Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8–2.9 up to 4.0–4.4 μm. The diameter of the hyphal elements remained constant, around 4 μm, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations.     

Characterization of two “Metabacterium” sp. from the gut of rodents

A new giant Gram-negative non-cultivatable symbiotic endospore-forming bacterium was found in the gut of the European hamster. This “Metabacterium” sp., provisionally named “Metabacterium criceti”, sp. n., has a length of approximately 20 μm and thickness of 4 μm. It forms 1 to 2 cylindrical endospores, approximately 9 μm long and 1.4 μm thick. TEM-micrographs show a cell wall structure characteristic of Gram-negative bacteria. Vegetative cells are filled with granules 0.3 μm in diameter which resemble starch granules. The reproduction occurs with binary fission and by formation of two endospores. Of thirteen biochemical components sought, four,i.e. glycogen, triacylglyceroles, peroxidase and alkaline phosphatase, were not found. Starch, acid mucosubstances, DNA, RNA, lipids, proteins, adenosine triphosphatase and acid phosphatase were found in different patterns, depending on the developmental stage of the bacterium. In the vegetative cell stage all these components, with the exception of starch, were found. In the endospore-bearing cell stage, only the starch-like cell component granulose could be detected. In free endospores only DNA, RNA and acid phosphatase were found. Some of the components,i.e. DNA, lipids, starch-like granulose, were linked to certain cell substructures, the distribution of others,viz. polysaccharides, RNA, adenosine triphosphatase and proteins was diffuse. The lipids, found only in vegetative cells, were associated with the cell wall. Presented in part during the19th Congress of the Czechoslovak Society for Microbiology, Košice (Slovakia) September 14–17, 1992. An erratum to this article is available at .     

Calcium-induced sperm fusion in Zea mays L.

 In this study, we examined morphological changes of isolated maize (Zea mays L.) sperm cells in the presence of Brewbaker and Kwack salts (BKS) or the individual components of BKS using light, transmission electron and scanning electron microscopy. Freshly isolated sperms are 7.5 μm in diameter. Treatment with BKS for 5 h resulted in large cells with a diameter up to 41 μm. Staining of sperm nuclei with 4′, 6-diamidino-2-phenylindole (DAPI) revealed two or more nuclei in a single cell, suggesting that BKS induces cell fusion. Treatment with each BKS component showed that cell fusion occurs only in the presence of calcium nitrate. Use of several calcium salts showed the same results, suggesting that the calcium ion, alone, is responsible for the observed cell fusion. Further studies were conducted to examine the relationship between calcium distribution and sperm location in pollen tubes using chlorotetracycline and DAPI. Growing maize pollen tubes exhibited a high membrane calcium region within 20–50 μm from the tip. The Sperms are found no closer than 90 μm to the tip of the tube, suggesting that sperms are located in a low calcium region prior to being released to the degenerating synergid. Received: 12 August 1996 / Revision accepted: 6 December 1996     

The production of nematode-immobilizing secretory cells by Climacodon septentrionalis

The ability of Climacodon septentrionalis to immobilize and kill a mycophagous nematode (Aphelenchoides sp.) in vitro is described for the first time. Two isolates produced droplets (20–45 μm in diameter) that formed at the apices of tall, stalked, and branching secretory cells (700–1,500 μm tall). On 2% modified malt extract agar, nematodes became enveloped in the droplets, which restricted their ability to move and resulted in complete immobilization and death within several hours of contact. The rate of decomposition of the nematodes varied considerably, with most individuals persisting for weeks whereas others were degraded within several days and appeared to be colonized by dense hyphal growth. This study provides the first documentation of a non-agaricoid fungus producing secretory cells that are able to immobilize nematodes.     

