Characterization of Chlorophyll–protein Complexes Isolated from Two Marine Green Algae, Bryopsis maxima and Ulva pertusa, Growing in the Intertidal Zone

Three Chl–protein complexes were isolated from thylakoid membranes of Bryopsis maxima and Ulva pertusa, marine green algae that inhabit the intertidal zone of the Pacific Ocean off the eastern coast of Japan by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. The slowest-moving fractions showed low Chl a/b and Chl/P-700 ratios, indicating that this fraction corresponds to complexes in PS I, which is large in both algae. The intermediate and fastest-moving fractions showed the traits of PS II complexes, with some associated Chl a/b–protein complexes and LHC II, respectively. The spectral properties of the separated Chl–proteins were also determined. The absorption spectra showed a shallow shoulder at 540 nm derived from siphonaxanthin in Bryopsis maxima, but not in Ulva pertusa. The 77 K emission spectra showed a single peak in Bryopsis maxima and two peaks in Ulva pertusa. Besides the excitation spectra indicated that the excitation energy transfer to the PS I complexes differed quite a lot higher plants. This suggested that the mechanisms of energy transfer in both of these algae differ from those of higher plants. Considering the light environment of this coastal area, the large size of the antennae of PS I complexes implies that the antennae are arranged so as to balance light absorption between the two photosystems. In addition, we discuss the relationships among the photosystem stoichiometry, the energy transfer, and the distribution between the two photosystems.     

Energy Transfer among Chlorophylls in Trimeric Light-harvesting ComplexⅡ of Bryopsis corticulans

A study on energy transfer among chlorophylls(Chls)in the trimeric unit of the major light-harvesting complex Ⅱ(LHC Ⅱ)from Bryopsis corriculan,was carried out using time-correlated singlephoton counting.In the chlorophyll Q region of LHC Ⅱ,six molecules characterized as Chlb_(628),Chlb_(646),Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683) were discriminated according to their absorption spectrumand fluorescence emission spectrum.Then,excited by pulsed light of 628 nm,fluorescence kinetics spectrain the chlorophyll Q region were measured.In accordance with the principles of fluorescence kinetics,thesekinetics data were analyzed with a multi-exponential model.Time constants on energy transfer were obtained.An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97picoseconds(ps),which shows that,before transferring energy to another Chl,the excited Chl might convertenergy to vibrations of a lower state with different multiplicity(intersystem crossing).Energy transfer at thelevel of approximately 10 ps was also obtained,which was interpreted as the excited Chls may go throughinternal conversion before transferring energy to another Chl.Although with a higher standard deviation,timeconstants at the femtosecond level can not be entirely excluded,which can be attributed to the ultrafastprocess of direct energy transfer.Owing to the arrangement and direction of the dipole moment of Chls inLHC Ⅱ,the probability of these processes is different.The fluorescence lifetimes of Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683)were determined to be 1.44ns,1.43 ns,636 ps and 713 ps,respectively.Thepercentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in thetrimeric unit of LHC Ⅱ.These results are important for a better understanding of the relationship between thestructure and function of LHC Ⅱ.     

Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll a/b Protein Complexes

We studied the difference in thermostability of photosystem Ⅱ （PSII） and leaf lipid composition between a T-DNA insertion mutant rice （Oryza sativa L.） VG28 and its wild type Zhonghuau. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSII photochemistry （FJ Fm） and oxygen-evolving activity of PSII in leaves significantly decreased with increasing temperature. However, the PSII activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of 16：1t and a marked increase in the content of 18：3 compared with the wild type. The effects of lipid composition and unsaturation of membrane lipids on the thermostability of PSII are discussed.     

Who Is in There? Exploration of Endophytic Bacteria within the Siphonous Green Seaweed Bryopsis (Bryopsidales,Chlorophyta)

Associations between marine seaweeds and bacteria are widespread, with endobiotic bacterial-algal interactions being described for over 40 years. Also within the siphonous marine green alga Bryopsis, intracellular bacteria have been visualized by electron microscopy in the early '70s, but were up to now never molecularly analyzed. To study this partnership, we examined the presence and phylogenetic diversity of microbial communities within the cytoplasm of two Bryopsis species by combining fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Sequencing results revealed the presence of Arcobacter, Bacteroidetes, Flavobacteriaceae, Mycoplasma, Labrenzia, Phyllobacteriaceae and Xanthomonadaceae species. Although the total diversity of the endobiotic communities was unique to each Bryopsis culture, Bacteroidetes, Mycoplasma, Phyllobacteriaceae, and in particular Flavobacteriaceae bacteria, were detected in several Bryopsis samples collected hundreds of kilometres apart. This suggests that Bryopsis closely associates with well-defined endophytic bacterial communities of which some members possibly maintain an endosymbiotic relationship with the algal host.     

