ICAR: endoscopic skull‐base surgery

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Chartae Latinae Antiquiores. Facsimile edition of the Latin charters 2nd series ninth century. Part L, Italy XXII, Cava dei Tirreni, ed. by G. Cavallo e G. Nicolaj, Dietikon-Zurich 1997

Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD) influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE). Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa) to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality

Amylopectin CLD (up to DP 30) prediction.

<p>It is predicted from changing the activity and transferred chain length (<i>X</i>₀ and <i>X</i>_min) of SBE (i) based on the Nipponbare endosperm amylopectin CLD. The CLD fitting of the Nipponbare amylopectin experimental CLD (A, green dots) is shown in red dots in panels A, B and C. The parameters used were β_(i) = 1.4, <i>X</i>_min(i) = 6 and <i>X</i>_0(i) = 7. The black curves were created by changing the <i>X</i>_min(i) (B) and β_(i) together <i>X</i>_min(i) (C).</p

The van der Waals surface of a structural model of wild-type mSBEIIa.

<p>The model was generated using SWISS-MODEL with the structure of rice (<i>Oryza sativa L</i>.) SBEI (PDB ID: 3AML) as a template. The model, truncated at amino acid 127, is orientated to show the front (A) and rear face (B) of the binding groove. The locations of the mutated residues are indicated by the arrows. The binding groove is highlighted in gray. Figures were generated using pyMOL version 1.6.9 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125507#pone.0125507.ref057" target="_blank">57</a>].</p

Native affinity gel.

<p>It shows the relative migration distance of the WT and mSBEIIa mutants in the presence of 0 mg mL^–1 and 2mg mL^–1 linear glucan compared to BSA. BSA was used as the standard for the comparison of migration rate of mSBEIIa. The experiment was repeated twice.</p

SEC characterization of the branched glucan products.

<p>The SEC weight distributions (arbitrarily normalized to the same maximum) are as functions of degree of polymerization, <i>X</i>, of the constituent branches of the branched glucan product after incubation with different mSBEIIa for different times. Black, blue, red, green, and purple lines are for CLDs after 0, 3, 6, 9, and 24 h incubation, respectively. The data shown correspond to one of the two independent experiments performed. In each case the difference between the two experiments was small.</p

The positions of Glu399, Tyr235, His467 and Arg342 (rice SBEI numbering) with respect to maltopentaose.

<p>The positions were produced after 25 ns simulation from (A) the WT rice SBEI and (B) the R342K rice SBEI. The dashed lines indicate distances between key functional groups. The figures were generated using pyMOL version 1.6.9 and orientated such that the backbone of the protein is superimposed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125507#pone.0125507.ref057" target="_blank">57</a>].</p

FACE characterization of the branched glucan products.

<p>These products were produced by WT mSBEIIa and mSBEIIa mutants using MAZACA amylopectin as the substrate. Top panel: CLD of MAZACA amylopectin expressed as molar % of each chain in MAZACA amylopectin. Lower panels: relative CLD of the products formed by wild-type mSBEIIa and mSBEIIa mutants enzymatic reactions at 30°C for 1, 3 and 6 h were subtracted from that in the substrate MAZACA amylopectin. The total number of chains in the substrate and products were normalized as 100. The data shown here are from one representative experiment chosen from two independent experiments. Both experiments showed the same trend.</p

A schematic of starch biosynthesis processes.

<p>It shows the key enzymatic steps involved in starch biosynthesis, elongation, branching (for simplicity, only inter-chain branching is shown), and debranching catalyzed by starch synthase (SS), starch-branching enzyme (SBE), and debranching enzyme (DBE), respectively. <i>X</i>₀ and <i>X</i>_min, respectively, are the minimum chain-length constraints of the residual and the transferred segments for the action of SBE.</p

Comparison of chain profiles of the branched glucan products from WT and R456K mSBEIIa.

<p>The chain profiles were from SEC weight distributions of the glucan products of WT and R456K mSBEIIa from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125507#pone.0125507.g003" target="_blank">Fig 3</a> and the FACE number distributions of the glucan products after 6 h incubation with WT and R456K mSBEIIa from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125507#pone.0125507.g004" target="_blank">Fig 4</a>. The different peaks are highlighted with their DPs in the SEC weight distributions. Black, blue, red, green, and purple lines in the SEC weight distribution are for CLDs after 0, 3, 6, 9 and 24 h incubation, respectively.</p