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At least 1,495 records · Page 83Linked to original sources
PubMed2002

Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein.

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.

3T3 Cells↗
PubMed2002

Demonstration of alpha-synuclein immunoreactivity in neuronal and glial cytoplasm in normal human brain tissue using proteinase K and formic acid pretreatment.

alpha-Synuclein (alphaS), a presynaptic nerve terminal protein, is now known to be a major component of neuronal and glial cytoplasmic inclusions in alpha-synucleinopathies (Lewy body disease and multiple system atrophy). However, alphaS has not been identified in either neuronal or glial cytoplasm in formalin-fixed, paraffin-embedded tissue sections from the normal human brain. Previous studies have shown that pretreatment with either proteinase K or formic acid enhances alphaS immunoreactivity. The aim of the present study was, therefore, to study the effects of pretreatment with proteinase K and formic acid on alphaS immunoreactivity in vibratome sections of brain tissue taken from normal human subjects. In addition to presynaptic staining, alphaS immunostaining was recognized in neuronal perikarya in the pretreated sections; this immunoreactivity was more intense in sections taken from the deeper layers of the cerebral neocortex, the CA2/3 region of the hippocampus, and the substantia nigra. This pattern of alphaS expression coincides with the distribution of intraneuronal inclusions in alphaS transgenic animals as well as in human autopsy tissue taken from patients with Lewy body disease. Furthermore, intense immunoreactivity was also found in the cytoplasm of astrocytes and oligodendrocytes throughout the brain. These findings suggest that a significant amount of alphaS is also present in the neuronal and glial cytoplasm in the normal human brain.

Aged↗
PubMed2001

Toxicity to bivalve hemocytes of pathogenic vibrio cytoplasmic extract.

Using a chemiluminescence (CL) test, it had been previously demonstrated that Vibrio pectenicida, which is pathogenic to Pecten maximus larvae, was able to inhibit completely the CL activity of P. maximus hemocytes and partially inhibit those of Crassostrea gigas. Conversely, a Vibrio sp. strain, S322, pathogenic to C. gigas larvae was more active in reducing the CL activity of oyster hemocytes than of scallop hemocytes. Using this same CL biotest, V. pectenicida and S322 cytoplasmic extracts were shown to reproduce CL inhibition while the cytoplasmic extract of a nonpathogenic strain (U1, Pseudoalteromonas) was without effect. Moreover, cytoplasmic extract as well as live V. pectenicida cells provoked, within a few hours, death of P. maximus hemocytes adhering to a glass slide. After partial purification, it was shown that toxic activities of V. pectenicida cytoplasmic extract was due to a toxin, named VHKT (for vibrio hemocyte-killer toxin), which is heat stable, acid and protease resistant, and less than 3 kDa in molecular weight. Attempts to purify VHKT by reverse-phase (C18) HPLC separated activity into the fraction eluted by water at a retention time of 4.02 min.

Animals↗
PubMed1994

The proton transfers in the cytoplasmic domain of bacteriorhodopsin are facilitated by a cluster of interacting residues.

The stepwise internal proton transfer reactions across the membrane, and the release and uptake at the surface, are the elementary steps that together constitute the transport mechanism in a proton pump. Although the proton donor and acceptor residues can be usually identified, the directionality and the energetics of the proton transfer must be determined to a large extent also by interactions of these with neighboring groups. We have examined the roles of residues D96, T46 and R227 in proton transfers during the photocycle of bacteriorhodopsin near its cytoplasmic surface, and in general the relationship between the reprotonation of the Schiff base and the subsequent proton uptake from the cytoplasmic side. The phenotypes of single and double mutants suggest close functional interaction among D96, T46, R227, and probably internal bound water. Measurements of the free energies of activation indicate that mechanistic interpretation of the rates changed by residue replacements is hindered by a general tendency toward lowered activation enthalpies in the mutated proteins. There is less ambiguity in the free energy levels of the photointermediates. It appears from these that the inhibitory and stimulatory influences of T46 and R227, respectively, on D96 as a proton donor compensate one another and ensure the effective reprotonation of the Schiff base. T46 and D96 mediate, in turn, proton uptake at the cytoplasmic surface. Although ultimately this will reprotonate D96, the observation of proton uptake from the bulk in R82Q without reprotonation of the aspartate residue suggests that the direct proton acceptor is not D96. The results thus indicate that the passage of the proton from the surface to the Schiff base is facilitated by multiple residue and water interactions in the cytoplasmic domain.

