Ultrastructure of hyphae and ascospores in the genus Eremascus eidam

Hyphae and ascospores of Eremascus fertilis and E. albus were studied in ultrathin sections. The lateral wall of the hyphae had a thick electron-light inner layer and a thin dark outer layer. The septa had a simple central pore with or without a plug, and there were Woronin bodies in the vicinity. The wall of the ascospores of E. fertilis showed a thick light inner layer and a thin dark outer layer. In the wall of the spores of E. albus a dark fibrillar layer was present between the light inner layer and the dark outer layer. The spores of this species germinated with a tube the wall of which was continuous with a newly formed layer inside the spore wall.This investigation was supported by the Netherlands Organization for the Advancement of Pure Research (Z. W. O.)     

Ultrastructure of the ascospores of the new yeast genus Sporopachydermia Rodrigues de Miranda

Development of the ascospores of Sporopachydermia lactativora and S. cereana was studied in ultrathin sections. The spores have a very thick wall consisting of a thin dark outer layer and a double light inner layer the outer part of which is very wide and often irregular. During germination, this part disappears, the outer dark layer breaks up and the inner part of the light layer remains around the protoplast during development to a vegetative cell.This investigation was supported by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Tissue-specific expression of Pa18, a putative lipid transfer protein gene,during embryo development in Norway spruce (Picea abies)

A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

Comparative studies onChlorella cell walls: Induction of protoplast formation

Among 12 strains ofChlorella ellipsoidea, C. vulgaris, andC. saccharophila tested, 4 strains (1,C. ellpsoidea; 2,C. vulgaris; 1,C. saccharophila) formed osmotically labile protoplasts after treatment with mixtures of polysaccharide degrading enzymes. The relationship between enzymatical digestibility and structure or composition ofChlorella cell walls were studied by electron microscopy and staining techniques with some specific dyes. The cell wall structures of the 12Chlorella strains were grouped into three types: (1) with a trilaminar outer layer, (2) with a thin outer monolayer, and (3) without an outer layer. Protoplasts were formed only from the strains with a cell wall of Type 2. In the strains with a cell wall of Type 1, the outer layer protected the inner major microfibrillar layer against enzymatic digestion. The cell wall of Type 3 was totally resistant to the enzymes; the chemical composition of the cell wall would be somewhat different from that of other types.     

Wall formation in the saprolegniales

Summary The primary and secondary cysts of Saprolegnia ferax and the secondary cysts of Dictyuchus sterile have a two layered wall structure, the outer layer of which bears various types of spines. These spines, and the outer wall layer are derived from preformed structures (bars) found in the cytoplasm prior to encystment. Golgi derived vesicles appear to contribute to the inner layer of the primary cyst wall of S. ferax. The outer surface of the secondary cyst walls of this species has fibrils which are not embedded in matrix material.     

Plasmid Encoded AcrAB–TolC Tripartite Multidrug-Efflux System in Acidiphilium symbioticum H8

Acidophilic bacterium, Acidiphilium symbioticum H8, is resistant to high levels of several heavy metals, hydrophobic agents, and organic solvents. The ~9.6 kb plasmid pASH8, was purified, digested with HindIII, and sub-cloned in pUC19 at the respective site. Three different fragment size clones were achieved. The clones were completely sequenced and analyzed. The first clone encodes for a single putative open reading frame (ORF), which showed significant homology to several rusticyaninA1 proteins. The second clone encodes for a 43-kDa protein, which has conserved domain homology with several outer envelop TolC proteins. The clone with pASH8 tolC gene can functionally complement an Escherichia coli tolC mutant strain, making it resistant to several toxic hydrophobic agents, earlier for which it was sensitive. The tolC gene was found to be essential for imparting resistance to the clone toward these toxic hydrophobic agents. The third clone encodes for a putative 318-aa AcrA (acriflavine resistance protein A) protein and the clone was resistance to plasmid curing dye acriflavine. The clone also has a truncated ORF, which showed significant homology to cation-efflux pump AcrB. This study is the first to report a multi-drug efflux system to be encoded on a plasmid of any Acidiphilium strain.     

Aminergic and Indoleamine Accumulating Neurons in the Retina of the River Lamprey (Lampetra fluviatilis)

Tissues were processed for fluorescence microscopy of biogenic amines according to the method of Falck and Hillarp. Normal animals, and animals injected with α-methylnoradrenaline or 5,6-dihydroxytryptamine were used. Catecholamine containing neurons (junctional cells) occur in the innermost rows of cell bodies of the inner nuclear layer (INL) and close to the vitreous surface. Catecholamine containing fibers occur in three layers: (1) an outer layer around the innermost perikarya of the INL, which is a condition not found in retinas of gnathostome chordates; (2) a middle layer within the outer third of the inner synaptic layer (ISL), separated from the outer layer by ganglion cell axons; (3) a sparse inner layer within the innermost third of the ISL. A few catecholamine containing fibers were seen to extend from the innermost region of the INL to the outer synaptic layer. The position of the junctional cells in the lamprey corresponds to that in gnathostome chordates, but whereas all catecholamine containing fiber layers in gnathostomes are located sclerally to the optic fiber layer and within the ISL, the middle and the inner fiber layers in the lamprey occur vitreally to the optic fiber layer. Indoleamine accumulating neurons occur in the innermost row of perikarya of the INL and close to the vitreous surface. Those of the INL send fine, varicose branches to the ISL forming a network which is somewhat denser at the inner and outer borders of the ISL than in its middle. The indoleamine accumulating terminals do not ramify within the INL in contrast to the catecholamine containing terminals.     

