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PubMed1979

Smooth muscle autoantibodies and autoantigens.

Smooth muscle autoantibody (SMA) was first found in the sera of patients with chronic active hepatitis and subsequently in the sera of patients with other autoimmune liver diseases, viral infections, certain cancers, heroin addicts and female infertility. SMA from patients with chronic active hepatitis reacts with many muscle and 'non-muscle' tissues while SMA from patients with other diseases usually reacts only with smooth muscle. These differences in immunofluorescent staining reactions suggest that SMA is a heterogeneous group of autoantibodies reactive with different smooth muscle autoantigens. As further evidence for this are findings that broad-reacting SMA can be absorbed out by actin, whereas autoantibodies reactive only with smooth muscle cannot, and that different SMAs give different immunofluorescent staining patterns using fibroblasts in tissue culture. Such staining patterns correspond to reactivity with either microfilaments, microtubules or intermediate filaments, ubiquitous cytoplasmic structures which make up the 'cytoskeleton'. Autoantibodies to actin-like microfilaments appear specific for chronic active hepatitis, autoantibodies to microtubules occur in infectious mononucleosis whereas autoantibodies to intermediate filaments occur in infectious hepatitis, chickenpox, measles and mumps. Predictably, future studies will show that presence of SMA with specificities for other proteins in the three types of cytoplasmic filaments, and given more information on antigenicity of the proteins and pathogenicity of the corresponding autoantibodies.

Actins↗
PubMed1979

Spike activity and histofluorescence correlated in the giant dopamine neurone of Planorbis corneus.

The relationship between catecholamine fluorescence and electrical activity of the nerve cell has been investigated in the giant dopamine neuron (GDN) of the left pedal ganglion of the European water snail, Planorbis corneus. Electrical recordings were performed in vitro with intracellular microelectrodes on 35 GDN. The ganglion was frozen to -195 degrees C with the electrode in situ and processed for histochemical microfluorimetry. The intensity of catecholamine fluorescence was measured over different places (42/cell) throughout the cytoplasm of the GDN. In order to investigate the temporal relation between histochemical and electrophysiological parameters, the activity of 21 GDN was changed by administration of nicotine to the bathing solution (10(-5)--10(-7) M). This treatment was followed by depolarization in most of the GDN, with increased firing in two thirds and decreased activity in one third of the spontaneously active cells, whereas hyperpolarization was seen in 4 GDN, accompanied by a decrease in firing. Acetylcholine (10(-5) M) tested on one GDN caused depolarization and increased firing. A signigicant positive correlation was found between the fluorescence intensity of individual GDN and the firing rate of these cells as observed during the last 60 sec or the last 10 sec before freezing. The correlation coefficient dropped markedly when the first rates of the 2nd, 3rd, or 4th and 5th min before freezing were correlated with cellular fluorescence intensity. Intensity was not correlated with the resting membrane potential recorded at the time of freezing. The intensity response to activation was not uniform throughout the cytoplasm of GDN. Neurons with increased mean fluorescence intensity regularly showed small clusters and cristae of intensely fluorescent material surrounded by less fluorescent parts of the cytoplasm. This morphological observation of increased intensity differences between cytoplasmic structures was confirmed by the statistics of the intensity values determined in individual cells. The present observations demonstrate the existence of a relation between cell firing and neuronal dopamine fluorescence at the level of the individual nerve cell. The activity immediately preceding the time of freezing of the cell appears to be most important. Certain components of the cytoplasm appear to react preferentially; their subcellular nature remains to be elucidated. The link between electrical activity and cellular dopamine fluorescene was found to be basically the same in the giatn invertebrate neuron and in the dopamine nerve cells of rat substantia nigra.

Animals↗
PubMed1999

Association of calmodulin with cytoskeletal structures at different stages of HeLa cell division, visualized by a calmodulin-EGFP fusion protein.

