The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Plöscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300–14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a non-competitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-β84-bound PCB in biliproteins by PEB.

Material flow and intermixing during dissimilar friction stir spot welding and friction stir seam welding are investigated. During friction stir spot welding, a ribbon of contiguous dissimilar lamellae is produced during each rotation of the tool and the number of intermingled lamellae contained in the intermixed region is determined by the tool rotational speed setting and the dwell time applied. When the rotating tool moves across the component, the ribbon of dissimilar contiguous lamellae continues to be produced and the linear distance in the traversing direction between dissimilar lamellae corresponds with the pitch distance [the travel speed (mm s−1) divided by the tool rotational speed (Hz)]. The material flow pattern produced when a threaded tool moves across a component is therefore a variant of that produced during the touch down period when the rotating tool is held stationary. It is suggested that the onion ring structures observed in similar and dissimilar friction stir seam welds made using threaded tools are produced by material incorporation from the locations beneath the tool shoulder and the bottom of the rotating pin and the creation of a helical vertical rotational flow within the intermixed region formed beside the periphery of the rotating pin.

为了研究鱼腥藻PCC7120（Anabaenasp.PCC7120）中别藻蓝蛋白（APC）α和β亚基（α-APC和β-APC）中藻蓝胆素（PCB）与脱辅基蛋白的生物合成,并在蓝藻体外对这两种色素蛋白PCB-ApcA和PCB-ApcB合成时聚集过程进行分析,通过多种组合的质粒在大肠杆菌体内共同表达进行重组。色素蛋白的吸收和荧光光谱以及Zn电泳表明,在大肠杆菌体内同时得到色素蛋白PCB-ApcA和PCB-ApcB,并且体内重组色素蛋白的细胞荧光光谱显示,色素蛋白以三聚体的形式存在,而破碎细胞后所得上清液所显示的光谱特征为单聚体的特征。

藻蓝蛋白β亚基(简称β-CPC)中有2个色素结合位点(Cys-84和Cys-155)分别以硫醚键与藻蓝胆素(PCB)连接[1,2].

BACKGROUND:Occlusion of the left atrial appendage (LAA) is thought to reduce the risk of thromboembolic events in patients with atrial fibrillation.OBJECTIVE:To examine the LAA and its relationship to neighbouring structures that may be put at risk when intervening to occlude its os.METHODS:31 heart specimens were examined grossly. Four of the LAAs were processed for histological examination and endocasts were made from 11 appendages. The diameters of the LAA os and proximity to the left superior pulmonary vein, mitral valve and left anterior descending artery were measured and areas of thin atrial wall in the vicinity were noted.RESULTS:The LAA orifice was oval shaped in all cases with a mean (SD) diameter of 17.4 (4) mm (range 10-24.1). The mean (SD) distances of the LAA orifice to the left superior pulmonary vein and mitral valve were 11.1 (4.1) mm and 10.7 (2.4) mm, respectively. The left anterior descending, circumflex artery and, in 6 cases, the sinus node artery, were in close proximity to the LAA. Pits or troughs and areas of thin atrial wall were found in 57.7% of hearts within a 20.9 mm radius from the os. Histology showed small crevices and areas of very thin wall within the trabeculated appendage.CONCLUSIONS:The LAA orifice is oval shaped and thin areas of appendage wall and atrial wall are common. Potentially, the left superior pulmonary vein, mitral valve and anterior descending coronary artery can be at risk during occlusion of the os.

Efforts to reduce vehicle weight and improve crash performance have resulted in increased application of advanced high strength steels (AHSS) and a recent focus on the weldability of these alloys. Resistance spot welding (RSW) is the primary sheet metal welding process in the manufacture of automotive assemblies. Friction stir spot welding (FSSW) was invented as a novel method to spot welding sheet metal and has proven to be a potential candidate for spot welding AHSS. A comparative study of RSW and FSSW on spot welding AHSS has been completed. The objective of this work is to compare the microstructure and mechanical properties of Zn coated DP600 AHSS (1·2 mm thick) spot welds conducted using both processes. This was accomplished by examining the metallurgical cross-sections and local hardnesses of various spot weld regions. High speed data acquisition was also used to monitor process parameters and attain energy outputs for each process. Results show a correlation found among microstructure, failure loads, energy requirements and bonded area for both spot welding processes.