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.     

Stratified microbial structure and activity within anode biofilm during electrochemically assisted brewery wastewater treatment

In a bioelectrochemical system (BES), microbial community of anode biofilm is crucial to BES performance. In this study, the stratified pattern of community structure and activity of an anode-respiring biofilm in a BES fueled with brewery wastewater was investigated over time. The anode biofilm exhibited a superior performance in the removal of ethanol to that of an open-circuit system. The electrical current density reached a high level of 0.55mA/cm² with a Coulombic efficiency of 71.4%, but decreased to 0.18mA/cm² in the late stage of operation. A mature biofilm developed a more active outer layer covering a less active inner core, although the activities of the outer and inner layers of biofilm were similar in the early stage. More Geobacter spp., typical exoelectrogens, were enriched in the outer layer than in the inner layer of biofilm in the early stage, while more Geobacter spp. were distributed in the inner layer than in the outer layer in the late stage. The inactive and Geobacter-occupied inner layer of biofilm might be responsible for the decreased electricity generation from wastewater in the late stage of operation. This study provides better understanding of the effect of anode biofilm structure on BES performance.     

Structure of the extraembryonic matrices around the benthic embryos of Patiriella exigua (Asteroidea) and their roles in benthic development: Comparison with the planktonic embryos of Patiriella regularis

The extracellular matrices (ECMs) surrounding the benthic embryos and larvae of the seastar Patiriella exigua and the planktonic embryos of Patiriella regularis were examined by transmission and scanning electron microscopy. Three ECMs surround unhatched embryos: An outer jelly coat, a fertilization envelope, and an inner hyaline layer. The ECMs of P. exigua are modified for supporting benthic development. The dense jelly coat attaches the embryo to the substratum, and the fertilization envelope forms a though protective case. In comparison, P. regularis has a less dense jelly coat and a thinner fertilization envelope. The hyaline layer of both species is comprised of three main regions: An intervillous layer overlying the epithelium, a supporting layer, and a coarse meshwork layer. Unhatched P. exigua have an additional outer amorphous layer that adheres to the fertilization envelope. As a result, the hyaline layer forms a continuous ECM that unites the embryonic surface with the fertilization envelope. Embryos of P. exigua removed from their fertilization envelopes lack the outer amorphous region, have a poorly developed hyaline layer, and do not develop beyond gastrulation. It appears that the substantial hyaline layer of P. exigua and its attachment to the fertilization envelope are essential for early development and that this ECM may function as a gelatinous cushioning layer around the benthic embryos. At hatching, the amorphous layer is discarded with the envelope. In contrast, an amorphous layer is absent from the hyaline layer of P. regularis. The demembranated embryos of this species have an ECM similar to that of controls and develop normally to the larval stage. © 1995 Wiley-Liss, Inc.     

Expression ofMycobacterium tuberculosis genes inEscherichia coli

TwoEscherichia coli clones expressingMycobacterium tuberculosis antigens were isolated from a gene-bank in the plasmid vector pBR 325. ‘Western blot’ analysis revealed the presence of a unique protein band of molecular weight 68,000 and 38,000, respectively in cellextracts from each clone. The 68,000 dalton antigen was found to be expressed onEscherichia coli outer surface. Plasmid DNA from a third clone could confer leucine independence on two differentleu B mutants ofEscherichia coli but not on mutants in otherleu genes, pointing to the possibility ofgenetic complementation. Thus,Mycobacterium tuberculosis DNA is capable of expression inEscherichia coli.     

Histochemical characteristics of spermatophore layers of Scylla serrata (Forskal) (Decapoda: Portunidae)

The spermatophores of S. serrata are protected by an outer thick chitinous layer and an inner thin non-chitinous one. Both layers are rich in acid mucopolysaccharides containing sulphated (outer layer) and carboxylic groups (inner layer). The proteins of the two layers show much tryptophan, but lack tyrosyl, sulfhydryl and disulphide groups. No phenols or phenol oxidases could be detected histochemically in either layer, suggesting the absence of phenolic tanning in the spermatophore. The physical properties, as revealed from treatment with acids and alkali, indicate the resistant nature of the outer layer; the inner layer easily shrinks or disrupts under such treatment. The outer layer, though resistant, is readily permeable to low molecular weight dye substances employed in permeability experiments. The mechanism of sperm release is recorded and discussed. It is suggested that, in S. serrata, the dehiscence of spermatophore may be caused by imbibing of low molecular weight substances by the sperm mass substances of the spermatophore while the latter is inside the spermatheca.     