Golgi-type I and Golgi-type II Neurons in the Ventral Anterior Thalamic Nucleus of the Adult Human: Morphological Features and Quantitative Analysis

The morphological and quantitative features of neurons in the adult human ventral anterior thalamic nucleus were studied in Golgi preparations. Two neuronal types were found and their quantitative features were studied. Golgi-type I neurons were medium to large cells with dense dendritic trees and dendritic protrusions and short hair-like appendages. They have somatic mean diameter of 30.8 μm (±9.4, n = 85). They have an average 100.3 dendritic branches, 48.97 dendritic branching points, and 58.85 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 3.1 μm (±1, n = 80), 1.85 μm (±0.8, n = 145), and 1.5 μm (±0.4, n = 160), respectively. Golgi-type II neurons were small to medium cells with few sparsely branching dendrites and dendritic stalked appendages with or without terminal swellings. They have somatic mean diameters of 22.2 μm (±5.8, n = 120). They have an average 33.76 dendritic branches, 16.49 dendritic branching points, and 21.97 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 1.6 μm (±0.86, n = 70), 1.15 μm (±0.55, n = 118), and 1 μm (±0.70, n = 95), respectively. These quantitative data may form the basis for further quantitative studies involving aging or some degenerative diseases that may affect cell bodies and/or dendritic trees of the Golgi-type I and/or Golgi-type II thalamic neurons.     

Photoreceptor ultrastructure in the Antarctic mussel shrimp Acetabulastoma (Crustacea; Ostracoda), a parasite of Glyptonotus antarcticus (Crustacea; Isopoda)

The ultrastructure of the nauplius eye of the tiny Antarctic ostracode Acetabulastoma sp. is described and conclusions about its possible function are drawn. Each of the three eye-cups measures approximately 20 μm in diameter and is optically isolated from its neighbour by screening pigments, which are contained in pigment cells behind a tapetum of concentrically arranged, ca. 1-μm-long and 0.1-μm-thick, crystals. Three and sometimes four separate rhabdoms with microvilli measuring 50–60 nm in diameter project from the concave side of the tapetum up to 5 μm deep into the eye-cup interior, which is filled by the retinula cell bodies with their spherical nuclei and various organelles. Desmosomes and microtubules are seen and light-induced cell or membrane damage was minimal. The observations suggest that the Acetabulastoma eye has photoreceptors that can tolerate an exposure to bright light and it may be used to inform its owner of the approach of danger, the depth of water, and/or the season. Accepted: 27 August 1998     

Tracheids in white spruce seedling's long lateral roots in response to nitrogen availability

This study examined how the availability of inorganic nitrogen (N) modified the anatomical characteristics of white spruce (Picea glauca (Moench) Voss) roots related to their hydraulic properties. Seedlings were grown for one growing season in 4 L capacity pots filled with sand under one of three N levels: low (10 ppm), medium (50 ppm) and high (125 ppm). First order lateral roots with intact tips were sampled from dormant seedlings in October. Root segments were collected from 4, 10, and 14 cm distances above the root tip for fixation and sectioning and for maceration. Additional specimens were collected from the 4 and 14 cm distances for maceration and scanning electron microscopy of xylem pits. Root diameter and surface area occupied by the xylem in root cross sections increased basipetally in all treatments but exceptions were found. Higher N-levels significantly increased root diameter and surface area occupied by the xylem. In the two higher N treatments secondary root development was more advanced near the root tip than in the low N treatment. There was a strong positive correlation between root diameter and cross-sectional root area occupied by the xylem (30–50% of the root cross section) but not in portions with little secondary development. Non-conducting space within the xylem occupied 10–13% of its cross-sectional surface. Tracheids of the primary xylem were larger, had larger lumens but thinner cell walls than those of the secondary xylem. Low N treatment seedling tracheids had smaller total cross-sectional area, less lumen, and less cell wall surface area than the two other N treatments. Tracheid diameter means were between 19–20 μm in the high and medium N treatments, and 15.2 μm in the low N treatment. The range was 4.5–51.3 μm. Tracheid length was not significantly affected by N. The average tracheid was about 1000 μm long, and the range was 110–3530 μm. Pit-border diameters ranged between 4.1–20.6 μm (average 10–11 μm) and were not affected by the N treatment. Pit aperture diameters were within 0.62–10.2 μm range (average between 3–4 μm) and were also not significantly affected by the N treatment, although tracheids from the medium N-treatment roots tended to have larger apertures. The pit border diameter equals that of the margo while the aperture size should be similar to that of the torus of the pit membrane. If the capacity for axial water transport in spruce roots is affected by N, it would be by its impact on conduit diameter and, possibly on the pit-membrane pore sizes but not by changes to conduit length and to the size of the pit membrane surface area. This revised version was published online in June 2006 with corrections to the Cover Date.     

