56 results on '"Joan Blanchette-Mackie"'
Search Results
2. Endothelium, the dynamic interface in cardiac lipid transport
- Author
-
Scow, Robert O., Joan Blanchette-Mackie, E., Dhalla, Naranjan S., editor, van der Vusse, Ger J., editor, and Stam, Hans, editor
- Published
- 1992
- Full Text
- View/download PDF
3. Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld/cld mice
- Author
-
Charles J. Schultz, E. Joan Blanchette-Mackie, and Robert O. Scow
- Subjects
lipoprotein lipase ,combined lipase deficiency ,immunofluorescence microscopy ,newborn mice ,Biochemistry ,QD415-436 - Abstract
Combined lipase deficiency (cld) is a recessive mutation in mice that causes a severe lack of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, hyperlipemia, and death within 3 days after birth. Earlier studies showed that inactive LPL and HL were synthesized by cld/cld tissues and that LPL synthesized by cld/cld brown adipocytes was retained in their ER. We report here a study of HL in liver, adrenal, and plasma of normal newborn and cld/cld mice. Immunofluorescence studies showed HL was present in extracellular space, but not in cells, in liver and adrenal of both normal and cld/cld mice. When protein secretion was blocked with monensin, HL was retained intracellularly in liver cell cultures and in incubated adrenal tissues of both groups of mice. These findings demonstrated that HL was synthesized and secreted by liver and adrenal cells in normal newborn and cld/cld mice. HL activities in liver, adrenal, and plasma in cld/cld mice were very low,
- Published
- 2000
- Full Text
- View/download PDF
4. Biological Implications of the Niemann-Pick C Mutation
- Author
-
Pentchev, Peter G., primary, Joan Blanchette-Mackie, E., additional, and Liscum, Laura, additional
- Published
- 1997
- Full Text
- View/download PDF
5. A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts
- Author
-
Stephen J. Demosky, Joan Blanchette-Mackie, Ulrike H. Schueler, Christine R. Kaneski, John A. Hanover, Roscoe O. Brady, Alan T. Remaley, and Nancy K. Dwyer
- Subjects
biology ,Cholesterol ,Globotriaosylceramide ,Enzyme replacement therapy ,medicine.disease ,Fabry disease ,Sphingolipid ,Article ,Cell biology ,chemistry.chemical_compound ,chemistry ,Biochemistry ,LDL receptor ,biology.protein ,medicine ,lipids (amino acids, peptides, and proteins) ,Apolipoprotein A1 ,Lipoprotein - Abstract
Fabry disease is an X-linked sphingolipid storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (AGA, EC 3.2.1.22) resulting in the intracellular accumulation of globotriaosylceramide (Gb3). We found that Gb3 storage also correlates with accumulation of endosomal-lysosomal cholesterol in Fabry fibroblasts. This cholesterol accumulation may contribute to the phenotypic pathology of Fabry disease by slowing endosomal-lysosomal trafficking. We found that LDL receptor expression is not downregulated in Fabry fibroblasts resulting in accumulation of both cholesterol and Gb3. 5A-Palmitoyl oleoyl-phosphatidylcholine (5AP) is a phospholipid complex containing a short synthetic peptide that mimics apolipoprotein A1, the main protein component of high-density lipoprotein (HDL) that mediates the efflux of cholesterol from cells via the ATP-binding cassette transporter. We used 5AP and HDL to remove cholesterol from Fabry fibroblasts to examine the fate of accumulated cellular Gb3. Using immunostaining techniques, we found that 5AP is highly effective for depleting cholesterol and Gb3 in these cells. 5AP restores the ApoA-1-mediated cholesterol efflux leading to mobilization of cholesterol and reduction of Gb3 in Fabry fibroblasts.
- Published
- 2015
6. The ABCA1 Transporter Modulates Late Endocytic Trafficking
- Author
-
Silvia Santamarina-Fojo, Nancy K. Dwyer, Christian A. Combs, Catherine L. Knapper, Joan Blanchette-Mackie, Stephen J. Demosky, Edward B. Neufeld, H. Bryan Brewer, John A. Stonik, Adele Cooney, Alan T. Remaley, and Marcella E. Comly
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,biology ,Endosome ,Endocytic cycle ,Wild type ,nutritional and metabolic diseases ,Cell Biology ,medicine.disease ,Biochemistry ,Cell biology ,Endocytic vesicle ,Tangier disease ,ABCA1 ,biology.protein ,medicine ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Sphingomyelin ,Molecular Biology - Abstract
We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.
- Published
- 2004
7. A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16
- Author
-
Vargheese M. Chennathukuzhi, Peter McNamara, E. Joan Blanchette-Mackie, Jerome F. Strauss, Rossana Sapiro, Mei Zhang, Anne Marie Curtis, Jeff Bray, Zhibing Zhang, Maja Bucan, and David Kapfhamer
- Subjects
Male ,Axoneme ,Microtubule-associated protein ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Immunocytochemistry ,Protozoan Proteins ,CHO Cells ,Microtubules ,Mice ,Cricetinae ,Two-Hybrid System Techniques ,Testis ,Animals ,Humans ,Amino Acid Sequence ,Cell Growth and Development ,Molecular Biology ,Gene ,Peptide sequence ,biology ,Tissue Extracts ,Chinese hamster ovary cell ,Algal Proteins ,Chlamydomonas ,Cell Biology ,biology.organism_classification ,Spermatozoa ,Fusion protein ,Molecular biology ,Cell biology ,Flagella ,Microtubule Proteins ,Microtubule-Associated Proteins ,Sequence Alignment - Abstract
cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.
- Published
- 2002
8. A Human Novel Gene DERPC Located on 16q22.1 Inhibits Prostate Tumor Cell Growth and Its Expression Is Decreased in Prostate and Renal Tumors
- Author
-
Shiv Srivastava, Mei Sun, E. Joan Blanchette-Mackie, Judd W. Moul, Lanfeng Ma, Mei Zhang, Zhiqiang Zou, Jia Li, Linda Xu, Mazen Makarem, Gyorgy Petrovics, Wei Zhang, and Isabell A. Sesterhenn
- Subjects
medicine.diagnostic_test ,Biology ,medicine.disease_cause ,medicine.disease ,Molecular biology ,Blot ,Prostate cancer ,Western blot ,LNCaP ,Genetics ,medicine ,Molecular Medicine ,Northern blot ,Nuclear protein ,Carcinogenesis ,Molecular Biology ,Genetics (clinical) ,Laser capture microdissection - Abstract
BACKGROUND: Deletion of chromosome 16q is frequently associated with diverse tumors. Numerous studies strongly suggest the presence of one or more tumor suppressor genes on chromosome 16q22 to 16qter including the widely studied cadherin gene family. However, the specific tumor suppressor genes residing in this region need better definition and characterization. MATERIAL AND METHODS: Standard molecular biology approaches have been used to clone and characterize the DERPC cDNA and its protein product on chromosome 16q22.1. Northern blotting was used to define the expression pattern in a multiple human tissue blots. DERPC expression was examined in multi-tumor array (Clontech, CA, USA) dot blot as well as in laser capture microdissection (LCM) derived prostate cancer (CaP) specimens by quantitative RT-PCR. Western blot analysis and a fluorescent microscopy were used to characterize the molecular size and the cellular location of green fluorescent protein (GFP)-tagged DERPC fusion proteins. A colony formation assay was conducted to determine the effects of DERPC expression on tumor cell growth. RESULTS: A novel gene DERPC (Decreased Expression in Renal and Prostate Cancer) was identified and characterized. DERPC encoded a strong basic, proline- and glycine-rich nuclear protein. DERPC was ubiquitously expressed, with abundant expression in kidney, skeletal muscle, testis, liver, ovary, and heart and moderate expression in prostate. DERPC expression was reduced in renal (67%) and prostate tumors (33%). Expression of DERPC has inhibitory potential on CaP cell growth. Further, overexpression of DERPC in LNCaP cells caused alterations of nuclear morphology. CONCLUSION: This study suggests that decreased expression of DERPC may be implicated in tumorigenesis of renal and CaPs.
- Published
- 2002
9. MLN64 Mediates Mobilization of Lysosomal Cholesterol to Steroidogenic Mitochondria
- Author
-
Federico Martinez, John A. Hanover, M E Comly, Toshio Fujimoto, E. Joan Blanchette-Mackie, Pei Liu, Nancy K. Dwyer, Mei Zhang, Jerome F. Strauss, and Lane K. Christenson
- Subjects
Time Factors ,Endosome ,Recombinant Fusion Proteins ,Blotting, Western ,Immunoblotting ,Endocytic cycle ,STARD3 ,CHO Cells ,Biology ,Transfection ,Endocytosis ,Biochemistry ,Cricetinae ,Animals ,Humans ,Molecular Biology ,Progesterone ,Genes, Dominant ,Microscopy, Confocal ,COS cells ,Cholesterol side-chain cleavage enzyme ,Cell Membrane ,Membrane Proteins ,Biological Transport ,Cell Biology ,Mitochondria ,Protein Structure, Tertiary ,Cell biology ,Transmembrane domain ,Cholesterol ,Microscopy, Fluorescence ,COS Cells ,lipids (amino acids, peptides, and proteins) ,Carrier Proteins ,Lysosomes ,Intracellular ,Plasmids ,Protein Binding - Abstract
This study demonstrates that the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing protein, MLN64, participates in intracellular cholesterol trafficking. Analysis of the intracellular itinerary of MLN64 and MLN64 mutants tagged with green fluorescent protein showed that the N-terminal transmembrane domains mediate endocytosis of MLN64 from the plasma membrane to late endocytic compartments. MLN64 constitutively traffics via dynamic NPC1-containing late endosomal tubules in normal cells; this dynamic movement was inhibited in cholesterol-loaded cells, and MLN64 is trapped at the periphery of cholesterol-laden lysosomes. The MLN64 START domain stimulated free cholesterol transfer from donor to acceptor mitochondrial membranes and enhanced steroidogenesis by placental mitochondria. Expression of a truncated form of MLN64 (ΔSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The ΔSTART-MLN64 dominant negative protein was located at the surface of the cholesterol-laden lysosomes. This dominant negative mutant suppressed steroidogenesis in COS cells expressing the mitochondrial cholesterol side chain cleavage system. We conclude that MLN64 participates in mobilization and utilization of lysosomal cholesterol by virtue of the START domain's role in cholesterol transport.
