The outcome of phagocytic cell division with infectious cargo depends on single phagosome formation

Given that macrophages can proliferate and that certain microbes survive inside phagocytic cells, the question arises as to the post-mitotic distribution of microbial cargo. Using macrophage-like cells we evaluated the post-mitotic distribution of intracellular Cryptococcus yeasts and polystyrene beads by comparing experimental data to a stochastic model. For beads, the post-mitotic distribution was that expected from chance alone. However, for yeast cells the post-mitotic distribution was unequal, implying preferential sorting to one daughter cell. This mechanism for unequal distribution was phagosomal fusion, which effectively reduced the intracellular particle number. Hence, post-mitotic intracellular particle distribution is stochastic, unless microbial and/or host factors promote unequal distribution into daughter cells. In our system unequal cargo distribution appeared to benefit the microbe by promoting host cell exocytosis. Post-mitotic infectious cargo distribution is a new parameter to consider in the study of intracellular pathogens since it could potentially define the outcome of phagocytic-microbial interactions.     

Histochemical analysis of sperm storage in Helicolenus dactylopterus dactylopterus (Teleostei: Scorpaenidae).

The bluemouth rockfish, Helicolenus dactylopterus dactylopterus (De la Roche, 1809), is a zygoparous species with internal fertilization. The male urogenital papilla acts as the copulating organ, and the females retain the spermatozoa in their ovaries for up to 10 months. The objective of this study is to extend our knowledge of the mechanisms that allow the sperm to be retained in the ovaries for prolonged periods. To this end, we analyze the histochemical properties of: 1) the epithelium of the testicular sperm duct, 2) the sperm of the males, 3) the internal epithelium of the ovary wall, 4) the ovarian fluid, and 5) the spermatozoa storage crypts of females. The PAS (Periodic acid-Schiff) and bright Coomassie blue positive reactions of the epithelium of the spermatic duct point to the secretion of polysaccharides and proteins that could promote the bundling of the spermatozoa. The internal epithelium of the ovarian wall secretes polysaccharides, protein, and lipid compounds throughout the storage and spawning period. The acid nature of the ovarian fluid during the storage period may maintain the bundling of spermatozoa when they enter the ovary and may also inhibit sperm motility until the moment of fertilization. The polysaccharide granules that come from the cryptal epithelium into the cavity where spermatozoa are maintained may supply them with nutrients for the storage period. The presence of glucosaminoglycans on the surface of the sperm is probably related to the inhibition of spermatic motility produced by the acidic environment. They are absent in the spermatozoa located in the testicular ducts, relatively scarce in those of the duct of the copulating organ, and abundant in those within the intraovarian cryptal structures.     

The resistant polymer of the walls of the hydrocarbon-rich alga Botryococcus braunii

The outer walls of the green alga Botryococcus braunii (main sites of hydrocarbon production and accumulation) show a complex constitution. They comprise a biopolymer highly resistant to non-oxidative degradation. The resistant polymer accounts for ca 9% of the cell dry wt and appears, along with hydrocarbons, as one of the major constituents of the alga. In addition to chemical resistance, B. braunii polymer exhibits other properties: mode of deposition and fluorescence, often used to identify sporopollenins. (Class of wall components generally regarded as originating from polymerization of carotenoid derivatives.) Nevertheless further studies, using IR spectroscopy and high resolution ¹³C NMR of solids, along with determination of elemental composition and unsaturation levels, indicate that the bulk of the resistant polymer from B. braunii outer walls does not derive from carotenoids; accordingly it cannot be considered, in this respect, as a sporopollenin. In fact the information obtained on the structure of this important constituent of the alga is consistent with its formation via oxidative polymerization of B. braunii dienic hydrocarbons.     

Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins.

The structure of the murine erythropoietin receptor was studied using antibodies against the intracellular part of the cloned erythropoietin receptor chain. These antibodies precipitated erythropoietin-receptor complexes from Triton X-100-solubilized cells. When the complexes were cross-linked by disuccinimidyl suberate, the 85- and 100-kDa erythropoietin-cross-linked proteins previously described were immunoprecipitated. However, these proteins were not precipitated when the complexes were denatured and reduced before immunoprecipitation. Using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide, we observed erythropoietin cross-linking with a protein of 66 kDa in addition to the 100- and 85-kDa proteins. Only the 66-kDa erythropoietin-cross-linked protein was immunoprecipitated by anti-receptor antibodies after denaturation and reduction of the complex. Thus, our results suggest that the 85- and 100-kDa proteins previously evidenced by cross-linking are associated with the cloned chain of the receptor to form a multimeric complex but these proteins seem immunologically unrelated to the cloned chain. We observed that reducing the length of molecules able to cross-link amino groups decreased the efficiency of cross-linking with the 100-kDa protein and only the 85-kDa protein was cross-linked with erythropoietin using 1,5-difluoro-2,4-dinitrobenzene. These results suggest that the 85- and 100-kDa proteins occupate slightly different positions relative to the erythropoietin molecule bound to the receptor.     

