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Articles 271 - 300 of 302

Full-Text Articles in Cell Biology

Foci Of Trinucleotide Repeat Transcripts In Nuclei Of Myotonic Dystrophy Cells And Tissues, Krishan L. Taneja, Mila Mccurrach, Martin Schalling, David Housman, Robert H. Singer Mar 1995

Foci Of Trinucleotide Repeat Transcripts In Nuclei Of Myotonic Dystrophy Cells And Tissues, Krishan L. Taneja, Mila Mccurrach, Martin Schalling, David Housman, Robert H. Singer

Open Access Articles

We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously ...


The Mechanisms And Mediators Of Tooth Eruption--Models For Developmental Biologists, Sandy C. Marks Jr., Jeffrey P. Gorski, Gary E. Wise Feb 1995

The Mechanisms And Mediators Of Tooth Eruption--Models For Developmental Biologists, Sandy C. Marks Jr., Jeffrey P. Gorski, Gary E. Wise

Open Access Articles

Tooth eruption is a localized process in the jaws which exhibits precise timing and bilateral symmetry. It involves resorption and formation of bone on opposite sides of the erupting tooth and these activities depend on the dental follicle, a thin connective tissue investment of the developing and erupting tooth. Biochemical studies have shown that during eruption cells, proteins and enzymes change in the dental follicle and several growth factors and proteins known to accelerate or retard eruption have been identified. This review discusses these aspects of tooth eruption and proposes testable hypotheses and strategies that can make studies of tooth ...


The Alpha Subunit Of The Saccharomyces Cerevisiae Oligosaccharyltransferase Complex Is Essential For Vegetative Growth Of Yeast And Is Homologous To Mammalian Ribophorin I, Susana Silberstein, Pallia G. Collins, Daniel J. Kelleher, Peter J. Rapiejko, Reid Gilmore Feb 1995

The Alpha Subunit Of The Saccharomyces Cerevisiae Oligosaccharyltransferase Complex Is Essential For Vegetative Growth Of Yeast And Is Homologous To Mammalian Ribophorin I, Susana Silberstein, Pallia G. Collins, Daniel J. Kelleher, Peter J. Rapiejko, Reid Gilmore

Open Access Articles

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae ...


Mediation Of Chemoattractant-Induced Changes In [Ca2+]I And Cell Shape, Polarity, And Locomotion By Insp3, Dag, And Protein Kinase C In Newt Eosinophils, Susan H. Gilbert, Kristine Perry, Fredric S. Fay Oct 1994

Mediation Of Chemoattractant-Induced Changes In [Ca2+]I And Cell Shape, Polarity, And Locomotion By Insp3, Dag, And Protein Kinase C In Newt Eosinophils, Susan H. Gilbert, Kristine Perry, Fredric S. Fay

Open Access Articles

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with ...


Sequences Responsible For Intracellular Localization Of Beta-Actin Messenger Rna Also Affect Cell Phenotype, Edward H. Kislauskis, Xiaochun Zhu, Robert H. Singer Oct 1994

Sequences Responsible For Intracellular Localization Of Beta-Actin Messenger Rna Also Affect Cell Phenotype, Edward H. Kislauskis, Xiaochun Zhu, Robert H. Singer

Open Access Articles

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity ...


A Double Leucine Within The Glut4 Glucose Transporter Cooh-Terminal Domain Functions As An Endocytosis Signal, Silvia Corvera, Anil Chawla, Ranjan Chakrabarti, Marguerite Joly, Joanne M. Buxton, Michael P. Czech Aug 1994

A Double Leucine Within The Glut4 Glucose Transporter Cooh-Terminal Domain Functions As An Endocytosis Signal, Silvia Corvera, Anil Chawla, Ranjan Chakrabarti, Marguerite Joly, Joanne M. Buxton, Michael P. Czech

Open Access Articles

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody ...


