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Ultrastructure of the fusion cell in Faucheocolax attenuata Setch. (Rhodophyta, Rhodymeniales).

The fusion cell in Faucheocolax attenuata Setch. is a highly lobed, thick-walled, multinucleate and irregularly shaped cell originating from the basal cell of the auxiliary cell branch. The formation of the fusion cell occurs by an incorporation of vegetative cells into the basal cell, after dissolution of septal plugs between these cell types. Thus the fusion cell is a syncytium containing only haploid nuclei, as well as unusual mitochondria and plastids. Mitochondria lack cristae and instead contain a tubular helical structure. Plastids are atypical with regard to thylakoid organization in red algae, because they lack the peripheral thylakoid and their photosynthetic thylakoids are aggregated to one side. In addition, they contain large osmiophilic bodies. Nuclear envelopes appear to produce large quantities of membrane cisternae. Floridean starch is absent and the cytoplasm contains few ribosomes. The plasma membrane is irregular and endoplasmic reticulum cisternae are situated parallel to it. Bundles of putative microfilaments were commonly found in nuclei and the cytoplasm. Structural evidence does not support any meristematic, nutritive or secretory functions previously ascribed to fusion cells in other genera.

Cell Fusion↗

Ultrastructural aspects of Peptichemio action on mouse sarcoma 180.

The electron microscope study of the cellular elements originating from mice experimental tumours (sarcoma 180) treated and non-treated with Peptichemio showed alterations of the nuclear and cytoplasmic structures in the animals treated. The data herein accord fully with the results obtained by other histological, biochemical and biomolecular techniques and make it possible once again to lay stress on the double alkylating and antimetabolic action of this antitumour drug, very likely taking place simultaneously both at nuclear and cytoplasmic level.

Animals↗

An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

Amino Acid Sequence↗

Theoretical estimates of mechanical properties of the endothelial cell cytoskeleton.

Current modeling of endothelial cell mechanics does not account for the network of F-actin that permeates the cytoplasm. This network, the distributed cytoplasmic structural actin (DCSA), extends from apical to basal membranes, with frequent attachments. Stress fibers are intercalated within the network, with similar frequent attachments. The microscopic structure of the DCSA resembles a foam, so that the mechanical properties can be estimated with analogy to these well-studied systems. The moduli of shear and elastic deformations are estimated to be on the order of 10(5) dynes/cm2. This prediction agrees with experimental measurements of the properties of cytoplasm and endothelial cells reported elsewhere. Stress fibers can potentially increase the modulus by a factor of 2-10, depending on whether they act in series or parallel to the network in transmitting surface forces. The deformations produced by physiological flow fields are of insufficient magnitude to disrupt cell-to-cell or DCSA cross-linkages. The questions raised by this paradox, and the ramifications of implicating the previously unreported DCSA as the primary force transmission element are discussed.

Actins↗

A novel 58-kDa protein associates with the Golgi apparatus and microtubules.

With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo function of 58K is to provide an anchorage site for microtubules on the outer surface of the Golgi.

Animals↗

Electro-optical imaging of F-actin and endoplasmic reticulum in living and fixed plant cells.

Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells. Fixation of plant tissues to preserve cytoplasmic structure poses special problems. When possible, emphasis should be placed on the imaging of structures in stained living cells over time. The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging. The first observable events after Nod factor stimulation occur in root hairs and are changes in rates of cytoplasmic streaming, nuclear movements, and changes in the shape of the vacuole. Within ten minutes, the endoplasmic reticulum shifts position towards the tip of the root hair. For comparison, the endoplasmic reticulum localization in pollen tubes and onion epithelial cells will be illustrated. The actin cytoskeleton undergoes a series of changes over a twelve hour period. These changes in the cytoskeleton are spatially and temporally correlated with the observed growth changes of the root hairs. This dynamic change of the actin filament and endoplasmic reticulum and associated secretory vesicles in these root hairs suggests a mechanism for the observed root hair growth changes.

Actins↗

Solution structure of the cytoplasmic region of Na+/H+ exchanger 1 complexed with essential cofactor calcineurin B homologous protein 1.

Na+/H+ exchanger 1 (NHE1) regulates intracellular pH, Na+ content, and cell volume. Calcineurin B homologous protein 1 (CHP1) serves as an essential cofactor that facilitates NHE1 exchange activity under physiological conditions by direct binding to the cytoplasmic juxtamembrane region of NHE1. Here we describe the solution structure of the cytoplasmic juxtamembrane region of NHE1 complexed with CHP1. The region of NHE1 forms an amphipathic helix, which is induced by CHP1 binding, and CHP1 possesses a large hydrophobic cleft formed by EF-hand helices. The apolar side of the NHE1 helix participates in extensive hydrophobic interactions with the cleft of CHP1. We suggest that helix formation of the cytoplasmic region of NHE1 by CHP1 is a prerequisite for generating the active form of NHE1. The molecular recognition detailed in this study also provides novel insight into the target binding mechanism of EF-hand proteins.