- Published
- 2002
10. Cellular Localization and Trafficking of the Human ABCA1 Transporter
- Author
-
M E Comly, Joan Blanchette-Mackie, Silvia Santamarina-Fojo, Adele Cooney, Edward B. Neufeld, Nancy K. Dwyer, Mei Zhang, John A. Stonik, Alan T. Remaley, H. Bryan Brewer, and Stephen J. Demosky
- Subjects
Endosome ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,CHO Cells ,Transfection ,Biochemistry ,chemistry.chemical_compound ,Cricetinae ,polycyclic compounds ,Animals ,Humans ,Cycloheximide ,Monensin ,Molecular Biology ,Cellular localization ,Apolipoprotein A-I ,biology ,nutritional and metabolic diseases ,Biological Transport ,hemic and immune systems ,Cell Biology ,Brefeldin A ,Immunohistochemistry ,Fusion protein ,Endocytosis ,Cell Compartmentation ,Cell biology ,Luminescent Proteins ,Sterols ,ATP Binding Cassette Transporter 1 ,Endocytic vesicle ,chemistry ,ABCA1 ,biology.protein ,ATP-Binding Cassette Transporters ,lipids (amino acids, peptides, and proteins) ,Intracellular ,HeLa Cells - Abstract
ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.
- Published
- 2001
11. Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld/cld mice
- Author
-
E. Joan Blanchette-Mackie, Charles J. Schultz, and Robert O. Scow
- Subjects
medicine.medical_specialty ,congenital, hereditary, and neonatal diseases and abnormalities ,Combined Lipase Deficiency ,lipoprotein lipase ,QD415-436 ,Immunofluorescence ,Biochemistry ,Endocrinology ,newborn mice ,Internal medicine ,medicine ,Extracellular ,Lipase ,Lipoprotein lipase ,biology ,medicine.diagnostic_test ,Chemistry ,Liver cell ,immunofluorescence microscopy ,Cell Biology ,respiratory system ,respiratory tract diseases ,Secretory protein ,biology.protein ,Hepatic lipase ,combined lipase deficiency - Abstract
Combined lipase deficiency (cld) is a recessive mutation in mice that causes a severe lack of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, hyperlipemia, and death within 3 days after birth. Earlier studies showed that inactive LPL and HL were synthesized by cld/cld tissues and that LPL synthesized by cld/cld brown adipocytes was retained in their ER. We report here a study of HL in liver, adrenal, and plasma of normal newborn and cld/cld mice. Immunofluorescence studies showed HL was present in extracellular space, but not in cells, in liver and adrenal of both normal and cld/cld mice. When protein secretion was blocked with monensin, HL was retained intracellularly in liver cell cultures and in incubated adrenal tissues of both groups of mice. These findings demonstrated that HL was synthesized and secreted by liver and adrenal cells in normal newborn and cld/cld mice. HL activities in liver, adrenal, and plasma in cld/cld mice were very low
- Published
- 2000
12. NPC1-Containing Compartment of Human Granulosa-Lutein Cells: A Role in the Intracellular Trafficking of Cholesterol Supporting Steroidogenesis
- Author
-
Jane M. Glick, Jerome F. Strauss, Shutish Patel, Hidemichi Watari, Peter G. Pentchev, E. Joan Blanchette-Mackie, Edward B. Neufeld, Gwo-Shing Sun, and Nancy K. Dwyer
- Subjects
Intracellular Fluid ,congenital, hereditary, and neonatal diseases and abnormalities ,8-Bromo Cyclic Adenosine Monophosphate ,Gene Expression ,CHO Cells ,Biology ,Filipin ,symbols.namesake ,chemistry.chemical_compound ,Niemann-Pick C1 Protein ,Cricetinae ,Luteal Cells ,hemic and lymphatic diseases ,Animals ,Humans ,Cells, Cultured ,Progesterone ,Granulosa Cells ,Membrane Glycoproteins ,Cholesterol ,Anticholesteremic Agents ,Vesicle ,Endoplasmic reticulum ,Intracellular Signaling Peptides and Proteins ,Proteins ,nutritional and metabolic diseases ,Biological Transport ,Cell Biology ,Golgi apparatus ,Phosphoproteins ,Cell biology ,Lipoproteins, LDL ,chemistry ,Biochemistry ,symbols ,Androstenes ,Female ,Steroids ,lipids (amino acids, peptides, and proteins) ,Progestins ,NPC1 ,Carrier Proteins ,Lysosomes ,Intracellular ,Lipoprotein - Abstract
Steroidogenic cells represent unique systems for the exploration of intracellular cholesterol trafficking. We employed cytochemical and biochemical methods to explore the expression, regulation, and function of the Niemann-Pick C1 protein (NPC1) in human granulosa-lutein cells. NPC1 was localized in a subset of lysosome-associated membrane glycoprotein 2 (LAMP-2)-positive vesicles. By analyzing the sensitivity of NPC1 N-linked oligosaccharide chains to glycosidases and neuraminidase, evidence was obtained for movement of nascent NPC1 from the endoplasmic reticulum through the medial and trans compartments of the Golgi apparatus prior to its appearance in cytoplasmic vesicles. NPC1 protein content and the morphology and cellular distribution of NPC1-containing vesicles were not affected by treatment of the granulosa-lutein cells with 8-Br-cAMP, which stimulates cholesterol metabolism into progesterone. In contrast, steroidogenic acute regulatory (StAR) protein levels were increased by 8-Br-cAMP. Incubation of granulosa-lutein cells with low-density lipoprotein (LDL) in the presence of the hydrophobic amine, U18666A, caused accumulation of free cholesterol in granules, identified by filipin staining, that contained LAMP-2 and NPC1. These granules also stained for neutral lipid with Nile red, reflecting accumulation of LDL-derived cholesterol esters. LDL-stimulated progesterone synthesis was completely blocked by U18666A, leaving steroid output at levels similar to those of cells incubated in the absence of LDL. The hydrophobic amine also blocked the LDL augmentation of 8-Br-cAMP-stimulated progesterone synthesis, reducing steroid production to levels seen in cells stimulated with 8-Br-cAMP in the absence of LDL. Steroidogenesis recovered after U18666A was removed from the culture medium. U18666A treatment caused a 2-fold or more increase in NPC1 protein and mRNA levels, suggesting that disruption of NPC1's function activates a compensatory mechanism resulting in increased NPC1 synthesis. We conclude that the NPC1 compartment plays an important role in the trafficking of LDL-derived substrate in steroidogenic cells; that NPC1 expression is up-regulated when NPC1 action is blocked; and that the NPC1 compartment can be functionally separated from other intracellular pathways contributing substrate for steroidogenesis.
- Published
- 2000
13. The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo
- Author
-
John P. Incardona, Adele Cooney, Roscoe O. Brady, Eugene D. Carstea, Calvin F. Roff, Nancy K. Dwyer, E. Joan Blanchette-Mackie, Marc C. Patterson, Peter G. Pentchev, Edward B. Neufeld, Jerome F. Strauss, Kousaku Ohno, Sundar Suresh, Shutish C. Patel, Marie T. Vanier, Meryl E. Wastney, and Jill A. Morris
- Subjects
Lysosomal transport ,Sucrose ,Molecular Sequence Data ,Biology ,Biochemistry ,Antibodies ,Receptor, IGF Type 2 ,Structure-Activity Relationship ,Antigens, CD ,Niemann-Pick C1 Protein ,Lysosomal-Associated Membrane Protein 2 ,Organelle ,Humans ,Amino Acid Sequence ,Molecular Biology ,Niemann-Pick Diseases ,Membrane Glycoproteins ,LAMP2 ,Vesicle ,Intracellular Signaling Peptides and Proteins ,Lysosome-Associated Membrane Glycoproteins ,Proteins ,Biological Transport ,Cell Biology ,Sterol transport ,Endocytosis ,Sterol ,Cell Compartmentation ,Cell biology ,Cholesterol ,Mutagenesis, Site-Directed ,Axoplasmic transport ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Carrier Proteins ,Lysosomes - Abstract
Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.
- Published
- 1999
14. Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization
- Author
-
E. Joan Blanchette-Mackie, Roscoe O. Brady, Nancy K. Dwyer, Jerome F. Strauss, Shutish C. Patel, Edward B. Neufeld, Hidemichi Watari, Jane M. Glick, and Peter G. Pentchev
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Lipid storage disorder ,Endosome ,Green Fluorescent Proteins ,CHO Cells ,Biology ,Endoplasmic Reticulum ,Transfection ,Niemann-Pick C1 Protein ,Cricetinae ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Filipin ,Sequence Deletion ,Niemann-Pick Diseases ,Membrane Glycoproteins ,Multidisciplinary ,Mannose 6-phosphate receptor ,Endoplasmic reticulum ,Chinese hamster ovary cell ,Intracellular Signaling Peptides and Proteins ,Proteins ,nutritional and metabolic diseases ,Biological Sciences ,medicine.disease ,Molecular biology ,Recombinant Proteins ,Cell biology ,Luminescent Proteins ,Cholesterol ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Carrier Proteins ,Lysosomes ,Niemann–Pick disease - Abstract
Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.
