Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence

The translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N- and C-terminal splice junctions and around the N-terminal one-third of VDE. We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His362, newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His736 assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE.     

A 43-Gb/s full-rate-clock 4:1 multiplexer in InP-based HEMT technology

This paper describes a full-rate-clock 4:1 multiplexer (MUX) in a 0.13-/spl mu/m InP-based HEMT technology for 40-Gb/s and above optical fiber link systems. To reduce output jitter, the serialized data are retimed at the final stage by a retimer, a D-type flip-flop, which has a symmetric layout with an optimized spacing to the ground that minimizes coupling capacitances. A phase adjuster, composed of an exclusive OR and a delay switch, uses external control signals to change each phase of the serialized data and clock entering the retimer and gives a correct timing for the clock to drive the retimer. A clock distributor with a simple wired splitter divides the clock into two clocks with high gain and low current. The MUX integrates 1355 HEMTs formed using electron beam lithography. A chip mounted in a test module operated at up to 47 Gb/s with a power consumption of 7.9 W for a single supply voltage of -5.2 V.     

High power with low electric power consumption activemulti-mode-interferometer laser diode for fibre-amplifier

Active multi-mode interferometer laser diodes (LDs), implemented in InGaAsP/InP, achieved a significant reduction of 40% (at 0.4 W output) in electric power consumption compared to regular single lobe LDs. With them, a high output power of 0.7 W was also obtained at 14XX nm     

Microstructures formed by secondary growth of fired ZSM-5 seed crystals

Oriented ZSM-5 seed crystals on an α-Al₂O₃ porous substrate were hydrothermally treated in a raw sol. The ZSM-5 membranes were fabricated via secondary growth of the seed crystals. For some samples, the seed-applied substrate was fired at 300 or 600 °C before the secondary growth in order to enhance adhesion between the substrate and the seed crystals. The influence of the firing on the subsequent secondary growth of the seed crystals was examined by XRD, SEM, and TEM. The TEM images of the sample fired at 300 °C showed that the resulting membrane was continuous, and in the membrane, large ZSM-5 particles were distributed in a porous matrix. The ZSM-5 particles were slightly smaller than the used seed crystals. HR-TEM observations showed that the porous matrix is comprised of ZSM-5 micro-crystals, and the part adjacent to the large ZSM-5 crystals has the same crystallographic orientation as the large crystals. The TEM images of the sample fired at 600 °C showed that the resulting layer is comprised of particles with a core-shell structure. The core consisted of ZSM-5 micro-crystals, whereas the shell was composed of large ZSM-5 rod-like crystals. It is inferred that the formation of these interesting microstructures is related to the degradation of the template agent, NPr₄OH, in the seed crystals by firing at 300 and 600 °C. The part including no template is dissolved by a hydrothermal treatment, and the dissolved species is re-crystallized via reaction with a template agent in the used raw sol, resulting in the formation of interesting microstructures.     

Involvement of actin and polarisome in morphological change during spore germination of Saccharomyces cerevisiae

We studied the morphological changes of Saccharomyces cerevisiae ascospores during germination. Initiation of germination is followed by polarization of actin patches, maintaining their localization to the site of cell surface growth. Loss of polarisome components, Spa2p, Pea2p, Bud6p or Bni1p, results in depolarization of actin patches. Green fluorescent protein-fused polarisome components exhibit the polarized localization, implying that polarisome is involved in the polarized outgrowth during germination. At the late stage of germination, we found that actin patches temporally depolarize before bud emergence. The observation that loss of Cla4p extends the polarized growth period suggests that Cla4p is involved in the actin-depolization step. Actin polarization in the initial stage is accelerated by overexpression of Ras2p, whereas hyperpolarization is continuously observed by overexpression of Rho1p. Thus, yeast spore germination is a morphological event that is regulated by a number of factors implicated in mitotic bud morphogenesis.     

Increased impact strength of biodegradable poly(lactic acid)/poly(butylene succinate) blend composites by using isocyanate as a reactive processing agent

