Tetrin polypeptides are colocalized in the cortex of Tetrahymena.

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Tetrin polypeptides are colocalized in the cortex of Tetrahymena

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Persistence of Oral Structures in the Sexual Process of Amicronucleate Paramecium tetraurelia

ABSTRACT In the sexual process, amicronucleate Paramecium tetraurelia , unlike micronucleates, fail to produce an oral apparatus, but resorb the pre-existing one. Exceptions were found in some amicronucleate cell lines in which about 1% of the cells possessed oral structures, including pieces of oral membranelles, sometimes complete with buccal cavity, after autogamy or conjugation. By following oral development in the sexual process in some detail, the present study supports the view that these oral structures are derived from the pre-existing oral apparatus and not newly developed from the oral primordium. The possible involvement of the micronucleus and the pre-existing oral apparatus in oral resorption is discussed. The possession of a functional oral apparatus after the sexual process may open up a new evolutionary avenue to the amicronucleates.     

Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439-4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM-1 and MPM-2, decorate throughout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno-electron microscopy showed that these antibodies labeled the electron-dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM-1 and MPM-2) and the epiplasm (MPM-1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.     

Biosynthesis of pigment-protein complex polypeptides in bacteriochlorophyll-less mutant cells of Rhodopseudomonas capsulata YS

Bacteriochlorophyll-less mutant cells of Rhodopseudomonas capsulata YS were capable of synthesizing pigment—protein complex polypeptides under conditions permitting the formation of the photosynthetic apparatus in the wild type. Individual polypeptides were identified by immunoprecipitation. Pulse-chase experiments revealed that the polypeptides did not accumulate in the membranes but underwent rapid degradation. The data suggest that bacteriochlorophyll is needed to stabilize the polypeptides of pigment—protein complexes.     

四膜虫中存在角蛋白样成分(简报)

中等纤维是脊椎动物细胞中普遍存在的一种细胞骨架成分,在一些无脊椎动物细胞中也发现有这类成分存在。据报道,枪乌贼的巨     

Intracellular transport, sorting, and turnover of acetylcholinesterase. Evidence for an endoglycosidase H-sensitive form in Golgi apparatus, sarcoplasmic reticulum, and clathrin-coated vesicles and its rapid degradation by a non-lysosomal mechanism

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.     

Association of degradation and secretion of three chimeric polypeptides in Escherichia coli.

We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.     

The role of cortical pattern in timing of cell division and morphogenesis in Stentor.

This paper describes experiments involving reciprocal exchange of the oral apparatus between a large (L) and a small (S) strain of Stentor coeruleus. Mature L-strain stentors given and S-strain oral apparatus formed an oral primordium in response to the presence of abnormally small oral structures and mostly divided although a few reorganized instead. Conversely, when S-strain stentors in early division were given a large L-strain oral apparatus, they resorbed the developing primordium and returned to interphase. When combined with previous results, these findings provide evidence for the hypothesis that the time of cell division in Stentor is determined by a "critical" ratio between the size of the oral apparatus and the size of the cell body (somatic) cortex. This ratio develops because the somatic cortex grows during interphase and the oral apparatus does not. Cell division in Stentor therefore appears to be initiated by a cortical pattern change resulting from cell surface growth during interphase.     

Involvement of Tetrahymena intermediate filament protein, a 49K protein, in the oral morphogenesis

To study the biological function of Tetrahymena intermediate-type filament protein (a 49K protein), we examined the immunofluorescence localization of 49K protein within Tetrahymena cells. The results showed that the immunofluorescence was localized in the oral apparatus, mitochondria and mucocysts. Among them, the fluorescence in the oral apparatus was of high interest in its unique region and vicissitude in the cell cycle: a tau-shaped region of the oral apparatus intensely fluoresced during interphase, but the fluorescence completely disappeared during dividing phase. The tau-shaped region corresponded to 'posterior connectives' and the root part of 'deep fiber', to the conjunction parts of microtubule bundles. In the those parts, there was electron-dense material in the microtubule bundles. Hence, it is conceivable that 49K protein corresponds to the dense material and has a function of microtubule bundle conjunction. On the other hand, disappearance of immunofluorescence from the old oral apparatus of most dividing cells reflected the oral apparatus regression and remodelling which have been known as necessary sequential events in the cell cycle. We observed that oral fluorescence disappeared concurrently with the onset of oral regression and of constriction of division furrow, whereas at a late dividing stage immunofluorescence began to appear simultaneously in both new and old oral apparatus. Thus, the 49K protein may play a crucial role(s) not only in the morphogenesis of oral primordia but also in the transient morphogenesis in the old oral system.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Structural determination of the photosystem II core complex from spinach

A photosystem II core complex was purified with high yield from spinach by solubilization with beta-dodecylmaltoside. The complex consisted of polypeptides with molecular mass 47, 43, 34, 31, 9 and 4 kDa and some minor components, as detected by silver-staining of polyacrylamide gels. There was no indication for the chlorophyll-a/b-binding, light-harvesting complex polypeptides. The core complex revealed electron-transfer activity (1,5-diphenylcarbazide----2,6-dichloroindophenol) of about 30 mumol reduced 2,6-dichloroindophenol/mg chlorophyll/h. The structural integrity was analyzed by electron microscopy. The detergent-solubilized protein complex has the shape of a triangular disk with a maximum diameter of 13 nm and a maximum height of 6.8 nm. The shape of this core complex differs considerably from that of cyanobacterial photosystem II membrane fragments, which are elongated particles. The structural differences between both the complexes of higher plants and cyanobacteria are discussed with special emphasis on their association with the antenna apparatus in the photosynthetic membranes.     

