The phenomenon of thunderstorm asthma in Bavaria, Southern Germany: a statistical approach.

Higher incidences of asthma during thunderstorms can pose a serious health risk. In this study, we estimate the thunderstorm asthma risk using statistical methods, with special focus on Bavaria, Southern Germany. In this approach, a dataset of asthma-related emergency cases for the study region is combined with meteorological variables and aeroallergen data to identify statistical relationships between the occurrence of asthma (predictand) and different environmental parameters (set of predictors). On the one hand, the results provide evidence for a weak but significant relationship between atmospheric stability indices and asthma emergencies in the region, but also show that currently thunderstorm asthma is not a major concern in Bavaria due to overall low incidences. As thunderstorm asthma can have severe consequences for allergic patients, the presented approach can be important for the development of emergency strategies in regions affected by thunderstorm asthma and under present and future climate change conditions.

New Insights into Weather and Stroke: Influences of Specific Air Masses and Temperature Changes on Stroke Incidence.

BACKGROUND/OBJECTIVES: Meteorological factors seem to influence stroke incidence, however, the complex association between weather and stroke remains unclear. Possible explanations from the literature do not categorize into subdivisions of ischemic strokes, only have small patient numbers, or refer to a selection of isolated weather elements without investigating weather changes and more. METHOD: In this exploratory trial, almost 18,000 stroke cases from a single stroke center in Southern Germany were analyzed from 2006 to 2015 and classified into the main subgroups of strokes and subdivisions of ischemic stroke etiologies applying the Trial of Org 10172 in Acute Stroke Treatment classification. For each stroke event, the air mass classification was determined from a subset of 7 air mass categories. Relative excess morbidities were derived for the 7 different air mass categories, taking into account the day of the event and up to 2 and 5 days preceding the stroke event. RESULTS: Statistically significant findings (α ≤0.1) reveal that dry tropical air masses were associated with a lower/higher risk for hemorrhagic (HEM)/macroangiopathic strokes (MAS), respectively. Dry polar air masses were associated with a higher risk for intracerebral bleedings and lower risk for ischemic stroke subtypes. Moist air masses were associated with a reduced incidence of MAS. A strong temperature increase 5 days prior to the event was associated with a lower risk of HEM strokes. Temperature increases were associated with lower risks for MAS and cardio-embolic strokes. Significant temperature decreases were associated with a higher risk of MAS. CONCLUSIONS: Temperature effects were dependent on both air masses and temperature changes within 5 days prior to the event and were associated with statistically relevant changes in stroke incidence. Decisive factors such as etiology, age, sex, and risk factors were also taken into account.

Silent Brain Infarctions and Leukoaraiosis in Patients With Retinal Ischemia: A Prospective Single-Center Observational Study.

BACKGROUND AND PURPOSE: We aimed to determine the incidence of co-occurring cerebral ischemia, extent of cerebral small vessel disease, and vascular risk profile of patients with acute retinal ischemia. METHODS: RETIS (Frequency of Acute Silent Brain Infarction and Systematic Evaluation of Stroke Risk in Retinal Ischemia) was a single-center, prospective, observational study comprising ophthalmologic examination, brain magnetic resonance imaging, and extensive diagnostic work-up of vascular risk factors and stroke cause. Silent brain infarctions were identified on diffusion-weighted imaging, leukoaraiosis was quantified on fluid-attenuated inversion recovery sequences, and carotid artery stenosis was assessed by carotid ultrasound. RESULTS: Of 112 patients with retinal ischemia, 77 (68.8%) had retinal arterial occlusion, and 35 (31.3%) presented with amaurosis fugax. Silent brain infarctions were found in 17 (15.1%) patients. Internal carotid artery stenosis was present in 19 (17.0%) and severe leukoaraiosis in 29 (25.9%) patients. Atrial fibrillation was detected in 14 (12.5%) patients. Patients with silent brain infarctions had higher rates of internal carotid artery stenosis (35.3% versus 13.7%; P=0.029) than those without, whereas leukoaraiosis and vascular risk factors were comparable between groups. Internal carotid artery stenosis was the only significant predictor of silent brain infarctions in multivariate analysis (odds ratio, 4.27; 95% confidence interval, 1.06-17.23). CONCLUSIONS: Silent cerebral ischemia is present in about 1 in 7 patients with retinal ischemia. The high rate of symptomatic internal carotid artery stenosis suggests that large artery atherosclerosis plays a major role in the pathogenesis of acute retinal ischemia.

