Two common fungi associated with farmer's lung: Fine structure of Aspergillus fumigatus and Aspergillus umbrosus

The fine structure of Aspergillus fumigatus and Aspergillus umbrosus by transmission electron microscopy (TEM) is described. The fine structure of the ascosporic and asexual stages of A. umbrosus is described for the first time. Dense, homogenous material and fibers were detected on the outer hyphal cell wall of the Aspergilli. Septal pores were found in the hypha of A. umbrosus. Two wall layers were detected in the cell wall of the conidia of the both Aspergilli. The ascospores of A. umbrosus contained thick cell wall and the surface of which was smoother than that of the conidia.     

狗脊孢壁发育超微结构的研究

鳞毛蕨型孢子类型众多,初步研究表明形态相似的孢子类型其孢壁发育特征存在差异,因此有必要对各代表类群的孢壁发育进行深入地研究。该文利用透射电镜对乌毛蕨科(Blechnaceae)狗脊(Woodwardia japonica)孢壁结构和发育的超微结构进行研究。结果表明:(1)狗脊孢子囊的结构由外向内分别为孢子囊壁细胞、两层绒毡层细胞和孢子母细胞;(2)狗脊孢子具乌毛蕨型(Blechnoid type)外壁,表面光滑,由两层构成,裂缝区域具辐射状的槽;(3)周壁属于空心型(cavity type),由四层构成,从内向外分别为P1、P2、P3和P4层,前三层叠合在一起,层间有不同程度的空隙,P4层与前三层之间具有明显而连续的空腔,并隆起形成片状褶皱纹饰;(4)有小球体和小杆共同参与孢子周壁的形成,周壁部分或全部来源于孢子囊壁细胞。综上所述,狗脊孢子与同属于鳞毛蕨型的贯众(Cyrtomium fortunei)和朝鲜介蕨(Dryoathyrium coreanum)孢壁的发育在周壁结构、周壁各层的发育顺序、周壁来源和参与成壁的特征物质等方面存在差异。该研究有利于进一步理解蕨类植物孢壁所蕴含的分类和演化上的科学意义和价值。     

Pollen Wall Structure Using a New Stripping-Sputtering Device for Scanning Electron Microscopy

A new method has been developed to elucidate pollen wall architecture by the separation of wall layers and the use of scanning electron microscopy (SEM). Separation of wall layers at natural boundaries, breakage across the wall and metal-coating of the specimen have been achieved by controlled ramming of free scattered ions produced by a novel “ion separating and coating – model A” instrument. The stripping treatment reveals interfaces and cross profiles of pollen walls and the sputtering treatment results in metal coating for examination with SEM. An advantage of the method is that it provides intact interfaces that are not eroded or damaged. The application of the method is exemplified by SEM analyses of pollen grains of Gossypium hirsutum L., Zea mays L., Sesamum indicum L. and Brassica napus L. var. oleifera. Interfaces between the tectum, column, foot, nexine-2 and intine layers of the pollen wall were all portrayed in G. hirsutum and to a great part in the other species. In G. hirsutum, it was possible to document the attachment point of surface spines, the appearance of individual baculae and the irregular labrum-operculum but regular inner labrum-aperture structure. No tectum was found in S. indicum. In all four species it was not possible to separate the intine from the sporoplast. The numbers of apertures were 20, 1, 10–14 and 3 in G. hirsutum, Z. mays, S. indicum and B. napus, respectively. The dumbell-shaped arrangement of apertures in G. hirsutum, the gear-shaped oblate sporoplast of S. indicum and the abundance of micropores on the intine of B. napus are characteristic features.     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

Musculature of the penial complex: A new criterion in unravelling the phylogeny of Hygrophila (Gastropoda: Pulmonata)

