Initial attachment ofCandida albicans cells to buccal epithelial cells

The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) ofCandida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion ofC. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer ofC. albicans responsible for adhesion to host cells.     

Anther ontogeny in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

Brachypodium distachyon has emerged as a model plant for the improvement of grain crops such as wheat, barley and oats and for understanding basic biological processes to facilitate the development of grasses as superior energy crops. Brachypodium is also the first species of the grass subfamily Pooideae with a sequenced genome. For obtaining a better understanding of the mechanisms controlling male gametophyte development in B. distachyon, here we report the cellular changes during the stages of anther development, with special reference to the development of the anther wall. Brachypodium anthers are tetrasporangiate and follow the typical monocotyledonous-type anther wall formation pattern. Anther differentiation starts with the appearance of archesporial cells, which divide to generate primary parietal and primary sporogenous cells. The primary parietal cells form two secondary parietal layers. Later, the outer secondary parietal layer directly develops into the endothecium and the inner secondary parietal layer forms an outer middle layer and inner tapetum by periclinal division. The anther wall comprises an epidermis, endothecium, middle layer and the secretory-type tapetum. Major documented events of anther development include the degradation of a secretory-type tapetum and middle layer during the course of development and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The tapetum undergoes degeneration at the tetrad stage and disintegrates completely at the bicellular stage of pollen development. The distribution of insoluble polysaccharides in the anther layers and connective tissue through progressive developmental stages suggests their role in the development of male gametophytes. Until sporogenous cell stage, the amount of insoluble polysaccharides in the anther wall was negligible. However, abundant levels of insoluble polysaccharides were observed during microspore mother cell and tetrad stages and gradually declined during the free microspore and vacuolated microspore stages to undetectable level at the mature stage. Thus, the cellular features in the development of anthers in B. distachyon share similarities with anther and pollen development of other members of Poaceae.     

Partial shoot reiteration in Wollemia nobilis (Araucariaceae) does not arise from 'axillary meristems'

Background and Aims

Conifers are characterized by the paucity of axillary buds which in dicotyledonous trees usually occur at every node. To compensate, conifers also produce ‘axillary meristems’, which may be stimulated to late development. In juvenile material of Wollemia nobilis (Araucariaceae: Massart''s model) first-order (plagiotropic) branches lack both axillary buds and, seemingly, axillary meristems. This contrasts with orthotropic (trunk) axes, which produce branches, either within the terminal bud or as reiterated orthotropic axes originating from axillary meristems. However, plagiotropic axes do produce branches if they are decapitated. This study investigated how this can occur if axillary meristems are not the source.

Methods

The terminal buds of a series of plagiotropic branches on juvenile trees were decapitated in order to generate axillary shoots. Shoots were culled at about weekly intervals to obtain stages in lateral shoot development. Serial sections were cut with a sliding microtome from the distal end of each sample and scanned sequentially for evidence of axillary meristems and early bud development.

Key Results

Anatomical search produced no clear evidence of pre-existing axillary meristems but did reveal stages of bud initiation. Buds were initiated in a group of small starch-rich cortical cells. Further development involved de-differentiation of these small cells and the development of contrasting outer and inner regions. The outer part becomes meristematic and organizes the apex of the new branch. The inner part develops a callus-like tissue of vacuolated cells within which vascular cambia are developed. This kind of insertion of a branch on the parent axis seems not to have been described before.

Conclusions

Axillary meristems in Wollemia characterize the leaf axils of trunk axes so that the origin of reiterated shoots is clear. Plagiotropic axes seemingly lack axillary meristems but still produce axillary branches by distinctive developmental processes. These observations demonstrate limited understanding of branch initiation in trees generally.     

Total and cell wall phosphorus content inBacillus megaterium in phosphate-limited media

A comparative study of the amount of total and cell wall phosphorus inBacillus megaterium ATCC 33085, grown in media with or without phosphate limitation was carried out. The phosphorus levels were investigated during six successive subcultures. A progressive decrease in total phosphorus was found in cells cultivated in a phosphate-limited medium. A decline in the cell wall phosphorus level was observed starting only from the third subculture in phosphate-limited medium, and no phosphorus was detected in the walls of cells in the fifth subculture.     

