Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p?=?0.048; CI 95%: 1.01-10.24; p?=?0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.     

Decomposition of old organic matter as a result of deeper active layers in a snow depth manipulation experiment

A snow addition experiment in moist acidic tussock tundra at Toolik Lake, Alaska, increased winter snow depths 2–3 m, and resulted in a doubling of the summer active layer depth. We used radiocarbon (?¹⁴C) to (1) determine the age of C respired in the deep soils under control and deepened active layer conditions (deep snow drifts), and (2) to determine the impact of increased snow and permafrost thawing on surface CO₂ efflux by partitioning respiration into autotrophic and heterotrophic components. ?¹⁴C signatures of surface respiration were higher in the deep snow areas, reflecting a decrease in the proportion of autotrophic respiration. The radiocarbon age of soil pore CO₂ sampled near the maximum mid-July thaw depth was approximately 1,000 years in deep snow treatment plots (45–55 cm thaw depth), while CO₂ from the ambient snow areas was ~100 years old (30-cm thaw depth). Heterotrophic respiration ?¹⁴C signatures from incubations were similar between the two snow depths for the organic horizon and were extremely variable in the mineral horizon, resulting in no significant differences between treatments in either month. Radiocarbon ages of heterotrophically respired C ranged from <50 to 235 years BP in July mineral soil samples and from 1,525 to 8,300 years BP in August samples, suggesting that old soil C in permafrost soils may be metabolized upon thawing. In the surface fluxes, this old C signal is obscured by the organic horizon fluxes, which are significantly higher. Our results indicate that, as permafrost in tussock tundra ecosystems of arctic Alaska thaws, carbon buried up to several thousands of years ago will become an active component of the carbon cycle, potentially accelerating the rise of CO₂ in the atmosphere.     

Differential water resource use by herbaceous and woody plant life-forms in a shortgrass steppe community

We conducted a study to test the predictions of Walter's two-layer model in the shortgrass steppe of northeastern Colorado. The model suggests that grasses and woody plants use water resources from different layers of the soil profile. Four plant removal treatments were applied in the spring of 1996 within a plant community codominated by Atriplex canescens (a C₄ shrub) and Bouteloua gracilis (a C₄ grass). During the subsequent growing season, soil water content was monitored to a depth of 180 cm. In addition, stem and leaf tissue of Atriplex, Bouteloua and the streamside tree Populus sargentii were collected monthly during the growing seasons of 1995 and 1996 for analysis of the δ¹⁸O value of plant stem water (for comparison with potential water sources) and the δ¹³C value of leaves (as an indicator of plant water status). Selective removal of shrubs did not significantly increase water storage at any depth in the measured soil profile. Selective removal of the herbaceous understory (mainly grasses) increased water storage in the top 60 cm of the soil. Some of this water gradually percolated to lower layers, where it was utilized by the shrubs. Based on stem water δ¹⁸O values, grasses were exclusively using spring and summer rain extracted from the uppermost soil layers. In contrast, trees were exclusively using groundwater, and the consistent δ¹³C values of tree leaves over the course of the summer indicated no seasonal changes in gas exchange and therefore minimal water stress in this life-form. Based on anecdotal rooting-depth information and initial measurements of stem water δ¹⁸O, shrubs may have also had access to groundwater. However, their overall δ¹⁸O values indicated that they mainly used water from spring and summer precipitation events, extracted from subsurface soil layers. These findings indicate that the diversity of life-forms found in this shortgrass steppe community may be a function of the spatial partitioning of soil water resources, and their differential use by grasses, shrubs, and trees. Consequently, our findings support the two-layer model in a broad sense, but indicate a relatively flexible strategy of water acquisition by shrubs. Received: 23 December 1997 / Accepted: 16 September 1998     

Capture and allocation of nitrogen byQuercus douglasii seedlings in competition with annual and perennial grasses

Summary The spatial overlap of woody plant root systems and that of annual or perennial grasses promotes competition for soil-derived resources. In this study we examined competition for soil nitrogen between blue oak seedlings and either the annual grassBromus mollis or the perennial grassStipa pulchra under controlled outdoor conditions. Short-term nitrogen competition was quantified by injecting¹⁵N at 30 cm depth in a plane horizontal to oak seedling roots and that of their neighbors, and calculating¹⁵N uptake rates, pool sizes and¹⁵N allocation patterns 24 h after labelling. Simultaneously, integrative nitrogen competition was quantified by examining total nitrogen capture, total nitrogen pools and total nitrogen allocation.Stipa neighbors reduced inorganic soil nitrogen content to a greater extent than didBromus plants. Blue oak seedlings responded to lower soil nitrogen content by allocating lower amounts of nitrogen per unit of biomass producing higher root length densities and reducing the nitrogen content of root tissue. In addition, blue oak seedlings growing with the perennial grass exhibited greater rates of¹⁵N uptake, on a root mass basis, compensating for higher soil nitrogen competition inStipa neighborhoods. Our findings suggest that while oak seedlings have lower rates of nitrogen capture than herbaceous neighbors, oak seedlings exhibit significant changes in nitrogen allocation and nitrogen uptake rates which may offset the competitive effect annual or perennial grasses have on soil nitrogen content.     