- Published
- 1999
15. Variegated transfer of recombinant glycosylphosphatidylinositol-anchored CD4 among cultured cells: Correlation of flow cytometric and microscopic observations
- Author
-
Nancy K. Dwyer, Jeffery L. Miller, Mara Giattina, and E. Joan Blanchette Mackie
- Subjects
Cell type ,Glycosylphosphatidylinositols ,Cell ,Cell Count ,Cell Communication ,Cell Line ,Pathology and Forensic Medicine ,Flow cytometry ,HeLa ,Fluorescence microscope ,medicine ,Animals ,Humans ,Cells, Cultured ,medicine.diagnostic_test ,biology ,General Medicine ,Flow Cytometry ,biology.organism_classification ,Coculture Techniques ,Recombinant Proteins ,Cell biology ,carbohydrates (lipids) ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Cytoplasm ,Cell culture ,CD4 Antigens ,lipids (amino acids, peptides, and proteins) ,Neural cell adhesion molecule - Abstract
Glycosylphosphatidylinositol-anchored proteins (GPI-proteins) expressed on the outer leaflet of cell membranes are involved in diverse physiologic as well as pathologic processes in humans. Previously, we demonstrated the intercellular transfer of overexpressed CD4-GPI in vitro from transduced HeLa cells to their parental cell line. In this report we present further information on the transfer process and the nature of the transferred GPI-proteins. In mixed-cell populations, the transfer of CD4-GPI was detectable within minutes at levels proportional to the ratio of donor and recipient cells. The amount of CD4-GPI detected with flow cytometry on the surface of the recipient cells varied according to cell type. Microscopy of mixed cell populations revealed discrete CD4-GPI containing aggregates on the target cells, whereas colocalized transfer of cytoplasm was not detected. Separation of cocultivated cells by semipermeable membranes largely prevented CD4-GPI transfer, but aggregates containing CD4-GPI were demonstrated by electron microscopy in supernatants passed through filters of 0.4-mm pore size.
- Published
- 1998
16. Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation
- Author
-
J M VandenBroek, L A Zech, Peter G. Pentchev, H B Brewer, R D Phair, C C Schwartz, Robert D. Shamburek, Edward B. Neufeld, Roscoe O. Brady, E D Carstea, P S Cooper, and Joan Blanchette-Mackie
- Subjects
Intermediate-density lipoprotein ,medicine.medical_specialty ,biology ,Cholesterol ,Sterol O-acyltransferase ,Reverse cholesterol transport ,QD415-436 ,Cell Biology ,Biochemistry ,chemistry.chemical_compound ,Endocrinology ,chemistry ,In vivo ,Internal medicine ,HMG-CoA reductase ,Cholesteryl ester ,medicine ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Lipoprotein - Abstract
Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.
- Published
- 1997
17. Niemann-Pick C1 Disease Gene: Homology to Mediators of Cholesterol Homeostasis
- Author
-
Rivka Carmi, Laura Liscum, Marcella E. Comly, Christine R. Kaneski, Marsha Zeigler, Adele Cooney, Roscoe O. Brady, Yiannis A. Ioannou, Peter G. Pentchev, Marc C. Patterson, James W. Nagle, E. Joan Blanchette-Mackie, Maureen E. Higgins, David B. Krizman, J Sokol, Raymond R. O'Neill, Jill A. Morris, Jerome F. Strauss, Stephen L. Sturley, Christiano Cummings, O. P. van Diggelen, Ta-Yuan Chang, Edward B. Neufeld, Danilo A. Tagle, Katherine G. Coleman, Jessie Z. Gu, Kousaku Ohno, Stacie K. Loftus, M H Polymeropoulos, Nancy K. Dwyer, David Markie, Melissa A. Rosenfeld, Marie T. Vanier, Anthony Brown, Eugene D. Carstea, Milan Elleder, Dana Zhang, William J. Pavan, and Clinical Genetics
- Subjects
7-Dehydrocholesterol reductase ,Positional cloning ,Molecular Sequence Data ,Receptors, Cell Surface ,Transfection ,Intracellular cholesterol transport ,chemistry.chemical_compound ,Niemann-Pick C1 Protein ,hemic and lymphatic diseases ,medicine ,Drosophila Proteins ,Homeostasis ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Polymorphism, Single-Stranded Conformational ,Niemann-Pick Diseases ,Membrane Glycoproteins ,Multidisciplinary ,Niemann–Pick disease, type C ,Sequence Homology, Amino Acid ,biology ,Cholesterol ,Intracellular Signaling Peptides and Proteins ,Chromosome Mapping ,Membrane Proteins ,Proteins ,nutritional and metabolic diseases ,Cholesterol, LDL ,medicine.disease ,Molecular biology ,chemistry ,Biochemistry ,Mutation ,HMG-CoA reductase ,biology.protein ,Insect Proteins ,Hydroxymethylglutaryl CoA Reductases ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Carrier Proteins ,Chromosomes, Human, Pair 18 ,Lysosomes ,Cholesterol storage - Abstract
Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)–derived cholesterol. By positional cloning methods, a gene (NPC1)with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-typeNPC1cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278–amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.
- Published
- 1997
18. Microtubules are not required for glucocorticoid receptor mediated gene induction
- Author
-
Nancy K. Dwyer, E. Joan Blanchette-Mackie, S. Stoney Simons, Daniele Szapary, and T. Barber
- Subjects
Transcriptional Activation ,Cytoplasm ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Fluorescent Antibody Technique ,Biology ,Transfection ,Microtubules ,Biochemistry ,Receptors, Glucocorticoid ,Endocrinology ,Glucocorticoid receptor ,Genes, Reporter ,Microtubule ,Gene expression ,medicine ,Animals ,Humans ,Receptor ,Cytoskeleton ,Molecular Biology ,PELP-1 ,Cell Nucleus ,Cell Biology ,Molecular biology ,Rats ,Cell biology ,Gene Expression Regulation ,Molecular Medicine ,Steroids ,Colchicine ,Glucocorticoid ,HeLa Cells ,medicine.drug - Abstract
Steroid-free glucocorticoid receptors are generally considered to reside in the cytoplasm of cells. After the binding of steroids, the receptors translocate into the nucleus in a manner that has been proposed to involve microtubules. However, some results with inhibitors of microtubule assembly argue to the contrary. In all of these studies, only the whole cell localization of receptors has been examined; the biological activity of these receptors has not been determined. We now report that steroid-induced gene expression is maintained in the absence of intact microtubules. This argues that microtubules are not required for either the nuclear translocation or biological activity of glucocorticoid receptors.
- Published
- 1994
19. The NP-C gene: a key to pathways of intracellular cholesterol transport
- Author
-
Peter G. Pentchev, E. Joan Blanchette-Mackie, and Eliezer A. Dawidowicz
- Subjects
Cholesterol ,Mechanism (biology) ,Cell Biology ,Biology ,Intracellular cholesterol transport ,Cell biology ,chemistry.chemical_compound ,Human disease ,chemistry ,Biochemistry ,lipids (amino acids, peptides, and proteins) ,Gene ,Intracellular ,Intracellular transport ,Lipoprotein - Abstract
Elucidation of the pathways for intracellular transport of cholesterol is an important yet elusive goal in cell biology. Analysis of the cellular defects in the human disease Niemann-Pick C (NP-C) is providing insights into this problem. Cholesterol derived from low-density lipoprotein accumulates in lysosomes of NP-C cells, apparently because intracellular movement of such cholesterol is blocked. Identification of the NP-C gene should provide crucial molecular clues to the mechanism of cholesterol transport within cells.
- Published
- 1994
20. Progesterone blocks cholesterol translocation from lysosomes
- Author
-
Raymond R. O'Neill, S Patel, M E Comly, Ehud Goldin, Adele Cooney, G Carstea, J D Butler, Joan Blanchette-Mackie, Calvin F. Roff, and Marc C. Patterson
- Subjects
medicine.medical_specialty ,biology ,Cholesterol ,Reverse cholesterol transport ,Sterol O-acyltransferase ,Cell Biology ,Intracellular cholesterol transport ,Biochemistry ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Low-density lipoprotein ,Internal medicine ,HMG-CoA reductase ,Cholesteryl ester ,biology.protein ,medicine ,Pregnenolone ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,medicine.drug - Abstract
Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.
- Published
- 1992
21. Niemann-Pick type-C disease: Deficient intracellular transport of exogenously derived cholesterol
- Author
-
Peter G. Pentchev, Roscoe O. Brady, Ehud Goldin, Adele Cooney, Marcella E. Comly, Calvin F. Roff, and Joan Blanchette-Mackie
- Subjects
Biological Transport, Active ,Fluorescent Antibody Technique ,Biology ,Lipidoses ,Intracellular cholesterol transport ,chemistry.chemical_compound ,Antigens, CD ,otorhinolaryngologic diseases ,medicine ,Humans ,Cells, Cultured ,Genetics (clinical) ,Niemann-Pick Diseases ,chemistry.chemical_classification ,Membrane Glycoproteins ,Esterification ,Cholesterol ,Lysosome-Associated Membrane Glycoproteins ,Metabolism ,medicine.disease ,Enzyme ,chemistry ,Biochemistry ,Niemann-pick type c disease ,lipids (amino acids, peptides, and proteins) ,Cholesterol Esters ,Lysosomes ,Niemann–Pick disease ,Homeostasis ,Intracellular transport - Abstract
NPC disease is an autosomal recessive neurovisceral storage disorder. A pleiotropic array of secondary enzymatic and storage abnormalities has in the past obscured a cohesive understanding of the underlying metabolic basis of this disorder. Recent findings, reviewed in this report, demonstrate that NPC disease is a cholesterol lipidosis resulting from defective intracellular cholesterol transport. The sequence of cellular events characteristic of NPC is 1) deficient intracellular transport of exogenously derived cholesterol resulting in retarded induction of cellular cholesterol homeostatic regulation; 2) accumulation of cholesterol in lysosomes; and 3) secondary cellular effects. Retarded esterification of exogenous cholesterol and accumulation of unesterified cholesterol in lysosomes is tightly coupled to the primary defect and serves as the basis for biochemical diagnosis of NPC.