Amino Acids↗
PubMed1994

Control of nucleo-cytoplasmic HLA-DRA mRNA partitioning by interaction of a retention signal with compartmentalized proteins.

Transport of mRNA from the nucleus to the cytoplasm is still a poorly understood process in which RNA signal sequences and cognate RNA-binding proteins may be involved. We have analysed the transport of the mRNA encoded by HLA-DRA, a member of the immunologically important MHC class II multigene family. We report that, in transient transfection experiments, HLA-DRA mRNA molecules encompassing a signal situated in the 3' untranslated region predominantly accumulate in the nucleus. We also show that the RNA sequence involved interacts with compartmentalized proteins of either nuclear or cytoplasmic origin. Deletion of the mRNA region encompassing this retention site results both in the abrogation of protein binding and in the release of HLA-DRA mRNA into the cytoplasm. In addition, we have found that the distribution of these HLA-DRA mRNA binding proteins is different in different cell types; in particular, their pattern of expression in Ntera-2, a human teratocarcinoma cell line, is distinct from that observed in Raji, a human B-lymphoma cell line, and is modulated by growth in retinoic acid. We conclude that recognition of a mRNA retention signal by proteins located in different compartments on either side of the nuclear membrane may regulate the nucleo-cytoplasmic partitioning of HLA-DRA transcripts and, perhaps, of MHC class II mRNA in general.

Base Sequence↗
PubMed1996

Molecular evolution of the C-terminal cytoplasmic domain of a superfamily of bacterial receptors involved in taxis.

Twenty-nine proteins from 16 different species of prokaryotes revealed an extensive sequence homology with the cytoplasmic domain of the Escherichia coli aspartate receptor. The high percentage of identity indicated that they constitute a superfamily of proteins. A consensus secondary structure consisting mostly of alpha-helices was predicted. The occurrence of a seven-residue repeat (a-b-c-d-e-f-g), in which both the a and d residues were hydrophobic with few exceptions, provided additional evidence for a conserved alpha-helical conformation. Sequence alignments, together with the predicted secondary structure, led to identification of the boundaries for the functional units constituting the cytoplasmic domain. Putative methylation sites were assigned for all the members of this superfamily. These proteins could be grouped into three classes based on the presence of 14-residue insertion/deletion regions found within both the signalling and the methylation functional units of the cytoplasmic domain. The gene coding for the C-terminal cytoplasmic domain of these proteins apparently evolved through gene duplication from a common ancestor in which the four original 14-residue insertion/deletion regions were deleted two by two during evolution.

Amino Acid Sequence↗
PubMed1999

Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1.

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site was inserted into the beta-globin reporter, Rev was able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. We conclude that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm.

Base Sequence↗
PubMed2000

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane.

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.

Amino Acid Sequence↗
PubMed1998

Malate-aspartate shuttle, cytoplasmic NADH redox potential, and energetics in vascular smooth muscle.

The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0. 4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2 consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism.

Aminooxyacetic Acid↗
PubMed2000

Biochemical and molecular analysis of the mammalian cytoplasmic dynein intermediate chain.

Cytoplasmic dynein is a multisubunit protein complex responsible for the intracellular movement of membranous organelles and other cargo along microtubules. The heavy chains contain the motor domains, while the intermediate chain and other subunits are important for binding to cargo. There are at least five different intermediate chain polypeptides, the products of alternative splicing of two genes. The cytoplasmic dynein intermediate chains are also phosphorylated. The expression of the different intermediate chain mRNAs is characterized by reverse transcription-polymerase chain reactions using oligonucleotide primers appropriate for the alternative splicing sites. The presence of the different intermediate chain polypeptide isoforms is determined by two-dimensional gel analysis of cytoplasmic dynein samples. The phosphorylation state of the polypeptides is determined by treatment of immunoprecipitated cytoplasmic dynein with protein phosphatase and analysis of changes in polypeptide spot distribution after two-dimensional gel electrophoresis.