Sound Source Localization through 8 MEMS Microphones Array Using a Sand-Scorpion-Inspired Spiking Neural Network.

Sand-scorpions and many other arachnids perceive their environment by using their feet to sense ground waves. They are able to determine amplitudes the size of an atom and locate the acoustic stimuli with an accuracy of within 13° based on their neuronal anatomy. We present here a prototype sound source localization system, inspired from this impressive performance. The system presented utilizes custom-built hardware with eight MEMS microphones, one for each foot, to acquire the acoustic scene, and a spiking neural model to localize the sound source. The current implementation shows smaller localization error than those observed in nature.

Wearable, multimodal, vitals acquisition unit for intelligent field triage.

In this Letter, the authors describe the characterisation design and development of the authors' wearable, multimodal vitals acquisition unit for intelligent field triage. The unit is able to record the standard electrocardiogram, blood oxygen and body temperature parameters and also has the unique capability to record up to eight custom designed acoustic streams for heart and lung sound auscultation. These acquisition channels are highly synchronised to fully maintain the time correlation of the signals. The unit is a key component enabling systematic and intelligent field triage to continuously acquire vital patient information. With the realised unit a novel data-set with highly synchronised vital signs was recorded. The new data-set may be used for algorithm design in vital sign analysis or decision making. The monitoring unit is the only known body worn system that records standard emergency parameters plus eight multi-channel auscultatory streams and stores the recordings and wirelessly transmits them to mobile response teams.

Sensitivity of proxies on non-linear interactions in the climate system.

Recent climate change is affecting the earth system to an unprecedented extent and intensity and has the potential to cause severe ecological and socioeconomic consequences. To understand natural and anthropogenic induced processes, feedbacks, trends, and dynamics in the climate system, it is also essential to consider longer timescales. In this context, annually resolved tree-ring data are often used to reconstruct past temperature or precipitation variability as well as atmospheric or oceanic indices such as the North Atlantic Oscillation (NAO) or the Atlantic Multidecadal Oscillation (AMO). The aim of this study is to assess weather-type sensitivity across the Northern Atlantic region based on two tree-ring width networks. Our results indicate that nonstationarities in superordinate space and time scales of the climate system (here synoptic- to global scale, NAO, AMO) can affect the climate sensitivity of tree-rings in subordinate levels of the system (here meso- to synoptic scale, weather-types). This scale bias effect has the capability to impact even large multiproxy networks and the ability of these networks to provide information about past climate conditions. To avoid scale biases in climate reconstructions, interdependencies between the different scales in the climate system must be considered, especially internal ocean/atmosphere dynamics.

A simple brain atrophy measure improves the prediction of malignant middle cerebral artery infarction by acute DWI lesion volume.

In patients with malignant middle cerebral artery infarction (MMI) decompressive surgery within 48 h improves functional outcome. In this respect, early identification of patients at risk of developing MMI is crucial. While the acute diffusion weighted imaging (DWI) lesion volume was found to predict MMI with high predictive values, the potential impact of preexisting brain atrophy on the course of space-occupying middle cerebral artery (MCA) infarction and the development of MMI remains unclear. We tested the hypothesis that the combination of the acute DWI lesion volume with simple measures of brain atrophy improves the early prediction of MMI. Data from a prospective, multicenter, observational study, which included patients with acute middle cerebral artery main stem occlusion studied by MRI within 6 h of symptom onset, was analyzed retrospectively. The development of MMI was defined according to the European randomized controlled trials of decompressive surgery. Acute DWI lesion volume, as well as brain and cerebrospinal fluid volume (CSF) were delineated. The intercaudate distance (ICD) was assessed as a linear brain atrophy marker by measuring the hemi-ICD of the intact hemisphere to account for local brain swelling. Binary logistic regression analysis was used to identify significant predictors of MMI. Cut-off values were determined by Classification and Regression Trees analysis. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the resulting models were calculated. Twenty-one (18 %) of 116 patients developed a MMI. Malignant middle cerebral artery infarctions patients had higher National Institutes of Health Stroke Scale scores on admission and presented more often with combined occlusion of the internal carotid artery and MCA. There were no differences in brain and CSF volume between the two groups. Diffusion weighted imaging lesion volume was larger (p < 0.001), while hemi-ICD was smaller (p = 0.029) in MMI patients. Inclusion of hemi-ICD improved the prediction of MMI. Best cut-off values to predict the development of MMI were DWI lesion volume > 87 ml and hemi-ICD ≤ 9.4 mm. The addition of hemi-ICD to the decision tree strongly increased PPV (0.93 vs. 0.70) resulting in a reduction of false positive findings from 7/23 (30 %) to 1/15 (7 %), while there were only slight changes in specificity, sensitivity and NPV. The absolute number of correct classifications increased by 4 (3.4 %). The integration of hemi-ICD as a linear marker of brain atrophy, that can easily be assessed in an emergency setting, may improve the prediction of MMI by lesion volume based predictive models.

