Photoacoustic Study of Changes in the Energy Storage of Photosystems I and II during State 1-State 2 Transitions

Using photoacoustic spectroscopy, state 1-state 2 transitions were demonstrated in vivo in intact sugar maple leaves (Acer saccharum Marsh.) by following the changes in energy storage of photosystems (PS) I and II. Energy storage measured with 650 nm modulated light (light 2) in the presence of background white light indicated the total energy stored by both photosystems (ES_t), and in the presence of background far-red light showed the energy stored by PSI (ES_psi). The difference between ES_t and ES_psi gave the energy stored by PSII (ES_psii). While ES_t remained nearly constant during state transitions, both ES_psi and ES_psii changed considerably. The ratio of ES_psii to ES_psi, an indicator of the energy distribution between the two photosystems, decreased or increased during transition to state 2 or state 1, respectively. State transitions were completed in about 20 min and were fully reversible. During transition from state 1 to state 2, the fraction of excitation energy gained by PSI was nearly equal to that lost by PSII. This fraction of excitation energy transferred from PSII to PSI accounted for about 5% of the absorbed light (fluorescence is not considered), 19% of ES_t, 34% of ES_psii, and 43% of ES_psi in state 2. NaF treatment inhibited the transition to state 1. Data in the present study confirm the concept of changes in absorption cross-section of photosystems during state transitions.     

Sulfite inhibition of photochemical activity of intact pea leaves

Sulfite treatment of pea leaf disks in light caused a significant decrease in the relative quantum yield of photosynthetic oxygen evolution and energy storage (ES) as measured by photoacoustic (PA) spectroscopy. The inhibition was concentration dependent and was less in darkness than in light, indicating light-dependent inhibitory site(s) on the photosynthetic electron transport chain. Further, in darksulfite-treated leaves, the energy storage was more affected than the relative quantum yield of oxygen evolution, suggesting that photophosphorylation and/or cyclic electron transport around PS I are sites of sulfite action in darkness. The R_fd values, the ratio of fluorescence decrease (fd) to the steady-state fluorescence (fs), decreased significantly in leaves treated with sulfite in light but were not affected in dark-treated ones, confirming the photoacoustic observations. Similarly, the ratio of variable fluorescence (F_v) to maximum fluorescence (F_m), a measure of PS II photochemical efficiency, was affected by sulfite treatment in light and not changed by treatment in darkness. An attempt was made to explain the mechanism of sulfite action on photosynthetic electron transport in light and in darkness.Abbreviations A_PT amplitude of photothermal signal - A_ox amplitude of oxygen signal - ES energy storage - fd fluorescence decrease - fs steady-state fluorescence - F_m maximum fluorescence - F_v variable fluorescence - PA photoacoustic(s)     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Energy storage and optical cross-section of PS I in the cyanobacterium Synechococcus PCC 7002 and a psaE- mutant

In vivo electron flow in the unicellular cyanobacterium Synechococcus sp. PCC 7002 was studied by pulsed, time- resolved photoacoustics (PTRPA). Using 1-s, 2 J or 10¹³ hcm-2 pulses at 695 nm, we observed large (42 ± 2%) Photosystem I (PS I) cyclic energy storage (ES) in the period of 2 to 12 ms after excitation with wild type (WT) intact cells. This cyclic ES was insensitive to flash interval from 0.3 to 10 s and to the presence of 1 m DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). At this low flash energy and in the absence of continuous background light (in the dark), antimycin A, carbonylcyanide-m- chlorophenylhydrazone (CCCP), 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), DCMU, 2- n-heptyl-4- hydroxyquinoline-N-oxide (HQNO), myxothiazol and N- ethylmaleimide (NEM) caused little or no inhibition of PS I cyclic electron flow. When continuous background far-red light ( > 715 nm) was added during the measurement, strong inhibition by DBMIB and NEM and less by HQNO was observed, the amplitude of which was related to both concentration and the intensity of the background light. Analysis of the data with DBMIB yields its binding constant, 1 m, and the turnover time of the system (> 20 ms). A turnover time of the uninhibited system of 2–3 ms was obtained by a pump-probe method. A dramatic lifting of the partial inhibition in the presence of far-red light was caused by antimycin A and a smaller effect by myxothiazol. The rescuing effect was assigned to a short circuiting of the electron flow about the cytochrome (cyt) b6/f system. Progressively increasing the laser pulse energy allowed us to calculate the PS I optical cross-section (54 ± 2 Å). Analysis by the sensitive method of convolutions revealed a possible energy loss on the few ms time scale by antimycin A in the dark. The analysis also revealed a similar effect or artifact in uninhibited samples using the same sample illuminated with saturating continuous light, the standard procedure in photoacoustics (PA). A psaE- mutant showed more inhibition in the dark by DBMIB and with far-red light by HQNO, but less inhibition in the far-red light by myxothiazol than the WT. Under normal growth conditions, maximum ES for the psaE- mutant (38 ± 2%) was similar to that of the WT (42 ± 2%). However, under mild heat stress, maximum ES for the psaE- mutant dropped to 26% while the WT maximum ES stayed unchanged at 41%, within batch-to-batch variation. These results would indicate that the PsaE protein is not essential for PS I cyclic electron flow under our experimental conditions but plays a stabilizing role in the PS I complex under a mild thermal stress.     

