Lysosomal component in the mechanism of the toxic effect of sporofusarin

Mutant spores of Dictyostelium discoideum, strain SG-10, differ from wild type spores in their ability to spontaneously germinate, to be activated with 5% Dimethyl Sulfoxide (DMSO), and to be deactivated with 0.2 M sucrose. Both heat-activated wild type and mutant spores began to swell after a lag of 60-75 min at ambient temperature. Suspension of heat activated spores in 5% DMSO resulted in blockage of spore swelling and a concomitant severe inhibition of respiration; removal of 5% DMSO allowed resumption of respiration and the spores began to swell after a lag of only 15 min. It was concluded that 5% DMSO allowed the early reactions (M) to proceed but blocked the later reactions (R) of post-activation lag. Treatment of one day old spores with 20% DMSO solution for 30-120 min quantitatively activated the population. The post-activation lag time was directly dependent on the time of 20% DMSO treatment. Spores activated with 20% DMSO treatment could be deactivated by incubation at 0 degrees C; the spores most quickly deactivated at 0 degrees C were those within 10 min of swelling. Mitochondrial transport inhibitors such as azide and cyanide caused deactivation in an analogous manner. It is hypothesized that spores proceed to the second portion of the lag phase called (R) before the environment determines if dormancy is reimposed or if germination will proceed. The sensitive strain (SG-10) showed a greater degree of "damage" than the wild type after supraoptimal treatment with 40% DMSO. The spores became more resistant with age to the "damaging" action of 40% DMSO. All the observed effects of DMSO treatment were compatible with our multistate model of activation which suggests that the early portion of the lag phase (M) may involve a relative uncoupling of oxidative phosphorylation while the later portion (R) may require tight coupling.     

Impact of Particle Aggregated Microbes on UV Disinfection. II: Proper Absorbance Measurement for UV Fluence

Ultraviolet (UV) absorbance measurements are subject to significant error using a standard spectrophotometer when particles or aggregates that scatter light are present. True UV absorbance for highly turbid waters should be measured using integrating sphere (IS) spectrophotometry that allows the collection of reflected and transmitted radiation simultaneously. This is especially important when the effects of scattering impact UV disinfection—such as with the presence of aggregates. The impact of light scattering of particle-aggregated microbes on UV disinfection was evaluated by comparing standard spectrophotometer and integrating sphere absorbance measurements for UV fluence determination. Spore–clay aggregates in simulated drinking waters and spore aggregates with natural particles from raw waters were induced by flocculation with alum. Coagulated systems significantly decreased the UV inactivation effectiveness compared to the noncoagulated system with the effects more pronounced for raw natural water. Absorbance measurement of suspensions and aggregates using standard spectrophotometry in the calculations of fluence resulted in overdosing whereas the use of IS spectroscopy did not. The results demonstrated that aggregation protected spores from UV disinfection, and that use of proper absorbance measurement techniques, accounting for particle scattering, is essential for correct interpretation of the results.     

Stress and coping among African American and Hispanic parents of deaf children

Phase-contrast microscopy coupled with image analysis has been used to study the germination of single spores of Clostridium botulinum and to investigate the variation of germination lag of individual spores in a population (biovariability). The experiment was repeated at five different temperatures between 20 degrees C and 37 degrees C to look at the effect of temperature on the biovaribility of the spore germination. Data analysis shows that the germination lag distribution is skewed, with a tail, and that its shape is affected by the temperature. The origin of this biovariability is not exactly known, but could be due to a distribution of characteristics (e.g. permeabilities) or molecules (e.g. lytic enzymes) in the spore population. The method developed in this study will help us to describe and better understand the kinetics of spore germination and how this is influence by different environmental factors such as temperature and other factors that influence germination.     