Combining magnetic resonance imaging within six-hours of symptom onset with clinical follow-up at 24 h improves prediction of 'malignant' middle cerebral artery infarction.

BACKGROUND: A large diffusion-weighted imaging lesion ≤six-hours of symptom onset was found to predict the development of 'malignant' middle cerebral artery infarction with high specificity, positive predictive value, and negative predictive value, but sensitivity was low. HYPOTHESIS: We tested the hypothesis that sensitivity can be improved by adding information from clinical follow-up examination after 24 h. METHODS: We analyzed data from a prospective, multicenter, observational cohort study of patients with acute ischemic stroke and middle cerebral artery occlusion studied by stroke magnetic resonance imaging ≤six-hours of symptom onset. We used the National Institutes of Health Stroke Scale to assess severity of symptoms after 24 h. We used the Classification and Regression Trees analysis to define the optimal thresholds of diffusion-weighted imaging lesion volume and the National Institutes of Health Stroke Scale after 24 h in patients developing 'malignant' middle cerebral artery infarction. We calculated sensitivity, specificity, positive predictive value, and negative predictive value for two simple predictive models based on acute diffusion-weighted imaging lesion volume alone and acute diffusion-weighted imaging lesion volume together with the National Institutes of Health Stroke Scale after 24 h. RESULTS: Of 135 patients, 27 (20%) developed a 'malignant' middle cerebral artery infarction. The Classification and Regression Trees analysis identified acute diffusion-weighted imaging lesion ≥78 ml and the National Institutes of Health Stroke Scale score after 24 h ≥22 as optimal cut-offs. Inclusion of the National Institutes of Health Stroke Scale score after 24 h in a simple two-step decision tree increased sensitivity from 0·59 to 0·79, while specificity, positive predictive value, and negative predictive value remained largely unchanged. CONCLUSION: Clinical follow-up examination after 24 h helps identify patients at risk of 'malignant' middle cerebral artery infarction that are missed by predictive algorithms based on early diffusion-weighted imaging lesion volume alone.

Outcome of MRI-based intravenous thrombolysis in carotid-T occlusion.

Low recanalization rates and poor clinical outcome have been reported after intravenous thrombolysis (IV-tPA) in carotid-T occlusion (CTO). We studied clinical outcome and imaging findings of MRI-based intravenous thrombolysis in CTO. Data of patients with acute ischemic stroke and CTO treated with IV-tPA within 6 h of symptom onset based on MRI criteria were retrospectively analyzed. Vessel occlusion was defined based on MR angiography. Acute diffusion and perfusion lesion volumes and final infarct volumes after 3-7 days were delineated. The National Institutes of Health Stroke Scale (NIHSS) was used to assess the neurological deficit on admission. Recanalization was evaluated after 24 h. Clinical outcome was assessed using the modified Rankin Scale (mRS) after 90 days. Clinical and imaging data were compared to patients with middle cerebral artery main stem occlusion (MCAO). A total of 20 patients with CTO and 51 patients with MCAO were studied. Onset to treatment time, NIHSS on admission, initial diffusion and perfusion lesion volumes, and recanalization rates after 24 h were similar between groups. Final infarct volume was larger for CTO (82 vs. 30 ml, p = 0.006). Although overall outcome was not significantly different between groups (p = 0.251), independent outcome (mRS 0-2) tended to be less frequent in CTO (17 vs. 39 %), while poor outcome (mRS 4-6) appeared more common (72 vs. 43 %). The proportion of patients with good clinical outcome after intravenous thrombolysis in CTO is small. Moreover, final infarct volume is larger and clinical outcome appears to be worse compared to MCAO.

Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: observation of the B-side radical pair P700(+)A1B(-) in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures.

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700(+)A1A(-) and P700(+)A1B(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700(+)A1A(-). A detailed comparison reveals different orientations of A1A(-) and A1B(-) in their respective binding sites such that formation of a strong hydrogen bond from A1(-) to the protein backbone is possible only in the case of A1A(-). We suggest that this is relevant to the rates of forward electron transfer from A1A(-) or A1B(-) to the iron-sulfur center F(X), which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.

Predictors of periprocedural brain lesions associated with carotid stenting.

BACKGROUND: Carotid stenting carries a risk of periprocedural stroke. We aimed at determining predictors of cerebral ischemic events associated with stenting for symptomatic carotid stenosis. METHODS: 127 patients who had been studied by diffusion-weighted MR imaging (DWI) before and on the day after carotid stenting were included. Six clinical variables and 5 variables characterizing the target carotid artery and aortic atherosclerosis were analyzed as potential risk factors for new ipsilateral DWI lesions after stenting. RESULTS: Among all variables assessed, only age, length of stenosis and carotid intima-media thickness (IMT) significantly modified the risk of new lesions after stenting. Age ≥68 years, stenosis ≥15 mm and IMT ≥1.3 mm were identified as the best thresholds to predict new lesions. In the subgroup of patients ≥68 years with carotid stenosis ≥15 mm in length and IMT ≥1.3 mm, the risk of new lesions was markedly higher than in patients to whom no more than two of these factors applied (odds ratio 7.250, 95% CI 1.612-34.513, p = 0.005). The use of this simple predictive model correctly identified patients who had new lesions after stenting with high specificity (0.96) and a negative predictive value (0.83), while the positive predictive value was moderate (0.60) and sensitivity was low (0.23). CONCLUSIONS: The risk of stenting for symptomatic carotid stenosis may vary with clinical and morphological patient characteristics. Further research is needed to validate these results and to evaluate the safety of stenting versus endarterectomy in specific patient subgroups.

Carotid plaque surface irregularity predicts cerebral embolism during carotid artery stenting.

BACKGROUND AND PURPOSE: Carotid artery stenting (CAS) is associated with the risk of periprocedural embolic events. The procedural risk may vary with plaque characteristics. We aimed at determining the impact of carotid plaque surface irregularity on the risk of cerebral embolism during CAS. METHODS: Solid microembolic signals (MES) during CAS for symptomatic carotid stenosis were assessed by means of dual-frequency transcranial Doppler ultrasound. Study endpoint was the number of solid MES during CAS in 12 patients with irregular carotid stenosis compared to 12 matched patients with smooth carotid stenosis. RESULTS: A total of 438 solid MES were detected. The cumulative number of solid MES was 329 in patients with irregular plaques and 109 in those with smooth plaques. The proportion of subjects in whom solid MES were detected was higher in the irregular plaque group (11/12) than in the smooth plaque group (5/12) (p = 0.030). The numbers of solid MES per CAS procedure and per hour of CAS procedure were both higher in patients with irregular plaques than in those with smooth plaques (p = 0.008 and 0.015, respectively). CONCLUSIONS: Carotid plaque surface irregularity predicts solid cerebral embolism during stenting of symptomatic carotid artery stenosis.

Hemin and magnesium-protoporphyrin IX induce global changes in gene expression in Chlamydomonas reinhardtii.

