秋水仙素诱导的野生大豆根类细胞超微结构变化

萌发了4d的野生大豆种子经秋水仙素处理3d以后,根尖分生区细胞的超微结构发生了一些显著变化：许多质体环绕细胞核分布,这些质体中含有数量不等的淀粉粒;液泡的体积明显增大,有几个大液泡分布在细胞核周围;少数细胞中形成网状粗面内质网聚集体;细胞核所占细胞体积的比例减小,有的细胞核形状变得极不规则。从超微结构分析,这些细胞已具备了分化细胞的特征,而不再处于有丝分裂之中。     

Reconstitution of endoplasmic reticulum in rapidly dividing cells of early Xenopus embryos

The cytology of early blastomeres of Xenopus laevis embryos was examined. Particular attention was given to the organization of the nuclear envelope of karyomeres (chromosome vesicles) and the endoplasmic reticulum (ER) at different stages in early cleavage cycles of frog development. Nuclear envelope formation was observed to occur rapidly around individual chromosomes during early anaphase, and karyomeres fused subsequently to yield the final nucleus during telophase. Endoplasmic reticulum in the perinuclear cytoplasm was observed to be vesicular during metaphase and cisternal in form during telophase. Following microinjection of rat liver rough microsomes into early blastomeres, heterologous ER components were identified by electron microscope immunocytochemistry. The foreign ER was observed as large, reconstituted cisternae at stages in the cell cycle when the nuclear envelope was intact. Therefore, transplanted ER maintained the capacity to reconstitute in the cytoplasm of a rapidly dividing cell. In an attempt to better assess ER structure at the metaphase stage of the cell cycle, we next slowed down the division process by treating Xenopus embryos with anti-microtubule agents. Treatment with critical concentrations of colchicine, nocodazole, or vinblastine led to cleavage arrest but not to inhibition of the nuclear cycle. Following such treatment, homologous ER was observed in a vesicular form at all stages of the nuclear cycle. Heterologous ER, however, identified by immunocytochemistry in microinjected cells treated with nocodazole, displayed both vesicular and cisternal forms. We conclude that microinjected ER membranes exhibit cell-cycle-specific behavior, which is different from that of the host cell ER.     

ULTRASTRUCTURE OF THE SHOOT APEX OF TOMATO (LYCOPERSICON ESCULENTUM)

The vegetative shoot apical meristem of tomato (Lycopersicon esculentum Mill.) was examined at the ultrastructural level. The meristem consisted of a surface layer that was different from the rest of the meristem and was unique among the dicotyledonous species. The cells of the surface layer contained large distal vacuoles with relatively large electron-dense inclusions, proplastids with membrane-bound inclusions (MB), and differentiating chloroplasts. In addition, periclinal and oblique divisions were observed in the surface layer cells along with anticlinal divisions. The cells of the subsurface layers contained small vacuoles with fewer inclusions as well as proplastids of various shapes but without MB. Differentiating chloroplasts were not observed in these cells, but autophagic vacuoles at various stages of development were present. The normal complement of cell inclusions, e.g., the mitochondria, golgi bodies, endoplasmic reticulum (ER), ribosomes, and microtubules were observed in subsurface layers, and in many cells the ER was observed to be continuous with the outer membrane of the nuclear envelope and with the plasmalemma. Further below in the meristem, cells contained both the proplastids and differentiating chloroplasts with MB. In the latter, the outer membrane of the MB was found to be continuous with the developing lamellae, suggesting that MB probably serve as the storage centers for lamellae membranes. Near the base of the meristem, in the pith-rib meristem, enlarged cells containing large vacuoles and differentiated chloroplasts were present.     

