Distribution In Subcellular Fractions Research Articles

Carbon Dot Blinking Enables Accurate Molecular Counting at Nanoscale Resolution.

Accurate counting of single molecules at nanoscale resolution is essential for the study of molecular interactions and distribution in subcellular fractions. By using small-sized carbon dots (CDs), we have now developed a quantitative single-molecule localization microscopy technique (qSMLM) based on spontaneous blinking to count single molecules with a localization precision of 10 nm, which can be accomplished on conventional microscopes without sophisticated laser control. We explore and adapt the blinking of CDs with diverse structures and demonstrate a counting accuracy of >97% at a molecular density of 500 per μm2. When applied to G-protein coupled receptors on a cell membrane, we discriminated receptor oligomerization and clustering and revealed ligand-regulated receptor distribution patterns. This is the first example of adapting nanoparticle self-blinking for molecular counting, and this demonstrates the power of CDs as SMLM probes to reliably decipher sub-diffraction structures that mediate crucial biological functions.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier invitro is dependent on its binding affinity.

The blood–brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re‐directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter‐study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (K d from 5 to174 nM) of the rat TfR‐binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics‐selected reaction monitoring and fluorescent imaging with markers of early‐ and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co‐localized with late‐endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co‐localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.Open Practices Open Science: This manuscript was awarded with the Open Materials Badge.For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

Influence of metal-contamination on distribution in subcellular fractions of the earthworm (Metaphire californica) from Hunan Province, China

Earthworms have the ability to accumulate of heavy metals, however, there was few studies that addressed the metals in earthworm at subcellular levels in fields. The distributions of metals (Cd, Cu, Zn, and Pb) in subcellular fractions (cytosol, debris, and granules) of earthworm Metaphire californica were investigated. The relationship between soil metals and earthworms were analyzed to explain its high plasticity to inhabit in situ contaminated soil of Hunan Province, south China. The concentration of Cd in subcellular compartments showed the same pattern as Cu in the order of cytosol > debris > granules. The distribution of Zn and Pb in earthworms indicated a similar propensity for different subcellular fractions that ranked as granules > debris > cytosol for Zn, and granules > cytosol > debris for Pb. The internal metal concentrations in earthworms increased with the soil metals (p<0.05). Significant positive correlations were found between soil Cd and Cd concentrations in cytosol and debris (p<0.01). Moreover, the soil Pb concentration significantly influenced the Pb concentrations in cytosol and debris (p<0.01), similar to that of Cd. The soil Cu concentrations was only associated with the Cu in granules (p<0.05). Soil Zn concentrations correlated with the Zn concentrations in each subcellular fraction (p<0.05). Our results provide insights into the variations of metals partitioning in earthworms at subcellular levels and the relationships of soil metals, which could be one of the detoxification strategies to adapt the long-term contaminated environment.