Impact of Particle Aggregated Microbes on UV Disinfection. I: Evaluation of Spore–Clay Aggregates and Suspended Spores

Aggregation of microbes with particles can reduce the effectiveness of ultraviolet (UV) disinfection. This study evaluated the comparative impact of dispersed spores, dispersed spores mixed with clay particles (nonaggregated), spore–spore aggregates, and spore–clay aggregates on UV disinfection performance in simulated drinking waters. Aggregates were induced by flocculation with alum and characterized by particle size analysis (count, volume, and surface area) of dispersed and aggregated systems, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. It was concluded that spores within aggregates of the spore–clay system were protected from UV irradiation compared to nonaggregated spores and the difference between these systems was found to be statistically significant throughout the UV range tested. In addition SEM-EDX analysis suggested that aggregate composition is nonhomogeneous with respect to the ratio of spores and clay particles among aggregates. It was estimated that 30–50% of the spores in the aggregates tested were protected from UV irradiation.     

Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores

Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.     

Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination

Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.     

Growth effects of regulatory peptides and intracellular signaling routes in human pancreatic cancer cell lines

Spore germination is a defined developmental process that marks a critical point in the life cycle of Dictyostelium discoideum. Upon germination the environmental conditions must be conducive to cell growth to ensure survival of emerged amoebae. However, the signal transduction pathways controlling the various aspects of spore germination in large part remain to be elucidated. We have used degenerate PCR to identify dhkB, a two-component histidine kinase, from D. discoideum. DhkB is predicted to be a transmembrane hybrid sensor kinase. The dhkB-null cells develop with normal timing to give what seem to be mature fruiting bodies by 22 to 24 h. However, over the next several hours, the ellipsoidal and encapsulated spores proceed to swell and germinate in situ within the sorus and thus do not respond to the normal inhibitors of germination present within the sorus. The emerged amoebae dehydrate due to the high osmolarity within the sorus, and by 72 h 4% or less of the amoebae remain as spores, while most cells are now nonviable. Precocious germination is suppressed by ectopic activation of or expression of cAMP-dependent protein kinase A. Additionally, at 24 h the intracellular concentration of cAMP of dhkB- spores is 40% that of dhkB+ spores. The results indicate that DHKB regulates spore germination, and a functional DHKB sensor kinase is required for the maintenance of spore dormancy. DHKB probably acts by maintaining an active PKA that in turn is inhibitory to germination.     

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).     

Autoactivation of spore germination in mutant and wild type strains of Dictyostelium discoideum

Freshly formed wild type Dictyostelium discoideum spores are constitutively dormant, and thus require an activation treatment to germinate. Wild type spores may germinate without an activation treatment (autoactivate) after a period of ageing (maturation) in the intact fruiting body. Mutants have been isolated which autoactivate without the need for ageing. Autoactivation of mutant and aged wild type spores appears to occur by identical mechanisms; thus the mutation may involve premature maturation. Autoactivation is mediated by autoactivator substances released from spores as they spontaneously swell. These factors are readily chromatographed, and elute from a Biogel P2 column in three peaks of activity. One activity peak appears only after spores have begun to germinate. No autoactivator substances are released from heat activated spores. Autoactivation is sensitive to cychloheximide, and may result from altered spore permeability. Autoactivation is likely to be the mechanism of D. discoideum spore germination in nature.     

Induction of chromosome shattering by ultraviolet irradiation and caffeine: comparison of whole-cell and partial-cell irradiation

Synchronized and asynchronously growing cells of a V79 sub-line of the Chinese hamster were either whole-cell irradiated ( gamma, 254 nm) or laser-UV-microirradiated ( gamma, 257 nm). Post-incubation with caffeine (1-2 mM) often resulted in chromosome shattering, which was a rare event in the absence of this compound. In experiments with caffeine, the following results were obtained. Shattering of all the chromosomes of a cell (generalized chromosome shattering, GCS) was induced by whole-cell irradiation at the first post-irradiation mitosis when the UV fluence exceeded a "threshold" value in the sensitive phases of the cell cycle (G1 and S). GCS was also induced by laser-UV-microirradiation of a small part of the nucleus in G1 or S whereas microirradiation of cytoplasm beside the nucleus was not effective. An upper limit of the UV fluence in the non-irradiated nuclear part due to scattering of the microbeam was experimentally obtained. This UV fluence was significantly below the threshold fluence necessary to induce GCS in whole-cell irradiation experiments. In other cells, partial nuclear irradiation resulted in shattering of a few chromosomes only, while the majority remained intact (partial chromosome shattering, PCS). G1/early S was the most sensitive phase for induction of GCS by whole-cell and partial nuclear irradiation. The frequency of PCS was observed to increase when partial nuclear irradiation was performed either at lower incident doses or at later stages of S. We suggest that PCS and GCS indicate 2 levels of chromosome damage which can be produced by the synergistic action of UV irradiation and caffeine. PCS may be restricted to microirradiated chromatin whereas GCS involves both irradiated and unirradiated chromosomes in the microirradiated nucleus.     