Effects of colchicine or demecolcine on cytoplasmic protrusions and assisted enucleation of golden hamster oocytes

To establish experimental protocols for cloning golden hamsters, optimal concentrations of colchicine and demecolcine were determined for inducing cytoplasmic protrusion (containing chromosomes) and assisting enucleation of their oocytes. Denuded oocytes at different ages were treated with 2.5–10 μg/ml of colchicine for 1–4 h or 0.02–0.6 μg/ml of demecolcine for 15–60 min. Cytoplasmic protrusions of oocytes were removed with a micromanipulation pipette. The results show that: 1) at 13.5–18 h post-hCG injection, ∼90% of oocytes treated for with 10 μg/ml of colchicine formed cytoplasmic protrusions, and in some oocytes enucleation occurred; 2) when treated with 0.4 μg/ml of demecolcine for 1 h, cytoplasmic protrusions 13.5–18 h post-hCG treatment were present in almost all oocytes; 3) after the protrusions induced by either treatment had been removed, the assisted enucleation rate was >80%, whereas it was ∼32% with blind enucleation.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

FINE STRUCTURE STUDIES OF PHLEUM ROOT MERISTEM CELLS. II. MITOTIC ASYMMETRY AND CELLULAR DIFFERENTIATION

Avers , Charlotte J. (Douglass Coll., Rutgers—The State U., New Brunswick, N.J.) Fine structure studies of Phleum root meristem cells. II. Mitotic asymmetry and cellular differentiation. Amer. Jour. Bot. 50(2): 140–148. Illus. 1963.—An electron microscopical study showed that ultrastructural differences distinguished the cell dividing symmetrically from that undergoing asymmetrical division in timothy grass epidermis. The spindle orientation led to cytokinesis which produced either equal- or unequal-sized sister cells, but the mitotic apparatus itself varied in the mitoses. In asymmetrical cells, the basal pole showed more extensive endoplasmic reticulum (ER) polarizations, which intruded into the spindle area during metaphase and anaphase. Such ER polarity was not obvious in symmetrical mitosis or in the apical end of asymmetrically dividing cells. The mitotic sequence is described photographically. Foci of ER were observed as early as prophase in the polar region, and it is suggested that there is a resemblance to astral ray foci seen in prophase of animal cell mitosis. Cell plate formation could be detected in anaphase by accumulations of vesicles and ER fragments along the spindle equator. Phragmosomes apparently were not involved in cell plate formation in Phleum, unlike Allium, cytokinesis. The mitotic asymmetry is discussed as a consequence of an intracellular gradient separate from the intercellular gradient of differentiation along the entire length of growing root tip epidermis.     

Morphological Alterations of Saccharomyces cerevisiae Induced by Benanomicin A,an Antifungal Antibiotic with Mannan Affinity

The effects of benanomicin A, a mannose-binding antifungal antibiotic, on yeast cells of Saccharomyces cerevisiae were studied by electron microscopy. Cytological studies using vital stain with methylene blue demonstrated that benanomicin A at 20 and 80 μg/ml killed buds in preference to parent cells. In confirmation, examination by TEM revealed that benanomicin A at 80 μg/ml damaged buds more severely than parent cells. The major effect on the ultrastructure was characterized by severe damage to the cell membrane. In addition, it caused expansion and vacuolation of the endoplasmic reticulum (ER), and partial fragmentation and disappearance of nuclear membranes. The membrane-disruptive activity of benanomicin A may be closely associated with its membrane affinity.     

Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle-stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go-nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley-Liss, Inc.     

Obtaining plantlets from apical meristem of the red alga Gelidium sp.

For the first time, plantlets were obtained from fragments and cell aggregates (CA) of apical meristem of the red alga Gelidium sp. After two months of cultivation, an initial weight of 100 mg of fragments and CA from fresh meristem produced 3 g of plantlets without rhizoids. During the same period of cultivation, 100 mg of meristem fragments and CA isolated from thalli by the freezing-thawing procedure produced more than 20 g of plantlets with rhizoids. It is assumed that our methods for obtaining plantlets from fragments and CA of fresh and frozen-thawed meristem could be used to generate mass planting material for cultivation of algae (plantlets with rhizoids) in the sea and for tank-bubbling cultivation (plantlets without rhizoids). We speculate that meristem cells of frozen-thawed algae might be natural “seedstock” in the Arctic and Antarctic seas.     