The fusion protein of calmodulin (CaM) with the enhanced green fluorescent protein EGFP has been expressed in a stably transfected HeLa cell line in order to visualise the localisation of calmodulin during the cell cycle on a continuous basis in live cells, and for immunofluorescence colocalisation with cytoskeletal structures. High-resolution images of CaM-EGFP in the mitotic apparatus show the characteristic strongly convoluted structure of the centrosome. CaM-EGFP also apparently associates with both polar and mitotic microtubules, and with a specific intracentrosomal structure. During cytokinesis, CaM-EGFP is also found decorating selected oriented filaments in close proximity to microtubules in the midbody region. In interphase cells, it is seen with filamentous and punctuate localisation at the nuclear envelope. The intensity and continuity of the CaM-EGFP images suggest that a significant fraction of the cellular calmodulin remains attached to cytoplasmic structures during the cell cycle.

Calmodulin↗
PubMed1989

The formation of multivesicular structures in the adipose cells of chick embryos.

Observation of the cytogenesis of adipose tissue of the chick embryo revealed a quantity of multiversicular structures (MVs) which were found in the intercellular space. Some of them were attached to the adipocytes and others were independently located in the intercellular space. The origin of those MVs appeared to be part of the degenerating mitochondria. Centrally located vesicles and vacuoles in degenerating mitochondria formed a group of short tubules and vacuoles which protruded through the cytoplasmic membrane or bulged out at the edge of the cytoplasmic process. The MVs then spread over the cytoplasmic membrane and finally were discharged from the cell surface as in the manner of apocrine secretion. An invisible barrier between the mass of vesicles and the rest of the cytoplasmic structures appeared to segregate the extruding MVs from the intercellular components such as ribosomes, microtubules, and microfilaments.

Adipose Tissue↗
PubMed2004

Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter IImannitol of the Escherichia coli phosphotransferase system.

The solution structure of the cytoplasmic B domain of the mannitol (Mtl) transporter (II(Mtl)) from the mannitol branch of the Escherichia coli phosphotransferase system has been solved by multidimensional NMR spectroscopy with extensive use of residual dipolar couplings. The ordered IIB(Mtl) domain (residues 375-471 of II(Mtl)) consists of a four-stranded parallel beta-sheet flanked by two helices (alpha(1) and alpha(3)) on one face and helix alpha(2) on the opposite face with a characteristic Rossmann fold comprising two right-handed beta(1)alpha(1)beta(2) and beta(3)alpha(2)beta(4) motifs. The active site loop is structurally very similar to that of the eukaryotic protein tyrosine phosphatases, with the active site cysteine (Cys-384) primed in the thiolate state (pK(a) < 5.6) for nucleophilic attack at the phosphorylated histidine (His-554) of the IIA(Mtl) domain through stabilization by hydrogen bonding interactions with neighboring backbone amide groups at positions i + 2/3/4 from Cys-384 and with the hydroxyl group of Ser-391 at position i + 7. Modeling of the phosphorylated state of IIB(Mtl) suggests that the phosphoryl group can be readily stabilized by hydrogen bonding interactions with backbone amides in the i + 2/4/5/6/7 positions as well as with the hydroxyl group of Ser390 at position i + 6. Despite the absence of any significant sequence identity, the structure of IIB(Mtl) is remarkably similar to the structures of bovine protein tyrosine phosphatase (which contains two long insertions relative to IIB(Mtl)) and the cytoplasmic B component of enzyme II(Chb), which fulfills an analogous role to IIB(Mtl) in the N,N'-diacetylchitobiose branch of the phosphotransferase system. All three proteins utilize a cysteine residue in the nucleophilic attack of a phosphoryl group covalently bound to another protein.

Amino Acid Motifs↗
PubMed1988

The acute response of Schwann cells to taxol after nerve crush.

The effect of taxol, an antimitotic drug which stabilizes microtubules and promotes their assembly, was studied with regard to Schwann cells over a 4-week period following a crush injury to rat sciatic nerve. A single intraneural injection of taxol in dimethyl sulfoxide (DMSO) was given immediately after the crush into the site of injury in one sciatic nerve and was compared with the other side which was crushed but injected with DMSO only. Sampled sites were taken proximal and distal to the lesion, as well as from the lesion itself, and studied by light and electron microscopy. The Schwann cell response was most marked during the degenerative phase immediately following the crush. At this time, there was a decrease of all cytoplasmic structures except microtubules and smooth endoplasmic reticulum. At the site of the crush lesion in taxol-treated nerves, Schwann cells possessed accumulations of myelin debris and lipid droplets. Mitotic Schwann cells were also engorged with myelin breakdown products. Multinucleated Schwann cells, believed to be the result of abnormal mitotic activity, were also apparent and were filled with large numbers of cytoplasmic microtubules. The latter were sometimes regularly arranged around phagocytosed or intracytoplasmic debris. Some recovery from the crush injury was noted with time, although the number of Schwann cells was much lower than would have been anticipated in the absence of taxol, in that long stretches of naked axon bundles were common and microtubule-related abnormalities persisted up to 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed2004

Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes.

Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domain-containing protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Cytoplasmic accumulation of cyclin B1 in human cells: association with a detergent-resistant compartment and with the centrosome.

Mitotic cyclins are thought to function as key regulatory subunits of the universal M-phase-promoting p34cdc2 protein kinase. Previous immunolocalization studies have demonstrated that a fraction of p34cdc2 undergoes cell cycle-dependent accumulation at the centrosome during G2/M. In order to identify the mitotic cyclins with which this p34cdc2 fraction interacts, we carefully examined the subcellular distribution of both cyclin A and B1 in HeLa cells. We show here that part of cyclin B1 is associated with duplicating centrosomes throughout its accumulation in the cytoplasm and up to metaphase. In contrast cyclin A does not exhibit centrosomal association except at the onset of mitosis, from preprophase up to metaphase. We also present cytological and biochemical evidence that cyclin B1 is preferentially accumulated as a detergent-insoluble protein independently of the state of microtubule assembly and under experimental conditions where most of p34cdc2 is soluble. Interestingly, the electrophoretic pattern of the minor insoluble p34cdc2 fraction was previously shown to be particularly enriched in slow-migrating and presumably hyperphosphorylated isoforms, known to interact specifically with cyclin B1 during interphase. From these results we propose that the interaction of cyclin B1 with the centrosomes and with the cytoplasmic structures is a constitutive feature of the mechanism whereby a fraction of p34cdc2 is recruited and subsequently targeted to the cyclin B-dependent activation pathway.

Blotting, Western↗
PubMed1980

[Ultrastructure of paired neuron contacts during the posthypoxic period].

Peculiar contacts between bodies of double neurons were revealed in the sensomotor cortex of the rat brain under ischemia and histotoxic hypoxia by means of the electron microscopic investigation. Ultrastructure of the contacting double neurons is not homogenous. In some cases, they do not differ from the normal, in other cases, certain signs of activation are revealed. In the contact area, peripheral focal chromatolysis is sometimes observed. In the double neurons the number of microtubules is often increased. In some cases, the cytoplasmic structure is altered (demonstrating certain activation in one of the cells) and combined with destructive processes in the neighbouring cell. The double neurons revealed can be divided into 3 groups: 1) pairs of morphologically identical neurons without specialized areas in the bordering membranes, 2) double neurons with specialized areas of membranes at places of their contacts, 3) double neurons with fusion of their cytoplasm at places where the cellular bodies come into contact with each other. The types of the double neurons described reveal some additional ways along which metabolism in neurons can occur.

Animals↗
PubMed1983

Maturation of the intracellular parasite and antigenicity.

The protein antigens synthesized by the malarial parasite change as the parasite matures, with a number of proteins showing strict stage-specificity. A detailed correlation between the stage-specificity of protein synthesis and parasite structure has yet to be established, but a number of proteins synthesized in the cycle are lost selectively during merozoite escape and reinvasion. These antigens are presumably associated with structures utilized and ultimately lost during this process. Particular interest has focused on some greater than 200K proteins identified as being on the surface of infected erythrocytes and internally and on the surface of merozoites. Smaller parasite proteins have also been identified in the erythrocyte membrane. The erythrocyte itself, including its membrane, is much modified by parasite growth. Changes include the presence of new cytoplasmic structures and differences in the surface labelling and isoantigenic characteristics of the surface membrane. An appreciation of the variability and specificity of exposed parasite antigens, and their relationship to newly exposed isoantigens, is central to our understanding of protective immunity to malaria.