Dynamic Changes In The Higher-Level Chromatin Organization Of Specific Sequences Revealed By In Situ Hybridization To Nuclear Halos, Michael G. Gerdes, Kenneth C. Carter, Phillip T. Moen, Jeanne B. Lawrence Jul 1994

Dynamic Changes In The Higher-Level Chromatin Organization Of Specific Sequences Revealed By In Situ Hybridization To Nuclear Halos, Michael G. Gerdes, Kenneth C. Carter, Phillip T. Moen, Jeanne B. Lawrence

Open Access Articles

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to ...


Nuclear Matrix Proteins Distinguish Normal Diploid Osteoblasts From Osteosarcoma Cells, Joseph P. Bidwell, Edward G. Fey, Andre J. Van Wijnen, Sheldon Penman, Janet L. Stein, Jane B. Lian, Gary S. Stein Jan 1994

Nuclear Matrix Proteins Distinguish Normal Diploid Osteoblasts From Osteosarcoma Cells, Joseph P. Bidwell, Edward G. Fey, Andre J. Van Wijnen, Sheldon Penman, Janet L. Stein, Jane B. Lian, Gary S. Stein

Open Access Articles

Interrelationships between nuclear architecture and gene expression were examined by comparing the representation of nuclear matrix proteins in ROS 17/2.8 rat and MG-63 human osteosarcoma cells with those in normal diploid osteoblasts. The tumor-derived cells coexpress genes which are expressed in a sequential and mutually exclusive manner during the progressive stages of osteoblast differentiation. In osteosarcoma cells two-dimensional electrophoretic analysis indicates a composite representation of nuclear matrix proteins characteristic of both the proliferative and postproliferative periods of osteoblast phenotype development. In addition, nuclear matrix proteins unique to the tumor cells and the absence of nuclear matrix proteins found ...


Gtp Hydrolysis By Complexes Of The Signal Recognition Particle And The Signal Recognition Particle Receptor, Timothy Connolly, Reid Gilmore Nov 1993

Gtp Hydrolysis By Complexes Of The Signal Recognition Particle And The Signal Recognition Particle Receptor, Timothy Connolly, Reid Gilmore

Open Access Articles

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP-dependent reaction during protein translocation is the SRP receptor-mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.


Exofacial Epitope-Tagged Glucose Transporter Chimeras Reveal Cooh-Terminal Sequences Governing Cellular Localization, Michael P. Czech, Anil Chawla, Chee-Wai Woon, Joanne M. Buxton, Michal Armoni, Wei Tang, Marguerite Joly, Silvia Corvera Oct 1993

Exofacial Epitope-Tagged Glucose Transporter Chimeras Reveal Cooh-Terminal Sequences Governing Cellular Localization, Michael P. Czech, Anil Chawla, Chee-Wai Woon, Joanne M. Buxton, Michal Armoni, Wei Tang, Marguerite Joly, Silvia Corvera

Open Access Articles

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked ...


Redistribution Of Clathrin-Coated Vesicle Adaptor Complexes During Adipocytic Differentiation Of 3t3-L1 Cells, Ranjan Chakrabarti, Marguerite Joly, Silvia Corvera Oct 1993

Redistribution Of Clathrin-Coated Vesicle Adaptor Complexes During Adipocytic Differentiation Of 3t3-L1 Cells, Ranjan Chakrabarti, Marguerite Joly, Silvia Corvera

Open Access Articles

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio ...


Isoform-Specific 3'-Untranslated Sequences Sort Alpha-Cardiac And Beta-Cytoplasmic Actin Messenger Rnas To Different Cytoplasmic Compartments, Edward H. Kislauskis, Zhifang Li, Robert H. Singer, Krishan L. Taneja Oct 1993

Isoform-Specific 3'-Untranslated Sequences Sort Alpha-Cardiac And Beta-Cytoplasmic Actin Messenger Rnas To Different Cytoplasmic Compartments, Edward H. Kislauskis, Zhifang Li, Robert H. Singer, Krishan L. Taneja

Open Access Articles

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and ...


