Effects of Fumidil B on the spore of Nosema apis and on lipids of the host cell as revealed by freeze-etching

The epithelial cells of the midgut of honey bees, Apis mellifera, infected with Nosema apis showed young and mature spores randomly distributed in the cytoplasm. In these cells, only mitochondria and protein granules were observed. After treating infected bees with Fumidil B, an ultrastructural alteration in the spore membrane, especially in the young spore, was observed. At the same time, lipid granules appeared in the cytoplasm, mostly around the spores. The number of protein granules also increased.     

Characterization of Bacillus anthracis Persistence In Vivo

Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.     

Effect of Heat on Spores of Rough and Smooth Variants of Bacillus stearothermophilus

Spores of variants of Bacillus stearothermophilus were subjected to activating and lethal temperatures. Spore suspensions which were incubated longer contained a higher percentage of spores of the rough variant. The effect of sublethal heat on spore suspensions containing mixed variants (rough and smooth) was difficult to measure at sublethal temperatures (110 C), since the rough variant was not as heat-resistant. While the rough variant was activated in a shorter time, the smooth variant was not activated; when the smooth variant was activated, the rough was killed. A higher percentage of the smooth variant was forced into dormancy after being held at 50 C for 30 hr than the rough variant. When mixed populations were subjected to a lethal temperature (120 C), the curves only reflected the smooth variant. Since the curves which represented the smooth variant or mixtures containing the smooth variant were not linear, this was thought to be due to activation overbalancing the lethal effect. This research emphasized the importance of variants in explaining differences in spore resistance among spore suspensions of the same strain.     

Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K

Dissemination of Bacillus anthracis from the respiratory mucosa is a critical step in the establishment of inhalational anthrax. Recent in vitro and in vivo studies indicated that this organism was able to penetrate the lung epithelium by directly entering into epithelial cells of the lung; however the molecular details of B. anthracis breaching the epithelium were lacking. Here, using a combination of pharmacological inhibitors, dominant negative mutants, and colocalization experiments, we demonstrated that internalization of spores by epithelial cells was actin-dependent and was mediated by the Rho-family GTPase Cdc42 but not RhoA or Rac1. Phosphatidylinositol 3-kinase (PI3K) activity was also required as indicated by the inhibitory effects of PI3K inhibitors, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and a PI3K dominant negative (DN) mutant Δp85α. In addition, spore entry into epithelial cells (but not into macrophages) required the activity of Src as indicated by the inhibitory effect of Src family kinase (SFK) inhibitors, PP2 and SU6656, and specific siRNA knockdown of Src. Enrichment of PI3K and F-actin around spore attachment sites was observed and was significantly reduced by treatment with SFK and PI3K inhibitors, respectively. Moreover, B. anthracis translocation through cultured lung epithelial cells was significantly impaired by SFK inhibitors, suggesting that this signaling pathway is important for bacterial dissemination. The effect of the inhibitor on dissemination in vivo was then evaluated. SU6656 treatment of mice significantly reduced B. anthracis dissemination from the lung to distal organs and prolonged the median survival time of mice compared to the untreated control group. Together these results described a signaling pathway specifically required for spore entry into epithelial cells and provided evidence suggesting that this pathway is important for dissemination and virulence in vivo.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Smooth Brucella strains invade and replicate in human lung epithelial cells without inducing cell death

Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar (A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5–3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity.     

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p?相似文献   

Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group

The polarity and magnitude of primary electric charges carried by basidiospores in the airborne state were investigated in living fungal fruiting bodies under natural forest conditions using a portable experimental device designed by the author. The operating principle was the falling of spores in the homogeneous horizontal electric field. The vertical and horizontal components of the trajectories of the spores were determined according to their deposition sites on electrodes (vertical metal plates). Altogether 33 samples of spores were examined for polarity, 10 of these samples (with 10⁴–10⁶ spores per sample) also were used to calculate the mean spore charge-to-mass quotient and the mean spore charge. The detection limits of spore charge-to-mass quotient varied in the range from (4.9±2.3)×10^?5 to (1.36±0.33)×10^?4 C kg^?1. Basidiospores (subglobose, smooth, diameter of 4–6 μm) of the closely related (sibling) species Phellinus alni, P. nigricans, P. populicola and P. tremulae (Hymenochaetales, Basidiomycota) carried positive electrical charges that have mean values from 48 to 305 elementary charges. The intraspecies variation of the spore charge could depend on the natural variation in spore size.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Fungus Spores as Uniform Reference Particles for Use in Absolute Cell Counts

