Pinpoint injection of microtools for minimally invasive micromanipulation of microbe by laser trap

This paper reports transportation of the target microbe by the laser trapped microtools with minimum laser irradiation to the target. The size of a microtool (MT) is around micrometer. The MTs are manipulated by the focused laser under the microscope to manipulate the target microbe. Here we propose a pinpoint injection method of MTs at the desired location in the microchamber, which is filled with liquid. At first, we classified the injection method of the MTs in four categories. Here we employed a new method to install the MTs inside the microchamber. We developed a MT holding chip to install the MTs. The MTs were injected in the microchamber, and were manipulated successfully by the laser scanning micromanipulator to transport the target microbe for separation. The proposed method is useful for the pinpoint injection of MTs and separation by the indirect micromanipulation.     

Poly(A) metabolism in Xenopus laevis embryos: substrate-specific and default poly(A) nuclease activities are mediated by two distinct complexes

The metabolism of the poly(A) tail is a process important for the translational regulation of maternal mRNAs in Xenopus laevis oocytes and early embryos. Two poly(A) nuclease (PAN) activities have been described in Xenopus embryo or activated egg extracts (Legagneux et al (1995) RNA 1, 1001-1008). These activities (default PAN and EgPAN) are distinguishable by their deadenylation kinetics and their substrate specificities. In this report, we show that these activities display different sensitivities to biochemical treatments. Urea and, to a lesser extent, spermidine, inhibit EgPAN at concentrations which have no effect on default PAN. Heparin activates default PAN but inhibits EgPAN. When extracts are fractionated by ultracentrifugation, the default activity is recovered in one unique fraction, whereas two fractions must be combined to reconstitute the EgPAN activity. Moreover, these two deadenylation activities are separable by size exclusion chromatography under native conditions. We conclude that these two deadenylation activities are mediated by two protein complexes.     

维拉诺瓦巴奎因城市公园

维拉诺瓦巴奎因城市公园坐落在里斯本市区，倚靠葡萄牙最长的河流——塔古斯河的右岸。在塔古斯河谷的大型综合项目中，包含了对这片10hm^2土地的改造，以重新评估它的城市和景观价值。     

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Construction of the plasmid PMEX8-HAK1 and random site-directed mutagenesis of human cytosolic adenylate kinase

The pMEX8-hAK1 vector was devised from the pAK plasmid (Kim J. H. et al., 1989, Protein Engineering 5, 379-386), which could directly express human adenylate kinase proteins without recombination and its single strand DNA could be withdrawn with helper phage for random site-directed mutagenesis. The conserved key residues at Lys21, Lys27, and Thr39 were engineered to obtain mutants for kinetic analysis. Three mutants were obtained as K21P, K27R, and T39S, their specific activities were strikingly reduced compared to those of wild type adenylate kinase. This pMEX8-hAK1 will be a powerful tool for site-directed mutagenesis to detect the substrate-enzyme interaction for human adenylate kinase including various other enzymes.     

Impulse breakdown voltages of triggered multistage vacuum gap

Several sealed-off triggered vacuum gaps are connected in series to improve hold-off voltage. The characteristics of impulse breakdown voltage of these series-connected gaps are investigated experimentally. The sum hold-off voltage of series-connected gaps decreases to a unit hold-off voltage when the maximum value of voltage division ratio across the gaps increases to unity. Self-breakdown probability of the series-connected gaps is always higher than that of a single gap under the same conditions. Hence, stage efficiency of the multistage gap decreases with increasing number of stages. Its value is 90 percent with 2-stage gap and 75 percent with 5-stage gap, respectively, under the same voltage division ratio and the same gap length (2.0 mm) in each stage. Triggered breakdown voltage of 2- or 3- stage gap is several hundred volts when all gaps are triggered simultaneously at the peak of the main impulse wave and a working voltage range is nearly 100 percent in this case. The working voltage range decreases with number of stages. Its value is 45 percent with 3-stage gap and 15 percent with 5-stage gap, respectively, when one triggered gap is fired for switching.     

Magnetic resonance imaging of disseminated sclerosis

Multiple sclerosis is a chronic demyelinating disease. Paraclinical examinations may contribute to the diagnosis of multiple sclerosis. Magnetic resonance imaging (MRI) has a very high sensitivity concerning multiple sclerosis, and has made it possible to visualize multiple sclerosis plaques in vivo, to follow each plaque over the course of time and in this way to obtain information about the pathogenesis. MRI has shown that the size of plaques may vary considerably, and that plaques are dynamic structures with the ability to change in size over few weeks. By using MRI and the contrast agent Gadolinium-DTPA, it is possible to distinguish a newly developed plaque from an older one. Therefore, MRI has become an important examination in therapeutic trials. Just now, MRI with Gadolinium-DTPA is being used to evaluate the efficacy of plasmapheresis and immunoglobulin treatment in a joint study between Rigshospitalet and Hvidovre Hospital.     

A high-speed halftoning processor for raster scanned images

A high-speed bilevel reproduction algorithm, called modified error diffusion (MED) algorithm, has been developed to provide high-quality halftoning images for continuous tone images and has been implemented in a CMOS LSI chip. The chip has been designed with standard-cell 1.5-μm CMOS technology using an optimum layout design. The chip has achieved a maximum processing speed of 60 ns/pixel     

Physical and functional association of cortactin with Syk in human leukemic cell line K562

Human leukemic cell line K562 is induced to differentiate into the megakaryocytic lineage by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate here that TPA stimulation increases tyrosine phosphorylation of an 80-kDa protein at an early stage of megakaryocytic differentiation and that this 80-kDa protein is identical with cortactin. Since tyrosine kinase Syk was activated by TPA stimulation, we examined the possibility that cortactin is a potential substrate of Syk in K562 cells. TPA-induced tyrosine phosphorylation of cortactin was decreased profoundly by overexpression of dominant-negative Syk. Furthermore, cortactin was associated with Syk even before TPA stimulation. Since cortactin was previously referred as an 80/85-kilodalton pp60src substrate, we examined the association between Src and cortactin, whereas its association could not be detected. These data suggest that Syk phosphorylates cortactin in K562 cells upon TPA treatment.