Amino Acid Sequence↗

[Ultrastructural changes in the mammary glands of white rats during the estrus cycle].

Secretory granules of milk protein were found in the apical cytoplasm of some ductal cells in the mammary gland of non-lactating albino rats. During the estrous period, the diestrous phase, the number of "coated" vesicles increased essentially. Some of the "coated" vesicles contained secretory granules and granular debris. The functional relationship of the "coated" vesicles to lysosomes and to some other cytoplasmic structures is discussed. The results suggest that during the estrous phase formation and release of secretory products prevail, whereas at the diestrous the process of their removal and destruction is predominant.

Animals↗

Histochemistry of glutamate metabolizing enzymes in the rat cerebellar cortex.

Applying catalytic enzyme histochemistry, glutamate dehydrogenase (GDH) and phosphate activated glutaminase (PAG) were demonstrated at the light microscopic level, and aspartate aminotransferase (AAT) was detected at the electron microscopic level. GDH staining appeared preferentially in glial cells (Bergmann glia and astrocytes), whereas AAT was localized only in neuronal structures. Cytoplasmic AAT was demonstrated in the perikarya and terminal plexus of basket cells, in the perikarya of stellate cells, in about 60% of the granule cells, in mossy fiber boutons, in numerous small boutons in the molecular layer, and in the axoplasm of numerous myelinated and unmyelinated fibers. PAG was observed in both neuronal structures (perikarya of granule and Purkinje cells) and in astrocytes and Bergmann glia.

Animals↗

The importance of the five phosphoribosyl-pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.

Phosphoribosyl-pyrophosphate synthetase (Prs) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.

Alkaline Phosphatase↗

A light & electron microscopic study on apoptotic bodies in acute aflatoxin B1 induced injury in the rat liver.

A subnecrogenic dose of aflatoxin B1 (AFB1) was injected intraperitoneally into 8-10 wk old male rats of the Holzman strain. Cytoplasmic and sinusoidal eosinophilic bodies were seen in the liver which appeared at 30 h and reached a maximum at 48 h. Electron microscopically some of the cytoplasmic structures were seen to be of mitochondrial origin whereas others resembled apoptotic bodies. The sinusoidal bodies were similar to apoptotic bodies.

Aflatoxin B1↗

Channel cytoplasmic loops alter voltage-dependent sodium channel activation in an isoform-specific manner.

1. The isoform-specific functional role of cytoplasmic structures of two voltage-gated sodium channel isoforms, the human cardiac channel (hH1) and the adult human skeletal muscle channel (hSkM1) was investigated through functional comparison of chimeras. 2. The voltage of half-activation (V(a)) for hH1 was shifted by > 20 mV in the hyperpolarised direction following internal papain treatment ('papain sensitive'), while V(a) for hSkM1 was unaffected ('papain insensitive'). 3. The hH1 region(s) responsible for this papain sensitivity was localised by testing a series of hH1/hSkM1 chimeras in which combinations of the large hH1 cytoplasmic loops joining the four transmembrane domains replaced analogous hSkM1 loops. Various chimeras were used to determine the smallest subset of loops that converted fully the papain-insensitive hSkM1 into a papain-sensitive channel. Then three converse chimeras were tested in which hSkM1 loops replaced hH1 loops to determine the smallest subset of loops necessary and sufficient to convert the papain-sensitive hH1 into a papain-insensitive channel. 4. Functional studies of this inclusive set of chimeras indicate that the first two cytoplasmic loops of the cardiac sodium channel that join domain I to II (loop A), and domain II to III (loop B), are both necessary, and together are sufficient to produce a papain-induced hyperpolarising shift in the voltage at which channels activate. When both loops are present (wild-type hH1 and the chimera hSkM1AB), V(a) for the channel shifts in the hyperpolarised direction by > 20 mV with papain treatment. When the analogous hSkM1 loops are present (wild-type hSkM1 and the chimera hH1AB), V(a) for the channel is not sensitive to treatment with papain. For channels that contain only one of the two hH1 loops, the effect of papain on V(a) is intermediary. 5. Experiments performed in the absence of papain showed that the activation voltages of the double loop chimeras, hSkM1AB and hH1AB, were shifted significantly from V(a) for hSkM1 and V(a) for hH1, respectively, indicating that these loops directly alter channel activation voltage. The resulting shifts in V(a) were in opposing directions, suggesting that cytoplasmic control of activation voltage is isoform specific. V(a) for hSkM1AB was about 20 mV more depolarised than V(a) for hSkM1, and V(a) for hH1AB was about 9 mV more negative than V(a) for hH1. 6. These data are the first to indicate isoform-specific cytoplasmic regions of the voltage-gated sodium channel that directly and differently alter the voltage of channel activation.

Animals↗

The molecular basis for membrane - cytoskeleton association in human erythrocytes.

