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M Melkonian

Publications and source records attributed to M Melkonian.

11 recordsLinked to original sources
PubMed1992

Structure of striated microtubule-associated fibers of flagellar roots. Comparison of native and reconstituted states.

Several fiber systems are associated with the flagella and basal bodies of eukaryotic cells. Apart from the contractile and Ca(2+)-sensitive system-II fibers, these include the noncontractile system-I fibers that run parallel to flagellar root microtubules. Using electron microscopy and image reconstruction, we have investigated the structure of the system-I fibers of the flagellate green alga Spermatozopsis similis. The fibers were observed in three different states: (1) in situ, (2) after isolation of the intact fibers, (3) after disassembly and reconstitution of fibers in vitro from their 34 kDa subunit protein. The fibers are highly ordered; they show a constant repeat of 28 nm, they are polar, and they contain several transverse and longitudinal striations. A model is discussed showing the system-I fiber to be built from rod-like molecules with a staggered arrangement and identical polarities.

Chlorophyta↗
PubMed1992

N-linked glycoproteins associated with flagellar scales in a flagellate green alga: characterization of interactions.

Glycoproteins associated with one type of flagellar scale (p-scale) isolated from the flagellate green alga Tetraselmis striata (Prasinophyceae) were shown to bind the mannose-specific lectin GNA (Galanthus nivalis agglutinin). Enzymatic deglycosylation of the glycoproteins with N-glycosidase F led to an electrophoretic mobility shift to lower molecular masses in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and abolished GNA-binding strongly indicating that most of the scale-associated glycoproteins contain asparagine-linked oligosaccharide side chains presumably of the high mannose type. To evaluate the significance of N-linked glycoproteins for scale structure and integrity, p-scales were digested with various proteases or extracted with 8 M urea and their ultrastructure and protein composition determined. The results show that while scale-associated N-linked glycoproteins do not determine the overall structure of the scale subunits (which consist of complex polysaccharides), they are apparently involved in mediating linkages between scale subunits; we have tentatively identified one glycoprotein of Mr 280,000 which may link outer scale subunits to one another. In addition, some scale-associated N-linked glycoproteins may provide connections between the layer of p-scales and the underlying flagellar membrane.

Chlorophyta↗
PubMed1991

Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga.

Isolation of intact photoreceptive organelles (eyespot apparatuses) involved in blue-light mediated photoresponses in a flagellate green alga (Spermatozopsis similis) allowed for the first time the identification of both 11-cis- and all-trans-retinal in a plant cell. Both isomers were identified by HPLC analysis in conjunction with UV spectra. Additionally, reconstitution of a distinct absorption band, centered around 540 nm, was achieved by addition of exogenous 9-cis-retinal to bleached, isolated eyespot apparatuses.

Carrier Proteins↗
PubMed1991

Scale formation in algae.

Scale biogenesis in algae represents a unique model system to study the transport of secretory macromolecules through the Golgi apparatus (GA) and their exocytosis. The larger scales can be visualized in the light microscope, and thus the kinetics of scale assembly, transport, and secretion can be studied in vivo. In addition, scales are osmiophilic and readily visible in conventional transmission electron microscopy; thus, details of scale assembly and sorting can be studied without invoking immunolabeling techniques. The following are distinctive features of scale biogenesis in algae: 1) transport of scales through the GA-stack occurs by cisternal progression; 2) scale secretion may be very rapid (in some cases a single GA-cisterna leaves the stack every 15-20 s); 3) sorting of different scale types does not occur in the GA, but in a post-GA-compartment. Recent progress in the analysis of scale formation in the green flagellates Tetraselmis and Scherffelia is reviewed.

Animals↗
PubMed1991

Striated microtubule-associated fibers: identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro.

Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross-striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4-labeling and autoradiography. Amino acid analysis of assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.

Amino Acids↗
PubMed1991

Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga.

We report on the isolation and purification of structurally intact eyespot apparatuses from the naked, biflagellate green alga Spermatozopsis similis. Two eyespot-enriched fractions, separated by sucrose gradient centrifugation, retained the typical reflective properties of eyespots in situ as demonstrated by reflection confocal laser scanning microscopy. Ultrastructurally, both fractions contained eyespot plates consisting of a single layer of lipid globules. Structurally intact eyespot apparatuses, including patches of plasma membrane and chloroplast envelope overlying the eyespot plate and a single thylakoid subtending the eyespot plate, were particularly enriched in one of the two fractions (fraction 2a). Measurement of several marker enzymes and chlorophyll content (less than 0.001% of total) established the absence of most other cell organelles from the eyespot fractions. The absorption spectra of the two fractions were dominated by carotenoids with an additional shoulder at 540 nm. Following extraction with organic solvents and sodium dodecyl sulfate polyacrylamide gel electrophoresis, several proteins were found to be considerably enriched in the two fractions. In addition to several proteins in the high Mr range, at least 4 polypeptides of 35, 29, 23, and 20 kDa are selectively enriched in fraction 2a with the 29 and 20 kDa proteins being the most prominent. The presence of glycoproteins in fraction 2a was demonstrated by binding of the mannose-specific lectin Galanthus nivalis agglutinin to several high molecular weight polypeptides. In addition, a hydrophobic component with abnormal electrophoretic mobility that reacts strongly with periodic acid-Schiff and thymol/sulfuric acid was prominent in both fractions. Mass isolation and purification of the intact phototactic apparatus of a flagellate green alga now greatly facilitates the biochemical and molecular characterization of the signal transduction chain involved in green algal phototaxis.