Proteome analyses of cellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone, a novel antibiotic candidate

The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC) values ranged from 31.25-62.5 μg/ml, and the minimal bactericidal concentration (MBC) was 250 μg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5-125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 μg/ml and 2 μg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 μg/ml) affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.     

The kinetics of resensitization of Tetrahymena following recovery from effects of cycloheximide

Synchronized cells of Tetrahymena pyriformis strain GL-C were exposed to cycloheximide (CHI) (0.2 μg per ml) from 40 to 140 minutes after the end of the heat synchronizing treatment. Recovery takes place during this treatment (Frankel, 1970). The CHI was washed out at 140 minutes. At various times after washout dividing cells were isolated in micro-drops under oil, and one daughter was transferred to a test drop containing CHI (0.2 μ per ml). The generation time of both daughters was recorded, and the “percent prolongation” of generation time brought about by the test exposure of one cell to CHI was computed for each cell-pair. This procedure was carried out for groups of cell-pairs at different times after the end of the CHI pretreatment. Comparable tests were performed with two control series, one which had not previously been exposed to CHI and another for which CHI was present continuously. Comparison of the prolongation observed in control and experimental series demonstrated that cells which have earlier undergone recovery in CHI gradually become resensitized following washout of the drug. Cells progressively lose most of their original resistance in a period of somewhat over three cell generations; however, a small but significant fraction of this resistance is still retained seven to eight generations after the CHI pretreatment.     

New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of the red alga Palmaria palmata

Isolation of meristematic tissue of the red alga Palmaria palmata by a freezing-thawing method and further maintenance of the tissue in culture showed the existence of groups of meristematic cells in superficial cortical layers of thallus forming wart-like outgrowths. For the first time, proliferations (plantlets) were obtained from meristematic tissue of sporophytic and male gametophytic fronds and tetraspores from submeristematic tissue of sporophytic fronds within a short period (6 weeks). Tissue fragments (1 × 1 mm²) from upper margins of fresh thalli and cell aggregates (10−100,000 cells) from marginal meristem and meristematic warts of fresh thalli and thalli after the freezing-thawing procedure were cultured for getting plantlets. Tissue fragments (TF) and cell aggregates (CA) from submeristematic tissue of fresh thalli were cultured for obtaining tetraspores. For mass getting proliferations (plantlets) and tetraspores we recommend to use CA from marginal tissue of fresh fronds because of fast growth, high numbers of proliferations and simple techniques of the method. The freezing-thawing method allows also to identify meristematic tissue and to obtain plantlets of red algae with apical meristem (e.g., Gelidium spp.).     

LEAF DEVELOPMENT IN DOXANTHA UNGUIS-CATI (BIGNONIACEAE)

Leaf structure in Doxantha unguis-cati is polymorphic. The usual mature compound leaf is composed of two lanceolate leaflets and a terminal tripartite spine-tendril. Leaf primordia are initiated simultaneously in pairs on opposite flanks of the shoot apical meristem by periclinal cell divisions in the third subsurface layer of the peripheral flank meristem. Two leaflet primordia are the first lateral appendages of the compound leaf. Initiation of these leaflet primordia occurs on the adaxial side of a compound leaf primordium 63–70 μm long. Lamina formation is initiated at the base of a leaflet primordium 70–90 μm long and continues acropetally. Mesophyll differentiation occurs in later stages of development of leaflets. The second pair of lateral appendages of the leaf primordium differentiate as prongs of the tendril. Initiation of the second pair of lateral appendages occurs on the adaxial side of a primordium approximately 168 μm long. Acropetal procambialization and vacuolation of cells extend to the apex of tendrils about 112 μm long, restricting the tendril meristem to the adaxial side of the primordium and resulting in curvature of the tendril. The tendril meristem is gradually limited to a more basipetal position as elongation of apical cells continues. Initiatory divisions and early ontogenetic stages of leaflets and tendrils are similar. Their ontogeny differs when the lateral primordia are approximately 70 μm long. Marginal and submarginal initials differentiate within leaflets but not in tendrils. Apical growth of tendrils ceases very early in ontogeny as compared with leaflets.     