A study of the process of synchronisation and micronucleation in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and the monitoring of an isolation procedure for micro-nuclei and micro-protoplasts by confocal laser scanning microscopy and flow cytometry

The process of synchronization and micro-nuclei induction in a suspension culture of Beta vulgaris, was induced by the sequential treatment with the DNA-synthesis inhibitor aphidicolin (30 μM, 24 h) and the spindle-toxin amiprophos-methyl (32 μM, 24 h). Mitotic arrest of divisions, spreading of G2-metaphase chromosomes, re-grouping of chromosomes, formation of a nuclear cell wall around single and re-grouped chromosomes and restitution of nuclei with a doubled DNA content was observed. The process of micro-nucleation was induced much less efficiently in Beta vulgaris than in Nicotiana plumbaginifolia. Cytological observations and monitoring of the process with flow cytometry and confocal laser scanning microscopy, was essential to follow up the course of events and to monitor the development of an efficient procedure for micro-protoplast isolation. Micro-nucleated protoplasts were fractioned by iso-osmotic Percoll gradient centrifugation to obtain heterogeneous micro-protoplast populations with cytoplasts and micro-protoplasts of different size. An enriched fraction with small sub-diploid micro-protoplasts was obtained with the equivalent DNA content of 1–4 chromosomes, as revealed by confocal laser scanning microscopy and flow cytometry. Sub-diploid micro-protoplasts with DNA amounts equivalent to 1–4 chromosomes were predominantly observed in the size classes of 1.8–10.2 μm at a frequency of 34.2–34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micro-nucleation followed the same course of cellular events as observed in N. plumbaginifolia. The correlation between DNA content and size of micro-nuclei and micro-protoplasts was not linear and affected by the degree of DNA condensation, total amount of DNA, and the presence of cytoplasm.     

Chromosomal unit fibers in Drosophila

Chromosomal unit fibers consisting of long, regular fibers of about 0.40 m diameter were obtained from disintegrated, isolated chromosomes of two Drosophila melanogaster cell lines. In one cell line with an essentially normal karyotype, three clearly defined size classes of 15, 13, and 11 m length were observed corresponding to the three larger chromosomes of Drosophila. In a cell line carrying an additional translocation between the two largest chromosomes a 19 m fiber derived from the translocation chromosome was observed. Direct determinations of the DNA content per m length of Drosophila unit fibers show that DNA is contracted by a factor of about 1400x in agreement with calculations based on the length of the unit fibers and the known DNA content of the individual Drosophila chromosomes. These findings support our previously proposed model for the unit fiber sub-structure of chromosomes as being derived by a hierarchy of coiling with the corresponding contraction ratios being 7 (100 Å string of nucleosomes), 5 to 6 (250–300 Å thick nucleohistone fiber), and about 40 (unit fiber), resulting in a total contraction of DNA in unit fibers in the order of 1400x.     

Hyphal anastomosis inPyricularia oryzae cav.

Summary Hyphal anastomosis inPyricularia oryzae occurs naturally in the lower epidermal cells and in the vascular bundles of young lesions on rice. In those cells the invading blast fungus are active. Two hyphal cells lying side by side start an anastomosis by forming two, one from each, very short penetration pegs which are opposite to each other. The cell wall of the pegs and the wall in the vicinity of them are then gradually eliminated and thus form a fusion aperture of 0.2–0.6 m in diameter that is big enough for the migration or exchange of nucleus and cytoplasm between two hyphal cells. The explanation of genetical variation inP. oryzae may be sought on the basis of the ultrastructural evidence of hyphal anastomoses presented in this paper.     