Animals↗
PubMed2005

Morphological anomalies in pollen tubes of Actinidia deliciosa (kiwi) exposed to 50 Hz magnetic field.

The role of the pollen grain, with respect to the reproductive process of higher plants, is to deliver the spermatic cells to the embryo sac for egg fertilisation. Delivery occurs through the pollen tube, a self produced organ that is generated when the pollen grain reaches the stigma surface. The effect of magnetic fields on pollen tube growth was reported in a recent publication by Germanà et al. Pollen tube growth is an interesting candidate for the detailed study of the effects of electromagnetic fields on cytoplasmic structures and organelles. In this research Actinidia deliciosa (kiwifruit) pollen grains were germinated in the presence of an alternating magnetic field (50 Hz). Our results, although of preliminary nature, show that pollen tube growth is affected by magnetic fields. The analysis of the observed anomalies in the pollen tube appear to be the result of changes in the ionic charges within the pollen tube cytoplasm.

Actinidia↗
PubMed2001

Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure.

Intracytoplasmic sperm injection (ICSI) is a delicate procedure requiring considerable skills of the person performing it. Theoretically, the injection procedure could damage cytoplasmic structures in the oocyte, resulting in sublethal cellular injury and/or numerical chromosomal abnormalities that could lead to impaired embryonic development. In the present study, features of the injection procedure were evaluated in a total of 2924 oocytes from 305 cycles. Development to the blastocyst stage was found to be compromised in a group of surplus embryos originating from oocytes in which >6 pl of cytoplasm was aspirated into the injection pipette during the ICSI procedure. Characteristics of the injection procedure as well as blastocyst development of surplus embryos was shown to be significantly different between the four technicians performing the ICSI. Neither the volume of cytoplasm aspirated during the injection procedure, nor the position of the polar body (6 o'clock or 12 o'clock) influenced the mean incidence of disomic cells per blastocyst as revealed by fluorescence in-situ hybridization using probes specific for chromosomes X, Y and 18. In conclusion, certain technical aspects of the injection procedure can affect subsequent embryonic development to the blastocyst stage, but do not seem to influence the rate of chromosomal abnormalities that occur in human pre-implantation embryos.

Blastocyst↗
PubMed1977

Outgrowth of human fibroblast aggregates on a substratum triggers a wide variety of morphogenetic properties in the cells.

Twenty-four-hour-old aggregates of human diploid skin fibroblasts are allowed to attach to a glass or plastic substratum. As a result of this attachment the cells in the aggregate demonstrate rapid and generalized changes in cell shape, cell surface and cytoplasm ultrastructure and in their ability to incorporate [3H]thymidine. Within 24 h they grow out on the substratum to attain the regular monolayer configuration. During the process of leaving the aggregate for the substratum a great number of different morphogenetic properties are displayed by the cells, resembling the properties of embryonic or epithelial cells. The simultaneous occurrence of this great variety of cell shape and cell surface changes, many of them unusual for fibroblasts, as well as the concurrent formation of organized cytoplasmic structures - microfilaments, microtubules - at localized areas of the cells, makes this system a potentially useful tool in the study of cell behaviour.

Cell Adhesion↗
PubMed1964

ELECTRON MICROSCOPY OF A FISSION YEAST, SCHIZOSACCHAROMYCES POMBE.

Maclean, Norman (University of Edinburgh, Edinburgh, Scotland). Electron microscopy of a fission yeast, Schizosaccharomyces pombe. J. Bacteriol. 88:1459-1466. 1964.-The structure of the fission yeast, Schizosaccharomyces pombe, was studied electron microscopically, with potassium permanganate and osmium tetroxide as fixatives. The cell was found to be bounded by a cell wall, 1,000 to 2,000 A thick, and a cell membrane. A layer of material was found between the cell membrane and the wall. The central nucleus, 2 to 3 mu in diameter, was bounded by a nuclear membrane, seen in some pictures to be double. Osmium tetroxide fixation revealed a granular body within the nucleus, identified as a nucleolus. Cytoplasmic structures included numerous vacuoles (probably normally containing lipid), a number of membranes and vesicles (which may represent a poorly organized mitochondrial system), and numerous granules (probably representing ribosomes).