Chlorotetracycline-binding surface regions in gemmalings of Riella helicophylla (Bory et Mont.) Mont.

The unistratose thallus of the gemmaling of Riella helicophylla is divided into an apical growth lobe with the meristem, an intermediate pillar, and a basal rhizoidal lobe. This organization can be correlated with a distinct fluorescence pattern of wall and membrane after treatment with chlorotetracycline. Mature cells of the growth lobe are distinguished by two chlorotetracycline-binding surface regions (CSR) with a diameter of 6–12 μm in the middle of both outer cell surfaces. Meristematic cells are devoid of CSR. The same is true for cells of the pillar which elongate under low light intensity. Rhizoid initials have an enlarged CSR on the site where the rhizoidal tube will emerge, whereas the opposite cell surface lacks any chlorotetracycline fluorescence. With the beginning of the extension of the rhizoid, CSR remains as a ring around the base of the tube. Darkness, plasmolysis, and sulfhydryl reagents inhibit the fluorescence of CSR, whereas calcium antagonists in addition suppress the fluorescence of the rhizoids. Cytochemical methods demonstrate that sulfhydryl proteins and anionic polysaccharides are involved in adsorbing chlorotetracycline in this region. Surface electron-microscopic preparations reveal a local depression of the wall covered with amorphous material.     

The chitin-glucan complex ofSaccharomyces cerevisiae

Differentiation of the cell wall ofSaccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.     

Identification and immunocytochemical localization of α-galactosidase in resting and germinated date palm (Phoenix dactylifera L.) seeds

α-Galactosidases (EC 3.2.1.22) from resting and germinated date (Phoenix dactylifera L.) seeds were compared and localized using immunocytochemical methods. The enzyme was present in both the endosperm and embryo of resting seeds, in the endosperm undergoing digestion where the greatest specific activity was present, and in the haustorium of seedlings. The enzyme had a molecular mass of 140000 as determined by gel filtration and a pH optimum of 4.5. At least seven forms of the enzyme with isoelectric points ranging from 3.85 to 5.2 were detected in the haustorium whereas only four of these forms were present in the endosperm. The relative activity levels of the various forms also differed between the two tissues. On Western blots all enzyme forms were recognized by antibodies raised against mung-bean (Vigna radiata) α-galactosidase. Using immunogold techniques, label was shown to be present in the protein bodies of the resting embryo cells but to decrease in this organelle as the reserve protein was mobilized and to appear diffusely in the cytoplasm in subsequent stages. In resting endosperm cells, label occurred in the protein bodies and in a thin region of inner wall. In endosperm undergoing digestion, where different stages of protoplast and wall breakdown occurred, immunogold staining was localized in the flocculent contents of vacuoles which resulted from storageprotein breakdown, then dense staining occurred in the inner wall of cell cavities formed by the complete dissolution of the cytoplasm, and finally, staining was uniformly diffuse throughout the remaining endosperm wall adjacent to the haustorium surface. These observations indicate that the α-galactosidase present in cell walls of the date palm endosperm during mannan mobilization is not secreted by the haustorium but instead is probably a pregermination product stored mainly in the protein bodies of resting endosperm and is released to the wall following loss of membrane integrity.     

Septoglomus jasnowskae and Septoglomus turnauae,two new species of arbuscular mycorrhizal fungi (Glomeromycota)

Phylogenetic analyses of SSU-ITS-LSU nrDNA sequences and morphological studies of spores and mycorrhizae confirmed our supposition of finding two new species of arbuscular mycorrhizal fungi of the genus Septoglomus in the phylum Glomeromycota. Morphologically, the first species, named S. jasnowskae, is distinguished by its pale yellow to brownish yellow, small spores with a 2-layered spore wall, of which the colourless outer layer 1 stains dark in Melzer’s reagent and layer 2 is laminate. The spores usually arise in loose clusters. The structures most distinguishing S. turnauae are its two coloured laminate layers in the 4-layered spore wall. In the field S. jasnowskae was associated with roots of Ammophila arenaria and an unrecognized plant species colonizing maritime dunes of the Mediterranean Sea near Thessalonica (Greece) and Calella (Spain), respectively, and S. turnauae formed mycorrhiza with a Cistus sp. (Cistaceae) growing in the soil of a mine located in Sulcis-Iglesiente, SW-Sardinia, Italy. In single-species cultures with Plantago lanceolata as host plant, the mycorrhiza of S. jasnowskae consisted of arbuscules, hyphae and vesicles, and that of S. turnauae comprised arbuscules and hyphae only.     