Transient Decrease of Light-harvesting Complex Ⅱ Phosphorylation Level by Hypoosmotic Shock in Dark-adapted Dunaliella salina

This study investigated the regulation of major light harvesting chlorophyll a/b protein （LHCⅡ） phosphorylation by hypoosmotic shock in dark-adapted Dunaliella salina cells. When the external NaCI concentration decreased in darkness, D. salina LHCⅡ phosphorylation levels transiently dropped within 20 min and then restored gradually to basal levels. The transient decrease in LHCII phosphorylation levels was insensitive to NaF, a phosphatase inhibitor. Inhibition of intracellular ATP production by addition of an uncoupler or an ATP synthase inhibitor increased LHCⅡ phosphorylation levels in D. salina cells exposed to hypoosmotic shock. Taken together, these results indicate that hypoosmotic shock inhibits the LHCⅡ phosphorylation process. The related mechanism and physiological significance are discussed.     

Impacts of Elevated CO2 Concentration on Biochemical Composition, Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations, Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2 concentrations within the range 3-186μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in ChloreUa pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2 enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.     

In silico Analysis of Sequential,Structural and Functional Diversity of Wheat Cystatins and Its Implication in Plant Defense

Phytocystatins constitute a multigene family that regulates the activity of endogenous and/or exogenous cysteine proteinases.Cereal crops like wheat are continuously threatened by a multitude of pathogens,therefore cystatins offer to play a pivotal role in deciding the plant response.In order to study the need of having diverse specificities and activities of various cystatins,we conducted comparative analysis of six wheat cystatins(WCs) with twelve rice,seven barley,one sorghum and ten corn cystatin sequences employing different bioinformatics tools.The obtained results identified highly conserved signature sequences in all the cystatins considered.Several other motifs were also identified,based on which the sequences could be categorized into groups in congruence with the phylogenetic clustering.Homology modeling of WCs revealed 3D structural topology so well shared by other cystatins.Protein-protein interaction of WCs with papain supported the notion that functional diversity is a consequence of existing differences in amino acid residues in highly conserved as well as relatively less conserved motifs.Thus there is a significant conservation at the sequential and structural levels;however,concomitant variations maintain the functional diversity in this protein family,which constantly modulates itself to reciprocate the diversity while counteracting the cysteine proteinases.     

Genetic Analysis of B2t, the Structural Gene for a Testis-Specific β-Tubulin Subunit in DROSOPHILA MELANOGASTER

Genetic analysis of the B2t locus has resulted in the recovery of four recessive mutations in the B2t structural gene and a deficiency that deletes the locus. Two of the mutations were recovered as suppressors of B2t^D, a dominant male sterile mutation at the locus, and two were induced on wild-type chromosomes. All four mutant genes encode β₂-tubulin subunits that are synthesized at normal rates but do not accumulate. All mutants are completely male sterile as homozygotes.     

Expression of aphidicolin, FUdR and caffeine‐induced fragile sites in lymphocytes of healthy Turkish individuals

The expression of common fragile sites (c‐fra) and frequency of chromosomal aberrations were studied in peripheral lymphocytes of 50 healthy Turkish individuals (26 males and 24 females from 1 to 87 years of age) after induction with aphidicolin (APC), 5′‐fluorodeoxyuridine (FUdR), and caffeine. A correlation was seen between age and the frequency of chromosomal aberrations in APC and caffeine treated cultures, but there were no significant differences in the frequencies of chromosomal aberrations between males and females in any of the treatments. The mean frequency of aberrations induced by FUdR was significantly higher than that induced by APC and caffeine. A chromosome aberration is defined as a fragile site when present in 1% of the cells analyzed from each culture and in at least 50% of the individuals studied. Using these criteria, 12 c‐fra were observed in the three treatments: 1p21, 1q21, 2p11‐q11, 3p14, 4q31, 6q26, 7q22, 7q32, 8q24, 11q23, 16q23, and Xp22. Sites 3p14, 16q23, and Xp22 were the most frequently observed c‐fra, with only the frequency of Xp22 being significantly increased in females in APC treated cultures. The results of these studies are important as a base against which the effects of other clastogenic and environmental agents, as well as genetic background, can be compared. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative Analysis of β-Amyloid Peptides Expressed in Human Cerebrospinal Fluid by an Improved Method of Antibody-Assisted Time-of-Flight Mass Spectrometry