Ultrastructure of Diplodia maydis grown on selected synthetic media

Of all the fixatives studied, glutaraldehyde followed by osmium was considered best. Fixation temperatures did not result in ultrastructural differences. Holdover for short periods (up to 7 days) in 70% alcohol and long periods (3 months) in glutaraldehyde did not result in ultrastructural differences from those processed without a hold-over period. Uranyl acetate increased contrast, as expected. Epon was selected over Epon-Araldite (more viscous) and Spurr's resin (less contrast). Generally, growth conditions did not alter gross hyphal morphology, although cytological changes were noted. Diplodia maydis is morphologically similar to ascomycetous fungi. It contained typical organelles as found in other fungi, e.g. cell wall, septa, Woronin bodies, nuclei, mitochondria, endoplasmic reticulum, lomasomes, plasma membranes, cytosomes, vacuoles, storage materials (lipids, carbohydrates and intracellular and extracellular electrondense substances), thickened mycelial strands, and intrahyphal hyphae. Bead-like structures and thickened nodes as observed by SEM were accounted for by structures observed by TEM. Mitochondria were dramatically elongated, and in one case, branched. Hyphae were organized in three zones: apical, subapical, and vacuolated. Apical vesicles were thought to account for cell extension. Evidence was presented that Woronin bodies originated from microbodies. It was suggested that the Golgi apparatus consists of isolated single cisternae. Free ribo- or polysomes (without ER) were suggested as the site of the majority of protein synthesis. Continuities and configurations of plasma and outer nuclear and mitochondrial membranes, ER, cytosomes, vacuoles and storage materials suggested that a system similar to Morre et al's endomembrane system is in operation in D. maydis to account for component flow and differentiation.     

Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major

In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis.     

Separation of processes associated with differentiation of two-celled Fucus embryos

Various inhibitors were used to separate the overlapping processes of polar axis fixation, intracellular localizations forming a polar cell, and cell division, all of which are essential for cellular differentiation in two-celled embryos of Fucus distichus L. Powell. Cycloheximide and sucrose delayed the appearance of a polar cell (rhizoid formation) without inhibiting the fixation of a polar axis. Cytochalasin B, at 10 μg/ml, reversibly inhibited rhizoid formation without altering cell division. At higher concentrations (50–100 μg/ml) given in short pulses, cytochalasin affected the orientation and delayed the fixation of a light-induced polar axis with no qualitative effect on cell division. Disruption of the mitotic apparatus and prevention of cell division by colchicine had no influence on rhizoid formation or on the photopolarization of the developmental axis.     

Differentiation of stomatal meristemoids and guard cell mother cells into guard-like cells in Vigna sinensis leaves after colchicine treatment

The temporary development of Vigna sinensis seedlings in the presence of colchicine results in the inhibition of stomata generation and the formation of numerous persistent stomatal meristemoids (P-SM) and guard cell mother cells (P-GMC). Before dividing differentially or becoming GMC, the untreated meristemoiidsundergo a preparatory differentiation, during which a synthesis of new densely ribosomal cytoplasm, an increase of nuclear size, and a detectable proliferation of all the organelles are observed. The same process appears depressed and delayed in treated meristemoids; the cells have usually undergone only part of it when they reach the C mitosis. After the inhibition of their division, the bulged meristemoids II and GMC increase further in size, synthesize new nonribosomal cytoplasm, and start vacuolating slowly. The plastids also increase in size, change in shape, and become able to synthesize large quantities of starch. The cells retain a ribosomal cytoplasm, rough ER membranes, and active dictyosomes for a long time. At the advanced stages of differentiation, the microtubules reappear in the cells even when the plant remains under colchicine treatment. When mature, the P-GMC and P-SM are quite similar to the guard cells and possess considerably thickened periclinal walls, numerous mitochondria, and small vacuoles, while the nucleus, the plastids, and the cytoplasm occupy significant parts of the cell volume. In the epidermis displaying open stomata in light, significant K⁺ quantities are detectable in guard cells and P-GMC or P-SM, while they are almost absent from their surrounding cells. When the stomata close in darkness, K⁺ is accumulated primarily in the subsidiary or typical epidermal cells surrounding these idioblasts and only minimally inside them. Besides, the P-GMC and P-SM, like the guard cells, retain the starch for a long time and build up considerable starch quantities from exogenously supplied sugars.Abbreviations P-GMC persistent guard cell mother cell - PSM persistent stomatal meristemoid - ER endoplasmic reticulum     

