The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus

Summary Cytoplasmic cleavage in the gametangia and zoosporangia ofA. macrogynus was studied using monensin, an ionophore known to disrupt several endomembrane functions in plant and animal cells. Monensin interfered with normal gamete and zoospore formation in a dose dependent manner such that at a 20 M concentration very abnormal cells were released from the reproductive structures. It was evident that monensin's effect was most pronounced during the first 25 minutes of gametogenesis and parallels in time the onset and continuation of the cytoplasmic cleavage events. Observations using fluorescence and differential interference contrast microscopy indicated that the ionophore inhibited normal cytoplasmic cleavage resulting in the production of multinucleate cells, many of which had either no flagella or multiple flagella. Transmission electron microscopy showed that the monensin-treated gametangia had many large vacuoles which contained amorphous electron-opaque material. X-ray microprobe analysis demonstrated that the elemental composition of the large vacuoles was identical to that of the dense globular inclusions seen in untreated gametangia, and morphological analysis confirmed the relationship between these endomembrane structures. Thus this swollen endomembrane component probably is not involved in the cleavage process. Single endomembrane cisternae which were very common in untreated gametangia were seldom seen in monensin-treated preparations. Instead, many smaller electron-transparent vacuoles were observed. These swollen cisternae may both represent monensin-modified Golgi apparatus equivalents and/or play a critical role during the process of gametogenesis and zoosporogenesis inA. macrogynus.     

Competition between Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134 (pJP4) during colonization of barley roots

Abstract: To use deliberately released beneficial microorganisms in the rhizosphere, we need a better understanding of the process of root colonization by seed-borne or soil-borne inocula. In this study, we determine the survival of Pseudomonas fluorescens Ag1 and Alcaligenes eutrophus JMP134, their colonization ability as affected by substrates, and the relative importance of migration versus competition for colonization of the root. Ag1 and the 2,4-dichlorophenoxy-acetic acid (2,4-D) degrader JMP134 were inoculated in sterile barley rhizosphere systems. After inoculation of seeds with individual strains, comparable population sizes were established in the rhizosphere as determined by immunofluorescence microscopic total cell counts. Both strains were motile and able to colonize the entire root system without percolating water to stimulate passive transport. Comparing immunofluorescence microscopic cell counts with colony-forming units demonstrated that a subpopulation of A. eutrophus JMP134 closely associated with the root was non-culturable in contrast to the population in rhizosphere soil. Hence, the sole use of culture-dependent methods may give misleading information about the distribution of bacteria in the rhizosphere. Colonization studies with both strains showed that co-inoculation of Ag1 and JMP134 caused a decrease of the population size of JMP134 if 2,4-D was not added to the soil as a specific carbon source for this strain. Thus, competition for limited carbon sources might influence the composition of the bacterial community in the rhizosphere. We also found that the presence of an established inoculum in the soil reduced subsequent root colonization by a seed-inoculated strain, probably by filling available niches, also indicating that competition from other bacteria may be an important factor determining the distribution of seed-borne inocula. This factor may be just as important for the distribution of bacteria as migration.     

Zoosporogenesis in Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

During sporulation of Pseudoperonospora cubensis on cucumber leaves ( Cucumis saliva ) zoosporangia are formed on the dichotomously branched sporangiophore. The mature zoosporangium has a preformed discharge papilla and the cytoplasm is uncleaved. The zoosporangium wall is decorated and the outer layer of the wall is electron opaque in ultrathin sections. As the zoosporangium is able to survive freezing (- 18°C) for prolonged periods of time (3–4 months) the zoosporangium may serve as the "resting" structure which survives overwintering in Northern latitudes in the absence of oospore formation.
Zoospore cleavage can be synchronized by placing freshly harvested zoosporangia in distilled water. Cleavage of the zoosporangial cytoplasm is by means of the fusion of small vesicles apparently derived from dictyosomes which become highly active after zoosporogenesis is induced.
Vesicles with an osmiophilic electron opaque content are the dominant type of vesicle found in the zoosporangia. The content of these vesicles undergoes dynamic changes during zoosporogenesis and during the late stages of sporogenesis the content becomes finely striated as is typical of these vesicles when observed in the zoospore. On the basis of the results presented here it is suggested that zoosporangium formation and zoosporogenesis in P. cubensis could serve as a model system for assays with obligate oomycetous plant pathogens, also in relation to fungicide mode of action studies.     