Establishment of diagnostic measures for early stage Alzheimer’s disease (AD) and mild cognitive impairment (MCI) is of crucial importance. Using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS), antibody-assisted MS of cerebrospinal fluid (CSF) has enabled quantitative analysis of the ratio of β-amyloid (Aβ) peptides, Aβ1-42/Aβ1-40, which has a diagnostic value for AD/MCI. To apply the MS analysis to a far wider range of CSF samples, we have established a method to analyze Aβ peptides expressed in 100 μl CSF samples quantitatively. Pretreatment of CSF samples by limit-filtration to condense peptides, and modified washing procedure using urea as denaturant, Aβ peptides of interest can be assessed with higher sensitivity by five to tenfolds to the original method. This improvement enables quantitative analysis of Aβ species from a residual amount of CSF samples, which will be occasionally obtained in case of lumbar anesthesia prior to operation and spinal tap performed for routine diagnostic purposes. Prevalence of the new procedure as laboratory test, especially among the elderly consulting for neurological clinic, will enhance the number of subjects diagnosed at early stage of AD/MCI.     

Spectral and Kinetic Analysis of the Energy Coupling in the PS I–LHC I Supercomplex from the Green Alga Chlamydomonas reinhardtii at 77 K

Energy transfer processes in the chlorophyll antenna of the PS I–LHCI supercomplexes from the green alga Chlamydomonas reinhardtii have been studied at 77 K using transient absorption spectroscopy with multicolor excitation in the 640–670 nm region. Comparison of the kinetic data obtained at low and room temperatures indicates that the slow ∼ ∼100 ps excitation equilibration phase that is characteristic of energy coupling of the LHCI peripheral antenna to the PS I core at physiological temperatures (Melkozernov AN, Kargul J, Lin S, Barber J and Blankenship RE (2004) J Phys Chem B 108: 10547–10555) is not observed in the excitation dynamics of the PS I–LHCI supercomplex at 77 K. This suggests that at low temperatures the peripheral antenna is energetically uncoupled from the PS I core antenna. Under these conditions the observed kinetic phases on the time scales from subpicoseconds to tens of picoseconds represent the superposition of the processes occurring independently in the PS I core antenna and the Chl a/b containing LHCI antenna. In the PS I–LHCI supercomplex with two uncoupled antennas the excitation is channeled to the excitation sinks formed at low temperature by clusters of red pigments. A better spectral resolution of the transient absorption spectra at 77 K results in detection of two ΔA bands originating from the rise of photobleaching on the picosecond time scale of two clearly distinguished pools of low energy absorbing Chls in the PS I–LHCI supercomplex. The first pool of low energy pigments absorbing at 687 nm is likely to originate from the red pigments in the LHCI where the Lhca1 protein is most abundant. The second pool at 697 nm is suggested to result either from the structural interaction of the LHCI and the PS I core or from other Lhca proteins in the antenna. The kinetic data are discussed based on recent structural models of the PS I–LHCI. It is proposed that the uncoupling of pigment pools may be a control mechanism that regulates energy flow in Photosystem I.     

Inhibition of Leishmania (Leishmania) amazonensis and Rat Arginases by Green Tea EGCG, (+)-Catechin and (?)-Epicatechin: A Comparative Structural Analysis of Enzyme-Inhibitor Interactions

Epigallocatechin-3-gallate (EGCG), a dietary polyphenol (flavanol) from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+)-catechin and (−)-epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L) and rat liver arginase (ARG-1). The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+)-catechin and (−)-epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+)-catechin (IC₅₀ = 0.8 µM) followed by (−)-epicatechin (IC₅₀ = 1.8 µM), gallic acid (IC₅₀ = 2.2 µM) and EGCG (IC₅₀ = 3.8 µM). Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC₅₀ values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.