The taxonomy of freshwater pulmonates (Hygrophila) has been in a fluid state warranting the search for new morphological criteria that may show congruence with molecular phylogenetic data. We examined the muscle arrangement in the penial complex (penis and penis sheath) of most major groups of freshwater pulmonates to explore to which extent the copulatory musculature can serve as a source of phylogenetic information for Hygrophila. The penises of Acroloxus lacustris (Acroloxidae), Radix auricularia (Lymnaeidae), and Physella acuta (Physidae) posses inner and outer layers of circular muscles and an intermediate layer of longitudinal muscles. The inner and outer muscle layers in the penis of Biomphalaria glabrata consist of circular muscles, but this species has two intermediate longitudinal layers separated by a lacunar space, which is crossed by radial and transverse fibers. The muscular wall of the penis of Planorbella duryi is composed of transverse and longitudinal fibers, with circular muscles as the outer layer. In Planorbidae, the penial musculature consists of inner and outer layers of longitudinal muscles and an intermediate layer of radial muscles. The penis sheath shows more variation in muscle patterns: its muscular wall has two layers in A. lacustris, P. acuta, and P. duryi, three layers in R. auricularia and Planorbinae and four layers in B. glabrata. To trace the evolution of the penial musculature, we mapped the muscle characters on a molecular phylogeny constructed from the concatenated 18S and mtCOI data set. The most convincing synapomorphies were found for Planorbinae (inner and outer penis layers of longitudinal muscles, three-layered wall of the penis sheath). A larger clade coinciding with Planorbidae is defined by the presence of radial muscles and two longitudinal layers in the penis. The comparative analysis of the penial musculature appears to be a promising tool in unraveling the phylogeny of Hygrophila.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

Body wall morphology of Pentapora foliacea (Ellis and Solander) (Bryozoa,Cheilostomata)

The ascophoran Pentapora foliacea was studied from epoxy sections of skeletal and soft (hard-soft) tissues. The basal wall is double, indicating the colony grew as two independent layers, back to back. The structure of the vertical walls and interzooidal communication organs indicates that zooids were budded in the usual way as in most encrusting cheilostomes. Secondary layers of the frontal wall are of acicular aragonite. The ovicell develops as a flattened cuticular bladder in early ontogeny; the aragonitic layer of the frontal wall later engulfs it. A median vesicle, an evagination of the vestibular wall, is present but the eggs may be supplied with sufficient yolk to nurture the embryo. The overall ovicell structure is similar to that of hyperstomial ovicells in other cheilostomes.     

BASIDIOSPOROGENESIS IN BOLETUS RUBINELLUS. I. STERIGMAL INITIATION AND EARLY SPORE DEVELOPMENT

Sterigmal initiation in Boletus rubinellus resembled hyphal tip growth. Four stages in early basidiospore development have been delineated based on gross morphology, and changes in wall layers and cytoplasm. Changes in wall layers and cytoplasm during spore development were stage-specific. During Stage 1 the spore wall consisted of two layers identical to those of the sterigmal wall with occasional pellicle remnants on the outer surface. The onset of wall differentiation began in Stage 2, and during Stage 3 wall layers characteristic of the mature spore developed. At Stage 4 there was a pronounced gradient in wall thickness from the apex to the base of the spore. Small vesicles (30–60 nm diam) were uniformly distributed in the cytoplasm of spherically enlarging spores (Stage 2), but during spore elongation (Stages 3 and 4) numerous larger vesicles as well as small vesicles aggregated at the spore apex. A variety of cytoplasmic organelles entered the spore during Stage 3; however, migration of storage materials and the nucleus to the spore did not occur until late basidiospore development. The hilar appendix body developed in the earliest spore primordium and persisted until Stage 3. Development of wall layers and their differential thickening, distribution of vesicles, and probable function of the hilar appendix body are discussed with reference to the control of spore shape. Systematic implications of the data are considered.     

The effects of xylanase type enzymes on the different layers of birch wood ORGANOSOLV pulp fibre walls

Some effects of the xylanase treatment on the separate birch ORGANOSOLV pulp fibre wall morphological layers were examined. These investigations were focused on the outer layers, i.e. the primary wall (P) and the outer layer of the secondary wall (S₁), as well as the central layers, i.e. the central layer of the secondary wall (S₂) and the tertiary wall (T). Step by step, the fractionation of the pulp components in the polar solvents N,N-dimethylformamide (DMFA), dimethylsulphoxide (DMSO) and DMSO/H₃PO₄ was used as a mild technique for the isolation of the lignin-carbohydrate complexes. The different residual amounts of lignin and hemicelluloses in the outer and central pulp fibre wall layers as well as the different lignin-hemicellulose ratios were determined. The size-exclusion chromatographical (SEC) analysis showed a higher initial lignin content in the region of the high molecular mass (MM) fibre wall fraction extracted with “DMSO/H₃PO₄” than the outer cell wall layers. In the central layers, the amounts of soluble lignin (calculated on the mass of total dissolved substance) were approximately the same for all the three solvents. The xylanase treatment brought the most considerable changes in the high MM part of the residual lignin (the lignin carbohydrate complex). This was true for both the P-S₁ and S₂-T layers. The careful brightness comparison of the outer and central fractions after the X-E-P-P bleaching sequence showed a surprisingly low bleachability of the outer layer fraction. The xylanase action depended on the composition of the lignin-carbohydrate complex (LCC) and the extent of the maintenance of the outer layers during the pulping process.     

