right olfactory nerve. The subcellular distribution of 63Ni2+ in the olfactory nerves and the olfactory epithelium of the pikes was studied in tissues subjected to homogenizations and centrifugations, and these methods were also used to examine the subcellular distribution of 63Ni2+ in tissues of the olfactory system of rats given the metal intranasally. It was found that the 63Ni2+, in both the pike and the rat, was present in the cytosol and also in association with various particulate cell constituents. Gel filtrations of the cytosols showed that the 63Ni2+ mainly was eluted at a Ve/Vo ratio corresponding to a MW of about 250. The same coefficient was obtained in gel filtrations performed with 63Ni2+ mixed with histidine in vitro. It is likely that the cytosolic nickel may be bound to histidine or possibly to other amino acids which are similar in size to histidine. Additionally, in the olfactory tissues of the rat the 63Ni2+ was partly present in the cytosol in association with a component with a MW of about 25,000. It is concluded that (i) 63Ni2+ is transported in the primary olfactory neurons by means of slow axonal transport, (ii) in this process the metal is bound to both particulate and soluble cytosolic constituents, and (iii) the metal shows this subcellular distribution also in other parts of the olfactory system.

Animals↗

PubMed1987

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ) by subcellular fractions and cells isolated from small intestine, kidney and liver of the rat.

The mutagenic activity of the pyrolysis products 2-amino-3-methyl-imidazo[4,5-f]-quinoline 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline in Salmonella typhimurium TA98 using rat intestinal and renal subcellular fractions as activation systems was approximately 1 and 5 revertants per nmol, respectively. This was 1,000 times less than the activity with a subcellular fraction from rat liver. The mutagenic activity of both compounds was considerably increased using intestinal, renal and hepatic preparations isolated from PCB (Aroclor 1254)-pretreated rats, compared to preparations from control animals. In addition, both compounds displayed a moderate direct-acting mutagenic activity at concentrations above 10(-5) M. Isolated cells from small intestine, kidney and liver incubated in nucleopore chambers were able to convert both compounds into products which mutated bacteria outside the chambers. The concentrations of chemicals required to yield responses of a similar magnitude were approximately 3 orders of magnitude higher in the intestinal and renal systems compared to the hepatic system. The formation of metabolites mutagenic for Salmonella typhimurium by hepatic subcellular and cellular systems was shown to be superior to the respective intestinal and renal systems.

Animals↗

PubMed1975

Subcellular compartmentalization of the luteal cell in the ovary of the dog.

The compartmentalization of the parenchyma of the corpus luteum in the dog was studied by both 100 and 1000 KV electron microscopy. The organells within the luteal cell are oriented with a high degree of consistency towards the pericapillary space. Characteristically, the avascular pole and the lateral margins of the cell posses predominantly stacked and whorled cisternae of agranular ER. In the central medial portions of the cell, pleomorphic mitochondria with tubulo-vesicular cristae and anastomosing tubules of agranular ER predominate. However, the distribution of organelles in this compartment is graded. Mitochondria predominate in the central medial areas while tubular ER is more dominant peripherally. Microfilaments are ubiquitous in this compartment and run a longitudinal course between and around the subcellular components towards the pericapillary space. The Golgi apparatus is large and prominent and is positioned over the pole of the nucleus that faces the basal lamina. Coated vesicles are abundant in the Golgi regions and along the lateral surface of the cell. Three distinct regional specializations of the cell surface exist. The basal surface contains long pleomorphic cytoplasmic folds that fill the pericapillary space, are interconnected by small gap junctions and contain abundant multivesicular bodies. The lateral cell surface is covered with microvilli and is organized into tortuous intercellular channels and canaliculi. These are interrupted at intervals by cytoplasmic protrusions that extend from one cell well into the cytoplasm of the next. Large, well-developed gap junctions line the margins of the cells furthest removed from the pericapillary space. Finally, the individual cells exhibit heterogeneity with respect to the amount one subcellular organelle or compartment is expressed relative to another. These observations are discussed in relation to the subcellular compartmentalization of progesterone synthesis and release.

Animals↗

PubMed1989

The regional and subcellular distribution of calcium activated neutral proteinase (CANP) in the bovine central nervous system.

Calcium-activated neutral proteinase (CANP) activity was determined in subcellular fractions and in different regions of bovine brain. The CANP specific activity in spinal cord and corpus callosum, areas rich in myelin, were almost six-fold greater than cerebral cortex and cerebellum. Treatment of whole homogenate and myelin with 0.1% Triton X-100 increased the CANP activity by tenfold. Subcellular fractions were prepared from bovine brain gray and white matter. Most of the CANP activity (70%) was in the primary particulate fractions P1 (nuclear), P2 (mitochondrial) and P3 (microsomal). On subfractionation of each particulate fraction, the majority of the activity (greater than 50%) was recovered in the myelin-enriched fractions (P1A, P2A, P3A) which separate at the interphase of 0.32 M- and 0.85 M-sucrose. The distribution of activity was P2A greater than P1A greater than P3A. Further purification of myelin (of P2A) increased the specific activity over homogenate by more than three-fold. The same myelin fractions contained the highest proportion (60%) and specific activity (five-fold increase) of CNPase. The enzyme activity in different regions of brain and in subcellular fractions was increased by 20-39% after the inhibitor was removed. Electron microscopic study confirmed that the myelin fractions were highly purified. The cytosolic fraction contained 20-30% of the total homogenate CANP activity. Other fractions contained low enzyme activity. CANP was identified in the purified myelin fraction by electroimmublot-technique. It is concluded that the bulk of CANP in CNS is tightly bound to the membrane, may be masked or hidden and is intimately associated with the myelin sheath.

