Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg

A transglutaminase (TGase) cDNA was cloned from carp ovary. It was highly homologous to zebrafish TGase. Immunoblot and enzymatical assay showed that TGase was present on the chorion and in the cytoplasm of carp eggs. Addition of TGase inhibitor, cadaverine or ethylene diaminetetracetic acid (EDTA) to the cortical reaction medium impaired the formation of the outer layer of fertilization envelope (FE(o)), the adhesive structure of carp egg. Fibroin-like substance (FLS), cystatin, cathepsin-like substance (CLS), and FEO-1 were the components of FE(o), wherein the majority of the former three were conjugated to form macromolecules of 90-205 kDa while the latter one was present in monomer of 22 kDa. Cadaverine interfered slightly the discharge of FLS conjugates out of the perivitelline space (PVS) but affected profoundly the recruitment of FLS conjugates to FE, whereas EDTA completely inhibited both the release and the recruitment of FLS conjugates to FE. Both EDTA and cadaverine did not inhibit the discharge of FEO-1 out of PVS but could inhibit the recruitment of FEO-1 to FE. The mechanism was studied. ZP2 and ZP3, the major constituents of inner layer of FE, were cross-linked during cortical reaction, which rendered FE hardened. In the presence of EDTA, the cross-linking of ZP2 and ZP3 were inhibited, thus FE remained soft. The PVS of an egg with a hardened FE was less expanded than an egg with a soft FE. It was assumed that a less expanded PVS would generate a higher fluid pressure than a more expanded PVS did. Therefore, the transportation of the macromolecules such as the FLS-cystatin-CLS conjugates out of PVS was facilitated in control and cadaverine-treated eggs whose FE were hardened but was blocked in EDTA-treated eggs whose FE were unhardened. On the other hand, the transportation of small molecules such as FEO-1 out of FE was not restrained, so they were discharged out of the PVS of the control and TGase inhibitor-treated eggs. In addition, TGase activity was also required for the recruitment of FLS conjugates to FE.     

Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum

Seven cDNA encoding silkworm fibroin homologues were cloned from a carp ovarian cDNA library. The encoded proteins are denoted as carp ovarian fibroin-like substances (FLS). FLS contain a repetitive domain consisting of tandem repeats of dipeptide of Gly-X, where X may be any amino acid. Each FLS has its own unique repeating sequence, such as GQGAGQGS, GQGMGQGM, GRGQGEGHGS, and GFGFGQGS, indicating a family of FLS genes exists in carp. FLS is exclusively expressed in oocytes and is stored in cortical granules. During cortical reaction, FLS is exocytosed to perivitelline space and then gradually added to the outer layer of the fertilization envelope (FEo). The FLS of fertilization envelope is conjugated with cystatin and cathepsin-like substance (CLS) and appears in multiple bands of molecular weights ranging from 40 to 205 kDa. After fertilization or artificial activation, carp eggs adhere firmly to the substratum via FEo. FLS is a major component of FEo. The presence of transglutaminase inhibitor, cadaverine or ethylene diaminetetraacetic acid, in the cortical reaction medium can impair or block the recruitment of FLS and other substances to FEo. As a consequence, FEo is not formed or is greatly reduced, resulting in a great reduction of egg adhesion.     

Excitation energy trapping in anoxygenic photosynthetic bacteria*

Various aspects of excitation energy conversion in anoxygenic photosynthetic bacteria are surveyed. This minireview discusses different models that have been proposed during the past 60 years to describe excitation energy transfer from an antenna molecule to the reaction center. First, a simple one-dimensional model was suggested, but over time the models became more detailed when structural and dynamic information was included. This review focuses mainly on the picture of purple bacteria and green sulfur bacteria developed during the past decades. This revised version was published online in June 2006 with corrections to the Cover Date.     

Membrane biogenesis in anoxygenic photosynthetic prokaryotes

Following the discovery of photosynthetic bacteria in the nineteenth century, technical developments of the 1950s led to their use in membrane biogenesis studies. These investigations had their origins in the isolation of subcellular particles designated as ‘chromatophores’ by Roger Stanier and colleagues, which were shown to be photosynthetically competent by Albert Frenkel, and to originate from the intracytoplasmic membrane (ICM) continuum observed in electron micrographs. These ultrastrucutral studies by the G. Drews group, Germaine Cohen-Bazire and others also suggested that the ICM originates by invagination of the cytoplasmic membrane, as later established in the biochemical and biophysical work of the R. Niederman and Drews groups. Through a combination of genetic approaches, first introduced in the early 1980s by Barry Marrs, and the atomic resolution structures determined for light-harvesting antennae and reaction centers, a detailed understanding is emerging of mechanisms regulating their levels in the membrane and the roles played by specific protein domains and additional factors in their assembly and supramolecular organization. Prospects for additional progress during the twenty-first century include further elucidation of molecular aspects of the assembly process and the application of newer spectroscopic probes to photosynthetic unit formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structure and function in the isolated reaction center complex of Photosystem II: energy and charge transfer dynamics and mechanism

The dynamics of energy and charge transfer in the Photosystem II reaction center complex is an area of great interest today. These processes occur on a time scale ranging from femtoseconds to tens of picoseconds or longer. Steady-state and ultrafast spectroscopy techniques have provided a great deal of quantitative and qualitative data that have led to varied interpretations and phenomenological models. More recently, microscopic models that identify specific charge separated states have been introduced, and offer more insight into the charge transfer mechanism. The structure and energetics of PS II reaction centers are reviewed, emphasizing the effects on the dynamics of the initial charge transfer. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