Spectrin, the major cytoskeletal protein in erythrocytes, is localized on the inner membrane surface in association with membrane-spanning glycoproteins and with intramembrane particles. The presence of a specific, high-affinity protein binding site for spectrin on the cytoplasmic surface of the membrane has been established by measurement of reassociation of spectrin with spectrin-depleted inside-out vesicles. A 72,000 Mr proteolytic fragment of this attachment protein has been purified, which bound to spectrin in solution and competed for reassociation of spectrin with vesicles. A 215,000 Mr polypeptide has been identified as the precursor of the spectrin-binding fragment. The membrane attachment protein for spectrin was named ankyrin, and has been purified and characterized. Ankyrin has been demonstrated to be tightly associated in detergent extracts of vesicles with band 3, a major membrane-spanning polypeptide, and to bind directly to a proteolytic fragment derived from the cytoplasmic domain of band 3. Ankyrin is thus an example of a protein that directly links a cytoplasmic structural protein to an integral membrane protein. The organization of the erythrocyte membrane has implications for more complex cell types since immunoreactive forms of ankyrin distinct from myosin or filamin have been detected by radioimmunoassay in a variety of cells and tissues. Indirect immunofluorescent staining of cultured cells reveals immunoreactive forms of ankyrin in a cytoplasmic meshwork and in a punctate distribution over nuclei. The staining changes dramatically during mitosis, with concentration of stain at the spindle poles in metaphase and intense staining of the cleavage furrow during cytokinesis.

Anion Exchange Protein 1, Erythrocyte↗

Possible involvement of some secondary metabolites in salt tolerance of sugarcane.

Accumulation of toxic ions in plant tissues modulates the levels of primary and secondary metabolites, which may be related to salinity tolerance. In this study two sugarcane clones, CP-4333 (tolerant) and HSF-240 (sensitive), were exposed to salinity levels at the formative stage, and evaluated three times at 10-day intervals. Although net rate of photosynthesis (Pn), leaf area, length and dry weight of shoots were decreased in both clones, the CP-4333 showed less reduction compared to HSF-240. Both clones displayed a general tendency to accumulate Na+ and Cl- and little K+, though CP-4333 accumulated less Na+ and more K+ compared to HSF-240, and thus showed a higher K+:Na+ ratio. The carotenoid (CAR) content remained steady, while total chlorophyll (CHL) was slightly reduced in the tolerant clone and significantly reduced in HSF-240. In contrast, soluble phenolics (PHE), anthocyanins (ANT) and flavones (FLA) levels were 2.5, 2.8 and 3.0 times greater in CP-4333 in comparison with HSF-240. The decrease in Pn and most secondary metabolites demonstrated by the sensitive clone, but not evidenced in the tolerant clones, suggest that the presence of those metabolites is related to increased salt tolerance of sugarcane. The increased synthesis of PHE, ANT and FLA seems to protect sugarcane from ion-induced oxidative stress, probably due to a common structural skeleton, the phenyl group, of those metabolites. CAR, as components of the light harvesting center (LHC) and biosynthesized in chloroplasts, may confer resistance to this organelle. The PHE, ANT and FLA synthesized in the cytosol may protect cells from ion-induced oxidative damage by binding the ions and thereby showing reduced toxicity on cytoplasmic structures.

Ion Transport↗

[The effect amniocen preparation on nucleic acid metabolism in rat liver cells].

Rates of DNA and RNA synthesis as well as transport of RNA into cytoplasmic structures of rat liver cells were studied after administration of amniocen preparation. The drug did not affect the rate of DNA and RNA synthesis in intact rat liver cells, while specific radioactivity of DNA was decreased in regenerating liver tissue 24 hrs after hepatectomy. Amniocen inhibited the rate of rRNA, mRNA and tRNA transport into cytoplasm but contributed to elevated penetration of nucleic acid precursors into rat liver cells. Effect of the drug on cell membrane system appears to be responsible for its biological action related to realization of genetic information.

Amnion↗

Hexachrome modification of Movat's stain.

A less than three-hour hexachrome modification of Movat's pentachrome stain is described, its various steps discussed, and its principal uses in histopathology presented. The hexachrome procedure consistently yields good results, with excellent and colorful differentiation of muscle, various connective tissue components, mucinous secretions and intra-cytoplasmic structures in human and animal tissues obtained surgically or at autopsy. Alternative abbreviated procedures for muscle-connective tissue differential staining and for study of nuclear detail are also described.

Animals↗

Exploring the use of soft X-ray microscopy for imaging subcellular structures of the inner ear.

The soft X-ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X-ray microscopy provides high-resolution visualization of hydrated, non-embedded and non-sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X-ray micrographs of structures isolated from the guinea-pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X-ray images of the acellular tectorial membrane and thin two-layered Reissner's membrane display a level of resolution comparable to low-power electron microscopy.

Animals↗

VIP36 localisation to the early secretory pathway.

VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localised to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al., 1994). Here we provide evidence that endogenous VIP36 is localised to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalise with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalisation with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.

1-Deoxynojirimycin↗