Sites of accumulation and composition of hydrocarbons in Botryococcus braunii

Raman spectrometry and electron microscopy show that, in the hydrocarbon-rich alga Botryococcus braunii, hydrocarbons accumulate in two distinct sites; internally in cytoplasmic inclusions and externally in successive outer walls and derived globules. No other classes of lipid are present in noticeable amounts in the cytoplasmic inclusions and in the external globules. The same hydrocarbons are observed in the internal and external pools but with different relative abundances, the shorter hydrocarbons being more abundant in the internal pool. The bulk of B. braunii hydrocarbons (ca 95%) is located in the external pool. Such an extracellular location allows this species to exhibit both an unusually high hydrocarbon content (15% of dry wt) and a normal level (0.75%) within the cells. The hydrocarbon pattern and location of B. braunii were compared with that of other organisms; a close relation appears between higher plant epidermal cells and this green alga. The trilaminar outer walls of B. braunii, at whose contact external hydrocarbon globules accumulate, contain a sporopollenin-like compound.     

Infection of Mice with Aerosolized Mycobacterium tuberculosis: Use of a Nose-Only Apparatus for Delivery of Low Doses of Inocula and Design of an Ultrasafe Facility

Aerosolized delivery of virulent or hypervirulent Mycobacterium tuberculosis requires careful consideration of methodology and safety. To maximize safety, we installed a nose-only aerosol apparatus that can reproducibly deliver a low dose (<100 CFU per mouse) of M. tuberculosis in a carefully designed biohazard facility.     

Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connection?

Fungi are relatively rare causes of life-threatening systemic disease in immunologically intact mammals despite being frequent pathogens in insects, amphibians, and plants. Given that virulence is a complex trait, the capacity of certain soil fungi to infect, persist, and cause disease in animals despite no apparent requirement for animal hosts in replication or survival presents a paradox. In recent years studies with amoeba, slime molds, and worms have led to the proposal that interactions between fungi and other environmental microbes, including predators, select for characteristics that are also suitable for survival in animal hosts. Given that most fungal species grow best at ambient temperatures, the high body temperature of endothermic animals must provide a thermal barrier for protection against infection with a large number of fungi. Fungal disease is relatively common in birds but most are caused by only a few thermotolerant species. The relative resistance of endothermic vertebrates to fungal diseases is likely a result of higher body temperatures combined with immune defenses. Protection against fungal diseases could have been a powerful selective mechanism for endothermy in certain vertebrates. Deforestation and proliferation of fungal spores at cretaceous-tertiary boundary suggests that fungal diseases could have contributed to the demise of dinosaurs and the flourishing of mammalian species.     

BACTERIAL INFLUENCE UPON GROWTH AND HYDROCARBON PRODUCTION OF THE GREEN ALGA BOTRYOCOCCUS BRAUNII1

Batch cultures of the hydrocarbon-rich alga Botryococcus braunii, Kütz. (axenic strains, non-axenic strains, associations with selected microorganisms) were examined with regard to total biomass and hydrocarbons at the onset of the stationary phase. Pronounced variations, related to the origin of the strains and to growth conditions, were observed with axenic cultures. It also appeared that the presence of microorganisms is not essential for high hydrocarbon production. Nevertheless, numerous bacteria were shown to exert considerable influence, antagonistic or beneficial, on B. braunii growth yield and hydrocarbon production. Such effects were strongly dependent on the species involved and on culture conditions. The presence of various microorganisms can influence not only the quantity of hydrocarbons produced, but also their level in the algal biomass and their relative abundance. However, their chemical structure is not affected. Intricate relationships were observed in B. braunii-bacteria systems and numerous factors (including, in some cultures, large positive effects due to bacterially produced CO₂) were implicated. Accordingly, specific associations should provide appropriate conditions for renewable hydrocarbon production via B. braunii large scale cultures.