Blotting, Western↗
PubMed1990

Reflection confocal laser scanning microscopy of eyespots in flagellated green algae.

The reflection properties of different types of eyespots in three unicellular, flagellated green algae (Tetraselmis chui, Chlamydomonas eugametos, Hafniomonas reticulata) were investigated using confocal laser scanning microscopy in the epireflection mode. The eyespots differed with respect to the number of eyespot lipid globule layers and surface appearance (concave/convex). A strong reflection signal was observed in all eyespots, and a detailed quantitative analysis by optical xy (horizontal) and xz (vertical) sectioning was performed. By applying both sectioning capabilities, multi- and single/double-layered eyespots as well as concave and convex eyespot surfaces could be distinguished using living, immobilized cells. Focusing of the reflected light was only observed in eyespots with concave surfaces. In xz series of multi-layered eyespots at reduced laser intensities (0.01%), the intensity profiles of the reflection revealed a series of alternating maxima and minima with increasing reflection intensities toward the cell surface. At very low laser intensities (0.001%), multi-layered eyespots exhibited about twice the reflection intensity at the presumptive photoreceptor site compared to single/double-layered eyespots. Our results provide the first experimental evidence to support the proposal that multi-layered eyespots act as interference reflectors in photoaxis of green algae.

Chlorophyta↗
PubMed1989

Identification of 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methyl-manno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid in the cell wall (theca) of the green alga Tetraselmis striata Butcher (Prasinophyceae).

The main constituent of the cell wall complex carbohydrate of the scaly green alga Tetraselmis striata Butcher is shown to be 3-deoxy-manno-2-octulosonic acid (42%). In addition two other 2-keto-sugar acids are present, namely, 3-deoxy-5-O-methyl-manno-2-octulosonic acid (7%), the first methylated derivative of 3-deoxy-manno-2-octulosonic acid found in nature, and 3-deoxy-lyxo-2-heptulosaric acid (11%). The characterization of the three 2-keto-sugar acids has been carried out on the corresponding methyl ester methyl glycosides using GLC-MS and 500-MHz 1H-NMR spectroscopy, and on the corresponding reduced alditol acetates using GLC-MS. Other monosaccharides occurring in the cell wall are D-galacturonic acid (14%), D-galactose (4%), D-gulose (2%), D-glucose (1%) and L-arabinose (1%).

Cell Wall↗
PubMed1987

Basal body reorientation mediated by a Ca2+-modulated contractile protein.

A rapid, Ca2+-dependent change in the angle between basal bodies (up to 180 degrees) is associated with light-induced reversal of swimming direction (the "photophobic" response) in a number of flagellated green algae. In isolated, detergent-extracted, reactivated flagellar apparatus complexes of Spermatozopsis similis, axonemal beat form conversion to the symmetrical/undulating flagellar pattern and basal body reorientation (from the antiparallel to the parallel configuration) are simultaneously induced at greater than or equal to 10(-7) M Ca2+. Basal body reorientation, however, is independent of flagellar beating since it is induced at greater than or equal to 10(-7) M Ca2+ when flagellar beating is inhibited (i.e., in the presence of 1 microM orthovanadate in reactivation solutions; in the absence of ATP or dithiothreitol in isolation and reactivation solutions), or when axonemes are mechanically removed from flagellar apparatuses. Although frequent axonemal beat form reversals were induced by varying the Ca2+ concentration, antiparallel basal body configuration could not be restored in isolated flagellar apparatuses. Observations of the photophobic response in vivo indicate that even though the flagella resume the asymmetric, breaststroke beat form 1-2 s after photostimulation, antiparallel basal body configuration is not restored until a few minutes later. Using an antibody generated against the 20-kD Ca2+-modulated contractile protein of striated flagellar roots of Tetraselmis striata (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, 1984, J. Cell Biol., 99:962-970), we have found the distal connecting fiber of Spermatozopsis similis to be immunoreactive by indirect immunofluorescence and immunogold electron microscopy. Electrophoretic and immunoblot analysis indicates that the antigen of S. similis flagellar apparatuses consists, like the Tetraselmis protein, of two acidic isoforms of 20 kD. We conclude that the distal basal body connecting fiber is a contractile organelle and reorients basal bodies during the photophobic response in certain flagellated green algae.

Calcium↗
PubMedDate not supplied

[Lipid peroxidation processes and the dynamics of shifts in the cholesterol level in plasma and erythrocytes under the influence of low-frequency acoustic oscillations].

Distinct alterations in lipid peroxidation, in content of alpha-tocopherol and cholesterol fractions were observed in rat blood plasma and erythrocyte membranes after long-term low-frequency acoustic oscillations (105 decibels, diurnally, within 3 months). Under these conditions activities of superoxide dismutase and erythrocyte Na+, K+-ATPase were also altered. The rate and direction of the enzymatic activity alterations correlated with the duration of the acoustic oscillation exposure and the patterns studied. Enrichment of blood plasma and erythrocyte membranes with alpha-tocopherol within 2-4 weeks of the acoustic oscillation is considered as an important phenomenon.

Animals↗