Poly(lactic acid) (PLA) blended with poly(butylene succinate) (PBS) was prepared in the presence of lysine triisocyanate (LTI) by using a twin‐screw extruder and injection molding machine. The physical properties, rheological behavior, compatibility, and morphology were investigated by using a tensile test, a Charpy impact test, melt mass‐flow rate (MFR) measurements, size exclusion chromatography (SEC), and laser scanning confocal microscopy (LSCM). The impact strength of PLA/PBS(90/10 wt %) blend composite was about 18 kJ/m² in the absence of LTI, and it increased to 50–70 kJ/m² in the presence of LTI at 0.5 wt %. The MFR value of PLA/PBS(90/10 wt %) decreased from 25 g/10 min at 200°C in the absence of LTI to approximately 3 g/10 min in the presence of LTI. These results imply that isocyanate groups of LTI reacted with both terminal hydroxyl or carboxyl groups of the polymers. Spherical particles at 1 μm were observed by using LSCM in the presence of LTI. These results indicate that the LTI is a useful reactive processing agent to increase the compatibility of PLA/PBS blend composites to increase the impact strength of PLA. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Effect of Processing Parameters on Physical Properties of Cadmium Stannate Thin Films Prepared by a Dip-Coating Technique

Thin films of dicadmium stannate spinel (Cd₂SnO₄) were deposited on glass substrates using a dip-coating technique. The films were transparent to visible light (90%) and electrically conductive. X-ray diffractometry showed that annealed films consisted of a single cubic spinel phase only when they were prepared from a solution with the composition of Cd:Sn = 2.5 and fired at a temperature of 400°–500°C. The Cd:Sn ratio, the firing temperature, and the post-deposition annealing sequence were crucial for the formation of a single phase, which is vital to obtain optimal optical and electrical properties. A resistivity as low as 3.3 × 10⁻⁴Ω·cm could be obtained after annealing.     

Distribution and diffusion coefficients of NaCl in polyamide (nylon‐6,6 and polyxylyleneadipamide) membranes

Thin membranes of an aliphatic polyamide (nylon‐6,6) and an aromatic polyamide (polyxylyleneadipamide) (PXAP) were prepared, and their distribution (K) and overall diffusion (D) coefficients of sodium chloride were measured with the unsteady‐state and steady‐state dialysis method. The overall diffusion coefficients at a zero concentration [D⁽⁰⁾] of sodium chloride for nylon‐6,6 and PXAP were 1.3–0.8 μm²/s (from 2 min of interfacial polymerization to 4 min) and 0.078, respectively. D⁽⁰⁾ for PXAP was about 3 times greater than that of a cellulose acetate (CA) membrane (0.024 μm²/s). The K values for nylon‐6,6 and PXAP were 0.7–0.5 from 2 to 4 min and 0.05, respectively. K for PXAP was almost the same as K for CA (0.06). A two‐part (dense and porous) model of the membrane structure was applied to obtain D_d (the diffusion coefficient in the dense part of the membrane) and D_p (the diffusion coefficient in the porous part of the membrane) for CA, PXAP, and nylon‐6,6 thin membranes. The values of D_d were almost the same for both nylon‐6,6 and PXAP (0.05–0.061 μm²/s) and about 10 times greater than the value for the CA membrane (5.6 × 10^?3 μm²/s). D_p for PXAP was almost the same as D_p for CA. However, D_p for the nylon‐6,6 membrane was 10–16 times greater than D_p for the PXAP membrane. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 2605–2612, 2002     

Silicalite-l Synthesis from Silicate Aqueous Solutions Including Amines as a Base

Aqueous silicate solutions could be prepared from a silicon alkoxide using amines as a base. The synthesis of silicalite-1 conducted by hydrothermally treating the solutions after the addition of NPrⁿ₄Br (TPABr), was examined focusing on triethylamine (NEt₃) as the amine. The aqueous solutions with chemical compositions of Si(OEt)₄ (TEOS) : NEt₃ : TPABr : H₂O = 1 : ≥0.5 : ≥0.8 : 200 led to silicalite-1 crystallization upon hydrothermal treatment at 130C for 4 days. The resulting crystals had a small thin coffin shape. With increasing the added amounts of NEt₃ and TPABr, the crystal size decreased. Among the investigated conditions, the smallest silicalite-1 crystal measuring about 1.0 × 0.7 × 0.4 um was obtained at starting compositions of NEt₃/TEOS ≥2.0 and TPABr/TEOS ≥3.2. The increase in the TPA content of the starting solutions accelerated the crystal nucleation of silicalite-1, so that the obtained crystals became smaller. During the hydrothermal synthesis, an amorphous silica gel was formed before the crystallization commenced. The increase in the NEt₃ content may suppress the formation of the silica gel, and/or facilitate its dissolution, thereby the supersaturation degree of silicate species in the solution was increased, giving small silicalite-1 crystals. When an aqueous suspension of the smallest silicalite-1 crystals was spread onto a glass substrate by a dip-coating method, an ordered mono-grain layer of the silicalite-1 crystals oriented with the straight channels normal to the substrate surface was obtained.