3-hydroxy fatty acids found in capsules of Cryptococcus neoformans

Using immunofluorescence confocal laser scanning microscopy, immunogold transmission electron microscopy and gas chromatography--mass spectrometry, we demonstrated the presence of 3-hydroxy fatty acids in Cryptococcus neoformans. Our results suggest that these oxylipins accumulate in capsules where they are released as hydrophobic droplets through tubular protuberances into the surrounding medium.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Distribution of 3-hydroxy oxylipins and acetylsalicylic acid sensitivity in Cryptococcus species

Using a well tested antibody specific for 3-hydroxy oxylipins, we mapped the presence of these oxylipins in selected Cryptococcus (Filobasidiella) species. Immunofluorescence microscopy studies revealed that these compounds are deposited on cell wall surfaces, appendages, and collarettes. In vitro studies revealed that growth of Cryptococcus species was inhibited by acetylsalicylic acid (which is known to inhibit mitochondrial function, including the production of 3-hydroxy oxylipins) at concentrations as low as 1 mmol/L. The results suggest that acetylsalicylic acid is effective in controlling the growth of tested pathogens, probably by targeting their mitochondria. This study further expands the known function of this anti-inflammatory drug as anti-fungal agent.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

The presence of 3-hydroxy oxylipins on the ascospore surfaces of some species representing Saccharomycopsis Schiönning

Through gas chromatography - mass spectrometry, the presence of oxylipins, mainly 3-hydroxy 9:1 and 3-hydroxy 10:1, was detected in Saccharomycopsis fermentans, Saccharomycopsis javanensis, and Saccharomycopsis vini. The distribution of these compounds was mapped using immunofluorescence microscopy, and they were found to be closely associated with the surfaces of aggregating ascospores.     

Oxylipin-coated hat-shaped ascospores of Ascoidea corymbosa

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

The influence of acetylsalicylic acid on oxylipin migration in Cryptococcus neoformans var. neoformans UOFS Y-1378

In this paper we report the influence of acetylsalicylic acid on oxylipin migration in Cryptococcus neoformans var. neoformans UOFS Y-1378, previously isolated from human bone lesion. Transmission electron microscopy suggests that osmiophilic material originates in mitochondria and is deposited inside the yeast cell wall, from which it is excreted into the environment, along capsule protuberances, or through capsule detachments. Previous studies using immunogold labeling indicate that these osmiophilic layers contain 3-hydroxy oxylipins. In this study, the addition of acetylsalicylic acid (an inhibitor of mitochondrial function) in increasing amounts to the cells abrogated the migration of osmiophilic material, as well as capsule detachment from cell walls, and hence, oxylipin excretion. Consequently, we hypothesize that 3-hydroxy oxylipins are produced in mitochondria, probably via incomplete beta-oxidation or fatty acid synthesis, from which they are deposited inside the cell wall and excreted through tubular protuberances attached to the surrounding capsules and (or) through detachment of these oxylipin-containing capsules.