Interplay between MAMP-triggered and SA-mediated defense responses

Plants respond to pathogen infection using an innate immune system with at least two distinct recognition mechanisms. One mechanism recognizes microbe-associated molecular patterns (MAMPs). The other is based on resistance (R) genes and specifically recognizes certain pathogen virulence factors, including those delivered through the type III secretion system (TTSS) of bacteria. Salicylic acid (SA)-mediated responses are an important part of the R gene-mediated defense. Substantial overlaps between MAMP-triggered and SA-mediated responses have been reported. However, interactions between MAMP-triggered and SA-mediated signaling mechanisms have not been well documented. Here we report intimate interactions between MAMP-triggered and SA-mediated signaling. We found that SA accumulated at a higher level 6 h after treatment with a MAMP, flg22 or inoculation with Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000) hrcC mutant, which is deficient in TTSS function. Disruptions of SA signaling components, such as SID2 and PAD4 , strongly affected MAMP-triggered responses monitored by expression profiling. We found two groups of genes that were induced by Pst DC3000 hrcC in an SA-dependent manner. One group was SID2 -dependent at all time points, whereas the other was SID2 -independent at early time points and SID2 -dependent at later time points. Thus, the expression of the latter genes responds to MAMPs through both SA-independent and SA-dependent signaling mechanisms. Strong resistance to Pst DC3000 hrcC was dependent on SA signaling. These results indicate that the SA increase triggered by MAMPs is a major component of the MAMP-triggered signaling mechanism, explaining the overlapping spectra of MAMP-triggered and SA-mediated responses.     

Multistep mass tagging coupled with 2D LC-MS--an approach to increasing the number of identified proteins.

Identification of large numbers of proteins from complex biological samples is a continuing challenge in the area of quantitative proteomics. We introduce here a simple and reliable multistep mass tagging technique using our recently developed solid phase mass tagging reagents. When coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D-LC/nano-ESI-MS), this method allows enhanced protein identification when tested on samples from prokaryotic and eukaryotic sources. The proteome of Escherichia coli D21 grown to either mid-exponential or stationary phase, and the membrane proteome from established breast cancer cell lines BT474 and MCF7 were used as model systems in these experiments. In both experiments, the numbers of total identified proteins are at least twice the numbers identified from a single tagging cycle. The sample complexity can be effectively reduced with corresponding increases in protein identification using the multistep method. The strategy described here represents a potentially powerful technique for large-scale qualitative and quantitative proteome research.     

Organisational downsizing,sickness absence,and mortality: 10-town prospective cohort study

Objective To examine whether downsizing, the reduction of personnel in organisations, is a predictor of increased sickness absence and mortality among employees.Design Prospective cohort study over 7.5 years of employees grouped into categories on the basis of reductions of personnel in their occupation and workplace: no downsizing (< 8% reduction), minor downsizing (8-18%), and major downsizing (> 18%).Setting Four towns in Finland.Participants 5909 male and 16 521 female municipal employees, aged 19-62 years, who kept their jobs.Main outcome measures Annual sickness absence rate based on employers'' records before and after downsizing by employment contract; all cause and cause specific mortality obtained from the national mortality register.Results Major downsizing was associated with an increase in sickness absence (P for trend < 0.001) in permanent employees but not in temporary employees. The extent of downsizing was also associated with cardiovascular deaths (P for trend < 0.01) but not with deaths from other causes. Cardiovascular mortality was 2.0 (95% confidence interval 1.0 to 3.9) times higher after major downsizing than after no downsizing. Splitting the follow up period into two halves showed a 5.1 (1.4 to 19.3) times increase in cardiovascular mortality for major downsizing during the first four years after downsizing. The corresponding hazard ratio was 1.4 (0.6 to 3.1) during the second half of follow up.Conclusion Organisational downsizing may increase sickness absence and the risk of death from cardiovascular disease in employees who keep their jobs.     

A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts

Surface plasmon resonance (SPR) spectroscopy has emerged as a powerful alternative to conventional biochemistry methods for studying protein-DNA interactions that involve recombinant proteins of known identity. There are, however, limited demonstrations of SPR detection of protein-DNA bindings in crude samples, e.g., cell extracts, where the challenge is to detect and identify specific DNA binding protein(s) among other protein components in a physiological setting. We have developed a two-step antibody approach for an SPR study of estrogen receptor α (ERα)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERα protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERα antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERα and amplify the signals, respectively. Through a series of experiments, we have demonstrated that the magnitude of the binding signals from the secondary antibody reflects the affinity by which ERα binds to different DNA sequences. The detection sensitivity is determined by the amount of nuclear extracts and the concentration of primary antibody used. The sequence specificity of the nuclear ERα measured using the two-step antibody approach is in agreement with that measured for recombinant ERα protein (using receptor binding signals).     

Compelling EPR evidence that the alternative oxidase is a diiron carboxylate protein

The alternative oxidase is a respiratory chain protein found in plants, fungi and some parasites that still remains physically uncharacterised. In this report we present EPR evidence from parallel mode experiments which reveal signals at approximately g = 16 in both purified alternative oxidase protein (g = 16.9), isolated mitochondrial membranes (g = 16.1), and in trypanosomal AOX expressed in Escherichia coli membranes (g = 16.4). Such signals are indicative of a dicarboxylate diiron centre at the active site of the enzyme. To our knowledge these data represent the first EPR signals from AOX present in its native environment.