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The cellular and subcellular distribution of [3H]-desipramine (DMI) in rat brain was studied by electron microscope (EM) autoradiography and by subcellular fractionation. A considerable proportion of label was found to be bound to the membranes of presynaptic nerve terminals, as well as to sites inside those terminals.
Rats were fed plant sterols containing campesterol and beta-sitosterol in the differerent proportions, and their distribution in serum lipoproteins and in liver subcellular fractions was determined. In serum lipoproteins, the percentage as well as the concentration of plant sterols increased with the increase in the density of lipoproteins. Thus, high density lipoprotein (HDL) contained the highest and very low density lipoprotein (VLDL), the lowest. Also, there were distinct differences in the ratio of campesterol to sitosterol among lipoproteins, it was the highest in VLDL and lowest in HDL. Quantitatively, more than 75% of campesterol and 80% of sitosterol were carried in HDL; the values were significantly different from those of cholesterol (ca. 70%) in relation to total cholesterol. The distribution of plant sterols in liver subcellular fractions was virtually the same with that of cholesterol. Both nuclei and microsomes contained approximately 40% of total plant sterols.
Day-old male broiler chickens were fed semipurified diets containing 5% lipid from one of four different lipid sources: corn oil (CO), partially hydrogenated soybean oil (HSBO), a spent restaurant grease (SRG) and a purified mixture of triolein, tripalmitin and tristearin (OPS). Diets CO and HSBO contained adequate amounts of linoleic acid, but diets SRG and OPS were deficient in linoleate. In addition, SRG and HSBO contained trans isomers of 16:1 and 18:1. The diets were fed for 3 wk to determine the effects of low linoleate levels and trans isomers on fatty acid profiles in liver microsomes, mitochondria and cytosol. Chicks fed HSBO had the highest body weights, while those fed SRG and OPS had the lowest. The incidence and severity of dermatitis were similar for all treatments. The proportions of linoleate and arachidonate in lipids from liver subcellular fractions were reduced significantly in chicks fed OPS and SRG; however, levels of 20:3 omega 9 were not increased. Feeding HSBO, which is high in both linoleate and linolenate, resulted in higher levels of 18:3 omega 3 and 20:5 omega 3 in liver subcellular fractions and lower levels of 20:4 omega 6 than those seen in chicks fed CO. The isomeric forms of 18:1 present in the partially hydrogenated fats (HSBO and SRG) appeared to be incorporated into the lipids of liver fractions. The results of this study show that dietary lipids influence fatty acid profiles of chick liver microsomes, mitochondria and cytosol. Decreases in linoleate and arachidonate in these organelles occur before overt essential fatty acid (EFA), deficiency signs in chicks fed EFA-deficient diets.
Studies were conducted examining the subcellular distribution of beta-carotene (BC), alpha-tocopherol (E) and retinol (A) in livers of control and BC-fed male White Leghorn chicks. Chicks were fed Cornell B chick starter diet with or without the addition of 0.5 g BC/kg diet. A first study involved liver fractionation by differential centrifugation in 0.25 M sucrose followed by high performance liquid chromatographic (HPLC) analyses of all fractions for quantitation of BC, E and A. A second study employed both intravenous injection of Triton WR-1339 four days prior to sacrifice and centrifugation in 1.0 M sucrose to separate mitochondria from lysosomes more efficiently. Fraction purity was assessed by marker enzyme analyses. Results showed that chick liver accumulated BC; BC-fed chicks had higher concentrations of BC in all fractions relative to controls, and the mitochondrial fraction contained the highest concentration of BC, followed by lysosomes, microsomes and nuclei, respectively. Plasma BC increased more than fivefold in BC-fed chicks. Dietary BC increased A and E levels in liver and in the mitochondrial and lysosomal fractions while the plasma E level was decreased. Plasma A changed little with BC feeding. While dietary BC had no effect on fatty acid composition of subcellular fractions, the increase in E resulted in a large increase in the molar ratio of E to polyunsaturated fatty acids. The incorporation of BC and increased amounts of E into cellular membranes presumably would result in increased resistance to peroxidative damage.
