UBIAD1 effectively alleviated myocardial ischemia reperfusion injury by activating SIRT1/PGC1α.

BACKGROUND AND OBJECTIVES: Myocardial ischemia-reperfusion injury (MIRI) threatens global health and lowers people's sense of happiness. Till now, the mechanism of MIRI has not been well-understood. Therefore, this study was designed to explore the role of UBIAD1 in MIRI as well as its detailed reaction mechanism. METHODS: The mRNA and protein expressions of UBIAD1 before or after transfection were measured using RT-qPCR and western blot. Western blot was also adopted to measure the expressions of signaling pathway-, mitochondrial damage- and apoptosis-related proteins. Moreover, mitochondrial membrane potential and ATP level were verified by JC-1 immunofluorescence and ATP kits, respectively. With the application of CCK-8, LDH and CK-MB assays, the cell viability, LDH and CK-MB levels were evaluated, respectively. In addition, the cell apoptosis was detected using TUNEL. Finally, the expressions of ROS, SOD, MDA and CAT were measured using DCFH-DA, SOD, MDA and CAT assays, respectively. RESULTS: In the present study, we found that UBIAD1 was downregulated in hypoxia-reoxygenation (H/R) -induced H9C2 cells and its upregulation could activate SIRT1/PGC1α signaling pathway. It was also found that UBIAD1 regulated mitochondrial membrane potential and ATP level via activating SIRT1/PGC1α signaling pathway. In addition, the injury of H/R-induced H9C2 cells could be relieved by UBIAD1 through the activation of SIRT1/PGC1α signaling pathway. Moreover, UBIAD1 exhibited inhibitory effects on apoptosis and oxidative stress of H/R-induced H9C2 cells through activating SIRT1/PGC1α signaling pathway. CONCLUSION: To sum up, UBIAD1 could alleviate apoptosis, oxidative stress and H9C2 cell injury by activating SIRT1/PGC1α, which laid experimental foundation for the clinical treatment of MIRI.

New Far-Red and Near-Infrared Fluorescent Phycobiliproteins with Excellent Brightness and Photostability.

Far-red and near-infrared fluorescent proteins can be used as fluorescence biomarkers in the region of maximal transmission of most tissues and facilitate multiplexing. Recently, we reported the generation and properties of far-red and near-infrared fluorescent phycobiliproteins, termed BeiDou Fluorescent Proteins (BDFPs), which can covalently bind the more readily accessible biliverdin. Far-red BDFPs maximally fluoresce at ∼670 nm, while near-infrared BDFPs fluoresce at ∼710 nm. In this work, we molecularly evolved BDFPs as follows: (a) mutations L58Q, S68R and M81K of BDFPs, which can maximally enhance the effective brightness in vivo by 350 %; (b) minimization and monomerization of far-red BDFPs 2.1, 2.2, 2.3, and near-infrared BDFPs 2.4, 2.5 and 2.6. These newly developed BDFPs are remarkably brighter than the formerly reported far-red and near-infrared fluorescent proteins. Their advantages are demonstrated by biolabeling in mammalian cells using super-resolution microscopy.

Quantification of the Masseter Muscle Hardness of Stroke Patients Using the MyotonPRO Apparatus: Intra- and Inter-Rater Reliability and Its Correlation with Masticatory Performance.

BACKGROUND Chewing dysfunction is one of the most common serious complications after a stroke. It may be influenced by the hardness of the masseter muscle and masticatory performance; however, the association between these 2 factors is not explicit. Thus, it is meaningful to explore the functional status of the masseter muscle among stroke patients. The main objectives of this study were to examine the intra- and inter-rater reliability of the MyotonPRO apparatus in measuring masseter muscle hardness in stroke patients and to investigate the correlation between the bilateral masseter muscle hardness and masticatory performance in these patients. MATERIAL AND METHODS A total of 20 stroke patients participated in our study. The hardness of the masseter muscle was measured by 2 physiotherapists using the MyotonPRO apparatus. Overall, each patient masticated 2 pieces of red-blue bicolor chewing gum for 20 chewing cycles each. The chewing pieces were analyzed using ViewGum software for masticatory performance. RESULTS The intra- and inter-rater reliability of the MyotonPRO apparatus for measuring bilateral masseter hardness of stroke patients was excellent. The correlation analysis showed that the hardness index of the masseter muscle on the affected side was moderately correlated with the masticatory performance of the same side. CONCLUSIONS The MyotonPRO device can be used for measuring the masseter muscle hardness of stroke patients, with excellent reliability. This study established the construct validity between the stiffness of the masseter muscle and masticatory performance.