水淹条件下秋华柳亚细胞中镉的分配特征

PDF HTML阅读 XML下载 导出引用 引用提醒 水淹条件下秋华柳亚细胞中镉的分配特征 DOI: 10.5846/stxb201612092537 作者: 作者单位: 作者简介: 通讯作者: 中图分类号: 基金项目: 国家国际科技合作专项（2015DFA90900）；三峡后续工作库区生态与生物多样性保护专项项目（5000002013BB5200002）；重庆市林业重点科技攻关项目（渝林科研2015-6） Cadmium distribution in the subcellular fractions of Salix variegata under flooding conditions Author: Affiliation: Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:选取秋华柳（Salix variegata）扦插苗为研究对象，通过设置0、0.5、2、10 mg/kg 4个镉胁迫浓度，研究了水淹条件下秋华柳根、枝、叶亚细胞中镉的分配特征。结果表明：（1）试验各处理组秋华柳存活率均为100%，表现出良好的镉和水淹耐受能力。（2）与对照相比，在水淹条件下，各处理组秋华柳根、枝和叶的细胞壁仍是镉最主要的富集部位。各处理组植株细胞壁中的镉含量显著高于其他组分，质体中镉含量次之，细胞核和线粒体组分中的镉含量始终处于较低水平。（3）水淹显著提高了秋华柳根细胞壁中的镉含量，显著降低了高浓度镉处理（10 mg/kg）下萌枝细胞壁中的镉含量，但对叶细胞壁中的镉含量没有显著影响。（4）水淹显著提高了秋华柳根细胞中质体中的镉含量，对萌枝、叶细胞质体中的镉含量没有显著影响。研究证明，水淹条件下，秋华柳根枝叶细胞壁仍然是镉积累富集的最主要部位，从而减少了重金属对植物细胞的伤害。秋华柳适用于三峡消落带镉污染区域的植物修复。 Abstract:The altered water regime of the Three Gorges reservoir in the Yangtze River in China has formed a hydro-fluctuation zone that has caused many serious environmental problems, such as vegetation degradation, soil erosion, and landscape deterioration. After water impoundment, heavy metals in the soil can dissolve into the water, thereby increasing the risk of water pollution. Cadmium (Cd) contamination is one of the most serious heavy metal pollutions in the Three Gorges reservoir area. Cd shows high biological activity; it can easily enter the food chain via absorption by plants and affect the ecosystem health. Revegetation is an eco-friendly measure in restoring ecological integrity of the hydro-fluctuation zone. However, it is a great challenge to successfully revegetate the degraded hydro-fluctuation zone because the plants need to exhibit tolerance to the altered water regime and Cd contamination. Salix variegata, a native species in the Three Gorges reservoir, exhibits tolerance to both long-term flooding and heavy metal stress and therefore, is a promising candidate for revegetation in this area. However, the ability of this species to tolerate and accumulate Cd under flooding conditions is still unclear. To explore the effect of flooding on Cd distribution in subcellular fractions of S. variegata, 60-d long factorial experimental treatments including two water regimes and four Cd concentrations were designed. The water treatments included ambient water supply (CK) and light flooding (FL), whereas the Cd treatments included control and low, middle, and high concentrations (0, 0.5, 2, and 10 mg/kg, respectively). Each treatment involved a group of planted S. variegata shoots. Plant cells were separated into subcellular fractions of cell walls, plastids, cell nuclei, and mitochondria and soluble fractions, by differential centrifugation. All subcellular fractions were digested using microwave digestion and the Cd content in different subcellular fractions was determined using an inductively coupled plasma-atomic emission spectrometer. The results showed that:(1) The survival rate of S. variegata in all treatments was 100%, which indicated high tolerance of this species to flooding and Cd stress. (2) Most of the accumulated Cd was more highly enriched in the cell walls of plant cells under FL than under CK. In contrast, the Cd content in the nucleus and mitochondria was always lower under FL than under CK. (3) Under FL, the Cd content significantly increased in root cell wall, decreased in stem cell wall under high Cd concentration, and showed no significant difference in leaf cell wall. (4) Flooding significantly increased Cd content in plastids of root cells; however, it did not affect the Cd content in plastids of stem and leaf cells. In conclusion, in S. variegata under flooding conditions, cell wall was still the main subcellular component enriched with Cd, which reduced the harmful effects of Cd stress on plants. These results indicated that there was no significant difference in the allocation strategy of Cd in subcellular fractions under flooding and ambient water supply for S. variegata, and that S. variegata could be suitable for phytoremediation of Cd in the hydro-fluctuation zone of the Three Gorges reservoir. 参考文献 相似文献 引证文献

Subcellular distribution of molybdenum, ultrastructural and antioxidative responses in soybean seedlings under excess molybdenum stress

Some studies have shown that excess molybdenum (Mo) could produce toxic effects on plants. However, little is known about the subcellular distribution of Mo and cell ultrastructure within plants under excess Mo stress. Here, we comprehensively analyzed the changes of Mo distribution in subcellular fractions, cell ultrastructure and antioxidant enzymes in leaves and roots of soybean seedlings in response to excess Mo stress. The results showed that roots exhibited higher Mo accumulation than leaves at the 100 mg L−1 Mo level, about 38.58-, 171.48- and 52.99-fold higher in cell walls, cell organelles and soluble fractions, respectively. Subcellular fractionations of Mo-containing tissues indicated that approximately 90% of Mo was accumulated in the soluble fractions and cell walls of the roots and leaves, and soluble fractions (accumulated 66.3–72.2% Mo) might serve as an effective storage site for excess Mo. Furthermore, excess Mo caused ultrastructural alterations in roots and leaves of soybean seedlings, leading to structural abnormality of chloroplast in leaf cells, plasmolysis, cellular deformity, vacuole enlargement and the swelling of cell wall and cytoplasm in root cells. Meanwhile, under excess Mo stress, the activity of POD, CAT and APX enzymes in roots was 1.43, 2.35 and 1.23 times that under standard Mo condition, while that of SOD and CAT enzymes in leaves was 1.23 and 1.94 times, respectively. This study provided novel insights into the mechanisms of excess Mo toxicity in soybean seedlings.