Spores of several different fungi were tested as microspheres in a recently described method of making absolute cell counts of bone-marrow cells. For a particular fungus to qualify for use in the counting method, it must be readily obtained and must produce large quantities of spores that are highly uniform in size. Thus far, the most satisfactory microspheres we prepared were the basidiospores of Calvatia gigantea, the giant puffball. This common field organism is a prolific spore producer. A simple procedure for obtaining spore suspensions was devised. Another source of microspheres is the conidia of species of Aspergillus and Penicillium. These widespread organisms were easily grown in the laboratory and also provided heavy spore harvests. It was suggested that conidia of these or additional species of fungi may be found useful in other kinds of research requiring small, highly uniform particles.     

Effect of edaphic factors and seasonal variation on spore density and root colonization of arbuscular mycorrhizal fungi in sugarcane fields

Sugarcane fields in 14 different study sites were analyzed for the presence of different arbuscular mycorrhizal fungal (AMF) spores. A total of 23 AMF species representing four genera were identified, among which Glomus fasciculatum and G. mosseae were the dominant species. The mean spore density in the root-zone soils of sugarcane plants varied from 119 to 583 per 100 g of soil, and the mean percentage root colonization varied from 60 to 89 %. A study of the effect of edaphic factors on AM spore density and percentage root colonization revealed a positive correlation between pH and AMF spore density and root colonization and a negative correlation between electrical conductivity, nitrogen, and phosphorus. A positive correlation was observed between AMF spore density and root colonization. Season was also found to play a vital role in determining AMF spore density and percentage root colonization, with high spore density and root colonization observed during the summer season and lower spore densities and root colonization during the winter season.     

Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.     

白色念珠菌对人口腔黏膜上皮角质细胞凋亡和增殖的影响

目的:探索白色念珠菌对人口腔黏膜上皮角质细胞(KB细胞)凋亡和增殖的影响。方法:分别培养孢子型白色念珠菌和菌丝型白色念珠菌,取健康志愿者的颊粘膜制备KB细胞,分别加入孢子型白色念珠菌(孢子组)和菌丝型白色念珠菌(菌丝组),另外取单纯KB细胞作为对照组。对比分析各组细胞凋亡率及各个周期的细胞数,统计分析各组细胞增殖指数(PI)。结果:菌丝组凋亡率显著高于对照组及孢子组(P0.05);菌丝组在G0/G1期的细胞比例均显著低于对照组及孢子组(P0.05);菌丝组在S期和G2/M期的细胞比例均显著高于对照组及孢子组(P0.05);菌丝组的PI显著高于孢子组和对照组(P0.05)。结论:菌丝型白色念珠菌可诱导KB细胞凋亡率的上升及改变KB细胞周期变化,并引起KB细胞的PI升高,对于临床上治疗口腔念珠菌病具有重要的指导意义。     

Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: <Emphasis Type="Italic">B. coagulans</Emphasis> and <Emphasis Type="Italic">B. subtilis</Emphasis> (natto)

Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2 h, followed by challenges with LPS for 3 and 18 h. Post-treatment assay was done by initial treatment of the cells with LPS for 18 h, followed by the spores for 3 and 6 h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3 h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.     

Evidence that the fertilization envelope blocks sperm entry in eggs of Xenopus laevis: interaction of sperm with isolated envelopes.

Single-celled myxamoebae undergo differentiation into either stalk cells or spore cells during a 24-hr period in Dictyostelium discoideum. This study employed ultramicrochemical techniques and enzymatic cycling to assess the presence of cell-specific events in spore and stalk cells. Freeze-dried sections of one organism were assayed in 0.1 μl of reaction mixture. This method was used to determine the extent of localization of trehalose in spore cells and stalk cells during development.Trehalose was low in the early stages of differentiation to about 20 hr when the level started to increase. In developing spore cells, the trehalose level increased sixfold during the last 5 hr of development. Likewise, the entire stalk contained trehalose when the stalk was first formed. At mature sorocarp, trehalose levels were the same in spores and the apex of the stalk. There was a decreasing gradient of trehalose down the stalk. The bottom one-fourth of the stalk was devoid of this disaccharide. Therefore, trehalose was degraded from an area of the stalk where it was localized earlier in development.The results of this investigation negate the assumption that trehalose is never present in the stalk. Although trehalose was found in spore cells, prestalk cells also contained high trehalose levels. The stalk cell-specific trehalose was not retained during differentiation, however, but was apparently degraded in the mature stalk cell.     