Pseudomonas phragmitis sp. nov., isolated from petroleum polluted river sediment.

A Gram-stain-negative, rod-shaped bacterium, motile by means of a single polar flagellum, designated S-6-2T, was isolated from petroleum polluted river sediment in Huangdao, Shandong Province, PR China. The 16S rRNA gene sequence analysis revealed that S-6-2T represented a member of the genus Pseudomonas, sharing the highest sequence similarities with Pseudomonas parafulva (97.5 %) and Pseudomonas fulva (97.5 %). Phylogenetic analysis based on 16S rRNA gene, concatenated 16S rRNA, gyrB, rpoB and rpoD genes and genome core-genes indicated that S-6-2T was affiliated with the members of the Pseudomonas pertucinogena group. The average nucleotide identity (ANI) and genome-to-genome distance between the whole genome sequences of S-6-2T and closely related species of the genus Pseudomonas within the P. pertucinogena group were less than 77.94 % and 20.5 %, respectively. Differences in phenotypic characteristics were also found between S-6-2T and the closely related species. The major cellular fatty acids (>10 %) were summed feature 8 (C18 : 1ω7c/ C18  : 1ω6c), C16 : 0, C17 : 0cyclo and C12 : 0. The predominant respiratory quinone was ubiquinone 9. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified lipid (L1), two unidentified phospholipids (PL1 and PL2) and an aminophospholipid (APL). The DNA G+C content of the genome of S-6-2T was 60.1 mol%. On the basis of the evidence from the polyphasic taxonomic study, strain S-6-2T can be classified as representative of a novel species of the genus Pseudomonas, for which the name Pseudomonas phragmitis sp. nov. is proposed. The type strain is S-6-2T (=CGMCC 1.15798T=KCTC 52539T).

Design of small monomeric and highly bright near-infrared fluorescent proteins.

Due to the low absorbance in the far-red (FR) and near-infrared (NIR) "optical window", NIR fluorescent proteins (FPs) are powerful tools for deep imaging. Here, we report three new, highly bright NIR FPs termed BDFP1.8, BDFP1.8:1.8 (tandem BDFP1.8) and BDFP1.9, which evolved from a previously reported FR FP, BDFP1.6: a derivative of ApcF2 from Chroococcidiopsis thermalis sp. PCC7203. ApcF2 binds phycocyanobilin (PCB) non-covalently, while BDFPs, the derivatives of ApcF2, can bind biliverdin (BV) covalently. We identified that dimeric BDFP1.8 and monomeric BDFP1.8:1.8 have a 2.4-and 4.4-fold higher effective brightness, respectively, than iRFP720, which has the highest effective brightness among the reported NIR FPs. Monomeric DBFP1.9 (17 kDa) has one of the smallest masses among highly bright FPs in the FR and NIR regions. Enhancing the affinity between the apo-proteins and the BV chromophore is an effective method to improve the effective brightness of biliprotein FPs. Moreover, BDFP1.8 and 1.9 exhibit higher stability to temperature, pH and light than iRFP720. Finally, the highly bright NIR BDFP1.8 together with FR BDFP1.6 could effectively biolabel cells in dual colors.

Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.

A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far-Red Fluorescent BDFP1.6.

Phycobiliproteins are constituents of phycobilisomes that can harvest orange, red, and far-red light for photosynthesis in cyanobacteria and red algae. Phycobiliproteins in the phycobilisome cores, such as allophycocyanins, absorb far-red light to funnel energy to the reaction centers. Therefore, allophycocyanin subunits have been engineered as far-red fluorescent proteins, such as BDFP1.6. However, most current fluorescent probes have small Stokes shifts, which limit their applications in multicolor bioimaging. mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and a high fluorescence quantum yield, and thus, can be used as a donor for energy transfer to a far-red acceptor, such as BDFP1.6, by FRET. In this study, mCherry was fused with BDFP1.6, which resulted in a highly bright far-red fluorescent protein, BDFP2.0, with a large Stokes shift (≈79 nm). The excitation energy was absorbed maximally at 587 nm by mCherry and transferred to BDFP1.6 efficiently; thus emitting strong far-red fluorescence maximally at 666 nm. The effective brightness of BDFP2.0 in mammalian cells was 4.2-fold higher than that of iRFP670, which has been reported as the brightest far-red fluorescent protein. The large Stokes shift of BDFP2.0 facilitates multicolor bioimaging. Therefore, BDFP2.0 not only biolabels mammalian cells, including human cells, but also biolabels various intracellular components in dual-color imaging.

