Histone acetyltransferase p300/CBP‐associated factor is an effective suppressor of secretory immunoglobulin synthesis in immature B cells

The histone acetyltransferase p300/CBP‐associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF‐deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H‐chain) synthesis. Remarkably, PCAF‐deficiency causes an increase in the amount of secretory IgM H‐chain mRNA, but not in that of IgM light chain and membrane‐bound IgM H‐chain mRNAs, resulting in dramatic up‐regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H‐chain synthesis.     

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

Energy transfer processes in Gloeobacter violaceus PCC 7421 that possesses phycobilisomes with a unique morphology

We examined energy transfer dynamics in phycobilisomes (PBSs) of cyanobacteria in relation to the morphology and pigment compositions of PBSs. We used Gloeobacter violaceus PCC 7421 and measured time-resolved fluorescence spectra in three types of samples, i.e., intact cells, PBSs, and rod assemblies separated from cores. Fremyella diplosiphon, a cyanobacterial species well known for its complementary chromatic adaptation, was used for comparison after growing under red or green light. Spectral data were analyzed by the fluorescence decay-associated spectra with components common in lifetimes with a time resolution of 3 ps/channel and a spectral resolution of 2 nm/channel. This ensured a higher resolution of the energy transfer kinetics than those obtained by global analysis with fewer sampling intervals. We resolved four spectral components in phycoerythrin (PE), three in phycocyanin (PC), two in allophycocyanin, and two in photosystem II. The bundle-like PBSs of G. violaceus showed multiple energy transfer pathways; fast (≈ 10 ps) and slow (≈ 100 ps and ≈ 500 ps) pathways were found in rods consisting of PE and PC. Energy transfer time from PE to PC was two times slower in G. violaceus than in F. diplosiphon grown under green light.     

The secondary electron acceptor of photosystem I in Gloeobacter violaceus PCC 7421 is menaquinone-4 that is synthesized by a unique but unknown pathway

The secondary electron acceptor of photosystem (PS) I in the cyanobacterium Gloeobacter violaceus PCC 7421 was identified as menaquinone-4 (MQ-4) by comparing high performance liquid chromatograms and absorption spectra with an authentic compound. The MQ-4 content was estimated to be two molecules per one molecule of chlorophyll (Chl) a', a constituent of P700. Comparative genomic analyses showed that six of eight men genes, encoding phylloquinone/MQ biosynthetic enzymes, are missing from the G. violaceus genome. Since G. violaceus clearly synthesizes MQ-4, the combined results indicate that this cyanobacterium must have a novel pathway for the synthesis of 1,4-dihydroxy-2-naphthoic acid.     

Excitation relaxation dynamics and energy transfer in fucoxanthin–chlorophyll a/c-protein complexes,probed by time-resolved fluorescence

In algae, light-harvesting complexes contain specific chlorophylls (Chls) and keto-carotenoids; Chl a, Chl c, and fucoxanthin (Fx) in diatoms and brown algae; Chl a, Chl c, and peridinin in photosynthetic dinoflagellates; and Chl a, Chl b, and siphonaxanthin in green algae. The Fx–Chl a/c-protein (FCP) complex from the diatom Chaetoceros gracilis contains Chl c₁, Chl c₂, and the keto-carotenoid, Fx, as antenna pigments, in addition to Chl a. In the present study, we investigated energy transfer in the FCP complex associated with photosystem II (FCPII) of C. gracilis. For these investigations, we analyzed time-resolved fluorescence spectra, fluorescence rise and decay curves, and time-resolved fluorescence anisotropy data. Chl a exhibited different energy forms with fluorescence peaks ranging from 677 nm to 688 nm. Fx transferred excitation energy to lower-energy Chl a with a time constant of 300 fs. Chl c transferred excitation energy to Chl a with time constants of 500–600 fs (intra-complex transfer), 600–700 fs (intra-complex transfer), and 4–6 ps (inter-complex transfer). The latter process made a greater contribution to total Chl c-to-Chl a transfer in intact cells of C. gracilis than in the isolated FCPII complexes. The lower-energy Chl a received excitation energy from Fx and transferred the energy to higher-energy Chl a. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus. Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits

The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C. The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation. The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al. (1985) J. Mol. Biol. 184, 257-277), which is related to the allophycocyanin trimer. The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits. The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3. The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors. Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores. L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes. L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes. L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores. By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.     

Metabolic engineering of the Chl d-dominated cyanobacterium Acaryochloris marina: production of a novel Chl species by the introduction of the chlorophyllide a oxygenase gene

In oxygenic photosynthetic organisms, the properties of photosynthetic reaction systems primarily depend on the Chl species used. Acquisition of new Chl species with unique optical properties may have enabled photosynthetic organisms to adapt to various light environments. The artificial production of a new Chl species in an existing photosynthetic organism by metabolic engineering provides a model system to investigate how an organism responds to a newly acquired pigment. In the current study, we established a transformation system for a Chl d-dominated cyanobacterium, Acaryochloris marina, for the first time. The expression vector (constructed from a broad-host-range plasmid) was introduced into A. marina by conjugal gene transfer. The introduction of a gene for chlorophyllide a oxygenase, which is responsible for Chl b biosynthesis, into A. marina resulted in a transformant that synthesized a novel Chl species instead of Chl b. The content of the novel Chl in the transformant was approximately 10% of the total Chl, but the level of Chl a, another Chl in A. marina, did not change. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d(P) by mass spectrometry and nuclear magnetic resonance spectroscopy. [7-Formyl]-Chl d(P) is hypothesized to be produced by the combined action of chlorophyllide a oxygenase and enzyme(s) involved in Chl d biosynthesis. These results demonstrate the flexibility of the Chl biosynthetic pathway for the production of novel Chl species, indicating that a new organism with a novel Chl might be discovered in the future.     

Porcine Model of Hemophilia A

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.     

Fluorescence properties of the allenic carotenoid fucoxanthin: Implication for energy transfer in photosynthetic pigment systems

The fluorescence spectrum of an allenic carotenoid, all-trans-fucoxanthin isolated from a brown alga, has been reported for the first time. This carotenoid is known to function efficiently as a primary photosynthetic antenna pigment in marine algae. The emission bands were located around 630, 685 and 750 nm in CS₂ at 20°C, absorption bands being located at 448, 476 and 505 nm. The energy difference between the 0-0 bands of absorption and emission spectra was about 3900 cm^-1 and location of the emission maximum was less sensitive to the polarizability of solvents than that of the absorption maximum. These clearly indicate that the emission originates from the optically forbidden singlet state (2A_g). This is in contrast to other carotenoids whose emission is assigned to 1B_u state, probably due to the symmetric structure of the conjugated double bond responsible for the absorption in the visible region. A rapid internal conversion from 1B_u to 2A_g state might be facilitated by distorted structure of the conjugated double bond of fucoxanthin. The energy level responsible for the emission is almost identical to the Q_y level of the acceptor molecule (Chl a), thus we propose an energy transfer pathway from the optically forbidden 2A_g state of the carotenoid to the Q_y transition of Chl a in algal pigment systems.