Structure, function and immunogenicity of streptococcal antigen I/II polypeptides

The antigen I/II family of cell-surface-anchored polypeptides in oral streptococci are structurally complex multi-functional adhesins, with multiple ligand-binding sites. Discrete regions within these polypeptides bind human salivary glycoproteins, other microbial cells, and calcium. Sequences within the N-terminal region bind preferentially fluid-phase glycoproteins, while the C-terminal half of the polypeptide contains species-specific adhesion-mediating sequences that bind surface-immobilized glycoproteins. These features may assist streptococcal adhesion to oral surface receptors despite the presence of excess fluid-phase receptors. Immunological studies reveal an array of T-cell and B-cell epitopes presented by antigen I/II polypeptides and suggest the occurrence of natural suppression of human antibodies to the adhesion-mediating sequences. The functional and immunological properties of antigen I/II proteins may account to a major extent for the success of oral streptococci colonizing and surviving within the human host.     

A novel rapid oral bacteria detection apparatus for effective oral care to prevent pneumonia

doi: 10.1111/j.1741‐2358.2011.00517.x A novel rapid oral bacteria detection apparatus for effective oral care to prevent pneumonia Objective: To clarify the oral environment, we evaluated the usefulness and clinical applicability of a new apparatus developed for the simple and rapid quantification of oral bacteria. Background: Professional oral health care can reduce the number of oral bacteria and days of fever and inhibit the development of pneumonia. A novel detection apparatus was developed by applying the dielectrophoretic impedance measurement method. Methods: First, to determine the accuracy of this apparatus, employing standard samples of Escherichia coli. Next, to evaluate the oral environment, samples were taken from the tongue in elderly (mean age: 86.6 years) in nursing home. Results: In the first study, a good correlation was observed between the two methods (R = 0.999). In the second study, there were significant correlations between measurement values obtained using this apparatus and those obtained by the culture method (R = 0.852), as well as those obtained by the FM method (R = 0.885). Conclusion: Our data showed that this rapid oral bacterial detection apparatus is effective in evaluating the oral hygiene to prevent pneumonia in the elderly.     

Highly selective binding of nascent polypeptides by an Escherichia coli chaperone protein in vivo.

Chaperone proteins bind to newly synthesized polypeptides and assist in various assembly reactions. The Escherichia coli chaperone protein SecB binds precursors of exported proteins and assists in export. In vitro, SecB can bind to many unfolded proteins. In this report, we demonstrate that SecB binding in vivo is highly selective; the major polypeptides that are bound by SecB are nascent precursors of the exported proteins maltose-binding protein (MBP), LamB, OmpF, and OmpA. These results support the hypothesis that the primary physiological function of SecB is to stimulate protein export. By interacting with nascent polypeptides, SecB probably stimulates their cotranslational association with the membrane-bound protein translocation apparatus.     

Preparatory changes and the development of the conjugation junction in a ciliate,Dileptus

Summary The present observations concern changes in the cortical structure of the ciliateDileptus prior to and during conjugation. The results can be summarized as follows: (1) Mating cells join each other in heteropolar configuration, i.e., the distal part of the oral apparatus of one partner faces the proximal part of the oral apparatus of another partner. (2) The structural changes prior to conjugation occur in the area of the oral apparatus. (3) The bonding area is situated within the oral apparatus, while the oral ciliature that encircles the oral apparatus remains outside the bonding area. (4) The fusion area is formed within the bonding area as a newly formed zone, without cortical organelles typically associated with the cell membrane. Two aspects of the formation of cell-to-cell union inDileptus are discussed: firstly, the heteropolar configuration of mating cells and its possible bearing upon an ability to form double cells when partners separation is inhibited. Secondly, the bonding area ofDileptus is compared to bonding areas of peniculine and hymenostome ciliates. A striking likeness in structural changes was found within the bonding areas of all three ciliates, in spite of the fact that the changes in question are localized within the oral apparatus ofDileptus, while inParamecium andTetrahymena the changed territory is situated between the oral apparatus and the anterior pole of the cell.     

Glycosylated polypeptides of soybean root endomembranes

Summary Endoplasmic reticulum, Golgi apparatus, plasma membrane and mitochondria vesicles were isolated from the roots of four-day-old dark-grown soybean [Glycine max (L.) Merr. cv. Wells II] seedlings and characterized by marker enzyme analyses. Glycoproteins of enriched membrane fractions were identified by concanavalin A (con A)-peroxidase staining of polypeptides separated by two-dimensional IEF-SDS-PAGE and transferred to nitrocellulose.Con A bound to many polypeptides in each endomembrane-enriched fraction with several glycopolypeptides common to all fractions. The mitochondria-enriched fraction possessed few glycopolypeptides and those appeared to be highly glycosylated contaminants of endomembrane origin. Comparison of the endomembrane con A-binding patterns revealed changes in relative stain intensity, molecular weight and isoelectric point of several membrane glycopolypeptides suggestive of processing reactions of the endomembrane complex.Abbreviations con A concanavalin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum     

Cell surface protein receptors in oral streptococci

Abstract Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells. These interactions are determined by the expression of cell-surface receptors some of which are species-specific. In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified. The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158–166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules. The LraI family of polypeptides are surface-associated lipoproteins (32–33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces . A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii . Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.