Animals↗

PubMed1976

Kinetics of the subcellular distribution of iron and cobalt in the intestinal mucosa of the rat.

A differential centrifugation technique was used to compare the subcellular distribution of labeled iron (Fe) and cobalt (Co) in iron-deficient animals (FeD), in which transport was active, with iron-loaded animals (FeL), in which transport was by diffusion. In FeL the amount of Fe and Co in each subcellular fraction was dependent upon dose; there was a linear relation between the amount of Fe or Co in each fraction and metal absorption. In FeD a curvilinear relation was noted between both Fe dose and absorption, and the amount of Fe in the particulate fractions I and II. In contrast, a linear relation was noted between both dose and absorption, and the smaller amounts of Fe in the other fractions. Most of the Co was in V ("soluble"), and the relation between Co in all the fractions and absorption was linear. The addition of Co to the Fe test dose was associated with a decrease in Fe in I and II. The results suggest that a constituent(s) in the particulate fractions I and II is involved in the enhanced absorption of Fe in FeD. The subcellular fraction responsible for the increased Co absorption in FeD was not identified.

Animals↗

PubMed1975

Essential fatty acid deficiency: metabolism of 20:3(n-9) and 22:3(n-9) of major phosphoglycerides in subcellular fractions of developing and mature mouse brain.

Essential fatty acid deficiency was initiated in young and mature mice. The metabolism of 20:3(n-9) and 22:3(n-9) in brain subcellular fractions was followed after the mice were switched from the deficient diet to a corn oil supplemented diet. After switching to the supplemented diet, the proportions of (n-9) polyunsaturated fatty acids in brain in both groups of mice decreased with time. The rate of disappearance of (n-9) polyunsaturated fatty acids was faster in the young groups than in the mature group. In the developing mice, the half-linves of the (n-9) polyunsaturated fatty acids in the total ethanolamine phosphoglycerides of brain microsomal, synaptosomal, and myelin fractions were 3, 10, and 15 days respectively. In the mature group, the half-lives for 20:3(n-9) in diacyl-glycerophosphorylethanolamine of microsome, synaptosome, and myelin fractions were 8-10, 10, and 22 days, respectively; and the half-lives for 22:3(n-9) in alkenylacyl-glycerophosphorylethanolamine of the same subcellular fractions were 8-12, 28, and rate of disappearance of 20:3(n-9) in brain was faster in the diacyl-glycerophosphorylethanolamine than in the alkenylacyl-glycerophosphorylethanolamine. These results demonstrate that the metabolism of (n-9) polyunsaturated fatty acid in brain phosphoglycerides during recovery from essential fatty acid deficiency not only varies with age, but also depends upon individual phosphoglycerides present in each subcellular fraction.

Aging↗

PubMed1979

Phospholipid exchange between subcellular organelles of rabbit lung.

A soluble protein fraction (PLEP) prepared from rabbit lung can catalyze the exchange of phospholipids between subcellular organelles of the lung and between these subcellular organelles and synthetic liposomes. Phospholipid exchange between microsomes and synthetic liposomes and between mitochondria and synthetic liposomes was stimulated 8-fold and 2.5-fold, respectively, in the presence of the protein fraction. Lung exchange protein could also catalyzed phospholipid exchange between subcellular organelles of the liver and synthetic liposomes. Phospholipid transfer between microsomes and lamellar bodies of the lung was stimulated 2-fold by the exchange protein. Both radiolabeled phosphatidylcholine (PC) and phosphatidylinositol (PI) were transferred from 32P-labeled microsomes to lamellar bodies, but the exchange protein exhibited no transfer activity for phosphatidylglycerol (PG) and that for phosphatidylethanolamine (PE) was insignificant compared to the transfer activity for phosphatidylcholine and phosphatidylinositol. While the physiological role of the phospholipid exchange proteins in the lung is unknown, it is possible that they participate in the distribution of the newly synthesized phospholipids from the site of synthesis to lamellar bodies and other membrane compartments of cells.

Animals↗

PubMed2001

Speciation and subcellular location of se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic fluorescence spectrometric detection.

Speciation of Se-containing proteins in the subcellular fractions of human liver was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by hydride generation-atomic fluorescence spectrometric (HG-AFS) detection. It was found that about 24 kinds of Se-containing proteins existed in subcellular fractions of normal human liver. The molecular weights (MW) of the subunits were mostly in the range 20-30 kDa and 50-80 kDa. Major Se-containing protein fractions at 61 kDa and 21 kDa are probably selenoprotein P and glutathione peroxidase, respectively. The 54 kDa protein is probably a thioredoxin reductase, which is presented in nuclei, mitochondria, lysosome, microsome and cytosol. We noticed that the Se-containing protein with the lowest MW of 9.3 kDa only existed in lysosome. Most of the proteins have not been identified and would require further investigation to characterize them. The specific subcellular distributions of different Se-containing proteins suggest that they could play important biological roles in each organelle.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope.

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2(n)x2(n) pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.

Animals↗