Inactivation of Bacillus Spores by Ultraviolet or Gamma Radiation

Bacillus anthracis spores represent an important bioterrorism agent that can be dispersed in air or water. Existing decontamination practices based on these spores have focused on chemical disinfectants; however, the basic characteristics of radiation-based disinfectants suggest potential advantages in their application for control of Bacillus spores. Experiments were conducted to examine the effectiveness of ultraviolet (UV254) radiation and γ radiation for inactivation of Bacillus spores. Spores of Bacillus cereus were used for most experiments because of their similarity to B. anthracis. A limited number of experiments were also conducted using B. anthracis Sterne spores. In aqueous suspension, B. anthracis Sterne spores were observed to be slightly more resistant to UV254 than the spores of B. cereus. For the conditions of culture and assay used in these experiments, both spore types were more sensitive to UV254 radiation in aqueous suspension than the spores of B. subtilis, which are commonly used to characterize the performance of UV disinfection systems for water. Dried spores on surfaces were observed to be more resistant to UV254 than the same spores in aqueous suspension; it is likely that the increased resistance to UV of the dried spores was attributable to surface characteristics (porosity and texture) of the solid materials. γ radiation was shown to accomplish similar rates of inactivation for spores in aqueous suspension and for dried spores on surfaces. Collectively, these results suggest that the application of UV or ionizing radiation may hold promise for decontamination following bioterrorism events.     

Structural roles of the spore coat proteins in Dictyostelium discoideum

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.     

Comparative Inactivation of Bacillus Subtilis Spores and MS-2 Coliphage in a UV Reactor: Implications for Validation

Biodosimetry is the currently accepted method for validation of fluence delivery in ultraviolet (UV) reactors for water disinfection. This method utilizes the inactivation of a surrogate microorganism to predict the reduction equivalent fluence and subsequent inactivation of a target pathogen. Two common surrogates—Bacillus subtilis spores and MS-2 coliphage—were used to examine the relationship between surrogate type and biodosimetry results. A pilot-scale LP UV reactor was investigated at two flow conditions (7.5 and 15?gpm) and four different UV 253.7?nm water transmittance (UVT, 1?cm) values between 82 and 91%. The calculated reduction equivalent fluence differed from a maximum of 30% at 7.5?gal./min and 15% at 15?gal./min between the surrogates tested, depending on the UVT. These differences were attributed to the sensitivity of organisms used, hydraulic inefficiences, and UV fluence distribution in the reactor, thus the choice of validation microbe may impact the determination of reduction equivalent fluence in UV reactors.     

Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551

During germination spores of Streptomyces viridochromogenes NRRL B-1551 excrete a compound, germicidin, which has an inhibitory effect on the germination of its own arthrospores at a concentration as low as 200 pM (40 pg/ml). At higher concentrations germicidin inhibits porcine Na+/K(+)-activated ATPase and retards the germination of the cress Lepidium sativum. Germicidin is the first known autoregulative inhibitor of spore germination in the genus Streptomyces and was isolated from the supernatant of germinated spores, but also from the supernatant of the submerged culture. Spectroscopic analysis and derivatization reactions revealed germicidin to be 6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone (C11H16O3). Crude isolates of germicidin from the supernatant of submerged culture, but not from the spores, contained a second, structurally very similar compound (C10H14O3), in which in contrast to germicidin a 2-propyl instead of the 2-butyl chain was bound to C-6 and which did not show any activity in the germination and ATPase assay. The germination assay was evaluated as a new screening model for specifically active compounds.     