Guanosine Diphosphatase Is Required For Protein And Sphingolipid Glycosylation In The Golgi Lumen Of Saccharomyces Cerevisiae, Claudia Abeijon, Ken Yanagisawa, Elisabet C. Mandon, Alex Hausler, Kelley W. Moremen, Carlos B. Hirschberg, Phillips W. Robbins Jul 1993

Guanosine Diphosphatase Is Required For Protein And Sphingolipid Glycosylation In The Golgi Lumen Of Saccharomyces Cerevisiae, Claudia Abeijon, Ken Yanagisawa, Elisabet C. Mandon, Alex Hausler, Kelley W. Moremen, Carlos B. Hirschberg, Phillips W. Robbins

Open Access Articles

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn ...


Concerted Control Of Multiple Histone Promoter Factors During Cell Density Inhibition Of Proliferation In Osteosarcoma Cells: Reciprocal Regulation Of Cell Cycle-Controlled And Bone-Related Genes, Fusinita M. I. Van Den Ent, Andre J. Van Wijnen, Thomas J. Last, Rita Bortell, Janet L. Stein, Jane B. Lian, Gary S. Stein May 1993

Concerted Control Of Multiple Histone Promoter Factors During Cell Density Inhibition Of Proliferation In Osteosarcoma Cells: Reciprocal Regulation Of Cell Cycle-Controlled And Bone-Related Genes, Fusinita M. I. Van Den Ent, Andre J. Van Wijnen, Thomas J. Last, Rita Bortell, Janet L. Stein, Jane B. Lian, Gary S. Stein

Open Access Articles

Cell density-induced growth inhibition of osteosarcoma cells (ROS 17/2.8) results in the shutdown of proliferation-specific histone H4 and H2B genes and the concomitant up-regulation of several osteoblast-related genes. In several respects, this reciprocal regulatory relationship is analogous to the proliferation/differentiation transition stage during development of the bone cell phenotype in normal diploid osteoblasts. Here, we comprehensively analyzed the promoter binding activities interfacing with key regulatory elements in the cell cycle-dependent histone and bone-specific osteocalcin genes. Similarly, we examined factors interacting with a series of general transcription regulatory elements that are present in a broad spectrum of promoters ...


Poly(A) Rna Codistribution With Microfilaments: Evaluation By In Situ Hybridization And Quantitative Digital Imaging Microscopy, Krishan L. Taneja, Lawrence M. Lifshitz, Fredric S. Fay, Robert H. Singer Dec 1992

Poly(A) Rna Codistribution With Microfilaments: Evaluation By In Situ Hybridization And Quantitative Digital Imaging Microscopy, Krishan L. Taneja, Lawrence M. Lifshitz, Fredric S. Fay, Robert H. Singer

Open Access Articles

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the ...


Protein Translocation Across The Er Requires A Functional Gtp Binding Site In The Alpha Subunit Of The Signal Recognition Particle Receptor, Peter J. Rapiejko, Reid Gilmore May 1992

Protein Translocation Across The Er Requires A Functional Gtp Binding Site In The Alpha Subunit Of The Signal Recognition Particle Receptor, Peter J. Rapiejko, Reid Gilmore

Open Access Articles

The signal recognition particle (SRP)-mediated translocation of proteins across the RER is a GTP dependent process. Analysis of the primary amino acid sequence of one protein subunit of SRP (SRP54), as well as the alpha subunit of the SRP receptor (SR alpha), has indicated that these proteins contain predicted GTP binding sites. Several point mutations confined to the GTP binding consensus elements of SR alpha were constructed by site specific mutagenesis to define a role for the GTP binding site in SR alpha during protein translocation. The SR alpha mutants were analyzed using an in vitro system wherein SR ...


Prevention Of Diabetes In Bb/Wor Rats By Intrathymic Islet Injection, Steven B. Koevary, Maureen Blomberg Feb 1992

Prevention Of Diabetes In Bb/Wor Rats By Intrathymic Islet Injection, Steven B. Koevary, Maureen Blomberg

Open Access Articles

The objective of this study was to determine whether the intrathymic injection of islets can prevent the development of diabetes in BB/Wor rats. Evidence suggests that a failure to induce islet thymic tolerance may be an etiological factor in the development of the disease. It was theorized that the introduction of islets into the thymus might directly induce islet tolerance and thus prevent disease. Islets from diabetes-resistant BB/Wor rats were injected into the thymuses of 23 young diabetes-prone BB/Wor rats; 25 sham-operated animals served as controls. Results showed that 22 of the 25 control rats became diabetic ...