To determine the subcellular distribution of disaturated phosphatidylcholine (DSPC) in lung tissue during perinatal development, fetal rabbits at 24, 26, 28 and 31 (term) days gestation and newborns were studied. Following alveolar lavage, fractions enriched in nuclei-cellular debris, mitochondria, microsomes, surfactant (lamellar bodies) and cytosol were prepared from the residual tissue homogenate, and their DSPC content was determined. The DSPC content of the unfractionated residual lung tissue homogenate progressively and significantly increased during fetal development, rising from 9.09 +/- 0.91 to 17.45 +/- 2.88 mg/g dry lung between 24 days gestation, and term. Between 24 and 26 days gestation the overall increase in tissue DSPC was due to a two-fold increase in the mitochondrial, microsomal and cytosolic pools. Lamellar bodies were first isolable at 26 days gestation. The DSPC content of this fraction increased six-fold (from 0.10 +/- 0.02 to 0.67 +/- 0.15 mg/g dry lung) between 26 and 28 days gestation and a further seven-fold (to 4.63 +/- 1.06 mg/g dry lung) by term, accounting for the overall increase in the tissue homogenate value during this time period. By the first postnatal day, microsomal and cytosolic DSPC increased another two-fold, but no significant change occurred in the other subcellular fractions. Alveolar lavage DSPC progressively increased over the time period studied. While there was no change in the lamellar body DSPC/total PC ratio during fetal development, each of the mitochondrial, microsomal and cytosolic ratios decreased between days 26 and 28 of gestation and then increased at term.(ABSTRACT TRUNCATED AT 250 WORDS)
In the presence of ATP, MgCl2 and CoASH, different subcellular membrane fractions isolated from rat cerebral cortex exhibited characteristic profiles for the incorporation of [1-14C]arachidonic acid into phospholipids and triacylglycerols. In general, uptake of label by phosphatidylcholines was higher in the synaptic membranes, and that by phosphatidylinositols was higher in the microsomes and somal plasma membranes. A substantial amount of the labeled arachidonate was also incorporated into triacylglycerols, especially in the somal plasma membranes and microsomes. Enzymes mediating the transfer of arachidonic acid to phospholipids were unstable with respect to sample storage and exposure to elevated temperatures. In contrast, the acyltransferase for triacylglycerols was more stable to these factors. Washing the membranes with bovine serum albumin resulted in an enhancement of the incorporation of label into phosphatidylinositols without affecting that of phosphatidylcholines, but the incorporation into triacylglycerols was inhibited. Treatment of synaptosomes and plasma membranes with saponin resulted in an enhancement in the labeling of phospholipids, but the labeling of triacylglycerols was inhibited. Thus, although labeled arachidonic acid was incorporated into phospholipids and triacylglycerols in brain subcellular membranes, these two types of acyltransferases exhibited different properties and responded differently to membrane perturbing agents.
The specific activity, tissue specificity, and subcellular distribution of alkaline phosphatase were studied in the fetal rat limb during initial cartilage calcification and bone formation. The pH optimum, Km, activation, and inhibition characteristics of the enzyme assayed for in 900 X g supernates of whole limb homogenates indicated that the activity represented a fetal bone alkaline phosphatase. Studies examining temporal changes of the enzyme in these preparations demonstrated a substantial increase in activity over each of the days during which they were studied (days 15-18). Fractions derived from the discontinuous density gradient centrifugation of the limb preparations were used to study the chronological subcellular distribution of the enzyme. Enzyme activity was found in all of the fractions with the greatest activity occurring in fractions consisting of ribosomes and small vesicles. The vesicular component was similar to the matrix vesicles dexcribed by others in calcifying tissues. The daily increase in activity measured in the curde supernate was further reflected in the distribution studies. The association of alkaline phosphatase with the vesicular structure is compatible with the theorized functions of matrix vesicles, and the substantial increase in activity between days 15 and 18 further demonstrates an intimate association of alkaline phosphatase with skeletal development.
1,25 Dihydroxyvitamin D3 has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)2D3-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)2D3 (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased 45Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)2D3. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)2D3-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed either in vivo or in vitro. The effects in vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)2D3-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)2D3 affects muscle calcium transport.
The application of immunoaffinity techniques to subcellular fractionation is reviewed and the basic principles underlying the various methods that have been successfully employed, identified. The requirement for organelle-specific antigens and high-avidity antibodies is discussed, as is the widespread use of indirect immunoadsorbents. Approaches for the optimization of immunoaffinity-based subcellular fractionation are suggested.