- Published
- 1992
22. Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors
- Author
-
John A. Chiorini, Nancy K. Dwyer, Stephanie Pack, David M. Harlan, William Jou, Roland A Owens, Michael Schmidt, Victor J McAlister, Oksana Gavrilova, Eric Liu, Klaus Pechhold, E. Joan Blanchette-Mackie, and Anthony T Craig
- Subjects
Transgene ,viruses ,Genetic Vectors ,Islets of Langerhans Transplantation ,Mice, SCID ,In Vitro Techniques ,Biology ,medicine.disease_cause ,Cell Line ,lcsh:Infectious and parasitic diseases ,Islets of Langerhans ,Mice ,Transduction (genetics) ,Multiplicity of infection ,Mice, Inbred NOD ,Transduction, Genetic ,Virology ,medicine ,Animals ,Humans ,lcsh:RC109-216 ,Rats, Wistar ,Adeno-associated virus ,Research ,Pancreatic islets ,Genetic Therapy ,Dependovirus ,Rats ,Transplantation ,Diabetes Mellitus, Type 1 ,Infectious Diseases ,medicine.anatomical_structure ,Helper virus ,Female ,Pancreatic islet transplantation - Abstract
Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.
- Published
- 2009
23. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism
- Author
-
E. Joan Blanchette-Mackie, Glenn L. Radice, Pei Liu, Igor Kostetskii, Federico Martinez, Nancy K. Dwyer, Jerome F. Strauss, Tatsuro Kishida, Steven U. Walkley, and Zhibing Zhang
- Subjects
Sterol O-acyltransferase ,STARD3 ,Biology ,Biochemistry ,chemistry.chemical_compound ,Mice ,Animals ,RNA, Messenger ,Molecular Biology ,Mice, Knockout ,Cholesterol ,Steroidogenic acute regulatory protein ,Gene Expression Profiling ,Sterol ester ,Biological Transport ,Cell Biology ,Phosphoproteins ,Lipids ,Sterol ,Sterol regulatory element-binding protein ,Protein Structure, Tertiary ,Sterols ,Fertility ,Phenotype ,chemistry ,Liver ,Mutation ,lipids (amino acids, peptides, and proteins) ,Female ,NPC1 - Abstract
The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.
- Published
- 2004
24. A human novel gene DERPC on 16q22.1 inhibits prostate tumor cell growth and its expression is decreased in prostate and renal tumors
- Author
-
Mei, Sun, Lanfeng, Ma, Linda, Xu, Jia, Li, Wei, Zhang, Gyorgy, Petrovics, Mazen, Makarem, Isabell, Sesterhenn, Mei, Zhang, E Joan, Blanchette-Mackie, Judd, Moul, Shiv, Srivastava, and Zhiqiang, Zou
- Subjects
Expressed Sequence Tags ,Male ,Base Sequence ,Molecular Sequence Data ,Nuclear Proteins ,Prostatic Neoplasms ,Proteins ,Kidney Neoplasms ,Gene Expression Regulation, Neoplastic ,Sequence Homology, Nucleic Acid ,Humans ,Female ,Genes, Tumor Suppressor ,Amino Acid Sequence ,RNA, Messenger ,Carrier Proteins ,Cell Division ,Chromosomes, Human, Pair 16 ,Research Article - Abstract
BACKGROUND: Deletion of chromosome 16q is frequently associated with diverse tumors. Numerous studies strongly suggest the presence of one or more tumor suppressor genes on chromosome 16q22 to 16qter including the widely studied cadherin gene family. However, the specific tumor suppressor genes residing in this region need better definition and characterization. MATERIAL AND METHODS: Standard molecular biology approaches have been used to clone and characterize the DERPC cDNA and its protein product on chromosome 16q22.1. Northern blotting was used to define the expression pattern in a multiple human tissue blots. DERPC expression was examined in multi-tumor array (Clontech, CA, USA) dot blot as well as in laser capture microdissection (LCM) derived prostate cancer (CaP) specimens by quantitative RT-PCR. Western blot analysis and a fluorescent microscopy were used to characterize the molecular size and the cellular location of green fluorescent protein (GFP)-tagged DERPC fusion proteins. A colony formation assay was conducted to determine the effects of DERPC expression on tumor cell growth. RESULTS: A novel gene DERPC (Decreased Expression in Renal and Prostate Cancer) was identified and characterized. DERPC encoded a strong basic, proline- and glycine-rich nuclear protein. DERPC was ubiquitously expressed, with abundant expression in kidney, skeletal muscle, testis, liver, ovary, and heart and moderate expression in prostate. DERPC expression was reduced in renal (67%) and prostate tumors (33%). Expression of DERPC has inhibitory potential on CaP cell growth. Further, overexpression of DERPC in LNCaP cells caused alterations of nuclear morphology. CONCLUSION: This study suggests that decreased expression of DERPC may be implicated in tumorigenesis of renal and CaPs.
- Published
- 2002
25. Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease
- Author
-
Dona C. Love, Edward B. Neufeld, Mei Zhang, Peter G. Pentchev, M E Comly, Adele Cooney, E. Joan Blanchette-Mackie, Nancy K. Dwyer, and John A. Hanover
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Endosome ,Endocytic cycle ,Blotting, Western ,Green Fluorescent Proteins ,Golgi Apparatus ,CHO Cells ,Endosomes ,Biology ,Endocytosis ,Endoplasmic Reticulum ,symbols.namesake ,Niemann-Pick C1 Protein ,hemic and lymphatic diseases ,Cricetinae ,Organelle ,medicine ,Animals ,Humans ,Niemann-Pick Diseases ,Multidisciplinary ,Membrane Glycoproteins ,Endoplasmic reticulum ,Intracellular Signaling Peptides and Proteins ,nutritional and metabolic diseases ,Golgi apparatus ,Biological Sciences ,medicine.disease ,Cell biology ,nervous system diseases ,Cell Compartmentation ,Luminescent Proteins ,Cholesterol ,Microscopy, Fluorescence ,symbols ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Niemann–Pick disease ,Carrier Proteins - Abstract
Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.
- Published
- 2001
26. Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice
- Author
-
Gilles Lambert, Liliane J. Striker, Alton Thomas, Gyorgy Csako, Alan T. Remaley, Silvia Santamarina-Fojo, Boris L. Vaisman, Chi-Chao Chan, Benoit Marteyn, Joan Blanchette-Mackie, Enrico Lupia, Gary E. Striker, H. Bryan Brewer, John N. Brady, Edward B. Neufeld, Rene Costello, Beverly Paigen, and Naohiko Sakai
- Subjects
Apolipoprotein E ,medicine.medical_specialty ,Apolipoprotein B ,Arteriosclerosis ,Lipoproteins ,Kidney ,Biochemistry ,Phosphatidylcholine-Sterol O-Acyltransferase ,chemistry.chemical_compound ,Mice ,High-density lipoprotein ,Internal medicine ,Cholesterylester transfer protein ,medicine ,Animals ,RNA, Messenger ,Molecular Biology ,Hypoalphalipoproteinemia ,DNA Primers ,Mice, Knockout ,biology ,Base Sequence ,Cholesterol ,Glomerulosclerosis, Focal Segmental ,Glomerulosclerosis ,Cell Biology ,medicine.disease ,Lipids ,Mice, Inbred C57BL ,Microscopy, Electron ,Endocrinology ,chemistry ,biology.protein ,Cholesteryl ester ,lipids (amino acids, peptides, and proteins) - Abstract
To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (−70 to −94%) and non-HDL cholesterol (−48 to −85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (−35% to −99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.
- Published
- 2001
27. Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes
- Author
-
S Patel, Hidemichi Watari, John A. Hanover, E. Joan Blanchette-Mackie, Nancy K. Dwyer, Dona C. Love, Adele Cooney, Edward B. Neufeld, Jerome F. Strauss, Mei Zhang, M E Comly, and Peter G. Pentchev
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,DNA, Complementary ,Endosome ,CHO Cells ,Endosomes ,Biology ,Transfection ,Biochemistry ,chemistry.chemical_compound ,Glycolipid ,Niemann-Pick C1 Protein ,hemic and lymphatic diseases ,Cricetinae ,Organelle ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,Membrane Glycoproteins ,Cholesterol ,Histocytochemistry ,Vesicle ,Intracellular Signaling Peptides and Proteins ,nutritional and metabolic diseases ,Biological Transport ,Cell Biology ,Compartment (chemistry) ,Intracellular Membranes ,Cell biology ,Cell Compartmentation ,Lipoproteins, LDL ,Protein Transport ,Endocytic vesicle ,chemistry ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Glycolipids ,Carrier Proteins ,Lysosomes ,Subcellular Fractions - Abstract
The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.