Dissimilar intermixing during friction stir spot welding of Al 5754 and Al 6111 sheets is investigated using a combination of experimentation and numerical modeling. Dissimilar intermixing is not observed in (a) spot welds made using a tool having a smooth pin with or without a dwell period and (b) spot welds made using a threaded tool without the application of a dwell period. It is proposed that dissimilar intermixing during the dwell period in spot welding results from the incorporation of upper (Al 5754) and lower (Al 6111) sheet materials at the top of the thread on the rotating pin. A ribbon of contiguous Al 5754 and Al 6111 lamella is moved downward via the pin thread as the tool rotates during the dwell period in spot welding. A helical vertical rotational flow is created during the dwell period in spot welding because the ribbon of contiguous Al 5754 and Al 6111 lamellae discharged from the bottom of the pin thread moves outward and upward before moving back toward the tool periphery and downward again.

AIM:To identify novel prebiotics that could be used to maintain persistence of three representative probiotic strains in vivo. METHODS AND RESULTS:Test mice were treated with prebiotics soybean oligosaccharide (SOS), fructooligosaccharide (FOS) or inulin, followed by probiotics Lactobacillus acidophilus LAFTI L10 (L10), Bifidobacterium lactis LAFTI B94 (B94) or Lactobacillus casei L26 LAFTI (L26). Faecal samples were then collected and analysed using selective medium and PCR analysis to determine the presence of the probiotic strains. In contrast to the control groups, in mice fed prebiotics, the survival and retention time of the test probiotics was increased extensively. SOS and FOS prolonged the retention period of L10 from 24 to 30 h. Of the three prebiotics, FOS gave the best result with B94, prolonging the retention period from 3 to > or =10 days. Of the three prebiotics, inulin gave the best result for L26, prolonging the retention period from 2 to > or =6 days. CONCLUSIONS:The prebiotics SOS, FOS and inulin significantly enhance survival and prolong the retention period of L10, B94 and L26 in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY:Our results demonstrate the potential use of FOS, inulin and SOS as prebiotics in conjunction with the probiotic strains L10, B94 and L26 for new synbiotic products.

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

Transient local melting can account for the remarkable decrease in the strain rate when the tool rotational speed, heating rate and cycle time produce temperatures exceeding the 475 °C in Al 7075-T6 spot welds. The calculated strain rate is about 20 s-1 in Al 7075-T6 spot welds produced using tool rotational speeds from 1500 RPM to 3000 RPM and dwell time of 4 s. However, the peak temperature is less than less than 475 °C when a low tool rotational speed setting (1000 RPM) is used; transient local melting and tool slippage are precluded and the calculated strain rate is high (about 650 s-1). It is proposed that transient local melted films directly influence formation of the stir zone microstructure in Al 7075- T6 spot welds. It is suggested that the stir zone microstructure in spot welds made using high tool rotational speed settings are determined by a combination of grain boundary sliding and the limitation of cavity interlinkages when transient local melted films form.

The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-β84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-β84 of the apo-β-subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 °C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3μm, and a kcat = 1.7 × 10-5 and 1.1 × 10-5 s-1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS.

Energy utilisation during spot welding is investigated using a combination of calorimetry, peak temperature measurement and plunge testing. When a steel tool, clamp and anvil support is used, only 12·6% of the energy generated during the spot welding is transferred into the welded Al 6111 sheets. In contrast, when a mica clamp and anvil support are used, 50% of the energy generated during spot welding transfers into the welded Al 6111 sheets. Only a small percentage of the energy generated during the friction stir spot welding operation is required for stir zone formation. During plunge testing of 6·3 mm thick Al 6061-T6 material, less than 4·03% of the energy which is generated during friction stir spot welding is required for stir zone formation. The remainder of the energy generated dissipates into the tool assembly, clamp, anvil support and the aluminium sheets which are being welded. The rotating pin produces around 70% of the energy generated during spot welding of 6·3 mm thick Al-6061 material, with the remainder being contributed by the tool shoulder.