Quantitation of chloramphenicol acetyl transferase (CAT) messenger RNA by filter hybridisation using a digoxigenin label

Summary Slot- and dot-blotting are commonly used to evaluate levels of messenger ribonucleic acid (mRNA). Quantitation of bacterially-expressed chloramphenicol acetyl transferase (CAT) mRNA by this method is highly dependent on total RNA immobilised onto the solid support as well as mRNA concentration. mRNA quantitation by comparison with a pure standard results in underestimation. An improved protocol for CAT mRNA detection is described.     

Body composition analysis by DEXA by using dynamically changing samarium filtration

Gotfredsen, Anders, Lene Bæksgaard, and Jannik Hilsted.Body composition analysis by DEXA by using dynamically changing samarium filtration. J. Appl. Physiol.82(4): 1200-1209, 1997.Dual-energy X-ray absorptiometry (DEXA)has a high accuracy for body composition analysis but is influenced bybeam hardening and other error sources in the extremes of measurement.To compensate for beam hardening, the Norland XR-36 introduces adynamically changing samarium filtration system, which depends on thecurrent-absorber thickness. With this system we found a good agreement(r = 0.99) between reference andmeasured amounts of tissue or fat percentages in a plastic phantom andin smaller (~0.5-4 kg) and larger (~5-20 kg) piles oftissue (ox muscle and lard). Scans of six healthy volunteers coveredwith combinations of beef and lard (~5-15 kg) showed a goodagreement (r = 0.99) between referenceand DEXA values of added soft tissue mass and fat percentage. Weconclude that the DEXA method (and, in particular, the Norland XR-36using dynamic filtration) has a high accuracy for body compositionanalysis. It has a potential for gaining status as a reference methodin the future and may presently be used as a supplement to thetraditional methods for body composition analysis.

     

The zoospore ofOlpidium brassicae

Summary The ultrastructure of the zoospore ofOlpidium brassicae is described and compared with observations made of other zoospores of the uniflagellatePhycomycetes. The zoospore ofO. brassicae is characterized by an extensive, cone-shaped rhizoplast and a lack of a nuclear cap, as well as a side-body complex or a rumposome. Vacuoles which contain osmiophilic material are termed gamma-like particles. Three-dimensional reconstructions based on serial sectioning were made of the organelles in the region of the nucleus, showing that the zoospore ofO. brassicae contains one or at most two elaborately branched mitochondria. Microbodies have a high degree of interconnection and are in intimate association with the mitochondrion, lipid drops, and the nuclear envelope.     

Pregnane X receptor-agonists down-regulate hepatic ATP-binding cassette transporter A1 and scavenger receptor class B type I

Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).     

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.     

Osmotic regulation of the uniflagellate phycomycete spore

In spores of the Blastocladiales there is a strict temporal correlation between the breakdown of the matrix of the gamma body and the swelling of the gamma body with the duration of the spore's motility. The swelling of the gamma body upon decay is interpreted as being due to water uptake. The swollen gamma bodies fuse with the plasmalemma and expel their content. We suggest that the swelling of the gamma body and its subsequent fusion with the plasmalemma of the spore are the means by which the spores of the Blastocladiales maintain osmotic balance with the medium in which they swim. The decay of the gamma body during the motile period of the spore is a separate and distinct process, not related to the mobilization of the remaining gamma bodies during encystment and cell wall formation.