Bright near-infrared fluorescence bio-labeling with a biliprotein triad.

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700-770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650-700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.

Effects of antagonistic bacteria B6 against the pathogens of apple replant disease on the biomass of Malus hupehensis Rehd seedlings and soil environment under replanting.

We isolated strains from the rhizosphere soil of apple trees with replanting disease and evaluated the biological control potential for the pathogens Fusarium proliferatum, F. moniliforme, F. oxysporum, and F. solani. The morphological, physiological and biochemical character, and 16S rDNA sequence of the strain with the highest inhibitory rate were analyzed. The effect of strain biofertilizer on the biomass of Malus hupehensis Rehd. seedlings and soil environment under replanting disease was evaluated in a pot experiment. The results showed that the strain B6 had the strongest antagonistic activity. The inhibitory rate of B6 for F. proliferatum, F. moniliforme, F. oxysporum, Fusarium solani reached 71.8%, 70.1%, 72.6% and 91.5%, respectively. The strain B6 was identified as Bacillus methylotrophicus according to the results of morphological, physiological and biochemical character and 16S rDNA sequence analysis. Compared with the control, the bacterial manure made from the strain B6 enhanced the biomass of Malus hupehensis Rehd. seedlings in replanting soil to different extent. The ground diameter, fresh and dry mass were significantly increased by 18.3%, 49.6% and 51.2%, respectively. The strain B6 dramatically increased the number of cultivable bacteria and actinomyces in replanting soil and reduced the abundance of fungus to 37.7%, which accelerated the conversion of fungal soil to bacterial soil. It also dramatically increased the activities of sucrase, phosphatase, ureaseandcatalase in soil by 37.3%, 24.0%, 42.9% and 49.4%, respectively. In conclusion, the B6 fertilizer could improve the structure of cultivable microbial communities in the continuous cropping soil of apple trees, increase the soil enzyme activity, and enhance the growth of Malus hupehensis seedlings.

Far-red acclimating cyanobacterium as versatile source for bright fluorescent biomarkers.

Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to regions of maximal transmission of most tissues, but present considerable engineering challenges. Far-red adapting cyanobacteria generate a novel set of biliproteins. One of them, ApcF2, from a thermophilic cyanobacterium was subjected to structure-guided, site-directed random and specific mutagenesis, and was screened for bright far-red emission. We report the generation of chromoproteins, termed BDFPs, that are small, bind auto-catalytically the ubiquitous biliverdin as chromophore, express well, and retain their fluorescence in mammalian cells and in the nematode, C. elegans. They are, moreover, photostable and tolerate high temperature, low pH and chemical denaturation. Homo-bichromophoric tandems of these proteins improve labeling, while hetero-bichromophoric systems with large Stokes shifts are suitable for applications like FRET, multi-channel or super-resolution microscopy. The BDFPs compare favorably to other biliproteins and provide a novel, extremely versatile labeling tool-box.

Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2.

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.

Chromophorylation (in Escherichia coli) of allophycocyanin B subunits from far-red light acclimated Chroococcidiopsis thermalis sp. PCC7203.