Aims: This study aimed to develop methods for the detection of the probiotic Lactobacillus casei LAFTI (R) L26 (L26) from mouse faeces, and to determine the survival and retention time of L26 in the mouse gastrointestinal tract.Methods and Results: A selective medium, de Man Rogosa Sharpe (MRS) + bromocresol green + vancomycin (MGV), was designed for the isolation and enumeration of L26 from faecal samples of mice. PCR primers were designed to confirm the identity of L26-like colonies on MGV. These primers did not produce PCR products from related organisms that grew on MGV. Following the administration of L26 to BALB/c mice, faecal samples were collected and analysed using the designed methods. Survival studies showed viable L26 cells to be present in the faeces of mice for > 48 h.Conclusions: Our results suggest that L26 is able to survive and be retained within the digestive tract of mice for at least 48 h following oral administration.Significance and Impact on the Study: MGV allows effective recovery of L26 from the background microbiota, including lactobacilli of mice. PCR was used to confirm that L26-like colonies were correctly identified as L26. Given the long retention time of L26 in the gastrointestinal tract of mice, it would appear that this probiotic strain may survive in the human gastrointestinal tract.

Material flow during friction stir spot welding is investigated. An examination of dissimilar Al 5754/Al 6111 spot welds was conducted to allow visualisation of material flow based on their differing etching characteristics. In addition, Al 6061–T6 spot welds containing Al2O3 tracer particles were examined to highlight the movement of material in different joint regions. It has been confirmed that upper sheet material is moved downwards into the lower sheet when a layer of upper sheet material is pushed ahead at the tip of the rotating pin, when upper sheet material becomes trapped at the root of the pin thread, and when an adhering layer of upper sheet material forms at the pin periphery during spot welding using a smooth pin. Lower sheet material is displaced upwards and outwards in a spiral motion when the rotating pin forms the keyhole. Two distinct zones of material flow are produced during friction stir spot welding: an inner flow zone close to the pin periphery where upper sheet material moves downwards in an anticlockwise direction with the rotating pin; and an outer flow zone where lower sheet material moves upwards and outwards in a spiral motion.

Self-assembled zinc sulfide (ZnS) of wurtzite structure nanocombs with a ZnO sheath have been grown using a simple catalyst-free thermal evaporation technique in the alumina furnace. The ZnS nanocombs are as long as to several tens of micrometers. The teeth with ZnO sheath are about 200nm in length and 30nm in width. The ZnO sheath is likely formed at cool-down stage due to the residual O2.

The mechanism of tool penetration during friction stir spot welding of Al-alloy and Mg-alloy sheet materials is investigated and is explained as a progression of wear events, from mild wear to severe wear and then to melt wear in material beneath the base of the rotating pin. Melt wear can also occur under the rotating tool shoulder provided that sufficient penetration of the upper sheet occurs during the spot welding operation.

The core-membrane linker, LCM, connects functionally the extramembraneous light-harvesting complex of cyanobacteria, the phycobilisome, to the chlorophyll-containing core-complexes in the photosynthetic membrane. Genes coding for the apoprotein, ApcE, from Nostoc sp. PCC 7120 and for a C-terminally truncated fragment ApcE(1-240) containing the chromophore binding cysteine-195 were overexpressed in Escherichia coli. Both bind covalently phycocyanobilin (PCB) in an autocatalytic reaction, in the presence of 4M urea necessary to solubilize the proteins. If judged from the intense, red-shifted absorption and fluorescence, both products have the features of the native core-membrane linker LCM, demonstrating that the lyase function, the dimerization motif, and the capacity to extremely red-shift the chromophore are all contained in the N-terminal phycobilin domain of ApcE. The red-shift is, however, not the result of excitonic interactions: Although the chromoprotein dimerizes, the circular dichroism shows no indication of excitonic coupling. The lack of homologies with the autocatalytically chromophorylating phytochromes, as well as with the heterodimeric cysteine-alpha84 lyases, indicates that ApcE constitutes a third type of bilin:biliprotein lyase.