- Published
- 2000
28. Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein
- Author
-
Nancy K. Dwyer, Edward B. Neufeld, Peter G. Pentchev, Shutish Patel, E. Joan Blanchette-Mackie, Michiko Watari, Jerome F. Strauss, and Hidemichi Watari
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Leucine zipper ,Glycosylation ,Glutamine ,Mutant ,CHO Cells ,Endosomes ,Biology ,medicine.disease_cause ,Transfection ,Biochemistry ,chemistry.chemical_compound ,Niemann-Pick C1 Protein ,hemic and lymphatic diseases ,Cricetinae ,medicine ,Animals ,Humans ,Point Mutation ,Molecular Biology ,Late endosome ,Niemann-Pick Diseases ,Mutation ,ATF3 ,Leucine Zippers ,Binding Sites ,Membrane Glycoproteins ,Point mutation ,Intracellular Signaling Peptides and Proteins ,nutritional and metabolic diseases ,Proteins ,Cell Biology ,Tunicamycin ,Intracellular Membranes ,Molecular biology ,Recombinant Proteins ,Molecular Weight ,Sterols ,Cholesterol ,Phenotype ,chemistry ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,lipids (amino acids, peptides, and proteins) ,NPC1 ,Asparagine ,Carrier Proteins ,Lysosomes - Abstract
Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.
- Published
- 1999
29. Induction of endocytic vesicles by exogenous C(6)-ceramide
- Author
-
Stephan Ladisch, E. Joan Blanchette-Mackie, and Ruixiang Li
- Subjects
Ceramide ,Time Factors ,Endosome ,Endosomes ,Biology ,Ceramides ,Biochemistry ,symbols.namesake ,chemistry.chemical_compound ,Mice ,Animals ,Molecular Biology ,Microscopy, Confocal ,Sphingosine ,Vesicle ,Cell Biology ,3T3 Cells ,Golgi apparatus ,Endocytosis ,Cell biology ,Endocytic vesicle ,chemistry ,Vital stain ,Second messenger system ,symbols ,Lysosomes - Abstract
Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis. Here, we present evidence that exogenously added C(6)-ceramide induces enlargement of late endosomes and lysosomes. 10 microM C(6)-ceramide caused the formation of numerous vesicles of varying sizes (2-10 micrometers) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C(6)-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 microM C(6)-ceramide. They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, approximately 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide. Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C(6)-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C(6)-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C(2)-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2, 3)-galactosyl-beta(1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously. Together, these studies demonstrate that exogenous C(6)-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.
- Published
- 1999
30. Combined lipase deficiency (cld/cld) in mice affects differently post-translational processing of lipoprotein lipase, hepatic lipase and pancreatic lipase
- Author
-
Jin-Woo Park, Charles J. Schultz, Robert O. Scow, and E. Joan Blanchette-Mackie
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,medicine.medical_specialty ,Combined Lipase Deficiency ,Adipose tissue ,Biochemistry ,Mice ,Internal medicine ,medicine ,Animals ,Secretion ,Lipase ,Molecular Biology ,Pancreas ,Lipoprotein lipase ,biology ,Chemistry ,Endoplasmic reticulum ,Liver cell ,Organic Chemistry ,nutritional and metabolic diseases ,Cell Biology ,respiratory system ,respiratory tract diseases ,Lipoprotein Lipase ,Endocrinology ,Liver ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Hepatic lipase ,Protein Processing, Post-Translational - Abstract
Lipoprotein lipase (LPL) and hepatic lipase (HL), which act on plasma lipoproteins, belong to the same gene family as pancreatic lipase. LPL is synthesized in heart, muscle and adipose tissue, while HL is synthesized primarily in liver. LPL is also synthesized in liver of newborn rodents. The active form of LPL is a dimer, whereas that of HL has not been established. Combined lipase deficiency (CLD) is an autosomal recessive mutation (cld) in mice which impairs post-translational processing of LPL and HL. Cld/cld mice have very low LPL and HL activities (< 5% of normal), yet normal pancreatic lipase activity. They develop massive hypertriglyceridemia and die within 3 days after birth. The CLD mutation allows synthesis, glycosylation and dimerization of LPL, but blocks activation and secretion of the lipase. Thus, dimerization per se does not result in production of active LPL. Immunofluorescence studies showed that LPL is retained in endoplasmic reticulum (ER) in cld/cld cells. Translocation of Golgi components to ER by treatment with brefeldin A (BFA) enabled synthesis of active LPL in cultured cld/cld brown adipocytes. Thus, production of inactive LPL in cld/cld cells results from inability of the cells to transport LPL from ER. The CLD mutation allows synthesis and glycosylation of HL, but blocks activation of the lipase. Immunofluorescence studies located HL mostly outside of cells in liver, liver cell cultures and incubated adrenal tissue of normal and cld/cld mice and mostly inside of cells in liver cell cultures and adrenal tissues treated with monensin (to block secretion of protein). These findings demonstrate synthesis and secretion of HL by both liver and adrenal cells of normal and cld/cld mice. Thus, the CLD mutation allows secretion of inactive HL by liver and adrenals. However, it does not block synthesis or secretion of active pancreatic lipase. Our findings indicate that LPL, HL and pancreatic lipase, although closely related, are processed differently.
- Published
- 1998
31. Cholesterol Distribution in Golgi, Lysosomes and Endoplasmic Reticulum
- Author
-
E. Joan Blanchette-Mackie and Peter G. Pentchev
- Subjects
Chemistry ,Cholesterol ,Endoplasmic reticulum ,Endocytic cycle ,Sterol O-acyltransferase ,Golgi apparatus ,Intracellular cholesterol transport ,Cell biology ,symbols.namesake ,chemistry.chemical_compound ,symbols ,lipids (amino acids, peptides, and proteins) ,Secretory pathway ,Cellular compartment - Abstract
Insight into the effect of exogeneously derived lipoprotein cholesterol on distribution of intracellular membrane cholesterol has been gained from structural studies on normal and Niemann Pick Type C human fibroblasts. Endocytic uptake of LDL enriches Golgi cholesterol in both normal and NPC cells. However, the NPC mutation and treatment of normal cells with progesterone during LDL uptake produces abnormal accumulation of cholesterol in lysosomes and trans Golgi cisternae. This lysosomal/Golgi block in cholesterol trafficking results in the inability of endocytosed cholesterol to induce cellular homestatic responses. In addition to lysosomes and Golgi the endoplasmic reticulum can also be a site along the intracellular cholesterol transport pathway that becomes a temporary depot for cholesterol. Specific inhibition of acyl CoA: cholesterol acyltransferase with S-58035 during endocytic uptake results in a reversable accumulation of cholesterol in membranes of ER. Thus the ER, normally low in intracellular cholesterol, has the capacity to act as a sink for endocytosed cholesterol when esterification is blocked. In contrast to the lysosomal/Golgi cholesterol sequestration, ER accumulation of cholesterol does not compromise but appears to enhance the induction of cellular homeostatic responses.
- Published
- 1998
32. Biological Implications of the Niemann-Pick C Mutation
- Author
-
E. Joan Blanchette-Mackie, Laura Liscum, and Peter G. Pentchev
- Subjects
Metabolic pathway ,Mutation ,medicine.anatomical_structure ,Membrane ,Chemistry ,Cell ,medicine ,Cellular lipid ,Compartmentalization (psychology) ,Intracellular cholesterol transport ,medicine.disease_cause ,Intracellular ,Cell biology - Abstract
The unique and varied membranes of a cell permit the compartmentalization of cellular functions. The synthesis of these distinct membrane domains is largely achieved through the protein and lipid sorting machinery of a cell. While an extensive experimental technology has evolved to study intracellular protein sorting (Rothman, 1994), cellular lipid topology has remained more a phenomenological science, with less progress made in elucidating specific molecular mechanisms. The relative paucity of experimental techniques available to study intracellular lipid trafficking has made the delineation of inherited metabolic defects particularly critical for delineating metabolic pathways of transport.
- Published
- 1997
33. Intracellular trafficking of cholesterol monitored with a cyclodextrin
- Author
-
Adele Cooney, Edward B. Neufeld, Josef Pitha, Eliezar A. Dawidowicz, Peter G. Pentchev, E. Joan Blanchette-Mackie, and Nancy K. Dwyer
- Subjects
Sterol O-acyltransferase ,Golgi Apparatus ,Biology ,Intracellular cholesterol transport ,Biochemistry ,chemistry.chemical_compound ,symbols.namesake ,Culture Techniques ,polycyclic compounds ,Humans ,Molecular Biology ,Cyclodextrins ,Cholesterol ,Endoplasmic reticulum ,Cell Membrane ,beta-Cyclodextrins ,technology, industry, and agriculture ,Biological Transport ,Cell Biology ,Cholesterol, LDL ,Brefeldin A ,Golgi apparatus ,Cell biology ,2-Hydroxypropyl-beta-cyclodextrin ,Cell Compartmentation ,Sphingomyelins ,carbohydrates (lipids) ,chemistry ,Microscopy, Fluorescence ,Molecular Probes ,symbols ,lipids (amino acids, peptides, and proteins) ,Sterol binding ,Sphingomyelin ,Lysosomes - Abstract
The sterol binding agent 2-hydroxypropyl-beta-cyclodextrin is shown to be a convenient and useful experimental tool to probe intracellular pathways of cholesterol transport. Biochemical and cytochemical studies reveal that cyclodextrin specifically removes plasma membrane cholesterol. Depletion of plasma membrane sphingomyelin greatly accelerated cyclodextrin-mediated cholesterol removal. Cholesterol arriving at the plasma membrane from lysosomes and the endoplasmic reticulum was also removed by cyclodextrin. Cellular cholesterol esterification linked to the mobilization of cholesterol from lysosomes was strongly attenuated by cyclodextrin, suggesting that the major portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum via the plasma membrane. Evidence for translocation of lysosomal cholesterol to the endoplasmic reticulum by a plasma membrane-independent pathway is provided by the finding that cyclodextrin loses its ability to suppress esterification when plasma membrane sphingomyelin is depleted. The Golgi apparatus appears to play an active role in directing the relocation of lysosomal cholesterol to the plasma membrane since brefeldin A also abrogated cyclodextrin-mediated suppression of cholesterol esterification. Using cyclodextrin we further show that attenuated esterification of lysosomal cholesterol in Niemann-Pick C cells reflects defective translocation of cholesterol to the plasma membrane that may be linked to abnormal Golgi trafficking.