Studies on perithecium of Ceratocystis stenoceras by a scanning electron microscope

The surface and cavity of the perithecium of Ceratocystis stenoceras were studied with a scanning electron microscope.The body was spherical, 96–231 in diameter, and the surface was covered with hyphae. The neck was a lanky hollow cylinder and the wall consisted of a bundle of tubular hyphae encircling a central canal. A whorl of hyaline hyphae was found at the tip of the neck. The surface of the neck from the body to a distance of about 1/6 was rough and warty. But the surface of the neck above that place was smooth, on which a regular design could be seen. The wall of the neck near the body consisted of 5–6 layers of cells. However, the wall at the tip was of 1–2 layers of cells. The cavity was filled with ascospores. They were like segments of an orange of 1 × 2 with smooth surface.     

On the morphology and ultrastructure of the cell wall of Spirulina platensis

The cell wall of the blue-green alga Spirulina platensis was studied with the electron microscope using ultra-thin sectioning, shadowing, carbon-replication or freeze-etching techniques for specimen preparation. The cell wall could be resolved into four layers, L-I through L-IV. The L-I and L-III layers contain fibrillar material. The septum is a three-layered wall: an L-II layer sandwiched between L-I layers. The shape in vitro of isolated septa might be an artifact due to the preparation technique used. Certain structural properties of the septum seem to allow tangential stretching; they might be reflected in the flexible gliding mobility of Spirulina species. The outer, L-IV layer contains material longitudinally arranged along the trichome axis.     

Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (K_d) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.     

BASIDIOSPORE INITIATION AND EARLY DEVELOPMENT IN COPRINUS CINEREUS

Early basidiospore development in Coprinus cinereus has been divided into four stages: 1) inception, 2) asymmetric growth, 3) equal enlargement, 4) elongation, all based on changes in spore size and shape, wall layering, and cytoplasm. The hilar appendix body formed on the adaxial side of the stage 1 basidiospore, persisted through all stages studied, and predicted the site of the hilar appendix. The hilar appendix formed in stage 2 by modification of certain wall layers. A band of peripheral endoplasmic reticulum covered an average of 38 % of the lower spore wall in stage 3 and was oriented around the axis of growth. Stage 4 was initiated by a break in wall layer 3 at the spore apex and the disappearance of the peripheral endoplasmic reticulum. A pore cap formed on the spore apex during spore elongation. The spore wall consisted at first of three layers and became six layered by deposition of layers between two of the initial layers. Cytoplasmic changes associated with spore growth included presence of small vesicles at stage 1 and larger Golgi vesicles later, absence of mitochondria and probable Golgi cisternae from the spore until stage 3, and presence of a zone nearly free of ribosomes and organelles under the spore apex in stage 4. Functions of the hilar appendix body, peripheral endoplasmic reticulum and the different wall layers in control of spore shape are discussed.     

Electron microscope studies of oogonial wall and elemental composition of oogonia in Phytophthora

Scanning electron microscopy of oogonia of Phytophthora spp. showed that the oogonial wall was smooth in P. cactorum, P. citricola, P. heveae, and P. palmivora; finely granular in P. megasperma and P. megasperma var. sojae; and coarsely granular in P. parasitica. Transmission electron microscopy demonstrated that the oogonial wall in Phytophthora was composed of three layers with the middle layer being the least or the most electron dense. A coat of amorphous material was found on the entire outer surface of the oogonial wall. Elemental analysis of oogonia by means of a SEM electron probe microanalyzer revealed similar emission spectra among Phytophthora spp. with a characteristic peak for calcium.     

Cell wall-associated peroxidase in cultured cells of liverwort,Marchantia polymorpha L. Changes of peroxidase level and its localization in the cell wall

Most of the peroxidase activities from cultured cells of Marchantia polymorpha L. were found in the cell wall. The activities increased markedly after the beginning of stationary growth. Cytochemical examination using an electron microscope indicated that the peroxidase was localized in the layers of the cell wall. The increase of peroxidase released from the cells into the culture medium was closely correlated with the increase of the peroxidase level in the cell wall. The release of peroxidase seemed to be caused by fragmentation of the cell wall stripped from cell.     