Ultrastructure of the ascospores of some species of the Torulaspora group

Development and germination of the ascospores in species of the Torulaspora group of yeasts have been described. Most species had warty spores which, in sections, showed a dark outer layer consisting of the outer unit membrane of the prospore wall and a layer underneath formed at an early stage of development of the spores. In mature spores the light inner layer of the wall was delimited at the outside by a thin dark layer. The warts often contained dark material. The ascospores of two Pichia and three Debaryomyces species were studied for comparison; they differed in sections from the Torulaspora spores. The taxonomic implications of the ultrastructural observations have been discussed.     

Stratification and age-related differences in blubber fatty acids of the male harbour porpoise (Phocoena phocoena)

Fatty acid composition of blubber was determined at four body sites of 19 male harbour porpoises. A total of 65 fatty acids were quantified in each sample. The array of fatty acids contained in harbour porpoise blubber was similar to those found in other marine mammals. While chemical composition of total blubber was uniform over the body, with the exception of the caudal peduncle, vertical stratification was evident between the deep (inner) and superficial (outer) blubber layers. Fatty acids with chain lengths shorter than 18 carbons were present in significantly greater amounts in the outer blubber layer, while the longer-chain unsaturated fatty acids were more prevalent in the inner layer. This distribution suggests that the inner blubber layer is more active metabolically than the outer layer in terms of lipid deposition and mobilization. The degree of stratification between the two layers appears to increase with age, indicating a predictable turnover in the blubber layer of male porpoises. Harbour porpoise blubber contained high levels (2–27%) of isovaleric acid in the outer blubber layer, and these levels were positively correlated with age.Abbreviations Caud caudal dorsal body site - GC gas chromatograph - FA fatty acid(s) - IUPAC International Union of Pure and Applied Chemistry - PUFA polyunsaturated fatty acid(s) - II dor II dorsal body site - III dor III Dorsal body site - II Ven II ventral body site     

The tapetum inSchizaea pectinata (Schizaeaceae) and a comparison with the tapetum inPsilotum nudum (Psilotaceae)

A combination tapetum consisting of a cellular, parietal component and a plasmodial component occurs inSchizaea pectinata. A single, tapetal initial layer divides to form an outer parietal layer which maintains its cellular integrity until late in spore wall development. The inner tapetal layer differentiates into a plasmodium which disappears after the outer exospore has developed. In the final stages of spore wall development, granular material occurs in large masses and is dispersed as small granules throughout the sporangial loculus. No tapetal membrane develops. Comparisons are drawn with the combination tapetum found inPsilotum nudum.     

ENDOPHYTE-HOST ASSOCIATIONS IN FORAGE GRASSES. VIII. HETEROTHALLISM IN EPICHLOË TYPHINA

Experiments conducted indicate the mating system in Epichloë typhina to involve simple bipolar heterothallism. Perithecia were formed when condia were transferred to a compatible stroma. Perithecial development followed the growth of mycelium over the surface of the stroma. The perithecial stroma consisted of two layers: an inner layer composed of the original mycelium of the conidial stroma and an outer layer that appears to be composed of mycelium growing from the inner stromal layer. Perithecia were initiated in the outer layer, just above the interface of the two layers. Natural onset of perithecial formation was associated with evidence of visitation by a prasitic fly (Phorbia phrenione).     

Periostracal mineralization in the gastrochaenid bivalve Spengleria

We investigated the spikes on the outer shell surface of the endolithic gastrochaenid bivalve genus Spengleria with a view to understand the mechanism by which they form and evaluate their homology with spikes in other heterodont and palaeoheterodont bivalves. We discovered that spike formation varied in mechanism between different parts of the valve. In the posterior region, spikes form within the translucent layer of the periostracum but separated from the calcareous part of the shell. By contrast those spikes in the anterior and ventral region, despite also forming within the translucent periostracal layer, become incorporated into the outer shell layer. Spikes in the posterior area of Spengleria mytiloides form only on the outer surface of the periostracum and are therefore, not encased in periostracal material. Despite differences in construction between these gastrochaenid spikes and those of other heterodont and palaeoheterodont bivalves, all involve calcification of the inner translucent periostracal layer which may indicate a deeper homology.     

The cell wall of the chlamydomonad flagellate,Gloeomonas kupfferi (Volvocales,Chlorophyta)

Summary The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.     

The fine structure of the pellicle ofEuglena gracilis as revealed by freeze-etching

Summary The ultrastructure of the pellicle ofEuglena gracilis (Klebs) Z strain was studied using the freeze-etching technique and the results were correlated with data obtained from thin sections of fixed material.Examination of freeze-etched pellicles reveals an outer particulate layer and an inner striated layer. The particles of the outer layer measure approximately 150 Å in diameter. The striations of the inner layer are about 50 Å wide and are separated from each other by about 35 Å. A broad repeating pattern is also visible with a periodicity of about 450 Å. When deep etching is employed, a smooth outer layer is seen covering the particulate layer. This is probably the outer surface of the plasma membrane. Mucilage is present on the outer surface of the cell and is seen as a substructure of threads superposed on the smooth layer of plasma membrane.Thin sectioning also shows a striated layer interior to the plasma membrane. This appears to be identical to the striated layer seen after freeze-etching.