Discrete Nuclear Domains Of Poly(A) Rna And Their Relationship To The Functional Organization Of The Nucleus, Kenneth C. Carter, Krishan L. Taneja, Jeanne B. Lawrence Dec 1991

Discrete Nuclear Domains Of Poly(A) Rna And Their Relationship To The Functional Organization Of The Nucleus, Kenneth C. Carter, Krishan L. Taneja, Jeanne B. Lawrence

Open Access Articles

The functional organization of the nucleus was studied using a fluorescence microscopy approach which allowed integration of positional information for RNA, DNA, and proteins. In cells from sea urchin to human, nuclear poly(A) RNA was found concentrated primarily within several discrete "transcript domains" which often surrounded nucleoli. Concentrations of poly(A) RNA were coincident with snRNP antigen clusters, providing evidence for the localization of pre-mRNA splicing at these sites. The spatial relationship of transcript domains with respect to various classes of DNA was established, in that the poly(A) RNA-rich regions coincided with discrete regions of low DNA density ...


Ribosome Binding To The Endoplasmic Reticulum: A 180-Kd Protein Identified By Crosslinking To Membrane-Bound Ribosomes Is Not Required For Ribosome Binding Activity, Pallia G. Collins, Reid Gilmore Aug 1991

Ribosome Binding To The Endoplasmic Reticulum: A 180-Kd Protein Identified By Crosslinking To Membrane-Bound Ribosomes Is Not Required For Ribosome Binding Activity, Pallia G. Collins, Reid Gilmore

Open Access Articles

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ...


Er Translocation Intermediates Are Adjacent To A Nonglycosylated 34-Kd Integral Membrane Protein, Kennan V. Kellaris, Sharon Bowen, Reid Gilmore Jul 1991

Er Translocation Intermediates Are Adjacent To A Nonglycosylated 34-Kd Integral Membrane Protein, Kennan V. Kellaris, Sharon Bowen, Reid Gilmore

Open Access Articles

We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cross-linked products of 45 and 64 kD were detected. The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45-kD product required both SRP and translocation competent microsomal membrane vesicles. In agreement with previous investigators, we suggest that the 64-kD product arises by cross-linking of ...


A Highly Polymorphic Dinucleotide Repeat On The Proximal Short Arm Of The Human X Chromosome: Linkage Mapping Of The Synapsin I/A-Raf-1 Genes, Cordula U. Kirchgessner, James A. Trofatter, Melanie M. Mahtani, Huntington F. Willard, Louis J. Degennaro Jul 1991

A Highly Polymorphic Dinucleotide Repeat On The Proximal Short Arm Of The Human X Chromosome: Linkage Mapping Of The Synapsin I/A-Raf-1 Genes, Cordula U. Kirchgessner, James A. Trofatter, Melanie M. Mahtani, Huntington F. Willard, Louis J. Degennaro

Open Access Articles

A compound (AC)n repeat located 1,000 bp downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA data-base comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. PCR amplification of this synapsin I/A-raf-1 associated repeat by using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a PIC value of .84 and a ...


Modulation Of Hexokinase Association With Mitochondria Analyzed With Quantitative Three-Dimensional Confocal Microscopy, Ronald M. Lynch, Kevin E. Fogarty, Fredric S. Fay Feb 1991

Modulation Of Hexokinase Association With Mitochondria Analyzed With Quantitative Three-Dimensional Confocal Microscopy, Ronald M. Lynch, Kevin E. Fogarty, Fredric S. Fay

Open Access Articles

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after ...


Actin Mrna Localizes In The Absence Of Protein Synthesis, Cynthia L. Sundell, Robert H. Singer Dec 1990

Actin Mrna Localizes In The Absence Of Protein Synthesis, Cynthia L. Sundell, Robert H. Singer

Open Access Articles

Actin mRNA is localized in chicken embryo fibroblasts to the distal regions of leading lamellae, but not within the ruffling edges. In this investigation we have addressed the role of actin translation in this process. The translocation of actin mRNA to the cell periphery was studied by monitoring the distribution of actin mRNA in cells during spreading. Within 90 min, actin mRNA moved from a perinuclear to a peripheral distribution. Formation of lamellipodia preceded actin mRNA localization, indicating that localization is not a prerequisite for this event. Neither puromycin (which dissociates ribosomes from mRNA) nor cycloheximide (which stabilizes ribosomes on ...