A New Pathway of Photoinactivation of Photosystem II. Irreversible Photoreduction of Pheophytin Causes Loss of Photochemical Activity of Isolated D1–D2–Cytochrome b559 Complex

A new pathway of photoinactivation of photosystem II (PS II) connected with irreversible photoaccumulation of reduced pheophytin (Ph) in isolated D1–D2–cytochrome b ₅₅₉ complexes of reaction center (RC) of PS II was discovered. The inhibitory effects of white light illumination on photochemical activity of D1–D2–cytochrome b ₅₅₉ complexes of RCs of photosystem II, isolated from pea chloroplasts, have been compared under anaerobic conditions in the absence and in the presence of sodium dithionite, electron transfer from which to the oxidized primary electron donor P680⁺ results in the photoaccumulation of anion-radical of the primary electron acceptor, PH. In both cases, prolonged illumination (1-5 min, 120 W/m²) led to a pronounced loss of the photochemical activity as it was monitored by measuring the amplitude of the reversible photoinduced absorbance changes at 682 nm related to the photoreduction of Ph. The extent of the photoinactivation depended on the illumination time and pH of the medium. At pH 8.0, the presence of dithionite during photoinactivation brought about a protective effect compared to that in a control sample. In contrast, lowering pH to 6.0 increased the sensitivity to photoinactivation in the dithionite containing samples. For 5 min irradiation, the photochemical activity in the absence and in the presence of dithionite decreased by 35 and 72%, respectively (this was accompanied by an irreversible bleaching of the pheophytin Q_x absorption band at 542 nm). Degradation of the D1 and D2 proteins was not observed under these conditions. A subsequent addition of an electron acceptor, potassium ferricyanide, to the illuminated samples restored neither the amplitude of the signal at 682 nm nor absorption at 542 nm. It is suggested that at pH < 7.0 the photoaccumulated PH is irreversibly converted into a secondary, most probably protonated form, that does not lead to destruction of the RCs but prevents the photoformation of the primary radical pair [P680⁺PH]. A possible application of this effect to photoinactivation of PS II in vivo is discussed.     

The Hearing Sensitivity of the Little Skate, Raja erinacea: A Comparison of Two Methods

We determined the hearing sensitivity of the little skate, Raja erinacea using two methods: Behavioral conditioning and the auditory brainstem response (ABR). This marks the first time that the hearing in any member of the Rajiformes has been examined and the first time that the ABR method has been used with an elasmobranch. We obtained audiograms of R. erinacea using each method and were found to be statistically similar. The best hearing sensitivity for R. erinacea was between 100 and 300Hz. We compared the audiograms to audiograms obtained from other species of elasmobranchs. This analysis showed that R. erinacea, a bottom-dwelling elasmobranch, has less sensitive hearing than the lemon shark, Negaprion brevirostris, and the bull shark, Carcharhinus leucas, a free-swimming, raptorial elasmobranch. However, R. erinacea showed sensitivity comparable to that of the horn shark, Heterodontus francisi, another bottom-dwelling elasmobranch; both species feed primarily on benthic prey. These findings are in agreement with Corwin's hypothesis (1978) that hearing sensitivity is correlated with feeding behavior. An examination of the macula neglecta of R. erinacea found a total count of 10000 hair cells, which is within the range of other bottom-dwelling elasmobranchs.     

Origin and evolution of transmembrane Chl-binding proteins: hydrophobic cluster analysis suggests a common one-helix ancestor for prokaryotic (Pcb) and eukaryotic (LHC) antenna protein superfamilies

All chlorophyll (Chl)-binding proteins constituting the photosynthetic apparatus of both prokaryotes and eukaryotes possess hydrophobic domains, corresponding to membrane-spanning alpha-helices (MSHs). Hydrophobic cluster analysis of representative members of the different Chl protein superfamilies revealed that all Chl proteins except the five-helix reaction center II proteins and the small subunits of photosystem I possess related domains. As a major conclusion, we found that the eukaryotic antennae likely share a common precursor with the prokaryotic Chl a/b antennae from Chl-b-containing oxyphotobacteria. From these data, we propose a global scheme for the evolution of these proteins from a one-MSH ancestor.     

Apical meristem organization and lack of establishment of the quiescent center in Cactaceae roots with determinate growth

Some species of Cactaceae from the Sonoran Desert are characterized by a determinate growth pattern of the primary root, which is important for rapid lateral-root formation and seedling establishment. An analysis of the determinate root growth can be helpful for understanding the mechanism of meristem maintenance in plants in general. Stenocereus gummosus (Engelm.) Gibson & Horak and Pachycereus pringlei (S. Watson) Britton & Rose are characterized by an open type of root apical meristem. Immunohistochemical analysis of 5-bromo-2-deoxyuridine incorporation into S. gummosus showed that the percentage of cells passing through the S-phase in a 24-h period is the same within the zone where a population of relatively slowly proliferating cells could be established and above this zone in the meristem. This indicated the absence of the quiescent center (QC) in S. gummosus. During the second and the third days of growth, in the distal meristem portion of P. pringlei roots, a compact group of cells that had a cell cycle longer than in the proximal meristem was found, indicating the presence of the QC. However, later in development, the QC could not be detected in this species. These data suggest that during post-germination the absence of the establishment of the QC within the apical meristem and limited proliferative activity of initial cells are the main components of a determinate developmental program and that establishment of the QC is required for maintenance of the meristem and indeterminate root growth in plants.Abbreviations QC quiescent center - RCP root cap-protoderm - BrdU 5-bromo-2-deoxyuridine - FITC fluorescein isothiocyanate - DAPI 4,6-diamidino-2-phenylindole