Accurate analysis of fusion expression of <Emphasis Type="Italic">Pichia pastoris</Emphasis> glycosylphosphatidylinositol-modified cell wall proteins

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in β-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.     

Microsporogenesis,pollen mitosis and pollen tube growth in Gagea villosa (Liliaceae)

In this study, Gagea villosa (Bieb.) Duby was investigated by using light microscopy methods in cytological and cytoembryological respects. Anthers were tetrasporangiate. Anther wall was formed with an epidermis, endothecium, middle layer and tapetum. Tapetum was glandular type and it began to degenerate when microspores released from tetrads. Tapetum cells generally have one or two nuclei. Mitosis seen in tapetum cells was generally normal but micronuclei were found in some of them. Fibrous thickenings were determined in endothecium. Microsporogenesis and pollen mitosis were generally regular. Cytokinesis was successive type. Meiosis in pollen mother cells was asynchronous in one anther locus. Mature pollen grains were 2-celled. Pollen sterility was found to be 24%. Some of the fertile pollen grains, smaller than the normal were seen at the end of the pollen mitosis. Microgametophyte development was examined in vivo and in vitro. Germination ratio of pollen grains in vitro was 4%. Generally swollen pollen tube tips and weak development of some curled pollen tubes were seen. Callose plug formation was seen only in vivo pollen tube growth.     

Regeneration of the cell wall of protoplasts of the fission yeastSchizosaccharomyces versatilis in liquid media and their reversion to cells

Regeneration of the cell wall and reversion of protoplasts with a completely regenerated cell wall to cells were studied by light and electron microscopy in protoplasts of the fission yeastsSchizosaccharomyces versatilis. On their surface the protoplasts regenerated a complete new wall even m liquid media The wall regeneration began with the formation of a thin irregular net of flat bundles of long microfibrils and the net was gradually filled with aggregates of short straight microfibrils and small piles of amorphous material. Osmotically resistant organisms with regenerated walls were detected after a 4–6 h cultivation Depending on the nutrient medium used 10–80 % of protoplasts with the regenerated wall were obtained that reverted subsequently to cells. The high percentage of the wall regeneration and reversion to cells was reached by combining cultivation in a poor medium with that in a rich medium Reversion to cells could only occur after the protoplasts had regenerated rigid cell walls These walled protoplasts underwent septation, and, by polar growth, produced cylindrical cells, further dividing by fission.     

Changes in the activities of catalase,peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N′-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Isolation and characterization of prosthecae ofAsticcacaulis biprosthecum

The budding process of the yeast form of Mucor rouxii was examined by electron microscopy of thin sections with particular reference to wall ontogeny. In most instances the bud wall is seen as a continuation of the inner layers of the parent cell wall. As the bud emerges it ruptures the outer layers of the parent wall. The bud wall is much thinner than the parent wall and remains so while the bud grows into a sphere of about one half the diameter of the parent cell. Then a septum begins to form centripetally, at the neck, by invagination of the plasmalemma. Before the neck canal is completely occuluded, electron-dense wall material is deposited into the septum space. Two separate septum walls are deposited, one on the parent side and one on the bud side of the invaginating plasmalemma. Septum wall formation extends to the surrounding neck walls. In this manner, the parent and bud cytoplasms become fully separated and each is surrounded by a continuous wall. The two cells remain attached to each other by the original neck wall; eventually, the bud abscisses leaving a birth scar on the bud cell and a more pronounced bud scar on the parent cell. In general, the mechanism of budding in this zygomycetous fungus resembles that of an ordinary ascomycetous yeast such as Saccharomyces cerevisiae.     

Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient hela cells

Although HeLa (BU25) cells are deficient in cytosol dT kinase activity, they contain two mitochondrial dT kinases with disc PAGE mobilities (R _m) of 0.4 and 0.6 and isoelectric points (pI) of 8.4 and 5.6, respectively. Mitochondrial extracts of parental HeLa S3 contain the two HeLa (BU25) activities, but also a cytosol-like enzyme (0.25 R _m, pI 9.8). The 0.6-R _m (pI 5.6) mitochondrial activity utilizes ribonucleoside 5′-triphosphates other than ATP (dATP) as phosphate donors and is sensitive to dCTP inhibition. The predominant HeLa S3 cytosol (0.25 R _m) enzyme and the 0.4 R _m mitochondrial enzymeefficiently utilize only ATP as a phosphate donor and are relatively insensitive to dCTP inhibition. Submitochondrial fractionation studies have shown that (1) 74–98% of the mitochondrial dT kinase is located in the matrix plus inner membrane fractions; (2) the matrix fraction has the highest specific activity, contains all the 0.6-R _m activity, most of the HeLa S3 0.25-R _m activity, and some 0.4-R _m activity; (3) the inner membrane fraction is the major site of the 0.4-R _m activity but the outer membrane fraction also contains the 0.4 R _m activity; and (4) all HeLa S3 submitochondrial fractions contain the 0.25-R _m dT kinase activity.     

Two-phase integrated sludge thickening and digestion (TISTD) reactor microbial diversity and community structure succession rules

A two-phase integrated sludge thickening and digestion (TISTD) reactor composed of an inner and an outer reactor was developed. Acidification of natural organic material was the primary process in the outer reactor, whilst methane production was the dominant bioreaction occurring in the inner one. The special structure of TISTD thus enables the effective separation of the acid production phase and methane production phase during sludge processing. Molecular biological technology, including 16S rRNA gene and PCR-TGGE, was utilized to investigate the overall microbial community structure and diversity, as well as the processes of dynamic change. Analysis was also conducted on succinate dehydrogenase and coenzyme F₄₂₀ change trends at each dosing ratio. The microbial community structure of the system exhibited disorder gradually and led to collapse when the dosing ratio increased above 30 %.     

Proteolytic activities in the developing seeds of ×Haynaldoticum sardoum

Aminopeptidase, carboxypeptidase and proteinase activities were measured in endosperms from unripe and ripe seeds of ×Haynaldoticum sardoum. Aminopeptidase and proteinase activities were high during the early maturation stages and then decreased. In contrast, carboxypeptidase activity increased with maturation. Localization studies demonstrated that aminopeptidase and carboxypeptidase activities were present in the three tissues examined (pericarp, green layer plus aleurone, and starchy endosperm). Proteinase activity against gliadin was located in the pericarp and in the green layer plus aleurone, but was absent in the starchy endosperm. The presence of proteolytic activities in the outer kernel layers might be correlated to the hydrolysis of transitory protein reserves during the senescence of the seed coat?. Aminopeptidase and carboxypeptidase activities located in the starchy endosperm could participate in the breakdown of protein reserves during the early phases of seed germination.     

The ultrastructure of spores ofClaviceps purpurea (Fr.) Tul.

The ultrastructure of saprophytic and parasitic spores of the AscomyceteClaviceps purpurea (Fr.) Tul. was studied. Considerable differences were found to exist between the saprophytic and parasitic spores as to morphology and fine structure. The reason for the different ultrastructural morphology is probably connected with the intensity of cell metabolism. Whereas the parasitic spores obtained from the honeydew possess the character of a resting cell with a thick electron-dense cytoplasm, abundant lipid bodies, few mitochondria, an underdeveloped and hence little active endoplasmic reticulum and with a homogenous thick cell wall, the saprophytic spores appear as cells with higher metabolic rate, containing more numerous mitochondria, a thinner cytoplasm, a highly developed endoplasmic reticulum, fewer lipid bodies and abundant large vacuoles as well as frequently a new wall layer.     