Cell Membrane↗
PubMed2001

U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids.

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

Animals↗
PubMed1999

Neuronal localization of a novel mosaic apolipoprotein E receptor, LR11, in rat and human brain.

A new type of mosaic protein was recently discovered as a new member of the low density lipoprotein receptor (LDLR) family, designated as LR11. The predominant expression of LR11 transcripts in brain tissue and the presence of elements found in neural adhesion molecules suggested a function(s) in the central nervous system (CNS). In order to gain insight about this complex receptor in the CNS, we raised a rabbit polyclonal antibody and examined immunohistochemically rat and human brain tissue. A strong LR11 immunoreactivity was found to be localized mainly in neurons throughout the brain in both species. A detailed mapping in the rat brain showed a distribution of LR11 immunoreactivity in a widespread population of neurons, though the intensity varied between different locations. The most prominent immunoreactivity was observed in neurons of the hippocampus, some nuclei of brain stem and Purkinje cells, whereas neurons of the thalamus and the hypothalamus showed weak staining. Uniquely, the single LR11 immunoreactive cytoplasmic puncta were observed in the proximity of apical dendrites, most conspicuously in the pyramidal neurons of hippocampus. In the human brain, one to four immunoreactive puncta were seen within individual neurons. The neuronal localization of LR11 and its unique association of cytoplasmic structure, presumably botrysome, may suggest the roles of LR11 in both the lipoprotein metabolism and intracellular trafficking in certain neuronal population of the CNS.

Adult↗
PubMed2003

hRUL138, a novel human RNA-binding RING-H2 ubiquitin-protein ligase.

Cellular as well as viral RNAs are usually found complexed with proteins. In an attempt to identify proteins that interact with transcripts of hepatitis B virus (HBV), a DNA virus that replicates through reverse transcription, a partial cDNA was isolated from a human cDNA expression library whose gene product bound to an HBV-derived RNA. Using an overlapping clone from a molecular hybridization screen a full-length cDNA was assembled. It contained a large open reading frame for a 1208 amino-acid protein of 138 kDa identical to the hypothetical product of the KIAA0675 clone. Closely related sequences are present in mouse cDNA libraries but not in the genomes of lower organisms. The protein sequence contained no known RNA-binding domain and, apart from a probable coiled-coil domain, the only significant homology involved a complete RING-H2 motif. This suggested that the protein might be a novel RNA-binding RING-dependent ubiquitin-protein ligase or E3 enzyme. A motif critical for RNA binding was experimentally mapped to a central Lys-rich region. Binding specificity is either broad or the protein has as yet unknown physiological targets; hence, at present, a potential importance for HBV biology remains open. The RING-H2 domain was functional in and essential for self- and trans-ubiquitylation in vitro and for proteasome-mediated turnover of the protein in vivo. We therefore termed it hRUL138 for human RNA-binding ubiquitin ligase of 138 kDa. hRUL138 mRNAs are expressed at low levels in most tissues. GFP-tagged hRUL138 derivatives were found associated with cytoplasmic structures, possibly the ER, but excluded from the nucleus. The combined presence of RNA binding and E3 activity in hRUL138 raises the possibility that both are mechanistically linked.

Amino Acid Motifs↗
PubMedDate not supplied

Curvilinear membranous formations in lymph node cells of an African AIDS patient with tuberculosis.

Tissue from an extirpated cervical lymph node of an African AIDS patient with tuberculosis was investigated with the electron microscope. A peculiar cytoplasmic structure was found in histiocytic cells of the lymph node tissue. This structure consisted of a loose aggregation of convoluted, folded curvilinear membranes, demonstrating C-shaped, U-shaped, and S-shaped profiles according to the plane of section. These curvilinear membranous formations (CMF) were formed by 2 parallel-running unit membranes separated by a narrow electron-lucent space. Continuities of CMF with rough-surfaced endoplasmic reticulum were sometimes observed. Occasionally, an electron-dense, blurred cytoplasmic area was seen between 2 neighboring CMF. Several CMF also formed a seastar-like configuration by mergence in such an area. It is unknown whether the HIV infection of the patient was the primary cause of the CMF.

AIDS-Related Opportunistic Infections↗