Tomato transcriptional changes in response to Clavibacter michiganensis subsp. michiganensis reveal a role for ethylene in disease development

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.     

Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters ( Enhdyra lutris nereis ) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.     

Interactions among mycorrhizae, atmospheric CO2 and soil N impact plant community composition

We examined plant community responses to interactions between arbuscular mycorrhizal (AM) fungi and availability of atmospheric CO₂ and soil N. Communities of 14 plant species were grown in mesocosms containing living or killed AM fungal inoculum, ambient or elevated atmospheric CO₂ and low or enriched soil N. After one growing season, significantly different plant communities existed in the different treatments. Plant species richness was lowest in +N mesocosms and highest in +AM + CO₂ mesocosms. At ambient CO₂, AM fungi reduced richness but at elevated CO₂ they increased it. This was caused by changes in mortality rates of several C₃ forbs and may suggest that CO₂ enrichment ameliorates the carbon cost of some AM symbioses. Soil moisture was higher in +CO₂ mesocosms but +AM counteracted this effect. These results suggest that AM symbioses may be important mediators of plant community responses to anthropogenic CO₂ and N enrichment.     

Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique.

We describe here a new stable isotope-mass spectrometric technique for measuring mitochondrial DNA (mtDNA) synthesis. Growing (2-4 mo old) and weight-stable (8-10 mo old) Sprague-Dawley rats were primed with (2)H(2)O (deuterated water) to 2.0-2.5% body water enrichment, via intraperitoneal injection, and then given 4% (2)H(2)O in drinking water for 3-11 wk. Mitochondria were isolated from cardiac and hindlimb muscle, and mtDNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides. PCR confirmed the absence of nuclear DNA contamination. The isotopic enrichment of the deoxyribose moiety of deoxyadenosine was determined by GC-MS analysis, and percent new mtDNA was calculated by comparison to genomic DNA enrichments in a tissue with nearly complete turnover (bone marrow). Initial label incorporation into deoxyadenosine of mtDNA was linear, and turnover of mtDNA was observed in nongrowing adult female rats (1.1-1.3% new mtDNA per day in cardiac and skeletal muscle). Die-away curves of mtDNA after discontinuing (2)H(2)O administration gave a similar turnover rate constant. Human subjects were also given (2)H(2)O for up to 6 wk, and mitochondria from platelets were isolated. Incubation with DNase removed any contaminating genomic DNA; platelet mtDNA exhibited linear incorporation from (2)H(2)O and reached plateau values identical to those in genomic DNA from fully turned over cells (circulating monocytes). In conclusion, replication of mtDNA can be directly measured in vivo in rodents and humans without the use of radioactivity. Use of this technique may allow improved understanding of the regulation of mitochondrial biogenesis in health and disease.     

Low Light Adaptation: Energy Transfer Processes in Different Types of Light Harvesting Complexes from Rhodopseudomonas palustris

Energy transfer processes in photosynthetic light harvesting 2 (LH2) complexes isolated from purple bacterium Rhodopseudomonas palustris grown at different light intensities were studied by ground state and transient absorption spectroscopy. The decomposition of ground state absorption spectra shows contributions from B800 and B850 bacteriochlorophyll (BChl) a rings, the latter component splitting into a low energy and a high energy band in samples grown under low light (LL) conditions. A spectral analysis reveals strong inhomogeneity of the B850 excitons in the LL samples that is well reproduced by an exponential-type distribution. Transient spectra show a bleach of both the low energy and high energy bands, together with the respective blue-shifted exciton-to-biexciton transitions. The different spectral evolutions were analyzed by a global fitting procedure. Energy transfer from B800 to B850 occurs in a mono-exponential process and the rate of this process is only slightly reduced in LL compared to high light samples. In LL samples, spectral relaxation of the B850 exciton follows strongly nonexponential kinetics that can be described by a reduction of the bleach of the high energy excitonic component and a red-shift of the low energetic one. We explain these spectral changes by picosecond exciton relaxation caused by a small coupling parameter of the excitonic splitting of the BChl a molecules to the surrounding bath. The splitting of exciton energy into two excitonic bands in LL complex is most probably caused by heterogenous composition of LH2 apoproteins that gives some of the BChls in the B850 ring B820-like site energies, and causes a disorder in LH2 structure.     