Retrograde signaling is a pathway of communication from mitochondria and plastids to the nucleus in the context of cell differentiation, development, and stress response. In Chlamydomonas reinhardtii, the tetrapyrroles magnesium-protoporphyrin IX and heme are only synthesized within the chloroplast, and they have been implicated in the retrograde control of nuclear gene expression in this unicellular green alga. Feeding the two tetrapyrroles to Chlamydomonas cultures was previously shown to transiently induce five nuclear genes, three of which encode the heat shock proteins HSP70A, HSP70B, and HSP70E. In contrast, controversial results exist on the possible role of magnesium-protoporphyrin IX in the repression of genes for light-harvesting proteins in higher plants, raising the question of how important this mode of regulation is. Here, we used genome-wide transcriptional profiling to measure the global impact of these tetrapyrroles on gene regulation and the scope of the response. We identified almost 1,000 genes whose expression level changed transiently but significantly. Among them were only a few genes for photosynthetic proteins but several encoding enzymes of the tricarboxylic acid cycle, heme-binding proteins, stress-response proteins, as well as proteins involved in protein folding and degradation. More than 50% of the latter class of genes was also regulated by heat shock. The observed drastic fold changes at the RNA level did not correlate with similar changes in protein concentrations under the tested experimental conditions. Phylogenetic profiling revealed that genes of putative endosymbiontic origin are not overrepresented among the responding genes. This and the transient nature of changes in gene expression suggest a signaling role of both tetrapyrroles as secondary messengers for adaptive responses affecting the entire cell and not only organellar proteins.

Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX.

Two Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

Classifications of atmospheric circulation patterns: recent advances and applications.

We review recent advances in classifications of circulation patterns as a specific research area within synoptic climatology. The review starts with a general description of goals of classification and the historical development in the field. We put circulation classifications into a broader context within climatology and systematize the varied methodologies and approaches. We characterize three basic groups of classifications: subjective (also called manual), mixed (hybrid), and objective (computer-assisted, automated). The roles of cluster analysis and principal component analysis in the classification process are clarified. Several recent methodological developments in circulation classifications are identified and briefly described: the introduction of nonlinear methods, objectivization of subjective catalogs, efforts to optimize classifications, the need for intercomparisons of classifications, and the progress toward an optimum, if possible unified, classification method. Among the recent tendencies in the applications of circulation classifications, we mention a more extensive use in climate studies, both of past, present, and future climates, innovative applications in the ensemble forecasting, increasing variety of synoptic-climatological investigations, and steps above from the troposphere. After introducing the international activity within the field of circulation classifications, the COST733 Action, we briefly describe outputs of the inventory of classifications in Europe, which was carried out within the Action. Approaches to the evaluation of classifications and their mutual comparisons are also reviewed. A considerable part of the review is devoted to three examples of applications of circulation classifications: in historical climatology, in analyses of recent climate variations, and in analyses of outputs from global climate models.

Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii.

A specific signaling role for H(2)O(2) in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H(2)O(2) but also on light. In the dark, the induction of the reporter gene by H(2)O(2) was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H(2)O(2) from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H(2)O(2) levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3'4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H(2)O(2) in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H(2)O(2) required to activate H(2)O(2)-dependent signaling pathways.

Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas.

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.

A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo.

A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions -81 to -149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced.

Identification of a plastid response element that acts as an enhancer within the Chlamydomonas HSP70A promoter.

Chloroplast-derived signals control a subset of nuclear genes in higher plants and eukaryotic algae. Among the types of signals identified are intermediates of chlorophyll biosynthesis such as Mg-protoporphyrin IX (MgProto). In Chlamydomonas reinhardtii, it was suggested that this tetrapyrrole mediates the light induction of chaperone gene HSP70A. Here we have analyzed cis elements involved in the regulation of HSP70A by MgProto and light. We identified two promoters and between their transcription start sites two regulatory regions that each may confer inducibility by MgProto and light to both HSP70A promoters. These regulatory regions, when cloned in front of basal non-light inducible heterologous promoters, conferred inducibility by MgProto and light. The orientation and distance independent function of these cis-regulatory sequences qualifies them as enhancers that mediate the response of nuclear genes to a chloroplast signal. Mutational analysis of one of these regulatory regions and an alignment with promoters of other MgProto-inducible genes revealed the sequence motif (G/C)CGA(C/T)N(A/G)N15 (T/C/A)(A/T/G) which, as shown for HSP70A, may confer MgProto responsiveness. This cis-acting sequence element is employed for induction of HSP70A by both MgProto and light, lending support to the model that light induction of this gene is mediated via MgProto.