Ultrastructure of the marine predatory flagellate <Emphasis Type="Italic">Metromonas simplex</Emphasis> Larsen et Patterson, 1990 (Cercozoa)

The ultrastructure of the marine predatory flagellate Metromonas simplex Larsen et Patterson was studied. The cell is surrounded by a low-contrast fibrous layer composed of thin hairs covered by a thin bilayer membrane and an outer layer of thin short fibers. The plasmalemma lies under these layers. The predator captures whole cells of the prey, usually bodonids or chrysomonads. The cytostome as a cell pocket is undetectable. The long flagellum bears very thin mastigonemes (hairs) with lengths of 0.8–1.0 μm; the short flagellum is naked and reduced in length. The transitional zone lacks spirals or other additional elements. The transversal plate is elevated on the cell surface. The flagellar root system is very simple and has one microtubular band which originates near the kinetosomes. The latter are parallel to each other and interconnected by fibrous bridges. The vesicular nucleus, Golgi apparatus, and endoplasmic reticulum are of typical structures. The oval mitochondria of 0.6–2.5 μm contain lamellar cristae. The cylindrical extrusomes (trichocysts) found in the cytoplasm have lengths of 1.0–1.4 μm and diameters of 0.12–0.08 μm. The trichocysts have a wheel-shaped structure with 13 spokes visible in cross-sections. The contractile vacuole is absent. The similarity that M. simplex shares with Metopion fluens Larsen et Patterson, cryothecomonads, and other predatory flagellates is discussed.     

Morphological differences among three species of newly settled pocilloporid coral recruits

 Investigation of the life history of corals is hampered by an inability to identify early recruits. In this study, the pattern of formation and morphology of the juvenile skeletons of three laboratory-reared pocilloporids, Seriatopora hystrix, Stylophora pistillata and Pocillopora damicornis, were compared to determine whether they could be reliably distinguished. The pattern of skeleton formation, including the origin and structure of the septa, columella and corallite wall was similar in all species. Following the completion of the primary corallite wall after 4–5 days, these species could be identified by differences in the diameter of the primary corallite. The mean diameter (±SE) of each species differed markedly: S. hystrix 400 ± 2.7 μm, range 325–450 μm; S. pistillata 505 ± 3.5 μm, range 400–550 μm; P. damicornis 697 ± 7.5 μm, range 492–885 μm. Values for the primary corallite diameter overlapped in only 3% of samples, demonstrating the potential utility of this feature as a tool for classifying recruits obtained from the field. Accepted: 4 January 2000     

The seasonal abundance and vertical distribution of the <3-μm phytoplakton in the north basin of Lake Biwa

The seasonal changes in the size-fractionated chlorophylla concentrations (<3 μm, 3 to 25 μm, and >25 μm) were investigated at a pelagic site of the north basin of Lake Biwa during June to December 1985. Autofluorescing plankton cells in the <3-μm fractions were also examined using the fluorescein isothiocyanate staining epifluorescence microscopic technique. The <3-μm phytoplankton (usually dominated by chroococcoid cyanobacteria except for a few cases dominated by small eukaryotes) showed a clearly different pattern of seasonal change compared with the larger fractions. That is, from August to early September, chlorophylla of the larger fractions declined considerably, while the <3-μm chlorophylla did not decrease significantly. Moreover, cyanobacterial cell density in the <3-μm fraction showed a maximum value (2–3.5×10⁵ cells·ml⁻¹) during this period. The relative contribution of the <3-μm chlorophylla to the total chlorophylla increased from <5% to 45% during the course of this change. No clear vertical trend in the distribution and composition of the <3-μm phytoplankton was found, except that relatively large cyanobacteria (>4 μm³) appeared at a depth of 15m but not at 0,5 and 10 m from late July to August. These large cells were also found in November and December. The drastic seasonal change of phytoplankton size structure occurring in this basin was discussed in relation to grazing, nutrient depletion and sinking. Contribution from Otsu Hydrobiological Station, Kyoto Univeristy (No. 308, foreign language series).     