- Published
- 1996
34. Cultured Skin Fibroblasts Derived from Patients with Mucolipidosis 4 Are Auto-Fluorescent
- Author
-
Goldin, Ehud, Blanchette-Mackie, E. Joan, Joan Blanchette Mackie, E., Dwyer, Nancy K., Pentchev, Peter G., and Brady, Roscoe O.
- Abstract
Mucolipidosis 4 (ML4) is an autosomal recessive disorder with both lipid and mucopolysaccharide storage. The disease is characterized by severe visual impairment and psychomotor retardation. In our effort to find a phenotypic marker for ML4 fibroblasts, living cells were stained with fluorescent compounds. The staining pattern in cells was complicated by autofluorescence. A careful study revealed that auto-fluorescence by itself was a sufficient marker for viable ML4 fibroblasts. ML4 cells in cultures obtained from four unrelated patients contain auto-fluorescent material. Auto-fluorescence was noted over a wide range of excitation wavelengths from approximately 365 to approximately 546 nm. The most intense fluorescence was observed in the lower wave-length range. Cultured fibroblasts from normal individuals or obligate ML4 heterozygotes did not fluoresce under adequately controlled culture conditions. High passage number of inadequate feeding caused a small proportion of fibroblasts obtained from normal individuals to auto-fluoresce. The auto-fluorescent material co-localized with phase-dense inclusion bodies, shown to be lysosomes by staining with LAMP-ab. These findings imply that fluorescence may relate to the specific compound(s) stored in the lysosomes. In a comparative study, neuronal ceroid lipofuscinosis fibroblasts were also fluorescent. Fibroblasts from other diseases such as Gaucher disease and glycogenosis type 2 did not show any fluorescence. These findings are currently used in our functional cloning strategy for determining the gene involved in ML4.
- Published
- 1995
35. Endothelium, the dynamic interface in cardiac lipid transport
- Author
-
E. Joan Blanchette-Mackie and Robert O. Scow
- Subjects
Very low-density lipoprotein ,Lipoprotein lipase ,biology ,Endothelium ,Insulin receptor ,medicine.anatomical_structure ,Biochemistry ,biology.protein ,Biophysics ,medicine ,Myocyte ,lipids (amino acids, peptides, and proteins) ,Lipid Transport ,Blood vessel ,Chylomicron - Abstract
Vascular endothelium is the dynamic interface in transport of lipid from blood to myocytes in heart and arteries. The luminal surface of endothelium is the site of action of lipoprotein lipase on chylomicrons and VLDL and the site of uptake of fatty acids from albumin. Fatty acids and monoacylglycerols are transported from the lumen in an interfacial continuum of endothelial and myocyte membranes. Lipoprotein lipase is transferred from myocytes to the vascular lumen, and is anchored there, by proteoheparan sulfate in cell membranes. Insulin, needed for synthesis of lipoprotein lipase and esterfication of fatty acids, is captured from the blood stream and delivered to myocytes by endothelial insulin receptors. Fatty acids, monoacylglycerols, lipoprotein lipase and insulin are transported along the same route, but by different mechanisms. The route involves the plasma membrane of endothelium and myocytes, the membrane lining transendothelial channels, and intercellular contacts. (Mol Cell Biochem 116: 181–191, 1992)
- Published
- 1992
36. Transport of fatty acids and monoacylglycerols in white and brown adipose tissues
- Author
-
Robert O. Scow and E. Joan Blanchette-Mackie
- Subjects
Lipoprotein lipase ,Chemistry ,General Neuroscience ,Cell Membrane ,Fatty Acids ,Adipose tissue ,Water ,Hormone-sensitive lipase ,Biological Transport ,Membranes, Artificial ,White adipose tissue ,Capillaries ,Glycerides ,Monoacylglycerol lipase ,medicine.anatomical_structure ,Biochemistry ,Adipose Tissue ,Adipose Tissue, Brown ,Solubility ,Brown adipose tissue ,Extracellular ,medicine ,Lipolysis ,Animals ,Carrier Proteins - Abstract
Long chain fatty acids (FA) and 2-monoacylglycerols (MG) are produced by lipoprotein lipase (LPL) from plasma triacylglycerols (TG) in capillaries of adipose tissue and transported to adipocytes for TG synthesis. It is widely proposed FA may be transported in cells by FA-binding protein. Mode of transport of MG has received little attention. Our findings in tissues and model membranes indicate that FA (as 1:1 acid-soaps) and MG can be transported in vivo by lateral movement in an interfacial continuum (IFC) of the outer leaflets of plasma and intracellular membranes of capillary endothelium and adipocytes. We postulate that FA and MG enter the IFC in capillaries and flow in the IFC across endothelium and extracellular space to sites in adipocytes where MG are hydrolyzed by MG-lipase (MGL) to FA and glycerol, and FA are esterified in endoplasmic reticulum or transferred to inner mitochondrial membrane for oxidation. FA and MG produced by hormone-sensitive lipase also enter the IFC. These MG flow in the IFC to sites of MGL activity, and the FA flow in the IFC to capillaries for transport to other tissues by albumin, or to mitochondria for heat production.
- Published
- 1991
37. Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol
- Author
-
Chris Kaneski, Ehud Goldin, Harold T. Pye, Peter G. Pentchev, Charles E. Argoff, Marie T. Vanier, Marcella E. Comly, Howard S. Kruth, Roscoe O. Brady, and Joan Blanchette-Mackie
- Subjects
Adult ,Male ,medicine.medical_specialty ,Oxysterol ,Sterol O-acyltransferase ,Biology ,Intracellular cholesterol transport ,Endoplasmic Reticulum ,Cell Line ,chemistry.chemical_compound ,Internal medicine ,medicine ,Homeostasis ,Humans ,Molecular Biology ,Niemann-Pick Diseases ,Esterification ,Cholesterol ,Histocytochemistry ,Endoplasmic reticulum ,Cell Membrane ,Biological Transport ,Cholesterol, LDL ,Fibroblasts ,Sterol transport ,Kinetics ,Endocrinology ,chemistry ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,Oxidation-Reduction ,Intracellular - Abstract
A uniquely attenuated disruption of cholesterol homeostasis has been characterized in certain Niemann-Pick, type C (NP-C) fibroblasts. Uptake of LDL-cholesterol by cultured fibroblasts derived from two clinically affected brothers with this variant biochemical phenotype led to less intracellular accumulation of unesterified cholesterol than found in other typical cell lines. This limited cholesterol lipidosis in the variant NP-C cells reflected cholesterol processing errors that differed from the cellular lesions in classical NP-C cells in the following ways: (1) a more limited intracellular distribution of the excessive unesterified cholesterol; (2) shorter and more transient delays in the induction of cholesterol-mediated homeostatic responses; and (3) more efficient intracellular transport of exogenously derived cholesterol to the plasma membrane and the endoplasmic reticulum. Activation of acyl-CoA cholesterol acyltransferase (ACAT) was greater than 100-fold in both control and NP-C fibroblasts when cell cultures were preconditioned with 25-hydroxycholesterol, but the subsequent esterification of exogenous non-lipoprotein [3H]cholesterol remained deficient in all NP-C cells. In the variant NP-C cells conditioned with the oxysterol, this esterification of exogenous [3H]cholesterol was less affected than in classical NP-C cultures. The NP-C mutation affects a broad spectrum of metabolic responses related to the processing of exogenously derived cholesterol. Among this pleiotropic array of deficient responses, retarded intracellular cholesterol transport appears most closely linked to the primary mutation. This conclusion is supported by two current observations: (1) the degree to which sterol transport is affected in mutant cells in turn reflects the extent to which cholesterol-homeostatic responses are compromised; and (2) sterol transport remains deficient despite concurrent normal activation of other cellular responses, such as cholesterol esterification.
- Published
- 1991
38. Type C Niemann-Pick disease: documentation of abnormal LDL processing in lymphocytes
- Author
-
Anthony Brown, Nancy K. Dwyer, Marcella E. Comly, E. Joan Blanchette-Mackie, Christine R. Kaneski, Roscoe O. Brady, Charles E. Argoff, and Peter G. Pentchev
- Subjects
medicine.medical_specialty ,Heterozygote ,Lymphocyte ,Biophysics ,Biology ,In Vitro Techniques ,Biochemistry ,Lesion ,chemistry.chemical_compound ,Internal medicine ,otorhinolaryngologic diseases ,medicine ,Humans ,Filipin ,Lymphocytes ,Molecular Biology ,Molecular lesion ,Niemann-Pick Diseases ,Cholesterol ,Homozygote ,Heterozygote advantage ,Cell Biology ,Cholesterol, LDL ,Intracellular Membranes ,medicine.disease ,Molecular biology ,Cell Compartmentation ,Lipoproteins, LDL ,stomatognathic diseases ,Microscopy, Electron ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Microscopy, Fluorescence ,Cholesteryl ester ,lipids (amino acids, peptides, and proteins) ,Cholesterol Esters ,medicine.symptom ,Niemann–Pick disease ,Intracellular - Abstract
Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (±5%) and 45% (±16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.