LIGNIFICATION DURING SECONDARY WALL FORMATION IN COLEUS: AN ELECTRON MICROSCOPIC STUDY

The fine structure of lignin deposition was examined in developing secondary walls of wound vessel members in Coleus. KMnO₄, which was used as the fixative, selectively reacts with the lignin component of the cell wall and thus can be used as a highly sensitive electron stain to follow the course of lignification during secondary wall deposition. Lignin was first detected as conspicuuos electron-opaque granules in the primary wall in the region where the secondary wall thickening arises and as fine granular striations extending into the very young secondary wall. As the secondary wall develops lignification becomes progressively more extensive. In cross sections the lignified secondary wall appears as concentric, fine granular striations; in tangent al or oblique sections it is seen as delicate, beaded fibrils paralleling the long axis of the thickening. High magnification of tangential or oblique sections shows that the fibrillar appearance is due to the presence of alternating light and dark layers each approximately 25-35 A wide. It is assumed that the light layers are the cellulose microfibrils and the dark regions contain lignin which fills the space between the microfibrils. KMnO₄, by selectively reacting with lignin, thus negatively stains the cellulose microfibrils revealing their orientation and dimensions.     

Electron Microscopic Studies on the Outer Layers of Staphylococcus aureus Using a Lytic Enzyme from Flavobacterium

The structure of the outer layers (cell wall and membrane) of Staphylococcus aureus was studied by electron microscope using a bacteriolytic enzyme from Flavobacterium sp. called the L-11 enzyme. Comparative studies on the morphology of bacteria before and after treatment with this enzyme and cell wall and membrane fractions obtained from bacteria after the enzyme treatment led to the following conclusions. (1) The cell wall of S. aureus is composed of morphologically distinct two layers which are both susceptible to the L-11 enzyme. (2) Between the cell wall and membrane, there is an electron opaque region which could not be stained using any of the methods tested. (3) Before treatment of bacteria with the enzyme the cell membrane could not be seen clearly. However, after enzyme treatment the membrane was clearly seen. (4) The infolding of the inner layer of the cell wall, forming a structure like a mesosome, was liberated by extensive enzyme treatment.     

A Histochemical Study of Cytoplasmic Changes During Wall Layer Formation in the Oospore of Albugo candida

The young multinucleate oogonium in Albugo is double-walled with an outer layer exhibiting a negative staining reaction for insoluble polysaccharides and an inner layer which is strongly PAS-positive. The oogonial nuclei exhibit an unusual staining behaviour with aniline blue showing an outer dark blue sheath of proteins surrounding a central hyaline nuclear core. Various histochemical localizations were performed for tracing the chronological sequence of development of the wall layers of the oospore. The first wall of the fertilized oospore was laid at the interphase of the periplasm and the ooplasm. Subsequent wall layers were formed both on the inner and outer side of the first oosporic wall. The second oosporic wall was formed just internal to the first one and exhibited faint PAS positivity. The third wall of the oospore was formed external to the first one and the PAS-negative material for this was apparently contributed by the periplasm. This wall layer at later stages acquired a ridged appearance and these ridges in a mature oospore appear as distinct “pegs”. The last wall to be formed is the innermost one and it completely surrounds the central ooplasm. This wall layer is callosic in nature.     

On the interpretation of twisted patterns in elongating plant cell wall: Informations obtained with ultracryotomy

Summary Ultracryotomy, which gives anin situ visualization of wall subunits, was used to study the texture of elongating wall of mung bean hypocotyl (Phaseolus aureus). The criss-crossed texture of the wall was confirmed. The controversed disposition of the subunits in twisted patterns was analyzed on the basis of observations of oblique sections on which a mild extraction may be performed. Despite the twisted appearance within one sectioning plane, no curved subunits were seen running between the criss-crossed layers. The results confirmed that the appearance of arcs is an illusion due to thin sectioning of successive strata in which the orientation of subunits rotates.     

An electron microscopic study on the mating tube formation in the heterobasidiomycete Tremella mesenterica

Ultrastructure of the mating tube formed in yeast haplont of the heterobasidiomycete Tremella mesenterica was studied by electron microscopy. Cell wall of the mating tube emerged as evagination of the inner layers, rupturing outer layers of the mother cell wall. Comparison with budding cells suggested that the tube emergence place at bud scar and the process of tube emergence was the same as that of bud emergence. Electron transparent vesicles of 0.1 m diameter were scattered in the cytoplasm of the mating tube. Nucleus-associated organelle was located at one side of the nuclear envelope which extended towards the mating tube. A few microtubules were detected in the mating tube, but their association with a nucleus was not clear. The cytoplasmic structure of the mating tube was discussed in comparison with that of hyphae of the filamentous fungi.