A study of the occurrence of regulator secondary structures in globular proteins

The distribution of regular secondary structures, viz. α-helices and β-strands, along the length of over 70 properties whose secondary structural details have been reported, has been analysed. The occurrence of these regular structures tends to be a maximum at the N- and C-termini. Our analysis suggests that both these free ends could possibly serve as nucleating centers for secondary structures and could play an important role in the folding of proteins.     

Cellulose-microfibril-orienting mechanisms in plant cells walls

A brief review is given of the changing views over the years, as knowledge of wall structure has developed, concerning the mechanism whereby cellulose chains may be oriented. This leads to an examination of current concepts, particularly those concerning microtubules. It is shown that none of the mechanisms suggested whereby microtubules might cause orientation of cellulose microfibrils is consistent with the known range of molecular architectures found in plant cell walls. It is further concluded that any mechanism which necessitates an indissoluble link between the plasmalemma and the cellulose-synthesising complex at the tip of a microfibril is unacceptable. A new proposal is presented in which it is speculated that both microtubules and microfibrils are oriented by a mechanism separate from both. It is shown that if two vectors are contemplated, one parallel to cell length and one at right angles, and a sensor exists on the plasmalemma surface which responds to changes in the vectors, then all known wall structures may be explained. The possible nature of the vectors and the sensor are considered.     

Oryzalin,a dinitroaniline herbicide,binds to plant tubulin and inhibits microtubule polymerization in vitro

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10^-8 M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10^-7 M oryzalin stopped migration abruptly. Oryzalin (1.0·10^-7 M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K _i ) of 2.59·10⁶ M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [¹⁴C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K _app) of 1.19·10⁵ M^-1. Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.Abbreviations MT microtubule - SIB sucrose isolation buffer - TO tubulin-oryzalin complex     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis. V. The cytoskeleton and the effects of microtubule poisons

Summary InStephanoeca diplocostata microtubules are located in four positions namely: within the flagellar axoneme; just beneath the plasmalemma; associated with the silica deposition vesicles (SDVs) during early stages of costal strip deposition; and in the mitotic spindle. At the anterior end of the cell the 50–60 peripheral microtubules, which are organized more or less parallel to the long axis of the cell, converge around the base of the emergent flagellum. A short second flagellar base is positioned between the nucleus and the base of the emergent flagellum. Developing costal strips are located individually within SDVs in the peripheral cytoplasm. During the early stages of silica deposition each SDV is curved and subtended longitudinally on its concave side by two microtubules. When a costal strip has achieved sufficient rigidity to withstand bending the SDV-associated microtubules are depolymerized. Treatment of exponentially growing cells with sublethal concentrations of microtubule poisons, such as colchicine, podophyllotoxin, griseofulvin andVinca alkaloids depresses growth. Treatment with these drugs also affects the length and morphology of developing costal strips perhaps by interfering with the shaping and supporting functions of SDV-associated microtubules. Instead of being long and crescentic with a standard radius of curvature, costal strips of treated cells are usually short and misshapen, with irregular bends. After drug treatment, juveniles produced as a result of cell division do not develop flagella but can still assemble a lorica although it is usually misshapen. The role of microtubules and microfilaments in lorica production is discussed.     

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O₂ evolution of the leaf tissue in saturating light and CO₂ was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO₂ assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.Abbreviations cyt f cytochrome f - chl chlorophyll - PS II photosystem II - Pn_max maximum photosynthesis - RuBCase Ribulose, 1-5,bisphosphate carboxylase     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall.Pas2G6 reacts with a component of basal bodies and the flagellar band in thePteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network.Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate.Abbreviations MT microtubule - PPB preprophase band     

MTOCs in higher plant cells: an immunofluorescent study of microtubule assembly sites following depolymerization by APM

Summary A one hour exposure to 3 M amiprophos-methyl (APM) depolymerizes all MT arrays in cells from higher plant suspension cultures. On removal of APM, MT repolymerization sites are detected using immunofluorescent staining. During interphase, Mt arrays return uniformly dispersed across the cell cortex with transverse arrays in elongated cells and random arrays in isodiametric cells. During cell division, MT arrays return as follows: Prophase-MT arrays return in association with the nuclear envelope. Metaphase-MTs return associated with chromosomes. Teleophase-MTs return in apparent association with the reforming nuclear envelope and as aberrant phragmoplasts. MTOCs in higher plant cells may be membrane associated at many stages in the cell cycle. Isolated, condensed chromosomes are capable of nucleating MTs, which can attain small, spindle-like configurations.Abbreviations APM Amiprophos-methyl - MT Microtubule - MTOC Microtubule organizing center - NS Nucleating site