- Published
- 1990
39. Microtubules are not required for glucocorticoid receptor mediated gene induction
- Author
-
Szapary, Daniele, primary, Barber, Therese, additional, Dwyer, Nancy K., additional, Joan Blanchette-Mackie, E., additional, and Stoney Simons, S., additional
- Published
- 1994
- Full Text
- View/download PDF
40. Continuity of intracellular channels with extracellular space in adipose tissue and liver: Demonstrated with tannic acid and lanthanum.
- Author
-
Joan Blanchette-Mackie, E. and Scow, Robert O.
- Published
- 1982
- Full Text
- View/download PDF
41. Scanning electron microscopic study of chylomicrons incubated with lipoprotein lipase
- Author
-
E. Joan Blanchette-Mackie and Robert O. Scow
- Subjects
Lipoprotein lipase ,Chromatography ,Aqueous solution ,Scanning electron microscope ,Chemistry ,Glyceride ,digestive, oral, and skin physiology ,Hydrogen-Ion Concentration ,Lipids ,Agricultural and Biological Sciences (miscellaneous) ,Rats ,Biochemistry ,Transmission electron microscopy ,Chylomicrons ,Microscopy ,Microscopy, Electron, Scanning ,Animals ,Lipolysis ,Female ,lipids (amino acids, peptides, and proteins) ,Anatomy ,Chylomicron - Abstract
The effects of lipolysis on the structure of chylomicrons were studied with the scanning electron microscope using rat chylomicrons incubated with purified bovine milk lipoprotein lipase for 20 minutes at pH 8.1. Since the amount of albumin added to the medium was limited, some of the free fatty acids and partial glycerides formed by lipolysis accumulated in the chylomicrons. Lipolyzed chylomicrons fixed with OSO4 at pH 7.4 appeared in scanning electron micrographs as spheres with multiply idented irregular surfaces, while those fixed at pH 5.5, AS WELL AS CONTROL CHYLOMICRONS FIXED AT BOTH PHs, appeared as spheres with smooth surfaces. Sections of OSO4-fixed specimens, viewed with the transmission electron microscope, showed that the core of lipolyzed chylomicrons fixed at pH 7.4 contained numerous circular electron-lucent areas at the periphery, thus accounting for the indented surfaces observed above, while the core surfaces of the other specimens were circular and smooth. These findings confirm an earlier report that aqueous spaces form in chylomicrons during lipolysis when albumin in the medium is limited, and that the aqueous spaces disappear when specimens are prepared at pH 5.5 for microscopy. Thin sections of specimens that had been prepared for scanning electron microscopy showed that the gold-palladium coating was desposited directly on the indented surface of the lipid core of lipolyzed chylomicrons fixed at pH 7.4. It is concluded that vacuum dehydration during specimen preparation ruptures the outer wall of the aqueous spaces in lipolyzed chylomicrons and thereby exposes the interior of the spaces to gold-palladium coating and viewing with the scanning electron microscope.
- Published
- 1976
42. Editorial Notes – Acknowledgement
- Author
-
R. Dhanireddy, J.R. Girard, Kazuhiko Tanaka, Hisashi Yamamoto, Koji Nakao, M. Hamosh, Teresa C. Lea, Malathy Singh, William Berman, Martha H. Stipanuk, Noriaki Oya, Deborah Christensen, Masao Asari, Peter B. Berendsen, I.D. Frasier, Joseph M. Samsa, Firmino F. Rubaltelli, Abdul M. Bhat, P. Ferre, Michele Felice, Farouk Karoum, V. Rubio, H.G. Button, N.M. Buckley, Bennett Lavenstein, Joan Blanchette-Mackie, Muriel Feigelson, Giuseppe Giancola, C.B. Tan, P. Hamosh, P. Turlan, D.K. Mullon, D.A. † Nixon, Nuria Torán, Shiu-Ming Kuo, Augusto Moragas, Yutaka Kano, L. Chuang, Antonio Fortunato, Morimi Shimada, P. Brazeau, Yasunobu Eguchi, Akiko Kosugi, Y.F. Smith, Kenneth M. Hoff, John W. Scanlon, J.W. Scanlon, Masako Yamamoto, D.P. Alexander, W.H.H. † Andrews, Bruno Granati, and Peter C. Baker
- Subjects
medicine.medical_specialty ,Medical education ,Pediatrics ,business.industry ,Pediatrics, Perinatology and Child Health ,Acknowledgement ,Alternative medicine ,Medicine ,business ,Developmental Biology - Published
- 1983
43. Effect of pH on visualization of fatty acids as myelin figures in mouse adipose tissue by freeze-fracture electron microscopy
- Author
-
E. Joan Blanchette-Mackie, Lynn M. Amende, Robert O. Scow, and Sidney S. Chernick
- Subjects
Male ,Lipid Bilayers ,Biophysics ,Adipose tissue ,Acid–base homeostasis ,Biochemistry ,Specimen Handling ,Fixatives ,Mice ,Myelin ,Endocrinology ,Lipid droplet ,Extracellular ,medicine ,Animals ,Freeze Fracturing ,Insulin ,Lipolysis ,Epididymis ,chemistry.chemical_classification ,Fatty Acids ,Isoproterenol ,Albumin ,Fatty acid ,Hydrogen-Ion Concentration ,Microscopy, Electron ,Glucose ,medicine.anatomical_structure ,Adipose Tissue ,chemistry - Abstract
We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 μM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1–9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures formed under these conditions are composed primarily of partially ionized fatty acids (acid-soaps), and that the smooth-surfaced droplets in the extracellular space are composed of un-ionized (protonated) fatty acids.
- Published
- 1985
44. Morphological Changes in Lipid Droplets within Cells of the Duodenal Transition Zone in Suckling Rats
- Author
-
Peter B. Berendsen and Joan Blanchette-Mackie
- Subjects
Duodenum ,Chemistry ,Age Factors ,Rats, Inbred Strains ,Anatomy ,Lipid Metabolism ,Small intestine ,Animals, Suckling ,Rats ,Milk ,medicine.anatomical_structure ,Animals, Newborn ,Lipid droplet ,Pediatrics, Perinatology and Child Health ,Transition zone ,medicine ,Ultrastructure ,Animals ,Developmental Biology - Abstract
This ultrastructural study compares the lipid droplets within epithelial cells at the bases of the duodenal villi in the transition zone between crypts and the lateral portions of the adjacent villi in newborn rats suckled 24 h, 10 days and 15 days and contrasts with cells in similar locations in newborn unsuckled and weaned rats. The mean diameters and numbers of lipid droplets per field decreased when cells from rats at 10 and 15 days of age were compared to those at 24 h of age. The range of sizes also narrowed. There was cell-to-cell variability in number and in opacity of the lipid droplets. Multiple membranous laminae enclosed the droplets. Cells in similar locations in newborn unsuckled and in weaned rats did not contain lipid droplets. It is concluded that the numbers and sizes of the large lipid droplets within cells in the transition zones at the bases of the villi decrease as cells at the same relative locations mature during suckling and as the infant rats mature.
- Published
- 1983
45. Uptake of plasma triacylglycerol by a muscular artery in the rat: an ultrastructural study
- Author
-
Alli Reinilä, E. Joan Blanchette-Mackie, and Robert O. Scow
- Subjects
Endothelium ,Biological Transport, Active ,Arteries ,Biology ,Mitochondrion ,Muscle, Smooth, Vascular ,Rats ,Microscopy, Electron ,medicine.anatomical_structure ,Membrane ,Biochemistry ,Lipid droplet ,medicine ,Ultrastructure ,Extracellular ,Biophysics ,Animals ,Lipolysis ,Female ,Triglycerides ,Intracellular - Abstract
The uptake of plasma triacylglycerol by the dorsalis pedis artery in the rat was studied using intravenous infusion of an emulsion of triacylglycerol at a rate of 2.3 mumol per min for 1.5 or 5 h. Electron microscopy revealed lipid droplets in the arterial lumen near the endothelium and in the medial smooth muscle cells (SMC), but not in the endothelial cells or in the extracellular space. Lamellar structures with a periodicity of 40 A developed in the arterial tissue when glutaraldehyde-fixed specimens were incubated at +25 degrees C before postfixation in osmium. Lamellae were present at the luminal and basal surfaces and within endothelial cells, and also in the medial extracellular space associated with the plasma membrane of SMC, in the intracellular channels and near and inside the mitochondria of the medial SMC. No lipid droplets or lamellae were found in the arterial tissue of the control rats. The findings indicate that plasma triacylglycerol is not taken up by the arterial tissue as intact lipid particles, but that these are hydrolyzed at the luminal surface of the endothelium, the lipolytic products then being transferred to the medial SMC for re-esterification and storage in the form of triacylglycerol. The lamellar structures found in the fixed and incubated arterial tissue are thought to represent fatty acids produced by the lipolysis of triacylglycerol during incubation, and we suggest that the transport of fatty acids from the arterial lumen to the medial SMC occurs by lateral movement in a continuum of cell membranes.
- Published
- 1984
46. Membrane continuities within cells and intercellular contacts in white adipose tissue of young rats
- Author
-
E. Joan Blanchette-Mackie and Robert O. Scow
- Subjects
Adipose tissue ,White adipose tissue ,Biology ,Basement Membrane ,Lipid droplet ,Extracellular ,medicine ,Animals ,Endothelium ,Molecular Biology ,Basement membrane ,Vesicle ,Cell Membrane ,Intracellular Membranes ,Animals, Suckling ,Capillaries ,Rats ,Cell biology ,Microscopy, Electron ,Intercellular Junctions ,Membrane ,medicine.anatomical_structure ,Adipose Tissue ,Biochemistry ,Anatomy ,Extracellular Space ,Intracellular - Abstract
White adipose tissue of suckling rats was examined for evidence of continuity of membranes within cells, and areas of contact between adipocytes and capillary endothelial cells. Extracellular space, invaginations of cell surfaces, and lumens of channels and vesicles within both types of cells were marked with electron-opaque material in tissues which were treated with tannic acid after being fixed with glutaraldehyde. The electron-opaque material also marked channels that extended from extracellular space to the surface of intracellular lipid droplets in fat cells. Presence of this material in channels and vesicles, but not in cytoplasm, indicates they were open to extracellular space during treatment with tannic acid and, furthermore, that their membranes were continuous with plasma membrane. Cell processes of adipocytes and endothelium extended through basement membrane to make contact with each other. Apparent continuity of the external leaflet of plasma membrane of one cell with that of another cell was observed. These findings demonstrate membrane continuity between plasma membrane and membranes of intracellular channels in adipocytes and endothelial cells, and intercellular contacts in adipose tissue.
