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PubMed1983

Small cytoplasmic RNAs from rat liver mRNP particles. Studies on their structure and function.

Free cytoplasmic 40S mRNP particles from rat liver were treated with EDTA and separated into two populations of RNP particles with sedimentation maxima of 20S and 35S, respectively. A characteristic set of distinct scRNAs is found for 20S and 35S RNP particles. The sequences of two of the most abundant scRNAs from 20S RNP particles with chain lengths of 104 (alpha 1-RNA) and 124 (beta 1-RNA) nucleotides, respectively, are presented. alpha 1-RNA shows a high sequence homology to the 3'-end of 18S rRNA. Since alpha 1-RNA carries a cap, it cannot be a degradation product of 18S rRNA. The beta 1-RNA is strongly post-transcriptionally modified, but uncapped. When the individual scRNAs of 20S and 35S RNP particles isolated from preparative polyacrylamide gels were assayed for their capability to inhibit in vitro protein synthesis, several potent translational inhibitory RNAs were detected. Particularly, the scRNAs of 147,203 and 263 nucleotide length associated with the 35S RNP particles turned out to be strong inhibitors of protein synthesis.

Animals↗
PubMed1989

Maturation at high frequency of germinal-vesicle-stage mouse oocytes after cryopreservation: alterations in cytoplasmic, nuclear, nucleolar and chromosomal structure and organization associated with vitrification.

After thawing and culture in vitro, greater than 90% of germinal-vesicle (GV)-stage mouse oocytes cryopreserved by vitrification, were capable of resuming meiosis and undergoing normal chromosomal and cytoplasmic maturation to metaphase II. This high frequency of development occurred against a background of profound alterations in the structure and organization of the cytoplasm, nucleus, nucleolus and chromatin during the dehydration stage of vitrification. Most, but not all, cytoplasmic and nuclear perturbations returned to a normal state during post-thaw culture. However, the results clearly demonstrate that vitrification is associated with chromosomal and cellular disorders that could adversely affect development after fertilization. Irreversible changes of potential developmental significance observed after vitrification at the GV stage include (i) premature chromosomal condensation, (ii) mixing of nucleoplasmic and cytoplasmic components prior to GV breakdown, and (iii) externalization of chromatin fragments into the cytoplasm after reformation of the oocyte nucleus, which shows the potential for the generation of fertilizable oocytes containing deleted segments of DNA.

Animals↗
PubMed1983

Effects of 5-iodo-2'-deoxyuridine on ultrastructural localization of viral antigens in cultured cells infected with herpes simplex virus.

Changes in viral morphology and localization of viral antigens induced by 5-iodo-2'-deoxyuridine (IDU) were studied using herpes simplex virus-infected monolayers of cultured rabbit corneal cells. A direct antibody enzyme method was employed for immunoelectron and light microscopy. There was no significant difference between the control and the IDU groups in the intracellular distribution of antigen up to 2 hours after infection. At 2 hours after infection, intracytoplasmic antigen began to appear in control and IDU-treated cells either in patchy distributions or in association with rough endoplasmic reticulum. Intranuclear staining appeared also at 2 hours after infection as a diffuse fine granular reaction. At 3 hours after infection, the intracytoplasmic reaction became more or less diffuse and there was slightly more cytoplasmic staining in the untreated group. At 6 hours after infection, reaction products increased in the cytoplasm and the nuclei in both groups but IDU-treated cells showed less intracytoplasmic and more intranuclear staining. The nuclear envelope was stained positive in both groups but IDU-treated cells showed a decreased reaction in the cytoplasmic membranous structure and fewer reaction deposits on the cell surface. At 12 and 18 hours after infection, the intranuclear accumulation of reaction products after IDU treatment became more evident. Progeny virus particles were significantly fewer in the presence of IDU: Nucleocapsids were found but in a greatly reduced number and only one incompletely enveloped particle was found. The morphology of the individual nucleocapsid, however, was not significantly altered by treatment with IDU. The preponderance of reaction substances in the nuclei compared to the cytoplasm in treatment groups may reflect distorted protein synthesis and may provide morphological evidence of the biochemical hypothesis of IDU efficacy: IDU exerts its antiviral effects through abnormal protein synthesis and the resultant defects in capsid assembly. Decreased reaction substances in the membranous structures of the cytoplasm may be related to reduced or abnormal production of envelope-associated viral antigens.

Animals↗
PubMed2002

Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state.