An analysis of the subcellular localization of platinum was conducted in Sprague-Dawley (SD) rats following administration of an i.v. dose of 6 mg/kg cisplatin (bolus and infusion). Biodistribution studies were carried out in the liver and kidney of control animals, as well as in these same organs and in the tumor (Walker 256 adenocarcinoma) of SD rats. The results obtained illustrate that in addition to the platination of DNA in these tissues, significant amounts of Pt are also incorporated into the chromosomal protein (CP) and cytosolic fractions. The localization of Pt in the cytosolic fractions was highest in the kidney, followed by the tumor, and lowest in the liver when determined as the fractional percentage of the total amount of injected drug (%ID/g). The significance of such cytosolic and CP localization of Pt is not known at this time, but they may be involved in the cytotoxic effects of cisplatin, as this drug is cytotoxic to tumors and kidneys but not to the liver. The localization of cisplatin in the subcellular fractions of liver, kidney, and tumor showed a trend toward being higher after i.v. infusion than after i.v. bolus administration of the drug.
It has been reported that isolated rat heart myocytes and cardiac mesenchymal cells convert arachidonic acid mainly into three types of prostaglandins (PGs): PGE2, PGF2 alpha, and PGI2. In addition, we have demonstrated that fresh atrial slices of patients with heart-valve disease contain appreciable quantities of PGE2 and PGF2 alpha. However, there have been no reports on the subcellular localization of the prostaglandin system in heart muscle tissue. The present study was performed to define the distribution of PGE2 in mitochondrial, microsomal, and cytosolic fractions, isolated by differential centrifugation from homogenates of fresh normal rat atrium and ventricle slices. In addition, we determined whether differences exist in PGE2 levels between atrial subcellular fractions and those of ventricular fractions. The results showed that PGE2 was located mainly in the high-speed cytosolic supernatant fraction of the heart homogenates analyzed. Furthermore, PGE2 concentrations (ng/mg protein) were significantly higher in fractions obtained from atrial than those of ventricular tissue.
This review covers recent developments in the preparation, manipulation, and analyses of subcellular environments. In particular, it highlights approaches for (1) separation and detection of individual organelles, (2) preparation of ultra-pure organelle fractions, and (3) utilization of novel labeling strategies. These approaches, based on innovative technologies such as microfluidics, immunoisolation, mass spectrometry and electrophoresis, suggest that subcellular analyses will soon become as commonplace as single cell and bulk cellular assays.
In our previous study, we found that mercaptopyruvate sulfurtransferase (MST) was evolutionarily related to mitochondrial rhodanese. To elucidate the difference between MST and rhodanese, the tissue, cellular, and subcellular distribution of rat MST was determined biochemically and immunohistochemically by using anti-MST antibody raised in rabbit. In an immunohistochemical study, tetramethyl rhodamine isothiocyanate-conjugated phalloidin against F-actin and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin as a secondary antibody to the anti-MST antibody were used for double fluorescent staining. They were detected by confocal laser fluorescence microscopy. In the immunoelectron microscopic study of hepatocyte and renal tubular epithelium, a postembedding immunogold method was used. Biochemical studies including western blot analyses of various tissues and subcellular fractions of the liver were also performed. MST was widely distributed in rat tissues but the cellular distribution was found to be different in each tissue. MST was predominantly localized in proximal tubular epithelium in the kidney, pericentral hepatocytes in the liver, cardiac cells in the heart, and neuroglial cells in the brain. This immunocytochemical study also found that MST was localized in both mitochondria and cytoplasm.