Immunolocalization of Sprouty-1 and Sprouty-2 in Developing Rat Lung

Objective: Sprouty, a common antagonist of fibroblast growth factor (FGF) and epidermal growth factor signaling, is a key player regulating tracheal branching and eye development in Drosophila. Four Sprouty homologs have been identified in vertebrates and all share a cysteine-rich region. However, the physiological function(s) of the individual Sprouty homologs is unknown. mRNA of Sprouty homologs is expressed during mouse lung development. In the present study, we investigated the immunolocalization of Sprouty proteins in rat lung at different stages of development. Methods: Rabbit antibodies were raised against peptides derived from rat Sprouty-1 and Sprouty-2 and were used in Western blot analysis to determine Sprouty distribution in subcellular fractions (pellets and supernatant centrifuged at 5,000 and 20,000 g) and bronchoalveolar lavage fluid (BAL) from adult rat lungs or used in immunohistochemistry. Results: Western blot analysis revealed a 30-kDa Sprouty-1 band and a 34-kDa Sprouty-2 band in the supernatant and pellet fractions centrifuged at 20,000 g. BAL contained a band of approximately 16 kDa with Sprouty-1 antibody derived from proteolytic fragmentation of Sprouty-1. In embryonic day (E) 14 and E16 lungs, Sprouty-1 and Sprouty-2 were expressed both in epithelial and peripheral mesenchymal cells. In adult rat lung, bronchiolar and alveolar type II epithelial cells showed staining for both Sprouty-1 and Sprouty-2. Sprouty-1 expression was also seen in alveolar type I epithelial cells. Conclusion: In light of the proximity of the distribution of Sprouty to that of FGF-10 (peripheral mesenchyme) and its receptor FGFR2IIIb (distal tubular epithelium) in lung development, and the finding that FGF-9, which is expressed in mesothelial cells, upregulates FGF-10, it appears that Sprouty expression in epithelial and mesenchymal cells during branching morphogenesis is closely related to signaling by FGF-9 and FGF-10.

A persistent change in subcellular distribution of calcineurin following fluid percussion injury in the rat

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. The effect of moderate, central fluid percussion injury on the subcellular distribution of this important neuronal enzyme was examined. Animals were sacrificed at several time points post-injury and calcineurin distribution in subcellular fractions was assayed by Western blot analysis and immunohistochemistry. A persistent increase in calcineurin concentration was observed in crude synaptoplasmic membrane-containing fractions. In cortical fractions, calcineurin immunoreactivity remained persistently increased for 2 weeks post-injury. In hippocampal homogenates, calcineurin immunoreactivity remained increased for up to 4 weeks. Finally, immunohistochemical analysis of hippocampal slices revealed increased staining in the apical dendrites of CA1 neurons. The increased staining was greatest in magnitude 24 h post-injury; however, staining was still more intense than control 4 weeks post-injury. The data support the conclusion that fluid percussion injury results in redistribution of the enzyme in the rat forebrain. These changes have broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

Intracellular redistribution of heme in rat liver under oxidative stress: the role of heme synthesis*

Heme distribution in subcellular fractions of rat liver was studied first hours under the action of several agents causing oxidative stress in vivo. Total and post-mitochondrial heme content in liver was found to depend on both the level of hemolysis products in blood and agent's capacity to modify heme and hemoproteins. The increase of activity of 5-aminolevulinate synthase (ALAS) and/or heme accumulation in mitochondria was accompanied by increase of tryptophan-2,3-dioxygenase (TDO) heme saturation. Membrane stabilisation by tocopherol or prevention of early ALAS induction by cycloheximide prevented both mitochondrial heme accumulation and increase of TDO heme saturation. Modification of heme fully prevented the alterations of total heme content even under severe hemolysis as well as the increase of TDO heme saturation if no increase of heme synthesis occurred. Thus heme synthesis can greatly contribute to heme intracellular redistribution under oxidative stress.

