Impact of sudden oak death on tree mortality in the Big Sur ecoregion of California

The Big Sur ecoregion in coastal California is a botanically and ecologically diverse area that has recently experienced substantial mortality of oak (Quercus spp.) and tanoak (Lithocarpus densiflorus) trees due to the emerging forest disease sudden oak death, caused by the invasive pathogen Phytophthora ramorum. In response to the urgent need to examine environmental impacts and create management response strategies, we quantified the impact of P. ramorum invasion on tree mortality across the Big Sur ecoregion using high-resolution aircraft imagery and field data. Using the imagery, we mapped all detectable oak and tanoak trees possibly killed by P. ramorum infection within redwood-tanoak forests and mixed oak woodlands. To validate and improve our remote assessment, we quantified the number, size, and infection status of host trees in 77 field plots (0.25 ha). The field data showed that our remote assessment underestimated mortality due to the occurrence of dead trees in the forest understory. For each forest type, we developed regression models that adjusted our remote assessments of tree mortality in relation to field observations of mortality and local habitat variables. The models significantly improved remote assessment of oak mortality, but relationships were stronger for mixed oak woodlands (r ² = 0.77) than redwood-tanoak forests (r ² = 0.66). Using the field data, we also modeled the amount of dead tree basal area (m²) in relation to the density of mapped dead trees in mixed oak woodlands (r ² = 0.73) and redwood-tanoak forests (r ² = 0.54). Application of the regression models in a GIS estimated 235,678 standing dead trees in 2005 and 12,650 m² of tree basal area removed from the ecoregion, with 63% of mortality occurring in redwood-tanoak forests and 37% in mixed oak woodlands. Integration of the remote assessment with population estimates of host abundance, obtained from an independent network of 175 field plots (0.05 ha each), indicated similar prevalence of mortality in redwood-tanoak forests (20.0%) and mixed oak woodlands (20.5%) at this time. This is the first study to quantify a realistic number of dead trees impacted by P. ramorum over a defined ecological region. Ecosystem impacts of such widespread mortality will likely be significant.

Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry

This study describes methods and equipment for measurement of metabolic heat rates of cells and tissues under conditions that provide simultaneous determinations of the flux rates of both O2 and CO2. Isothermal measurement of metabolic heats are conducted in a sealed ampule. A trapping solution is employed to absorb metabolic CO2. Absorption of CO2 produces heat at a rate proportional to the rate of CO2 production. Under these conditions, O2 consumption by the tissue results in a decrease in the partial pressure of O2 within the sealed ampule. The decrease in pressure can be monitored with a pressure sensor and related to O2 consumption rates. The combined measurements of heat rates, CO2, and O2 fluxes provide important information on bioenergetic efficiency of cell metabolism. These data can also suggest possible shifts in metabolic pathways or substrate sources as cells develop, or are exposed to effectors, inhibitors, and environmental factors.     

DNA sequence divergence and functional conservation at the STB locus of yeast 2 microns circle variants.

2 microns DNA isolated from industrial Saccharomyces cerevisiae yeasts exhibited extensive restriction fragment length polymorphisms. At least five 2 microns species were identified from eleven [cir+] strains. Southern hybridization mapped restriction fragment length polymorphisms at STB, a cis-acting locus essential for plasmid partitioning. Some 2 microns variants (e.g., 4110-2 microns and 4108-2 microns) had an altered number of 125-bp consensus repeats at STB. However, the corresponding region of 7754-2 microns has only approximately 70% nucleotide sequence homology with the 125-bp STB consensus repeat. YRp plasmids containing 7754-2 microns STB behave as YEp plasmids in laboratory yeasts, thereby indicating STB sequence divergence coupled to conservation of function.     

Modification and Inheritance of Pleiotropic Cross Resistance and Collateral Sensitivity in SACCHAROMYCES CEREVISIAE

A meiotic segregant (oli^PR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oli^PR1 has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethylphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton-X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oli^PR1 phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested. Resistant diploids that produced four resistant ascospores were the result of mitotic recombination prior to meiosis. A partial revertant phenotype (sensitive to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was shown to be due to a single nuclear gene causing partial suppression of oli^PR1. Anaerobic pretreatment, 37° and 0.5 M KCl were observed to reduce the growth of oli^PR1 when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,-chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho—] state. A revertant of oli^PR1 (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin and neomycin. Continuous monitoring of oxygen uptake by the revertant after anaerobic pretreatment revealed that anaerobiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oli^PR1 is a mutation resulting in the alteration of plasma membrane premeability to many diverse inhibitors.     