The effects of cholinergic and alpha-adrenergic stimulation on calcium concentrations in rat submandibular gland acinar cells were evaluated at the subcellular level by electron probe X-ray microanalysis, and the subcellular distribution of receptors for inositol 1,4,5-trisphosphate (IP(3)Rs) was investigated by electron microscopic immunohistochemistry. For measurement of calcium concentrations, experiments were carried out both in vivo and in vitro. In in vivo experiments, fragments of glands were removed and slam-frozen 3 min after intraperitoneal injection of an agonist. In in vitro experiments, the gland tissue was enzymatically dispersed, treated with an agonist in the presence or absence of extracellular Ca(2+), and slam-frozen. The frozen specimens were cut into ultrathin cryosections, which were then freeze dried. Spectra were collected from secretory granules, the perinuclear cytoplasm containing endoplasmic reticulum (ER), and the nucleus of the acinar cells. A clear decrease in calcium content in secretory granules was observed after cholinergic stimulation both in vivo and in vitro in the presence or absence of extracellular Ca(2+). In the presence of extracellular Ca(2+), cholinergic stimulation following alpha-adrenergic stimulation reduced the calcium content in secretory granules to the same extent as cholinergic stimulation alone. No significant changes in the calcium content of the perinuclear cytoplasm and the nucleus were noticed after cholinergic stimulation. alpha-adrenergic stimulation did not significantly affect the calcium concentrations in any of the three compartments studied. IP(3)Rs were localized to ER lamellae, but not to secretory granule membranes or to nuclear membranes. Our findings suggest that: (1) in rat submandibular acini, Ca(2+) can be released from secretory granules by exposure to cholinergic, but not alpha-adrenergic, stimuli, (2) there are, however, no IP(3)Rs present in the granular membrane and the trigger for the Ca(2+) release remains under study, and (3) the response to either type of stimulus does not involve changes in the Ca(2+) content of the nucleus or of the perinuclear cytoplasm/ER, despite the presence of IP(3)Rs in the latter structure. This may be the result of limitation in the technique used, which cannot measure changes in the proper compartment of the ER.
Electron microscopy was used to quantify the subcellular distribution of the GLUT-1 isoform of the glucose transporter in developing microvessels of the brain of embryonic rats from E (embryonic stage) 13 to E19 and in adult rats. Gold-conjugated secondary antibodies were used to localize, on ultrathin sections of brain, a rabbit polyclonal antiserum (anti-GLUT-1) raised against a synthetic peptide encoding 13 amino acids of the C-terminus of the human glucose transporter. Staining was weak at E13 but increased in density during development into adulthood. The increase represented an increase in the absolute amount of transporter per vessel profile, with a concomitant decrease in vessel size with the narrowing of the wall. At early stages, the percentages of total particles per profile of lumenal membrane, ablumenal membrane, and cytoplasm were approximately equivalent. The ratio of lumenal to ablumenal particle density then shifted from below 1 at E13 to above 2 at E19 and to 4 in the adult. In contrast, vessels of the choroid plexus were devoid of labeling, but the choroid plexus epithelium stained as early as E15. In the brain, no astrocytes, neurons, or pericytes were stained at any stage examined. Developmental upregulation of the GLUT-1 glucose transporter therefore seems to occur at the blood-brain barrier, and the modulation of the subcellular distribution of the transporter can be correlated with other observed changes in the microvessels as they develop the blood-brain barrier phenotype.
The tissue distribution, preferentially in the human male genital system, and the subcellular localization of the lysosome-associated membrane protein 2 (lamp 2) was studied immunohistochemically using a mouse monoclonal antibody, 2D5. Strong immunoreactivity was present in the tubular system of the kidney, in acinar cells of salivary glands and pancreas, prostate, mammary glands, placenta and in cutaneous sweat glands. Moderate immunoreactivity was observed in cerebral neuronal cells, epidermal cells, testis, epididymis, seminal vesicle and endometrium. Very low immunoreactivity was found in liver. In some of the tissues mentioned, the distribution pattern of immunoreactivity is smooth and homogeneous, while in others it is granular and concentrated in the supra- or perinuclear cytoplasm. The subcellular distribution was studied on ultracryosections and on pre-embedding-processed chopper sections of human prostate. In the latter gland, the protein is not restricted to epithelium, but is also present in stromal cells. Ultrastructurally, the immunoreactivity in secretory cells was localized in electron-translucent vacuoles and granules, including the secretory granules. A close association with cell membranes was not generally the case. Only part of the immunoreactive material was linked to the apical plasma membrane pointing to a biosynthesis independent from an association step with the apical plasma membrane. As shown by immunoelectron microscopy and Western blotting, a high amount of lamp 2 is secreted and is found in so-called prostasomes. The findings indicate that in the human prostate most of the membrane-bound lamp 2 is released from the secretory cells, presumably in an apocrine fashion.
Arachidonate 12-lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-lipoxygenase cDNA or a vector without 12-lipoxygenase cDNA, these structural changes were not observed.
Janus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the "conventional" in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2's enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.