Effect of Dexamethasone Stress on Concentrations of Zinc in Blood Plasma and in Sub-Cellular Fractions of Various Tissues of Neonatal Buffalo Calves

Effect of chemical stress of daily administration of glucocorticoid (dexamethasone @0.125 mg./calf/day) injections on plasma zinc levels, Zn status of body tissues and its distribution in sub cellular fractions, was studied in neonatal buffalo calves. Daily i/m injections of dexamethasone, starting at the completion of 1 week of age and continued till 8th week, led to a significant decline in plasma Zn concentration from 3rd week onwards, which then persisted throughout the rest of the experimental period. In control group, liver had the highest concentration of zinc, followed by heart, muscle, spleen, kidney and testis. In all these tissues, cytosolic fractions had the highest (>60%) zinc levels followed by nuclear, mitochondrial and microsomal fractions. In dexamethasone treated calves, there was a significant increase in the Zn uptake by the tissues of liver and muscle. This increase in zinc concentration was observed in all the sub cellular fractions of liver and muscle, however about 80% of this increase was in cytosolic fraction. It was concluded that glucocorticoid-induced stress caused increase in Zn levels of liver/muscles and decrease in blood plasma zinc, thus indicating a redistribution of Zn in body. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 7 : 1022-1025)

Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts.

thi1 has been recently isolated from Arabidopsis thaliana and is probably involved in both thiamine biosynthesis and as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggests a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thaliana thi1 cDNA complements a yeast strain disrupted for the homologous gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic alpha-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. To define the putative role of the two predicted targeting sequences in tandem, we produced two chimeric genes encompassing the chloroplastic THI1 TP and either 4 or 27 (including the putative mitochondrial presequence) N-terminal residues of the mature THI1, both linked to the reporter (gusA) gene. Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construct retaining only 4 residues of mature THI1, GUS was found in the chloroplastic fraction. Extension of the THI1 transit peptide to 27 residues of the mature protein allowed import and processing of GUS into both mitochondria and chloroplasts. Direct analysis by immunogold-labeling with an anti-THI1 polyclonal antibody identified THI1 in both organelles in Arabidopsis. We also provide evidence that the precursors of both organellar isoforms are encoded by a single nuclear transcript. Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.

Investigation of selenium distribution in subcellular fractions of human liver by neutron activation analysis.

Selenium is an important and essential trace element to living systems. In the article, two methods of instrumental neutron activation analysis and hydride generation-atomic fluorescence spectrometry were applied to determine Se in biological samples and the accuracy was evaluated by several reference materials. The subcellular distribution of selenium in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation combined with INAA. Selenium was mainly enriched in mitochondria, nuclei, and cytosol. Almost half of the total Se content existed in nuclei as a result of the large amount in liver and the high Se concentration. Generally, the highest Se concentration in the mitochondrial fractions of each liver sample suggested that Se had important functions in this liver component.

Cloning and expression of mouse legumain, a lysosomal endopeptidase.

Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe- 125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients.Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.

The phorbol ester TPA induces a translocation of the insulin sensitive glucose carrier (GLUT4) in fat cells

Insulin activates the glucose transport in isolated fat cells through a translocation of the insulin sensitive glucose carrier subtype (GLUT4) and by activation of glucose carriers in the plasma membrane. Protein kinase C stimulating phorbol esters are able to mimick partially the insulin effect on glucose transport. In order to determine whether this phorbol ester effect occurs through a translocation of the insulin sensitive glucose carrier (GLUT4) we used a monoclonal antibody against GLUT4 to determine its distribution in subcellular fractions of rat adipocytes. We found that the phorbol ester TPA is able to increase the amount of GLUT4 in the plasma membrane fraction about two-fold.

Isolation of alveolar epithelial cells from lung tissue obtained at autopsy.