- Published
- 1981
47. EFFECTS OF LIPOPROTEIN LIPASE ON THE STRUCTURE OF CHYLOMICRONS
- Author
-
E. Joan Blanchette-Mackie and Robert O. Scow
- Subjects
Glycine ,Serum albumin ,Fatty Acids, Nonesterified ,Biology ,Tritium ,Article ,chemistry.chemical_compound ,Hydrolysis ,Chylomicrons ,Glycerol ,Animals ,Lipolysis ,Carbon Radioisotopes ,Serum Albumin ,Triglycerides ,Lipoprotein lipase ,Chromatography ,Triglyceride ,Lipid Mobilization ,digestive, oral, and skin physiology ,Albumin ,food and beverages ,Cell Biology ,Hydrogen-Ion Concentration ,Rats ,Microscopy, Electron ,chemistry ,Biochemistry ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Lymph ,Chylomicron - Abstract
Chylomicrons isolated from rat lymph were complexed with lipoprotein lipase of post-heparin plasma (chylomicrons-LPL) in order to study the effects of lipolysis on the structure of chylomicrons. Triglyceride in the chylomicron core was readily hydrolyzed to free fatty acids (FFA) and glycerol when chylomicrons-LPL were incubated at pH 8.3 in medium containing albumin. Although most of the FFA were immediately released to the medium, some were retained within chylomicrons when FFA-binding sites on albumin were not available. These observations suggest that albumin may have a specific role in the transfer of FFA across the chylomicron surface film. Chylomicrons-LPL assumed many different shapes as they were depleted of triglyceride by the lipolytic action of the enzyme, and total removal of core triglyceride resulted in empty sacks of surface film. The surface film was visualized in sections of OsO4-fixed chylomicrons-LPL as a thin electron-opaque line, 25–30 Å wide, in areas where the underlying electron-opaque core had been replaced by zones of decreased electron opacity, and in folds of surface film extending outward from chylomicrons partially depleted of core lipid. The findings demonstrate that chylomicrons consist of a core of liquid triglyceride enveloped by a pliable and durable monolayer surface film, and that lipoprotein lipase reduces the triglyceride core without disrupting the surface film.
- Published
- 1973
48. SITES OF LIPOPROTEIN LIPASE ACTIVITY IN ADIPOSE TISSUE PERFUSED WITH CHYLOMICRONS
- Author
-
E. Joan Blanchette-Mackie and Robert O. Scow
- Subjects
Glycerol ,Tris ,Time Factors ,Endothelium ,Glyceride ,Adipose tissue ,Vascular permeability ,Palmitic Acids ,Fatty Acids, Nonesterified ,Biology ,Tritium ,Article ,Thoracic Duct ,Capillary Permeability ,Nitrophenols ,chemistry.chemical_compound ,Chylomicrons ,medicine ,Animals ,Triglycerides ,Inclusion Bodies ,Aldehydes ,Carbon Isotopes ,Lipoprotein lipase ,Histocytochemistry ,Tissue Extracts ,Hydrolysis ,Histological Techniques ,Rats, Inbred Strains ,Cell Biology ,Capillaries ,Culture Media ,Rats ,Perfusion ,Lipoprotein Lipase ,Microscopy, Electron ,medicine.anatomical_structure ,Adipose Tissue ,Biochemistry ,chemistry ,Female ,Glutaraldehyde ,Chylomicron - Abstract
Lipoprotein lipase activity was studied in rat parametrial adipose tissue perfused with chylomicrons and in gelatin blocks containing postheparin plasma and chylomicrons. The tissues and blocks were fixed in glutaraldehyde and incubated in 0.035 M CaCl(2)-0.1 M Tris medium (pH 8.3) at 38 degrees C. The doubly labeled chylomicron triglycerides (glycerol-(3)H and palmitate-(14)C) in the tissues and blocks were hydrolyzed during incubation to free fatty acids (FFA) and the FFA remained in the specimens; hydrolysis was inhibited by 0.004 M diethyl paranitrophenyl phosphate (E-600). Incubated blocks and tissue were treated with 0.05 M Pb(NO(3))(2), postfixed in OsO(4), dehydrated with acetone, embedded in Epon, and examined by electron microscopy. The incubated blocks contained electronlucent areas and granular and laminar precipitates at sites of hydrolysis. Similar precipitates were found in incubated tissue, within vacuoles and microvesicles of capillary endothelium, and in the subendothelial space (between the endothelium and pericytes), but not in the capillary lumen or in or near fat cells. The cytochemical reaction was greatly reduced, in blocks and tissues incubated with E-600. It is concluded that plasma glycerides are hydrolyzed by lipoprotein lipase in capillary endothelial cells and in the subendothelial space of adipose tissue and that glycerides across the endothelial cells within a membrane-bounded system.
- Published
- 1971
49. Continuity of intracellular channels with extracellular space in adipose tissue and liver: demonstrated with tannic acid and lanthanum
- Author
-
Robert O. Scow and E. Joan Blanchette-Mackie
- Subjects
Adipose tissue ,White adipose tissue ,Biology ,Ion Channels ,Diabetes Mellitus, Experimental ,chemistry.chemical_compound ,Adipose Tissue, Brown ,Lanthanum ,Lipid droplet ,Brown adipose tissue ,Tannic acid ,medicine ,Extracellular ,Animals ,Endoplasmic reticulum ,Intracellular Membranes ,Agricultural and Biological Sciences (miscellaneous) ,Hydrolyzable Tannins ,Animals, Suckling ,Rats ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Adipose Tissue ,Liver ,Biophysics ,Female ,Anatomy ,Extracellular Space ,Intracellular - Abstract
Tannic acid was used to demonstrate continuity of intracellular channels with extracellular space in white adipose tissue of adult rats, brown adipose tissue of suckling rats, and liver of diabetic rats. Electron-opaque material resulting from treatment of glutaraldehyde-fixed tissue with tannic acid was found in extracellular space, invaginations of cell surfaces, vesicles, and intracellular channels. Electron-opaque material was present in channels that surrounded lipid droplets in both white and brown adipocytes and in hepatocytes. The small distance between the lumen of marked channels and lipid droplets in adipocytes indicates that a monolayered structure, perhaps a leaflet of membrane lining the channel, separates the lipid droplet from the lumen of the channel, suggesting that the lipid droplet may be located between leaflets of the membrane lining the channel. Similar findings were obtained in brown adipose tissue using lanthanum instead of tannic acid to mark intracellular channels continuous with extracellular space. Since endoplasmic reticulum is the primary site of triacylglycerol synthesis in adipocytes, marked channels near lipid droplets may be elements of endoplasmic reticulum. Some of the channels marked with tannic acid in hepatocytes contained lipoprotein particles, whereas others were located, in relation to mitochondria and lipid droplets, in the same sites as endoplasmic reticulum in untreated tissue. This indicates that some of the channels marked with tannic acid in hepatocytes are endoplasmic reticulum. Presence of electron-opaque material in intracellular channels and vesicles, but not in cytoplasm, of treated tissue indicates the channels and vesicles were open to extracellular space during treatment with tannic acid or lanthanum and, furthermore, that their membranes were continuous with plasma membrane.
- Published
- 1982
50. Electron microscopic visualization of fatty acids in tissues
- Author
-
E. Joan Blanchette-Mackie and Lynn M. Amende
- Subjects
chemistry.chemical_classification ,Lipoprotein lipase ,biology ,Epoxy Resins ,Cell Membrane ,Fatty Acids ,Fatty acid ,Adipose tissue ,Hydrogen-Ion Concentration ,Rats ,Hydrolysis ,Fixatives ,Microscopy, Electron ,chemistry ,Biochemistry ,Adipose Tissue ,Lipid droplet ,Amphiphile ,Chylomicrons ,biology.protein ,Ultrastructure ,Animals ,lipids (amino acids, peptides, and proteins) ,Anatomy ,Lipase - Abstract
Long-chain fatty acids are amphipathic molecules with important structural and metabolic functions in tissues. Fatty acids are derived from triacylglycerol-rich particles in capillaries (chylomicrons and very-low-density lipoproteins) and from triacylglycerol stored in cells (lipid droplets) by the hydrolytic activity of tissue lipases. The identification and localization of fatty acids in tissues has been considered difficult to obtain by using conventional ultrastructural techniques. However, structural findings from our studies on fatty acid transport in tissue became interpretable due to the use of many overlapping techniques. We present here these ultrastructural techniques developed to study fatty acids in tissues and review data which demonstrate lipase activity and fatty acid production from triacylglycerol in aldehyde-fixed tissue. Accumulations of fatty acid in tissue are present as lamellar structures with periodicity of 40-50 A in sections of resin-embedded tissue and as hydrated myelin figures in freeze fracture replicas of unfixed and fixed tissue. Finally, a new method, using the ionization properties of fatty acids combined with freeze fracture, locates these amphipathic molecules to leaflets of membrane bilayers.
- Published
- 1987
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.