CYCLES OF REDUPLICATION IN MEGAKARYOCYTE NUCLEI

1. Estimates of the number of dividing megakaryocytes with mitotic apparatus have been obtained in intact and in phenylhydrazine-papain treated rats. 2. In treated rats chromosome morphology was studied using colchicine and hypotonic treatment. 3. In bone marrow smears and in cultures two systems for nuclear reduplication were demonstrated: C-mitosis, and multipolar mitosis without cytokinesis. 4. In phenylhydrazine-papain treated rats, the mitotic index of each bone marrow cell series was greatly increased.     

Effect of auxin on the mitotic cell cycle in cultured leaf segments at different stages of development in wheat

Young leaves of Triticum timopheevi Zukh. show a defined gradient of development. One-mm-long sections from such leaves were cultured in vitro. At a low concentration of exogenous auxin, cells in the most basal, highly meristematic explants divided readily in culture, but in the absence of auxin they soon ceased dividing and were arrested in G1 and G2 of the mitotic cell cycle. In the region adjoining the meristem, where most cells were arrested in G1, very high concentrations of auxin had to be applied to reinitiate cell division, i.e. stimulate transitions from G1 to S-phase and from G2 to mitosis. Above this potentially auxin-responsive region, which represented less than 50% of the total leaf length, there followed tissue, which, when excised, showed nuclear DNA replication in a number of cells in the absence of auxin. However, the cells did not complete the mitotic cycle, either in the absence or presence of exogenous auxin. We suggest this loss of responsiveness is correlated with an uncoupling of auxin from the control of the cell cycle.     

Studies on induced changes in growth patterns and polarity in roots of Vicia faba L.

Summary Roots of Vicia faba were treated with colchicine (0.025%), or IAA (4.7×10^-6 M), or both, for 3 hours and fixed at various intervals over the following 11 days. The axis of spindle orientation and the distribution of mitotic figures, lateral root primordia and xylem vessel elements was examined in the apical 10 mm of median longitudinal sections of these roots.No effect of IAA was found on the orientation of the spindle. However, evidence was obtained indicating that the systems controlling the polarity of cell division and cell expansion differ in some way.The number of lateral root primordia formed was greater in roots treated with IAA or colchicine than in control roots. These primordia were always initiated adjacent to a xylem vessel. Thus, no primordium was closer to the apex than the most apical xylem vessel, suggesting that an endogenous factor involved in primordia initiation is transported in the xylem. The primordia which develop after colchicine treatment grow out as lateral roots; this is in contrast with those which form after IAA treatment and which do not undergo elongation. These results, which it must be emphasized apply only to the apical 1 cm of treated roots, indicate that lateral root primordia become sensitive to IAA at a certain stage in their development. Exogenous IAA acts as an inhibitor.The new meristem, which forms in the primary root apex after colchicine treatment, contains both diploid and polyploid cells, i.e. it was formed from cells that were unaffected and from cells that were affected by colchicine. Following colchicine treatment the size of the meristem shrinks and this can be prevented by treatment with IAA. This and other evidence presented here, suggests that IAA is a factor involved in the control of the size of the apical meristem in normal roots.