Lung alveolar epithelial cells have been studied in a variety of laboratory animal models, and studies of human alveolar epithelial cells are important for comparison to information obtained from animal studies. Autopsy material is a source of human cells for study. Studies of human autopsy material revealed variables that negatively affected the yield of viable cells. For specimens from adults, these included death greater than 12 h before cell isolation, obvious severe lung fibrosis, longstanding metabolic disorders, and lung congestion indicated by weight of the right middle lobe greater than 150 g. Samples from children yielded significant numbers of viable cells up to 18 h after death. For 17 specimens that conformed to the above criteria, approximately 8.5 x 10(6) alveolar cells were obtained per gram of tissue (tissue weights ranged from 30 to 108 g) using a procedure involving instillation of proteases into the airways. The cells could be further fractionated, and 10 to 15% of the mixed cells obtained were type II pneumocytes. Analysis of NADPH cytochrome-c-reductase distribution in subcellular fractions provided evidence that the cells obtained were intact. Phospholipid enzyme activities and synthetic activity were within the ranges previously found in laboratory studies of freshly obtained animal lungs. These results suggest that significant numbers of viable and functional human lung cells, including type II pneumocytes, can be obtained from autopsy material.

Early changes in the tissue distribution of cadmium after oral but not intravenous cadmium exposure

The kinetics of 109Cd distribution in tissues of male and female mice were measured at intervals of 5 min to 15 days after oral (100 μg Cd/kg; by gavage) or intravenous (1 μg Cd/kg; i.v.) administration of 109CdCl 2. Unexpectedly, the ratio of 109Cd in liver to that in kidneys was ⩾10 within 1 h after administration by either route. However, after 4 h, route-dependent differences in distribution between liver and kidney became apparent. In mice receiving oral cadmium, the liver:kidney 109Cd ratio decreased with time to approximately 4 at 72 h after gavage. In contrast, in mice receiving IV cadmium, the liver:kidney 109Cd ratio remained high and relatively constant during the same time period. The time-dependent decrease in the liver:kidney 109Cd ratio after oral cadmium administration was caused by a 4–5-fold increase in cadmium content of the kidney that occurred between 30 min and 72 h after oral but not i.v. administration. During this time, there was no change in cadmium distribution in subcellular fractions of either liver or kidney. These results could be explained by the existence of 2 separate pathways for cadmium deposition after oral exposure. Early after exposure, cadmium may leave the intestine, bind to serum albumins or other high molecular weight proteins, and accumulate primarily in liver, as is also observed after IV cadmium administration. With time, cadmium may leave the intestinal mucosa bound to metallothionein and deposit primarily in the kidney. The different pathways of deposition after oral vs. i.v. exposure may in part explain why acute parenteral cadmium exposure causes liver toxicity, but chronic oral exposure causes renal toxicity.

Subcellular localization of a cysteine proteinase from Trypanosoma cruzi

Epimastigotes of Trypanosoma cruzi, Tulahuén strain, Tul 2 stock, contain a cysteine proteinase able to degrade azocasein at pH 5. This enzyme activity was extracted from whole cells by digitonin concentrations higher than those required for cytosolic markers, lower than those required for glycosomal and mitochondrial markers, and very similar to those required for solubilization of the acidic α-mannosidase. Both, the azocasein-degrading proteinase and the α-mannosidase, showed similar latency and distribution in subcellular fractions (the large granule fraction was the most active), and the same behavior in isopycnic sucrose gradient centrifugation; a broad peak centered at an equilibrium density of about 1.15 g cm −3, with a shoulder between 1.07 and 1.10 g cm −3, was obtained for both enzymes. The results suggest the cysteine proteinase activity is placed in the lysosomes.

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained ≈ 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.

Mechanisms of absorption of inorganic mercury from rat small intestine. I. Solvent drag effect on absorption of inorganic mercury.

A correlation of mercury absorption with water absorption was investigated by using the perfusion of rat small intestine with buffers containing 10(-4) M HgCl2. With a decrease in the osmolarity of the buffers or with increases in the sodium ion and urea concentrations in the buffers, absorption of water and mercury and accumulation of mercury in the intestinal tissue increased, and the increases in mercury absorption and accumulation were found to correlate with the increase in water absorption. Both the decrease in osmolarity and the increase in sodium ion concentration increased mercury accumulation in the epithelial cell after the perfusion. But these changes did not alter the mercury distribution in subcellular fractions. The results suggest that the increase in water absorption due to the hyptonicity or the increase in concentration of sodium ion or urea increases the mercury absorption and accumulation in the epithelial cell without change in the distributional pattern of mercury in the cell.