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0–8.5 × 10⁷ spores/cm²-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R², 0.653–0.999).     

Microanatomy and ultrastructure of the foot of the infaunal bivalve Tegillarca granosa (Bivalvia: Arcidae)

In this study, the morphology and ultrastructure of the foot of Tegillarca granosa was compared with the bivalves from different habitats. The sediment of habitat of T. granosa is mostly a mixture of sand (68.93%) and mud (24.12%). The foot is wedge-shaped with multiple projections on the surface and covered with ciliary tufts. The epithelial layer is simple and composed of ciliated columnar epithelia and mucous cells. Although the mucous cells are distributed mostly in the epithelial layer, they are developed even in the connective tissues and muscle layers, and the mucous cells mostly contain acidic carboxylated mucosubstances. From the TEM observation, secretory cells are classified into three types. Type A secretory cell has a goblet form and is most widely distributed among the three types. Type B secretory cell has an oval form and the secretory granule has fibrous substance. Type C secretory cell has an elongated elliptic form and membrane-bounded secretory granules. The muscle fiber bundles are composed mainly of smooth muscle fibers. The smooth muscle fibers can be divided into two types. Type A muscle fibers have evenly distributed thick microfilaments between the thin microfilaments of cytoplasm. Type B muscle fiber has cluster of condensed microfilaments in the medulla cytoplasm while the cortical cytoplasm has loose distribution of thin microfilaments.     

STRUCTURE OF THE TOAD'S URINARY BLADDER AS RELATED TO ITS PHYSIOLOGY

The structure of the urinary bladder of the toad Bufo marinus was studied by light and electron microscopy. The epithelium covering the mucosal surface of the bladder is 3 to 10 microns thick and consists of squamous epithelial cells, goblet cells, and a third class of cells containing many mitochondria and possibly representing goblet cells in early stages of their secretory cycle. This epithelium is supported on a lamina propria 30 to several hundred microns thick and containing collagen fibrils, bundles of smooth muscle fibers, and blood vessels. The serosal surface of the bladder is covered by an incomplete mesothelium. The cytoplasm of the squamous epithelial cells, which greatly outnumber the other types of cells, is organized in a way characteristic of epithelial secretory cells. Mitochondria, smooth and rough surfaced endoplasmic reticulum, a Golgi apparatus, "multivesicular bodies," and isolated particles and vesicles are present. Secretion granules are found immediately under the plasma membranes of the free surfaces of the epithelial cells and are seen to fuse with these membranes and release their contents to contribute to a fibrous surface coating found only on the free mucosal surfaces of the cells. Beneath the plasma membranes on these surfaces is an additional, finely granular component. Lateral and basal plasma membranes are heavily plicated and appear ordinary in fine structure. The cells of the epithelium are tightly held together by a terminal bar apparatus and sealed together, with an intervening space of only 0.02 mµ near the bladder lumen, in such a way as to prevent water leakage between the cells. It is demonstrated in in vitro experiments that water traversing the bladder wall passes through the cytoplasm of the epithelial cells and that a vesicle transport mechanism is not involved. In vitro experiments also show that the basal (serosal) surfaces of the epithelial cells are freely permeable to water, while the free (mucosal) surfaces are normally relatively impermeable but become permeable when the serosal surface of the bladder is treated with neurohypophyseal hormones. The permeability barrier found at the mucosal surface may be represented, structurally, either by the filamentous layer lying external to the plasma membrane, by the intracellular, granular component found just under the plasma membrane, or by both of these components of the mucosal surface complex. The polarity of the epithelial sheet is emphasized and related to the physiological role of the urinary bladder in amphibian water balance mechanisms.