Effect Of Nocodazole On Vesicular Traffic To The Apical And Basolateral Surfaces Of Polarized Mdck Cells, Philip P. Breitfeld, Wendy C. Mckinnon, Keith E. Mostov Dec 1990

Effect Of Nocodazole On Vesicular Traffic To The Apical And Basolateral Surfaces Of Polarized Mdck Cells, Philip P. Breitfeld, Wendy C. Mckinnon, Keith E. Mostov

Open Access Articles

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic ...


Differential Distribution Of Myosin Isoforms Among The Myofibrils Of Individual Developing Muscle Fibers, Geraldine E. Gauthier Mar 1990

Differential Distribution Of Myosin Isoforms Among The Myofibrils Of Individual Developing Muscle Fibers, Geraldine E. Gauthier

Open Access Articles

Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly ...


Detection Of Hiv-1-Infected Cells From Patients Using Nonisotopic In Situ Hybridization, Robert H. Singer, Kevin S. Byron, Jeanne B. Lawrence, John L. Sullivan Nov 1989

Detection Of Hiv-1-Infected Cells From Patients Using Nonisotopic In Situ Hybridization, Robert H. Singer, Kevin S. Byron, Jeanne B. Lawrence, John L. Sullivan

Open Access Articles

We have demonstrated that a sensitive, nonisotopic in situ hybridization (ISH) assay can be used to detect HIV-infected cells from seropositive, asymptomatic individuals. Our assay is based on the detection of a biotinated HIV DNA probe hybridized to human immunodeficiency virus (HIV)-infected peripheral blood lymphocytes (PBL) using streptavidin and alkaline phosphatase to identify positive cells. This assay is rapid in that it can be performed within a day and is sensitive enough to unambiguously identify a rare, single, positive cell. Patient samples derived from HIV-seropositive hemophiliacs and HIV-seropositive infants were analyzed before and after coculture with normal PBL. The ...


Structural Changes Induced In Ca2+-Regulated Myosin Filaments By Ca2+ And Atp, Ling-Ling Young Frado, Roger W. Craig Aug 1989

Structural Changes Induced In Ca2+-Regulated Myosin Filaments By Ca2+ And Atp, Ling-Ling Young Frado, Roger W. Craig

Open Access Articles

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP ...


Ultrastructural Visualization Of Cytoskeletal Mrnas And Their Associated Proteins Using Double-Label In Situ Hybridization, Robert H. Singer, Gary L. Langevin, Jeanne B. Lawrence Jun 1989

Ultrastructural Visualization Of Cytoskeletal Mrnas And Their Associated Proteins Using Double-Label In Situ Hybridization, Robert H. Singer, Gary L. Langevin, Jeanne B. Lawrence

Open Access Articles

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed ...


Periodic Organization Of The Contractile Apparatus In Smooth Muscle Revealed By The Motion Of Dense Bodies In Single Cells, Gary J. Kargacin, Peter H. Cooke, Steven B. Abramson, Fredric S. Fay Apr 1989

Periodic Organization Of The Contractile Apparatus In Smooth Muscle Revealed By The Motion Of Dense Bodies In Single Cells, Gary J. Kargacin, Peter H. Cooke, Steven B. Abramson, Fredric S. Fay

Open Access Articles

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns ...


Access Of Proteinase K To Partially Translocated Nascent Polypeptides In Intact And Detergent-Solubilized Membranes, Timothy Connolly, Pallia G. Collins, Reid Gilmore Feb 1989

Access Of Proteinase K To Partially Translocated Nascent Polypeptides In Intact And Detergent-Solubilized Membranes, Timothy Connolly, Pallia G. Collins, Reid Gilmore

Open Access Articles

We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while ...