Although a number of plant carotenoid cleavage dioxygenase (CCD) genes have been functionally characterized in different plant species, little is known about the biochemical role and enzymatic activities of members of the subclass 4 (CCD4). To gain insight into their biological function, CCD4 genes were isolated from apple (Malus×domestica, MdCCD4), chrysanthemum (Chrysanthemum×morifolium, CmCCD4a), rose (Rosa×damascena, RdCCD4), and osmanthus (Osmanthus fragrans, OfCCD4), and were expressed, together with AtCCD4, in Escherichia coli. In vivo assays showed that CmCCD4a and MdCCD4 cleaved β-carotene well to yield β-ionone, while OfCCD4, RdCCD4, and AtCCD4 were almost inactive towards this substrate. No cleavage products were found for any of the five CCD4 genes when they were co-expressed in E. coli strains that accumulated cis-ζ-carotene and lycopene. In vitro assays, however, demonstrated the breakdown of 8′-apo-β-caroten-8′-al by AtCCD4 and RdCCD4 to β-ionone, while this apocarotenal was almost not degraded by OfCCD4, CmCCD4a, and MdCCD4. Sequence analysis of genomic clones of CCD4 genes revealed that RdCCD4, like AtCCD4, contains no intron, while MdCCD, OfCCD4, and CmCCD4a contain introns. These results indicate that plants produce at least two different forms of CCD4 proteins. Although CCD4 enzymes cleave their substrates at the same position (9,10 and 9′,10′), they might have different biochemical functions as they accept different (apo)-carotenoid substrates, show various expression patterns, and are genomically differently organized.

Long-term exposure to air pollution increases cardiopulmonary morbidity and mortality, but it is not clear which components of air pollution are the most harmful, nor which time window of exposure is most relevant. Further studies at low exposure levels have also been called for. We analyzed two Swedish cohorts to investigate the effects of total and source-specific particulate matter (PM) on incident cardiovascular disease for different time windows of exposure. Two cohorts initially recruited to study predictors of cardiovascular disease (the PPS cohort and the GOT-MONICA cohort) were followed from 1990 to 2011. We collected data on residential addresses and assigned each individual yearly total and source-specific PM and Nitrogen Oxides (NOx) exposures based on dispersion models. Using multivariable Cox regression models with time-dependent exposure, we studied the association between three different time windows (lag 0, lag 1-5, and exposure at study start) of residential PM and NOx exposure, and incidence of ischemic heart disease, stroke, heart failure and atrial fibrillation. During the study period, there were 2266 new-onset cases of ischemic heart disease, 1391 of stroke, 925 of heart failure and 1712 of atrial fibrillation. The majority of cases were in the PPS cohort, where participants were older. Exposure levels during the study period were moderate (median: 13µg/m3 for PM10 and 9µg/m3 for PM2.5), and similar in both cohorts. Road traffic and residential heating were the largest local sources of PM air pollution, and long distance transportation the largest PM source in total. In the PPS cohort, there were positive associations between PM in the last five years and both ischemic heart disease (HR: 1.24 [95% CI: 0.98-1.59] per 10µg/m3 of PM10, and HR: 1.38 [95% CI: 1.08-1.77] per 5µg/m3 of PM2.5) and heart failure. In the GOT-MONICA cohort, there were positive but generally non-significant associations between PM and stroke (HR: 1.48 [95% CI: 0.88-2.49] per 10µg/m3 of PM10, and HR: 1.50 [95% CI: 0.90-2.51] per 5µg/m3 of PM2.5, in the last five years). Effect estimates were stronger for women, non-smokers, and higher socioeconomic classes. Exposure in the last five years seemed to be more strongly associated with outcomes than other exposure time windows. Associations between source-specific PM air pollution and outcomes were mixed and generally weak. High correlations between the main pollutants limited the use of multi-pollutant models. The main PM air pollutants were associated with ischemic heart disease and stroke (in women) at the relatively low exposure levels in Gothenburg, Sweden. The associations tended to be stronger for women than for men, for non-smokers than for smokers, and for higher socioeconomic classes than for lower. The associations could not be attributed to a specific PM source or type, and differed somewhat between the two cohorts. The results of this study confirm that further efforts to reduce air pollution exposure should be undertaken in Sweden to reduce the negative health effects in the general population.

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth. A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in ∼74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4–6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate andN-methyl-d-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.

Dentate granule cells become synaptically interconnected in the hippocampus of persons with temporal lobe epilepsy, forming a recurrent mossy fiber pathway. This pathway may contribute to the development and propagation of seizures. The physiology of mossy fiber–granule cell synapses is difficult to characterize unambiguously, because electrical stimulation may activate other pathways and because there is a low probability of granule cell interconnection. These problems were addressed by the use of scanning laser photostimulation in slices of the caudal hippocampal formation. Glutamate was released from a caged precursor with highly focused ultraviolet light to evoke action potentials in a small population of granule cells. Excitatory synaptic currents were recorded in the presence of bicuculline. Minimal laser photostimulation evoked an apparently unitary excitatory postsynaptic current (EPSC) in 61% of granule cells from rats that had experienced pilocarpine-induced status epilepticus followed by recurrent mossy fiber growth. An EPSC was also evoked in 13–16% of granule cells from the control groups. EPSCs from status epilepticus and control groups had similar peak amplitudes (∼30 pA), 20–80% rise times (∼1.2 ms), decay time constants (∼10 ms), and half-widths (∼8 ms). The mean failure rate was high (∼70%) in both groups, and in both groups activation of N-methyl-d-aspartate receptors contributed a small component to the EPSC. The strong similarity between responses from the status epilepticus and control groups suggests that they resulted from activation of a similar synaptic population. No EPSC was recorded when the laser beam was focused in the dentate hilus, suggesting that indirect activation of hilar mossy cells contributed little, if at all, to these results. Recurrent mossy fiber growth increases the density of mossy fiber–granule cell synapses in the caudal dentate gyrus by perhaps sixfold, but the new synapses appear to operate very similarly to preexisting mossy fiber–granule cell synapses.