Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

Competitive adherence as a mechanism of bacterial interference

To determine whether competition among bacteria for specific attachment sites on host cells can explain bacterial interference, Staphylococcus aureus strain 502A was tested in turn against two different nasal coryneforms, a strain of Pseudomonas aeruginosa, and a virulent strain of S. aureus, all in the presence of nasal mucosal cells. Particularly examined was the influence of sequence in which bacteria were presented to the nasal cells in comparison with initial mixtures and individual suspensions. Results paralleled those observed in clinical prophylaxis: the bacterium first to adhere to the epithelial cells was able, under uniform conditions, to interfere with the colonization of subsequently added bacteria. Secondary adherence was not eliminated but substantially reduced, and was probably related to steric blockage by the initial colonizer and its particular ability to dissociate from the host cell.     

Cold stress proteins induced in Listeria monocytogenes in response to temperature downshock and growth at low temperatures.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at refrigerator temperatures. Twelve cold shock proteins (Csps) with apparent M(r)s of 48,600, 41,000, 21,800, 21,100, 19,700, 19,200, 18,800, 18,800, 17,200, 15,500, 14,500, and 14,400 were induced by cold shocking L. monocytogenes 10403S from 37 to 5 degrees C, as revealed by labeling with L-[35S]methionine followed by two-dimensional gel electrophoresis. Strain SLCC53 showed a similar response. Cold acclimation proteins were observed in cultures of strain 10403S growing at 5 degrees C, and four of these proteins, with apparent M(r)s 48,000, 21,100, 19,700, and 18,800, were also Csps. Two cold-sensitive transposon-induced mutants were labeled less efficiently than the parent strain, but the Csp response of the mutant examined was very similar to that of the parent strain.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Cold shock and its effect on ribosomes and thermal tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0 degrees C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 +/- 0.1 degrees C (mean +/- standard deviation) to 72.1 +/- 0.5 degrees C (P < or = 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 +/- 0.4 degrees C to 66.8 +/- 0.5 degrees C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

Arginine Deiminase in Staphylococcus epidermidis Functions To Augment Biofilm Maturation through pH Homeostasis

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.     

SRAP Technique Efficiently Generates Polymorphisms in Puccinia striiformis Isolates

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f.sp. tritici (PST), is a major disease of wheat in temperate‐cold climates. The identification of new markers would ease the procedure for evaluating the ongoing pathogen evolution. Twelve single pustule isolates were generated from samples of PST obtained in UK during 1987–2001. They were evaluated for their pathogenic behaviour on a set of differential cultivars and were analysed by sequence‐related amplified polymorphisms (SRAP) technique, to identify polymorphisms useful to evaluate variability among isolates. This is the first report of the application of SRAP technique to Uredinales order.