Clostridium butyricum reduce lipogenesis through bacterial wall components and butyrate

Intervention strategies for obesity are global issues that require immediate attention. The objective of this study was to assess the possibility that Clostridium butyricum and its potential components could reduce lipogenesis. Co-culture experiments of Caco-2 cells and 1?×?10⁶, 1?×?10⁷, and 1?×?10⁸ CFU/ml of C. butyricum were set up to monitor the cytotoxicity of C. butyricum and the changes of angiopoietin-like protein 4 (ANGPTL4) mRNA expression. It was found that cell viability was not affected by C. butyricum, and ANGPTL4 mRNA expression in Caco-2 cells was highly induced by 1?×?10⁷ CFU/ml of C. butyricum. Co-culture experiment of Caco-2 cells and potential components of C. butyricum were set up to monitor any ensuing alteration in ANGPTL4. It was observed that bacterial wall components and potentially secreted factors from C. butyricum could induce ANGPTL4 mRNA expression and protein secretion. To determine whether butyrate could affect the ANGPTL4 production in Caco-2 cells, the role of monocarboxylate transporter 1 (MCT1) in mediating potentially secreted factors from C. butyricum-induced ANGPTL4 production in Caco-2 cells and the effect of 0.1 mM of butyrate on ANGPTL4 production in Caco-2 cells were investigated. It is confirmed that butyrate was the factor secreted by C. butyricum to stimulate ANGPTL4 production. Besides, the soluble factors secreted by live C. butyricum-Caco-2 cells interaction, bacterial wall components-Caco-2 cells interaction, and the main metabolites butyrate-Caco-2 cells interaction reduced lipogenic gene expression in HepG2 cells. In conclusion, 1?×?10⁷ CFU/ml of C. butyricum could reduce lipogenesis through the bacterial wall components and the metabolites such as butyrate.     

Changes in Inner and Outer Retinal Layer Thicknesses after Vitrectomy for Idiopathic Macular Hole: Implications for Visual Prognosis

Purpose

To investigate sequential post-operative thickness changes in inner and outer retinal layers in eyes with an idiopathic macular hole (MH).

Methods

Retrospective case series. Twenty-four eyes of 23 patients who had received pars plana vitrectomy (PPV) for the closure of MH were included in the study. Spectral domain optical coherence tomography C-scan was used to automatically measure the mean thickness of the inner and outer retinal layers pre-operatively and up to 6 months following surgery. The photoreceptor outer segment (PROS) length was measured manually and was used to assess its relationship with best-corrected visual acuity (BCVA).

Results

Compared with the pre-operative thickness, the inner layers significantly thinned during follow-up (P = 0.02), particularly in the parafoveal (P = 0.01), but not perifoveal, area. The post-operative inner layer thinning ranged from the ganglion cell layer to the inner plexiform layer (P = 0.002), whereas the nerve fiber layer was unaltered. Outer layer thickness was significantly greater post-operatively (P = 0.002), and especially the PROS lengthened not only in the fovea but also in the parafovea (P < 0.001). Six months after surgery, BCVA was significantly correlated exclusively with the elongated foveal PROS (R = 0.42, P = 0.03), but not with any of the other thickness parameters examined.

Conclusions

Following PPV for MH, retinal inner layers other than the nerve fiber layer thinned, suggestive of subclinical thickening in the inner layers where no cyst was evident pre-operatively. In contrast, retinal outer layer thickness significantly increased, potentially as a result of PROS elongation linking tightly with favorable visual prognosis in MH eyes.     

Pattern formation and the fine structure of the developing cell wall in colonies of Pediastrum boryanum

A single-layered disc of peripheral pronged cells and central prongless cells impart the typical gear shape to colonies of Pediastrum, while the walls of each cell have a characteristic reticulate triangular pattern. The two-layered wall forms in the cells during colony formation following zoospore aggregation and adhesion. The uniformly thin outer layer reflects contours resulting from differential thickening in the reticulate pattern of the inner, thicker, more fibrillar and granular wall layer. The reticulate pattern thus imparted to the outer wall layer persists in empty zoosporangia following the release of zoospores. Columns of electron-dense material extend through the outer wall layer except at the ridges and centers of the reticulum. Following mitosis and cleavage, the resulting zoospores are extruded within a vesicle membrane consisting of the inner wall layer. Separation of this membrane from the parent cell occurs in material of the inner layer adjacent to the outer wall. Vesicles containing swarming zoospores also contain a granular material which appears to become associated with the aggregating and adhering cells of new colonies. Microtubules occur in zoospores prior to adherence but are absent during wall deposition.