Cyanobacterial phycobilisomes funnel the harvested light energy to the reaction centers via two terminal emitters, allophycocyanin B and the core-membrane linker. ApcD is the α-subunit of allophycocyanin B responsible for its red-shifted absorbance (λmax 665 nm). Far-red photo-acclimated cyanobacteria contain certain allophycocyanins that show even further red-shifted absorbances (λmax > 700 nm). We studied the chromophorylation of the three far-red induced ApcD subunits ApcD2, ApcD3 and ApcD4 from Chroococcidiopsis thermalis sp. PCC7203 during the expression in E. coli. The complex behavior emphasizes that a variety of factors contribute to the spectral red-shift. Only ApcD2 bound phycocyanobilin covalently at the canonical position C81, while ApcD3 and ApcD4 gave only traces of stable products. The product of ApcD2 was, however, heterogeneous. The major fraction had a broad absorption around 560 nm and double-peaked fluorescence at 615 and 670 nm. A minor fraction was similar to the product of conventional ApcD, with maximal absorbance around 610 nm and fluorescence around 640 nm. The heterogeneity was lost in C65 and C132 variants; in these variants only the conventional product was formed. With ApcD4, a red-shifted product carrying non-covalently bound phycocyanobilin could be detected in the supernatant after cell lysis. While this chromophore was lost during purification, it could be stabilized by co-assembly with a far-red light-induced ß-subunit, ApcB3.

A Simple Preparation Method for Phytochromobilin.

Phytochromobilin (PΦB), the chromophore of plant phytochromes, is difficult to isolate because phytochromes occur at very low concentrations in plants. It is, therefore, frequently replaced in plant phytochrome studies by phycocyanobilin, which is abundant in cyanobacteria. PΦB is also an attractive chromophore for far-red emitting chromoproteins. In this work, we design and optimize a simple method to efficiently isolate useful quantities of PΦB: The chromophore is generated in Escherichia coli and transiently bound to a tailored chromophore-binding domain of ApcE2, the apo-protein of a core-membrane linker, from which it can subsequently be released. The ease and effectiveness of this method hinges not only on the enhanced biosynthesis of PΦB in the presence of the ApcE2 construct from Synechococcus sp. PCC7335, but also on the noncovalent binding of the pigment to its apo-protein. The isolated PΦB was successfully incorporated into phytochrome-related assemblies, and furthermore, the noncovalently bound PΦB could be transferred directly from the ApcE2 construct to the apo-proteins of phytochromes, cyanobacteriochromes and phycobiliproteins, without loss of relevant biological activity.

Far-red light photoacclimation: Chromophorylation of FR induced α- and ß-subunits of allophycocyanin from Chroococcidiopsis thermalis sp. PCC7203.

Cyanobacterial light-harvesting complexes, phycobilisomes, can undergo extensive remodeling under varying light conditions. Acclimation to far-red light involves not only generation of red-shifted chlorophylls in the photosystems, but also induction of additional copies of core biliproteins that have been related to red-shifted components of the phycobilisome (Gan et al., Life 5, 4, 2015). We are studying the molecular basis for these acclimations in Chroococcidiopsis thermalis sp. PCC7203. Five far-red induced allophycocyanin subunits (ApcA2, ApcA3, ApcB2, ApcB3 and ApcF2) were expressed in Escherichia coli, together with S-type chromophore-protein lyases and in situ generated chromophore, phycocyanobilin. Only one subunit, ApcF2, shows an unusual red-shift (λAmax~675nm, λFmax~698nm): it binds the chromophore non-covalently, thereby preserving its full conjugation length. This mechanism operates also in two Cys-variants of the induced subunits of bulky APC. All other wild-type subunits bind phycocyanobilin covalently to the conventional Cys-81 under catalysis of the lyase, CpcS1. Although three of them also show binding to additional cysteines, all absorb and fluoresce similar to conventional APC subunits (λAmax~610nm, λFmax~640nm). Another origin of red-shifted complexes was identified, however, when different wild-type α- and ß-subunits of the far-red induced bulky APC were combined in a combinatorial fashion. Strongly red-shifted complexes (λFmax≤722nm) were formed when the α-subunit, PCB-ApcA2, and the ß-subunit, PCB-ApcB2, were generated together in E. coli. This extreme aggregation-induced red-shift of ~90nm of covalently bound chromophores is reminiscent, but much larger, than the ~30nm observed with conventional APC.

Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335.

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.

The terminal phycobilisome emitter, LCM: A light-harvesting pigment with a phytochrome chromophore.

Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, L(CM). The chromophore domain of L(CM) forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in L(CM) by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of L(CM). Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.

The structure of allophycocyanin B from Synechocystis PCC 6803 reveals the structural basis for the extreme redshift of the terminal emitter in phycobilisomes.