Bovine rhodopsin was specifically labeled on the cytoplasmic surface at cysteine 140 (the first residue of the loop connecting helices III and IV) or at cysteine 316 (in the loop connecting helix VII and the palmitoylation sites) with the fluorescent labels fluorescein and Texas Red. These loops are involved in activation and signal transduction. The time-resolved fluorescence depolarization was measured in the dark state and in the M(II) state, with labeled samples consisting of rhodopsin-octylglucoside micelles or rod outer segment (ROS) membranes. In this way the diffusional dynamics of the flexible loops of rhodopsin were measured for the first time directly on the nanosecond time scale. Control experiments showed that the large number of weak excitation pulses required in these single photon counting experiments leads to <5% bleaching of the sample. Rhodopsin was trapped in the activated M(II) state for the duration of the fluorescence experiments ( approximately 20 min) after illumination at pH 6 and 5 degrees C. For both types of samples and at both labeled positions the dynamics of the label and loop motion as monitored by the time constants of the depolarization were not significantly different in the two states of the receptor. The end-anisotropy increased, however, from 0.09 in the dark to 0.16 in the M(II) state for ROS samples labeled at C140. The corresponding numbers for the C316 position are 0.06 and 0.12. Light-induced activation in M(II) is thus associated with a large increase in the loop steric hindrance due to a changed loop domain structure on the cytoplasmic surface. These results are supported by fluorescence quenching experiments with I(-), which indicate a significant decrease in the collisional quenching constant k(q) and in accessibility in the M(II) state at both positions. The rotational correlation time of the rhodopsin micelles increased from 48 ns in the dark state to 60 ns in M(II). This increase is caused by a change in volume and/or shape and is consistent with a structural change. These results demonstrate that time-resolved fluorescence depolarization is a powerful tool to study the changes in conformation and dynamics of the cytoplasmic loops that accompany the activation of rhodopsin and other G-protein coupled receptors.

Animals↗
PubMed1990

Immunohistochemical and electron microscopic findings in a case of mixed hamartoma of the liver.

A case of mixed hamartoma of the liver in a 36 day old female infant is reported herein. The resected tumor was well demarcated, measured 7.5 X 7.0 cm and weighed 179 gm. Microscopically, the tumor was composed of loose fibrous stroma intermingled with spindle and polygonal cells and ductular structures. The spindle and polygonal tumor cells displayed the distinct cytoplasmic staining for alpha-1-antitrypsin. Most of the tumor cells showed the characteristic ultrastructural features of immature hepatocytes, having numerous cytoplasmic microvillous structures, abundant rough and smooth endoplasmic reticulum and mitochondria. These findings suggest that embryonic liver cells, which are capable of transforming into biliary structures and hepatocytes, serve as essential elements of mixed hamartoma of the liver.

Adult↗
PubMed2004

Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring components.

An understanding of the involvement of bacterial cytoplasmic filaments in cell division requires the elucidation of the structural organization of those filamentous structures. Treponemal cytoplasmic filaments are composed of one protein, CfpA, and have been demonstrated to be involved in cell division. In this study, we used electron tomography to show that the filaments are part of a complex with a novel molecular organization that includes at least two distinct features decorating the filaments. One set of components appears to anchor the filaments to the cytoplasmic membrane. The other set of components appears to bridge the cytoplasmic filaments on the cytoplasmic side, and to be involved in the interfilament spacing within the cell. The filaments occupy between 3 and 18% of the inner surface of the cytoplasmic membrane. These results reveal a novel filamentous molecular organization of independent filaments linked by bridges and continuously anchored to the membrane.

Bacterial Proteins↗
PubMed1984

Temperature dependence of action of polymyxin B on Escherichia coli.

The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes. At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s. The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C. The uptake of TPP+ also increased after polymyxin addition. The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously. In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C. This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes. The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E. coli. Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability. The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.

Calcium↗
PubMed1987

Microbial structures in a patient with sporadic non-A, non-B fulminant hepatitis treated by liver transplantation.

Double-shelled virus-like particles (60 nm) and long cytoplasmic tubular structures were found in the cytoplasm of hepatocytes from areas of collapsed and regenerating areas of hepatectomised liver in a 13-year-old boy who received a liver graft for fulminant hepatitis attributed to sporadic non-A, non-B hepatitis. The patient died on the ninth postoperative day from acute graft failure. Although virus-like particles were not found, instead, gram-negative rods were identified in the necrotic graft and the most likely cause of death was a gram-negative septicaemia with a Shwartzman-like reaction localized to the liver.

Adolescent↗
PubMed2006

Andersen's syndrome mutation effects on the structure and assembly of the cytoplasmic domains of Kir2.1.