Binge Ethanol-induced Neurodegeneration in Rat Organotypic Brain Slice Cultures: Effects of PLA2 Inhibitor Mepacrine and Docosahexaenoic Acid (DHA)

Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures, we examined whether phospholipase A2 (PLA2) activity is involved in binge alcohol (ethanol)-induced neurodegeneration, and whether docosahexaenoic acid (DHA; 22:6n-3), a fish oil-enriched fatty acid that is anti-inflammatory in brain damage models, is neuroprotective. Assessed with propidium iodide and lactate dehydrogenase (LDH) leakage, neurodamage from ethanol (6 days 100 mM ethanol with four withdrawal periods) was prevented by the PLA2 pan-inhibitor, mepacrine. Also, ethanol-dependent neurodegeneration—particularly in the entorhinal region—was significantly ameliorated by DHA supplementation (25 μM); however, adrenic acid, a 22:4n-6 analog, was ineffective. Consistent with PLA2 activation, [³H] liberation was approximately fivefold greater in [³H]arachidonic acid-preloaded HEC slice cultures during ethanol withdrawal compared to controls, and DHA supplementation suppressed [³H] release to control levels. DHA might antagonize PLA2 activity directly or suppress upstream activators (e.g., oxidative stress); however, other DHA mechanisms could be important in subdueing ethanol-induced PLA2-dependent and independent neuroinflammatory processes.     

Anionic PAMAM dendrimers as drug delivery vehicles for transition metal-based anticancer drugs

The use of anionic half-generation poly(amidoamine) dendrimers as drug delivery vehicles for [Pt(S,S-dach)(5,6-Me₂phen)]²⁺ (56MESS) (where S,S-dach = 1S,2S-diaminocyclohexane; 5,6-Me₂phen = 5,6-dimethyl-1,10-phenanthroline) and [{Δ,Δ-Ru(phen)₂}₂(μ-bb7)]⁴⁺ (Rubb₇) (where phen = 1,10-phenanthroline; bb7 = 1,7-bis[4-(4′-methyl-2,2′-bipyridyl)heptane]) has been studied by nuclear magnetic resonance spectroscopy. From one- and two-dimensional ¹H NMR spectra both 56MESS and Rubb₇ were found to bind to the surface of generation 3.5, 4.5, 5.5 and 6.5 dendrimers through electrostatic interactions. The higher charge and larger size of Rubb₇ resulted in stronger binding to all dendrimer generations (K_b ? 2 × 10⁵ M⁻¹) compared with 56MESS (K_b ? 1 × 10⁴ M⁻¹). Interestingly, there appeared to be no observable trend between dendrimer size and binding constant strength. The size of the free and 56MESS-bound dendrimers were examined using pulsed-gradient spin-echo NMR. The dendrimers ranged in hydrodynamic diameter from 11 to 20 nm and in all cases were larger than their corresponding full-generation dendrimer. Upon the addition of 56MESS the diameter of the dendrimers increased, consistent with surface binding.     

Identifying patterns in the diet of mackerel icefish (<Emphasis Type="Italic">Champsocephalus gunnari)</Emphasis> at South Georgia using bootstrapped confidence intervals of a dietary index

Ontogenetic, inter-annual and regional variations in diet were investigated for mackerel icefish, Champsocephalus gunnari, in three successive summer seasons around South Georgia. Stomach contents from 2239 C. gunnari (130–560 mm total length) were examined. A bootstrapping technique was used to calculate confidence intervals for an index of relative importance of prey categories (% IRI_DC). Diet varied significantly between years and age classes but there was little regional difference in diet. In general, diet was dominated by krill, Euphausia superba and by the amphipod Themisto gaudichaudii. Smaller (younger) fish tended to prey on a higher proportion of T. gaudichaudii and small euphausiids such as Thysanoessa sp. and took smaller quantities of E. superba. In a season of poor krill availability (summer of 2003–2004) the proportion of krill in the diet, stomach fullness and fish condition (indicated by length–weight relationships) were significantly lower than in the other summer seasons. A large reduction (>80%) in the estimated annual (2005) biomass of the C. gunnari stock directly followed the season of poor krill availability. This decline was largely because of mortality of 2+ and 3+ fish, which were more krill dependent than 1+ fish. Younger fish appear to have survived, leading to an increase in the estimated population biomass in 2006.