Regulation of DNaseY activity by actinin-alpha4 during apoptosis

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.     

Validation of a voxel-based FE method for prediction of the uniaxial apparent modulus of human trabecular bone using macroscopic mechanical tests and nanoindentation

Assessment of the mechanical properties of trabecular bone is of major biological and clinical importance for the investigation of bone diseases, fractures and their treatments. Finite element (FE) methods are getting increasingly popular for quantifying the elastic and failure properties of trabecular bone. In particular, voxel-based FE methods have been previously used to calculate the effective elastic properties of trabecular microstructures. However, in most studies, bone tissue moduli were assumed or back-calculated to match the apparent elastic moduli from experiments, which often lead to surprisingly low values when compared to nanoindentation results. In this study, voxel-based FE analysis of trabecular bone is combined with physical measures of volume fraction, micro-CT (microCT) reconstructions, uniaxial mechanical tests and specimen-specific nanoindentation tests for proper validation of the method. Cylindrical specimens of cancellous bone were extracted from human femurs and their volume fraction determined with Archimede's method. Uniaxial apparent modulus of the specimens was measured with an improved tension-compression testing protocol that minimizes boundary artefacts. Their microCT reconstructions were segmented to match the measured bone volume fraction and used to create full-size voxel models with 30-45 microm element size. For each specimen, linear isotropic elastic material properties were defined based on specific nanoindentation measurements of its embedded bone tissue. Linear FE analyses were finally performed to simulate the uniaxial mechanical tests. Additional parametric analyses were performed to evaluate the potential errors on the predicted apparent modulus arising from variations in segmentation threshold, tissue modulus, and the use of 125-mm(3) cubic sub-regions. The results demonstrate an excellent correspondence between experimental measures and FE predictions of uniaxial apparent modulus. In conclusion, the adopted voxel-based FE approach is found to be a robust method to predict the linear elastic properties of human cancellous bone, provided segmentation of the microCT reconstructions is carefully calibrated, tissue modulus is known a priori and the entire region of interest is included in the analysis.     

A simulation test bed for hypotheses of genome evolution

MOTIVATION: Microbial genomes undergo evolutionary processes such as gene family expansion and contraction, variable rates and patterns of sequence substitution and lateral genetic transfer. Simulation tools are essential for both the generation of data under different evolutionary models and the validation of analytical methods on such data. However, meaningful investigation of phenomena such as lateral genetic transfer requires the simultaneous consideration of many underlying evolutionary processes. RESULTS: We have developed EvolSimulator, a software package that combines non-stationary sequence and gene family evolution together with models of lateral genetic transfer, within a customizable birth-death model of speciation and extinction. Here, we examine simulated data sets generated with EvolSimulator using existing statistical techniques from the evolutionary literature, showing in detail each component of the simulation strategy. AVAILABILITY: Source code, manual and other information are freely available at www.bioinformatics.org.au/evolsim. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Distinctive architecture of the chloroplast genome in the chlorophycean green alga Stigeoclonium helveticum

The chloroplast genome has experienced many architectural changes during the evolution of chlorophyte green algae, with the class Chlorophyceae displaying the lowest degree of ancestral traits. We have previously shown that the completely sequenced chloroplast DNAs (cpDNAs) of Chamydomonas reinhardtii (Chlamydomonadales) and Scenedesmus obliquus (Sphaeropleales) are highly scrambled in gene order relative to one another. Here, we report the complete cpDNA sequence of Stigeoclonium helveticum (Chaetophorales), a member of a third chlorophycean lineage. This genome, which encodes 97 genes and contains 21 introns (including four putatively trans-spliced group II introns inserted at novel sites), is remarkably rich in derived features and extremely rearranged relative to its chlorophycean counterparts. At 223,902 bp, Stigeoclonium cpDNA is the largest chloroplast genome sequenced thus far, and in contrast to those of Chlamydomonas and Scenedesmus, features no large inverted repeat. Interestingly, the pattern of gene distribution between the DNA strands and the bias in base composition along each strand suggest that the Stigeoclonium genome replicates bidirectionally from a single origin. Unlike most known trans-spliced group II introns, those of Stigeoclonium exhibit breaks in domains I and II. By placing our comparative genome analyses in a phylogenetic framework, we inferred an evolutionary scenario of the mutational events that led to changes in genome architecture in the Chlorophyceae.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Nucleotide sequence data reported are available in the GenBank database under the accession number DQ630521.