Animals↗
PubMed2000

The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging.

Deletion of the cytoplasmic tails of the influenza A virus spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), has previously been shown to result in markedly defective virion morphogenesis (Jin et al., 1997, EMBO J. 16, 1236-1247). We have found that influenza A virus preparations lacking the HA and NA cytoplasmic tails (HAt-/NAt-) have a reduced vRNA to protein content, contain an increase in cellular RNA contaminants, and exhibit increased resistance to ultraviolet (UV) inactivation. There is also a direct correlation between abnormal virion morphology and reduced infectivity. The data suggest that the HAt-/NAt- virion population contains a broader range of number of packaged RNA segments than wild-type (wt) virus. Sucrose gradient centrifugation analysis indicated the presence of a subpopulation of virions with pronounced deformation in virion morphology and reduced infectivity. The role of the HA and NA cytoplasmic tails was examined further by using a trans-complementation assay and it was found that expression of wt HA and NA from cDNAs followed by HAt-/NAt- virus infection caused the formation of a pseudotype virus with wt sedimentation properties. Taken together the data indicate that the HA and NA cytoplasmic tails affect not only virion morphology but also proper genome packaging.

Animals↗
PubMed1975

Biogenesis of chloroplast membranes in Chlamydomonas reinhardi: Chloroplast-controlled transfer of cytoplasmic proteins to the developing chloroplast membranes as visualized by quantitative radioautography.

The light-induced formation of the photosynthetic membranes (greening) in y-1 mutant of Chlamydomonas reinhardi requires synthesis of new proteins which become incorporated into the growing membranes. It has been shown previously (Eytan and Ohad, 1970) that proteins synthesized by both chloroplast and cytoplasmic ribosomes concur in the formation of functional photosynthetic membranes, indicating the presence of a mechanism permitting the specific transfer of membrane proteins synthesized in the cytoplasm into the chloroplast. Transfer of such proteins cannot yet be identified by the usual biochemical techniques unless they become part of the growing photosynthetic membranes. However, it is possible to follow their synthesis and translocation between the different cellular compartments by use of quantitative electron microscopic radioautography. In the present work, the radioautographic grain distribution among chloroplast, chloroplast membrane, nucleus, mitochondria, and the remainder of the cytoplasm (cytosol) was carried out following short radioactive pulse-labeling and chase during greening of dark-grown mutants in the presence or absence of protein synthesis inhibitors. The results indicate that transport of some of the proteins of cytoplasmic origin to their final location within the chloroplast is at least partially controlled by concomitant synthesis of proteins by the chloroplast ribosomes.

Arginine↗
PubMed1992

Allele-specific expression of the cytoplasmic exon of HLA-DQB1 gene.

The beta chain of the HLA-DQ molecule is shorter by eight amino acid residues than other major histocompatibility complex class II beta chains due to elimination of the fifth exon coding for part of the cytoplasmic domain. This elimination is caused by one base substitution in the splice acceptor site of the exon. We found that two HLA-DQB1 alleles, DQB1*0503 and DQB1*0601, did not have this substitution, and the exon was utilized in these two alleles. However, two forms of HLA-DQB mRNA, with or without exon 5, were generated in Epstein-Barr virus-transformed cell lines homozygous for DQB1*0503 or DQB1*0601, indicating alternative mRNA splicing. The alternative splicing of DQB1*0601 mRNA was also found in peripheral blood lymphocytes and L cell transfectants. To investigate the functional relevance of the allele-specific long cytoplasmic tail of HLA-DQ beta chain, we developed three types of L cell transfectants expressing exclusively the HLA-DQw6 molecules with short cytoplasmic tail, long cytoplasmic tail, or both forms of the beta chain, and used them as antigen presenting cells for streptococcal cell wall antigen-specific T cell lines. These three types of transfectants could function almost equally well as antigen presenting cells. It was thus demonstrated that both forms of HLA-DQ beta chain, with or without eight amino acid residues coded for by the exon 5, can be associated with the HLA-DQ alpha chain, be expressed on the cell surface, and function as restriction molecules in antigen recognition by the CD4+ T cells.

Alleles↗
PubMed1989

Cytoplasmic actin and cochlear outer hair cell motility.

Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaining cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.

Actins↗
PubMed1983

Cytoplasmic modification of nuclear gene expression.

A review is presented on 1) the autonomous nature of mammalian cell cytoplasm and 2) the cytoplasmic modification of nuclear gene expression. Topics include a discussion of cytoplasmic suppression of tumorigenicity. It is proposed that alterations in DNA methylation patterns may be a possible mechanism to explain cytoplasmic modification of nuclear gene expression.

Animals↗
PubMed1975

Intranuclear and cytoplasmic annulate lamellae in grasshopper spermatocytes (genus Melanoplus).

Intranuclear and cytoplasmic annulate lamellae were studied in grasshopper spermatocytes (Melanoplus) with the electron microscope. Although cytoplasmic annulate lamellae were observed in all three species examined, intranuclear annulate lamellae were found in only one species. The intranuclear annulate lamellae encompass certain nuclear material adjacent to the nuclear envelope forming a vesicle that is extruded into the spermatocyte cytoplasm. In this same species, cytoplasmic annulate lamellae are seen contiguous with granular masses of varying size. These structures were noted as being morphologically indistinguishable from the "yolk nuclei" of dragonfly oocytes (Kessel and Beams, 1969; Kessel, 1973).

Animals↗
PubMed1990

A chimeric gene containing the 5' portion of atp6 is associated with cytoplasmic male-sterility of rice.

Three ATPase subunit 6 (atp6) genes of rice mitochondria were isolated, one from normal and two from cms-Bo male-sterile cytoplasms, in order to determine whether the extra atp6 gene in cms-Bo rice plays a role in cytoplasmic male-sterility (CMS). The nucleotide sequences of all three genes were determined and analysis showed a chimeric atp6 gene (urf-rmc) as well as a normal atp6 gene in cms-Bo cytoplasm, but only the normal atp6 gene in normal cytoplasm. The urf-rmc gene is completely homologous to the normal atp6 gene from at least position -426 in the 5' flanking region to position +511 downstream from the initiation codon ATG: however, the following downstream sequence shows no homology with the normal rice atp6 gene, or any other reported sequence. Introduction of the restorer of fertility gene altered transcription of the urf-rmc gene but not the atp6 gene, indicating participation of the chimeric gene in the expression of CMS. Southern blot analysis showed that the urf-rmc gene was generated by an intramolecular recombination event in mitochondrial DNA, and the homologous recombination point between the atp6 gene and the opposite ancestral sequence was identified as 5'-TTCCCTC-3'.

Adenosine Triphosphatases↗
PubMed1984

Disobutamide-induced cytoplasmic vacuoles in cultured dog coronary artery muscle cells.

Smooth muscle cells released by protease from the coronary artery of dogs were cultured in M199 supplemented with 10% fetal calf serum. Cells were grown on glass coverslips to semiconfluence and exposed to disobutamide at 0, 1, 2, 3, 6, 8, and 10 X 10(-4) M for 24, 48, and 72 h, examined in situ by light microscopy, then fixed in 100% methanol, stained with May-Grünwald Giemsa and examined by light microscopy. Cells were also exposed to drug for 24 h, pelleted, fixed and prepared routinely for electron microscopy. Control cells and cells exposed to 6 X 10(-4) M disobutamide were examined. Vacuole formation was dose and time dependent between 2 X 10(-4) M and 10(-3) M. By light microscopy, morphologic alterations induced by the drug were clear cytoplasmic vacuoles in live cells, vacuoles in dead cells and dead cells without vacuoles. Small round vacuoles were an early change. Dark granules were dispersed among the vacuoles. The vacuoles increased in size at higher doses or longer times. Eventually all the cytoplasm was occupied by vacuoles and the cells were enlarged. By electron microscopy, the vacuoles were round, primarily membrane-bound, contained mostly electron-lucent material and occasionally small flocculent bodies. There was vacuolar coalescence. The dose response of vacuole induction in confluent and semiconfluent cells was similar. Cytoplasmic vacuoles without cell death can be induced by disobutamide at 2 or 4 X 10(-4) M during a 3-day exposure. Cell culture is a suitable biological system for studying cytoplasmic vacuoles of the type induced by disobutamide.

Animals↗