Kir2.1 channels play a key role in maintaining the correct resting potential in eukaryotic cells. Recently, specific amino acid mutations in the Kir2.1 inwardly rectifying potassium channel have been found to cause Andersen's Syndrome in humans. Here, we have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and delta314-315 and have found multiple effects on the ability of the cytoplasmic domains in Kir2.1 channels to form proper tetrameric assemblies. For the R218Q mutation, we identified a second site mutation (T309K) that restored tetrameric assembly but not function. We successfully crystallized and solved the structure (at 2.0 A) of the N- and C-terminal cytoplasmic domains of Kir2.1-R218Q/T309K(S). This new structure revealed multiple conformations of the G-loop and CD loop, providing an explanation for channels that assemble but do not conduct ions. Interestingly, Glu303 forms both intra- and intersubunit salt bridges, depending on the conformation of the G-loop, suggesting that the E303K mutant stabilizes both closed and open G-loop conformations. In the Kir2.1-R218Q/T309K(S) structure, we discovered that the DE loop forms a hydrophobic pocket that binds 2-methyl-2,4-pentanediol, which is located near the putative G(betagamma)-activation site of Kir3 channels. Finally, we observed a potassium ion bound to the cytoplasmic domain for this class of K+ channels.

Amino Acid Substitution↗
PubMed2003

The vaccinia virus I3L gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental DNA.

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogeneous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

Cytoplasm↗
PubMed1986

Crystalline arrays of the Escherichia coli sn-glycerol-3-phosphate acyltransferase, an integral membrane protein.

The gene encoding the Escherichia coli sn-glycerol-3-phosphate acyltransferase, plsB, was inserted into hybrid plasmids under transcriptional control of the lambda PL and tac promoters. Enzymatic activities 35-50-fold above wild type and a large increase in glycerol-P acyltransferase polypeptide were obtained. Thin section electron microscopy of the cells overproducing the glycerol-P acyltransferase revealed 235-245-A diameter tubular structures associated with the cytoplasmic membrane. These structures were released from the cell by osmotic lysis and purified on Matrex Gel Green A. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the dominant protein constituent of the tubules was the glycerol-P acyltransferase. Analysis of tubule-enriched fractions isolated by differential centrifugation revealed a decreased phospholipid to protein ratio as compared to total and cytoplasmic membrane fractions. At high magnification, negative stained tubules displayed ordered arrays of stain-excluding components projecting 50-60 A from the cytoplasmic surface. Optical diffraction patterns from the micrographs contained intense layer lines at (1/78 A) and (1/39 A) along the tubule axis and a prominent spot at (1/62 A) near the equator. From compositional and structural data, 18-37% of the polypeptide volume is estimated to lie within the hydrophobic domain of the tubule membrane.

Acyltransferases↗
PubMed1977

The perineural sheath in the urodele amphibian.

The perineural epithelial cells of the peripheral nerve sheath in triturus are elongated flat cells with long and thin cytoplasmic processes. Structural characteristics of the processes include a relative absence of common cytoplasmic organelles of the perinuclear cytoplasm but an abundance of vesicular profiles similar to fully formed pinocytotic vesicles. Unlike pinocytotic vesicles, vesicles of the processes often contain an 8-nm thick filament attached to their cytoplasmic face. Vesicles are also interconnected by similar filaments or by tubules. In addition, almost all vehicles appear to have openings at the cell surface, a feature not seen for pinocytotic vesicles. The concentration of exogenous peroxidase in the vesicles, and the subsequent appearance of peroxidase in the endoneural fluid, myelin sheath and axon, support the notion that the vesicles function in the exchange of substances between extraneural and endoneural fluids.

Animals↗
PubMed1997

Cytoplasmic chaperonin containing TCP-1: structural and functional characterization.

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the cytoplasmic chaperonin containing TCP-1 (CCT, also called TRiC) and, in the case of alpha- and beta-tubulin, additional protein cofactors. It has been previously demonstrated that nucleotide exchange and ATP hydrolysis act to switch CCT between conformations that interact either strongly or weakly with unfolded substrates [Melki, R., & Cowan, N.J. (1994) Mol. Cell. Biol. 14, 2895-2904]. The present study further documents the conformational changes and function of CCT. It is first shown, by the use of a range of labeled denatured substrate proteins and a radiolabeled total soluble HeLa cell extract, that CCT in the absence of nucleotides can bind any of a large number of proteins in vitro with high affinity. Second, by the use of denatured labeled beta-actin and beta-tubulin as model substrates for binding to CCT, we demonstrate that the CCT particle can contain two substrate protein chains simultaneously. Third, by electron microscopy, sedimentation velocity, and intrinsic fluorescence measurements, we document the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate protein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target proteins.

Actins↗
PubMed1976

Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane.