Grazing by the Antarctic sea-ice ciliate Pseudocohnilembus

Potential uptake and clearance rates of fluorescent microspheres (FM) from 0.25 to 4.05 μm diameter were determined for the non-loricate ciliate Pseudocohnilembus sp. from Antarctic sea ice. The percentage of ciliate cells that ingested FM after 20 min incubation decreased with increasing particle diameter. Pseudocohnilembus sp. ingested FM between 0.25 and 4.05 μm in diameter. We offered FM at concentrations less than natural concentrations for plankton plus detrital material and obtained clearance rates less than those previously reported for bactivorous ciliates. Clearance rates were 3.6–5.4 nl cell⁻¹ h⁻¹ for FM 0.5 and 1 μm diameter, respectively, but decreased to 1.1 nl cell⁻¹ h⁻¹ for 1.97 μm diameter and 1.4 nl cell⁻¹ h⁻¹ for 4.05-μm-diameter FM. Clearance and uptake rates of FM 0.5 and 1 μm diameter indicate that Pseudocohnilembus sp. principally grazes on bacteria-sized particles. However, it can also ingest organisms as large as nanoplankton and may graze particles as small as femtoplankton and colloids. This suggests a feeding strategy that may suit the temporal and spatial changes in food availability in the sea-ice habitat. Accepted: 13 August 2000     

Secondary Intracerebral Blastomycosis with Giant Yeast Forms

Secondary central nervous system (CNS) blastomycosis is an unusual manifestation of blastomycosis. We report a case of recurrent intracerebral blastomycosis that presented histopathologically with giant yeast-like cells and multinucleation that mimicked Coccidioides immitis. The yeast forms of Blastomyces dermatitidis usually range in size from 8 to 20 μm in diameter. Large or giant yeast forms (20–40 μm) are rare. The four cases previously reported in the literature involving giant yeast cell forms of B. dermatitidis are reviewed here. Intracerebral blastomycosis should be suspected in patients with signs and symptoms of CNS lesions and histories of primary blastomycosis, or treatment with corticosteroids, or comprised immune systems. The diagnosis should be confirmed by culture which presents typical biphasic microbiologic features.     

Asterostroma species (Basidiomycota) from mangrove forests in Japan

A new homobasidiomycete, Asterostroma macrosporum, was found in mangrove forests of Iriomote Island, Japan. This species is morphologically characterized by having resupinate basidiomata, a monomitic (asterodimitic) hyphal system, simple septate generative hyphae, dextrinoid asterosetae, four sterigmate basidia and globose, tuberculate and amyloid basidiospores measuring 8.5–11 × 7.5–9 μm. It is similar to A. muscicola, but basidiospores in the latter are smaller (7–8 × 5.5–7 μm). Furthermore, phylogenetic analysis using internal transcribed spacer (ITS) region revealed that A. macrosporum is distinctly separated from A. muscicola. In Japan, A. muscicola is widely distributed in warm-temperate to subtropical regions, growing on a variety of broadleaved trees including mangroves, while A. macrosporum has been found only on mangroves.     

The effects of exopolymers on cell morphology and culturability of Leuconostoc mesenteroides during starvation