Allophycocyanin B (AP-B) is one of the two terminal emitters in phycobilisomes, the unique light-harvesting complexes of cyanobacteria and red algae. Its low excitation-energy level and the correspondingly redshifted absorption and fluorescence emission play an important role in funnelling excitation energy from the hundreds of chromophores of the extramembraneous phycobilisome to the reaction centres within the photosynthetic membrane. In the absence of crystal structures of these low-abundance terminal emitters, the molecular basis for the extreme redshift and directional energy transfer is largely unknown. Here, the crystal structure of trimeric AP-B [(ApcD/ApcB)3] from Synechocystis sp. PCC 6803 at 1.75 Šresolution is reported. In the crystal lattice, eight trimers of AP-B form a porous, spherical, 48-subunit assembly of 193 Šin diameter with an internal cavity of 1.1 × 10(6) Å(3). While the overall structure of trimeric AP-B is similar to those reported for many other phycobiliprotein trimers, the chromophore pocket of the α-subunit, ApcD, has more bulky residues that tightly pack the phycocyanobilin (PCB). Ring D of the chromophores is further stabilized by close interactions with ApcB from the adjacent monomer. The combined contributions from both subunits render the conjugated rings B, C and D of the PCB in ApcD almost perfectly coplanar. Together with mutagenesis data, it is proposed that the enhanced planarity effectively extends the conjugation system of PCB and leads to the redshifted absorption (λmax = 669 nm) and fluorescence emission (679 nm) of the ApcD chromophore in AP-B, thereby enabling highly efficient energy transfer from the phycobilisome core to the reaction centres.

Structure and mechanism of the phycobiliprotein lyase CpcT.

Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein ß-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped ß-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the ß- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965-983).

High glucose levels increase the expression of neurotrophic factors associated with p-p42/p44 MAPK in Schwann cells in vitro.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus. One contributing factor to DPN is altered neurotrophism due to changes in the synthesis and expression of neurotrophins. Schwann cells (SCs) are the myelin-forming cells of the peripheral nervous system that promote nerve regeneration through the expression and secretion of neurotrophic factors (NTFs). Therefore, in this study, using SCs cultured in the presence of high levels of glucose for 24 h, with and without the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor, PD98059, we investigated the effect of high glucose levels on SCs over a short period of time. The cultured cells were evaluated using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst staining, immunocytochemistry, reverse transcriptase-polymerase chain reaction and western blot analysis. High glucose levels did not promote morphological abnormalities or decrease the viability of SCs. However, high glucose levels enhanced the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and induced the activation of p42/p44 MAPK in cultured SCs in a dose-dependent manner. Additionally, the phosphorylation of p42/p44 MAPK may be associated with the expression of NTFs by SCs exposed to high glucose conditions; the excessive activation of p42/p44 MAPK inhibited the expression of NTFs. These observations demonstrate that exposure to high glucose levels lead to acutely elevated levels of NGF and BDNF in SCs over a short period of time, which may be involved in the p42/p44 MAPK pathway.

Time-dependent changes in BDNF expression of pentylenetetrazole-induced hippocampal astrocytes in vitro.

Pentylenetetrazole (PTZ), a γ-aminobutyric acid (GABA(A)) receptor antagonist, has been used extensively to induce seizures in animal models of epilepsy. The aim of the present study was to investigate the effects of PTZ on hippocampal astrocytes. Cells were incubated with 10, 20, or 40 mM PTZ for 24h and viability and apoptosis were examined using an MTT assay and Hoechst staining. The high concentration of PTZ (20 and 40 mM) resulted in a significant decrease in viability (MTT: 83.6 ± 7.8% and 69.3 ± 4.2%, respectively) (P<0.01), whereas the lower concentration of PTZ (10mM) did not induce cell apoptosis or reduce viability. When cells were treated with 10mM PTZ for 0, 0.5, 2, 4, 8, 12, or 24h, the level of brain-derived neurotrophic factor (BDNF), both protein and mRNA, was significantly reduced at 2 to 12h of culture (P<0.01), with maximal reduction detected at 8h; expression was restored to near control levels after 24h. Collectively, our results suggest that astrocytes may participate in epilepsy through a marked, but transient decrease in BDNF expression.