Identification of several unique, low-molecular-weight basic proteins in dormant spores of clastridium bifermentans and their degradation during spore germination

Two acid-soluble, low-molecular-weight basic proteins comprise approximately 20% of the protein in dormant spores of Clostridium bifermentans. Both of these proteins are rapidly degraded during spore germination.     

Dyed Microspheres for Quantification of UV Dose Distributions: Photochemical Reactor Characterization by Lagrangian Actinometry

Lagrangian actinometry represents a new method of photochemical reactor characterization. The method is based on an application of dyed microspheres, which were developed by attachment of (E)-5-[2-(methoxycarbonyl)ethenyl]cytidine (hereafter referred to as S) to polystyrene microspheres. S is a nonfluorescent molecule that when subjected to ultraviolet (UV) irradiation yields a single product, 3-β-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine (hereafter referred to as P), which displays a strong fluorescence signal. Dyed microspheres were subjected to UV irradiation under a collimated beam and using a single-lamp, monochromatic (low pressure Hg), continuous-flow reactor. In parallel with these experiments, a biodosimetry experiment was conducted using Bacillus subtilis spores as the challenge organism. Particle-specific fluorescence intensity measurements were conducted on samples from the collimated-beam experiments and the flow-through reactor experiments by flow cytometry. Estimates of the dose distribution delivered by the flow-through reactor for each operating condition were developed by deconvolution of data resulting from flow cytometry analysis of these samples. In conjunction with these experiments, a numerical model was developed to simulate the behavior of the reactor system. A commercially available computational fluid dynamics package was used to simulate the flow field, while line-source integration was used to simulate the irradiance field. A particle-tracking algorithm was employed to interrogate the flow and irradiance field simulations for purposes of developing particle-specific (Lagrangian) estimates of dose delivery. Dose distribution estimates from the microspheres assays and the numerical simulations were combined with the measured dose–response behavior of B. subtilis spores to yield estimates of spore inactivation in the flow-through experiments. For the range of operating conditions used in these experiments, predictions of spore inactivation based on dose distribution estimates from both methods were in good agreement with each other, and with the measured spore inactivation behavior. Lagrangian actinometry is capable of yielding accurate, detailed measurements of dose delivery by continuous-flow UV systems. This method represents a substantial improvement over existing experiment-based methods of UV reactor characterization (e.g., biodosimetry) in that it yields a measurement of the dose distribution for a given operating condition. This method also represents an improvement over existing methods for validation of numerical simulations. Specifically, because this method yields a measurement of the dose distribution, it is possible to compare these measurements with predicted dose distributions from the numerical simulation. The combined application of biodosimetry, numerical modeling, and Lagrangian actinometry represents an extremely robust approach to reactor characterization and validation.     

Polychromatic UV Fluence Measurement Using Chemical Actinometry, Biodosimetry, and Mathematical Techniques

With the growing acceptance of ultraviolet (UV) irradiation for disinfection of water and wastewater, the use of medium pressure Hg-based UV lamps and nonmercury, pulsed UV lamps emitting polychromatic light, are being considered in place of low-pressure, monochromatic UV lamps. However, traditional methods of fluence measurement developed for monochromatic light sources are not appropriate for polychromatic light sources. New approaches for estimation of effective germicidal fluence for polychromatic UV sources were investigated integrating chemical actinometry (uridine and iodide/iodate) with mathematical modeling. Actinometric fluence measurements as well as radiometry were referenced against microbial biodosimetry. Combining traditional chemical actinometry with mathematical analysis improved the accuracy of incident and germicidal polychromatic UV fluence measurement. Uridine actinometer germicidal fluence measurement, with or without mathematical correction, was just outside of the 95% confidence interval of that measured by biodosimetry. Similarly, an iodide/iodate actinometer was observed to accurately measure incident fluence, and coupled with mathematical corrections, it measured germicidal fluence within the 95% confidence interval of that measured by biodosimetry. These methods have potential to develop into flexible, convenient ways to measure germicidally effective UV fluence from any type of UV lamp.     

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.