Biofilm formation by bacterial cells can be used to modify the subsurface permeability for the purpose of microbial enhanced oil recovery, bio-barrier formation, and in situ bioremediation. Once injected into the subsurface, the bacteria undergo starvation due to a decrease in nutrient supply and diffusion limitations in biofilms. To help understand the starvation response of bacteria in biofilms, the relationship between exopolymer formation and cell culturability was examined in a batch culture. The average cell diameter was observed to decrease from 0.8 μm to 0.35 μm 3 days after starvation began. Cell chain fragmentation was also observed during starvation. Cells that underwent starvation in the presence of insoluble exopolymers showed a slower rate of decrease in cell diameter and in cell chain length than cells without insoluble exopolymers. The rate of decrease in the average cell diameter and cell chain length were determined using a first order decay model. Cells starved in the presence of exopolymers showed greater culturability than cells starved without exopolymers. After 200 days starvation, 2.5 × 10⁻³% cells were culturable, but no increase in cell number was observed. During starvation, the exopolymer concentration remained constant, an indication that the exopolymer was not consumed by the starving bacteria as an alternative carbon or energy source. Received: 8 April 1999 / Received revision: 16 July 1999 / Accepted: 6 August 1999     

Cell culture density dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B-cells

Murine pre-B-cells grown in the presence of lower (1 μM) or higher (5 μM) concentration of cadmium chloride were separated into 13 fractions by centrifugal elutriation. The rate of DNA synthesis after cadmium treatment determined in permeable cells was dependent on cell culture density during cadmium treatment. Cell cycle analysis revealed a shift in the profile of DNA synthesis from replicative to repair DNA synthesis upon cadmium treatment. The study of the relationship between cell culture density and cell diameter at lower and higher cell densities in the presence of 1 μM cadmium chloride concentration showed that a. at 5×10⁵ cell/ml or lower densities cells were shrinking indicating apoptotic changes, b. at higher cell culture densities the average cell size increased, c. the treatment of cells with low CdCl₂ concentration (1 μM) at higher cell culture density (>5×10⁵ cell/ml) did not change significantly the average cell diameter. At 5 μM cadmium concentration and higher cell culture densities (>5×10⁵ cell/ml) the average cell size decreased in each elutriated fraction. Most significant inhibition of cell growth took place in early S phase (2.0–2.5 C value). Apoptotic chromatin changes in chromatin structure after cadmium treatment were seen as large extensive disruptions, holes in the nuclear membrane and stickiness of incompletely folded chromosomes.     

Morphological changes of <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis> induced by CF66I,an antifungal compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The morphological effects of CF66I, an antifungal compound produced by Burkholderia cepacia, on growing hyphae of Fusarium oxysporum were studied by fluorescence microscopy (FM) and transmission electron microscopy (TEM). At 20 μg/ml, CF66I strongly inhibited growth and induced significant changes of the hyphal morphology. These changes included swelling of hyphae with considerable thickening cell wall and abnormal chitin deposition, which was indicative of the alterations in cell wall structure. Furthermore, fluorescein diacetate (FDA) staining indicated the loss of intracellular esterase activity. CF66I probably inhibits fungal growth by interfering with the cell metabolic pathways. At 120 μg/ml, CF66I killed F. oxysporum (accompanied by propidium iodide permeation, intracellular cytoplasm leakage and crushing of hyphal tips), probably by direct damage to the cell membrane. Thus, there are two different antifungal mechanisms of CF66I, depending on its concentration, and further studies on this compound might be useful for us to develop a new class of antifungal agents.     

Karyotyping of the narrow-leafed lupin (Lupinus angustifolius L.) by using FISH, PRINS and computer measurements of chromosomes

BAC (bacterial artificial chromosome) clones from the genomic BAC library of the narrow-leafed lupin (Lupinus angustifolius) were used for cytogenetic mapping of mitotic metaphase chromosomes of that species by the BAC-FISH technique. Location of the clones, together with cytogenetic markers localised earlier by FISH (fluorescencein situ hybridisation) and PRINS (primedin situ DNA labelling), was combined with computer-aided chromosome measurements, to construct the first idiogram of the narrow-leafed lupin. The chromosomes are meta- or submetacentric; the mean absolute chromosome lengths range from 1.9 μm to 3.8 μm, and mean relative lengths from 1.6% to 3.3%. Data concerning linkage of resistance to 2 fungal pathogens as well as assignment of the second linkage group to the appropriate chromosome are given for the first time.