A structure which appears to organize cytoplasmic microtubules in interphase mouse 3T3 cells can be visualized by immunofluorescence microscopy. Purified monospecific antibody against homogeneous tubulin from brain visualizes, in addition to cytoplasmic microtubules, a cytoplasmic polar structure as the focal point from which the microtubules seem to radiate. The structure is preserved after treatments that depolymerize cytoplasmic microtubules, i.e., exposure of cells to mitotic drugs or to low temperature. When cells recover from these treatments one end of each microtubule organizing structure acts as a nucleating center from which cytoplasmic microtubules grow toward the plasma membrane. Thus cytoplasmic microtubules assemble in vivo in an ordered unidirectional manner, and therefore the cell must be able to avoid the assembly of unwanted, unoriented, and disconnected microtubules. These results suggest that the assembly of tubulin into microtubules is regulated in vivo.

Cell Line↗
PubMed1965

Messenger RNA in early sea-urchin embryos: cytoplasmic particles.

Three structures containing messenger RNA can be demonstrated in the cytoplasm of early sea-urchin embryos: (i) particles that sediment more slowly than ribosomes and contain newly synthesized DNA-like RNA, (ii) light polyribosomes, which also contain this newly synthesized RNA, and (iii) heavy polyribosomes, which seemingly contain only already existing or "maternal" messenger RNA and account for the bulk of the synthesis of protein.

Animals↗
PubMed2002

Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway.

Brain microvascular endothelial cells (BMVECs) present an incomplete barrier to human immunodeficiency virus type 1 (HIV-1) neuroinvasion. In order to clarify the mechanisms of HIV-1 invasion, we have examined HIV-1 uptake and transcellular penetration in an in vitro BMVEC model. No evidence of productive infection was observed by luciferase, PCR, and reverse transcriptase assays. Approximately 1% of viral RNA and 1% of infectious virus penetrated the BMVEC barrier without disruption of tight junctions. The virus upregulated ICAM-1 on plasma membranes and in cytoplasmic vesiculotubular structures. HIV-1 virions were entangled by microvilli and were taken into cytoplasmic vesicles through surface invaginations without fusion of the virus envelope with the plasma membrane. Subsequently, the cytoplasmic vesicles fused with lysosomes, the virions were lysed, and the vesicles diminished in size. Upon cell entry, HIV-1 colocalized with cholera toxin B, which targets lipid raft-associated GM1 ganglioside. Cholesterol-extracting agents, cyclodextrin and nystatin, and polyanion heparin significantly inhibited virus entry. Anti-CD4 had no effect and the chemokine AOP-RANTES had only a slight inhibitory effect on virus entry. HIV-1 activated the mitogen-activated protein kinase (MAPK) pathway, and inhibition of MAPK/Erk kinase inhibited virus entry. Entry was also blocked by dimethylamiloride, indicating that HIV-1 enters endothelial cells by macropinocytosis. Therefore, HIV-1 penetrates BMVECs in ICAM-1-lined macropinosomes by a mechanism involving lipid rafts, MAPK signaling, and glycosylaminoglycans, while CD4 and chemokine receptors play limited roles in this process.

Blood-Brain Barrier↗
PubMed1976

An IgG-Fc receptor induced in cytomegalovirus-infected human fibroblasts.

Cytomegalovirus- (CMV) infected cultured human fibroblasts incubated with normal human serum were shown to react with IgG by direct and indirect fluorescent antibody tests. The binding of IgG to infected cells was unrelated to the presence of anti-CMV complement fixing (CF) antibodies and was not observed with uninfected cells. The reaction was first observed 36 hours after infection as diffuse cytoplasmic staining which by 72 hr became localized into a dense perinuclear structure. The cytoplasmic fluorescence was not detected with anti-IgA or anti-IgM fluorescent conjugates. The reaction was seen with purified IgG and Fc fragments but was only minimally detectable with Fab fragments. It occurred in fibroblasts infected with three standard laboratory strains of CMV and seven recent CMV isolates from patients and was observed in six separate lots of human foreskin fibroblast cultures as well as in WI-38 cells. We conclude that CMV infection induces the formation of a IgG receptor in human fibroblasts.

Adult↗
PubMedDate not supplied

[The use of monoclonal antibodies for detecting the influenza virus].

The possibilities of using influenza A (Leningrad) 385/80 (H3N2) virus matrix protein-specific FITC-labeled D8 monoclonal antibodies in immunofluorescence assays were investigated. The virus antigen accumulation was detected in chorioallantoic cells of chick embryos. Exhibiting the type-specific properties, the fluorescent antibodies stain the perinuclear space, cytoplasmic membrane, and granular structures in the cytoplasm of infected cells. The haemagglutination test tires in the corresponding specimens were at least 1:16.

Animals↗