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.     

A HIERARCHICAL ANALYSIS OF GENETIC DIFFERENTIATION IN A MONTANE LEAF BEETLE CHRYSOMELA AENEICOLLIS (COLEOPTERA: CHRYSOMELIDAE)

Herbivorous insects that use the same host plants as larvae and adults can have a subdivided population structure that corresponds to the distribution of their hosts. Having a subdivided population structure favors local adaptation of subpopulations to small-scale environmental differences and it may promote their genetic divergence. In this paper, I present the results of a hierarchical study of population structure in a montane willow leaf beetle, Chrysomela aeneicollis (Coleoptera: Chrysomelidae). This species spends its entire life associated with the larval host (Salix spp.), which occurs in patches along high-elevation streams and in montane bogs. I analyzed the genetic differentiation of C. aeneicollis populations along three drainages in the Sierra Nevada mountains of California at five enzyme loci: ak-1, idh-2, mpi-1, pgi-1, and pgm-1, using recent modifications of Wright's F-statistics. My results demonstrated significant differentiation (F_ST = 0.043) among drainages that are less than 40 kilometers apart. One locus, pgi-1, showed much greater differentiation than the other four (F_ST = 0.412), suggesting that it is under natural selection. C. aeneicollis populations were also subdivided within drainages, with significant differentiation 1) among patches of willows (spanning less than three kilometers) and 2) in some cases, among trees within a willow patch. My results demonstrate that this species has the capacity to adapt to local environmental variation at small spatial scales.     

Modelling temperature effects on growth–respiration relations of maize

The temperature dependence of plant growth rate is related to the temperature dependence of respiratory metabolism. To determine how the effects of temperature on respiration rate and efficiency are transmitted to growth, this study measured the dark metabolic heat rate ( q ) and CO₂ production rate ( R _CO2) in excised shoots of seedlings of 14 maize cultivars ( Zea mays L.) at several temperatures. The temperature coefficients of q and R _CO2 differ within a given cultivar and also differ among the cultivars. Both q and R _CO2 exhibit an isokinetic temperature of 20 ± 3 °C. The measured temperature dependences of q and R _CO2 were used to model the temperature dependences of both growth and substrate carbon conversion efficiency. This procedure may be useful in determining the suitability of cultivars for growth in a given climate and in understanding metabolic adaptation to climate.     

Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure–function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1–PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure–function relationships of natural and artificial PARP1 variants.     

A transient duplication of the acetolactate synthase gene in a cell culture of Datura innoxia

Summary A 2.0 kb fragment of the yeast ILV2 gene, which codes for the target enzyme acetolactate synthase (ALS) of the herbicide chlorsulfuron, was shown to hybridize to the nuclear DNA of a haploid cell culture of Datura innoxia P. Mill. Nuclear DNA of a chlorsulfuron resistant line of D. innoxia, CSR6, gave a prominent 2.65 kb band when cleaved by either EcoRI or HindIII. The 2.65 kb band has been shown to hybridize with the yeast ILV2 probe. A herbicide resistant line descended from CSR6 by continuous culture resulted in the loss of the 2.65 kb restriction fragment. These observations suggest that CSR6 resulted from a large tandem duplication of the ALS gene and that a point mutation for herbicide resistance in an ALS gene repeat unit of the duplication was selected during subsequent growth of the resistant line.     

Implementation of a thermal biosensor in a process environment: on-line monitoring of penicillin V in production-scale fermentations.

The production of penicillin V was monitored in 0.5 m3 and 160 m3 bioreactors. The thermal biosensor was an enzyme thermistor modified for split-flow analysis. The heat signal generated in the enzyme column was corrected for any nonspecific heat with the use of an identical but inactive reference column. The on-line monitoring was performed in the fermentation pilot plant and in a fermentation plant of Novo Nordisk A/S. Immobilized beta-lactamase was used to monitor three consecutive 0.5 m3 penicillin fermentations. Broth samples were continuously filtered through a tangential flow filtration unit in a sterile external loop. The on-line penicillin V values were 10% higher than those obtained by off-line HPLC analysis. Alternatively a polypropylene filtration probe was inserted into a 160 m3 bioreactor and samples were withdrawn at 0.5 ml/min. The same experiments were repeated with purified and immobilized penicillin V acylase. The on-line penicillin V values obtained with this enzyme correlated very well with those from HPLC analysis. The on-line monitoring was controlled and analysed by a software program written in Labtech Notebook.