Pronghorn (Antilocapra americana) enamel phosphate δ18O values reflect climate seasonality: Implications for paleoclimate reconstruction

Stable oxygen isotope (δ¹⁸O) compositions from vertebrate tooth enamel are widely used as biogeochemical proxies for paleoclimate. However, the utility of enamel oxygen isotope values for environmental reconstruction varies among species. Herein, we evaluate the use of stable oxygen isotope compositions from pronghorn (Antilocapra americana Gray, 1866) enamel for reconstructing paleoclimate seasonality, an elusive but important parameter for understanding past ecosystems. We serially sampled the lower third molars of recent adult pronghorn from Wyoming for δ¹⁸O in phosphate (δ¹⁸O_PO4) and compared patterns to interpolated and measured yearly variation in environmental waters as well as from sagebrush leaves, lakes, and rivers (δ¹⁸O_w). As expected, the oxygen isotope compositions of phosphate from pronghorn enamel are enriched in ¹⁸O relative to environmental waters. For a more direct comparison, we converted δ¹⁸O_w values into expected δ¹⁸O_PO4* values (δ¹⁸O_W‐_PO4*). Pronghorn δ¹⁸O_PO4 values from tooth enamel record nearly the full amplitude of seasonal variation from Wyoming δ¹⁸O_W‐PO4* values. Furthermore, pronghorn enamel δ¹⁸O_PO4 values are more similar to modeled δ¹⁸O_W‐PO4* values from plant leaf waters than meteoric waters, suggesting that they obtain much of their water from evaporated plant waters. Collectively, our findings establish that seasonality in source water is reliably reflected in pronghorn enamel, providing the basis for exploring changes in the amplitude of seasonality of ancient climates. As a preliminary test, we sampled historical pronghorn specimens (1720 ± 100 AD), which show a mean decrease (a shift to lower values) of 1–2‰ in δ¹⁸O_PO4 compared to the modern specimens. They also exhibit an increase in the δ¹⁸O amplitude, representing an increase in seasonality. We suggest that the cooler mean annual and summer temperatures typical of the 18th century, as well as enhanced periods of drought, drove differences among the modern and historical pronghorn, further establishing pronghorn enamel as excellent sources of paleoclimate proxy data.     

Differences in Species Composition and Feeding Ecology of Catostomid Fishes in Two Distinct Segments of the Missouri River, North Dakota, U.S.A.

In 1997 and 1998, we sampled the Missouri River, North Dakota to determine if anthropogenic disturbances had influenced catostomid species composition and feeding ecology. We compared two distinct river segments, the Missouri River between the mouth of the Yellowstone River and Lake Sakakawea (the Yellowstone–Sakakawea segment (YSS)), a moderately altered segment and the Missouri River between Garrison Dam and Lake Oahe (the Garrison–Oahe segment (GOS)), a highly altered segment. The segments exhibited greatly different sucker communities. Bigmouth buffalo, Ictiobus cyprinellus, smallmouth buffalo, Ictiobus bubalus, and river carpsucker, Carpiodes carpio, represented 94% of the sucker catch in the YSS, whereas in the GOS, white sucker, Catostomus commersoni, and longnose sucker, Catostomus catostomus, constituted 98% of the sucker catch. In the YSS, high zooplankton densities led to greater sucker zooplanktivory and food niche overlap than in the GOS. Intense anthropogenic disturbances to the GOS are associated with the differences in sucker species composition, prey density and composition, and sucker feeding ecology between the two segments.     

Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity

The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu²⁺, Zn²⁺, Mn²⁺, and Co²⁺ on Zpl (Prnp ^-/-) and ZW (Prnp ^+/+) hippocampus-derived mouse neuronal cells. Prnp ^-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.     

From Intra-plant to Regional Scale: June Temperatures and Regional Climates Directly and Indirectly Control Betula nana Growth in Arctic Alaska

Ecosystems - Tundra shrubs reflect climate sensitivities in their growth-ring widths, yet tissue-specific shrub chronologies are poorly studied. Further, the relative importance of regional climate...     

Membranes of Bacillus stearothermophilus: Factors Affecting Protoplast Stability and Thermostability of Alkaline Phosphatase and Reduced Nicotinamide Adenine Dinucleotide Oxidase

Protoplasts of Bacillus stearothermophilus NCA 1503-4R are resistant to osmotic rupture and are not sensitive to mechanical manipulation. Protoplast stability is maintained by divalent cations. The thermostability of protoplasts is enhanced when the cells are grown at elevated temperatures. The membrane content of the cell and the protein-to-lipid ratio of the membrane increases as the growth temperature is increased. The membrane-bound nicotinamide adenine dinucleotide (reduced form) oxidase system from cultures grown at 70 C was more thermostable than the same enzyme system from cultures grown at 55 C. Alkaline phosphatase was resistant to thermal inactivation in the intact protoplast. The extent of this protection is dependent on protoplast stability.