Preparation of long sticky ends for universal ligation-independent cloning: Sequential T4 DNA polymerase treatments

Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.     

Preparation of cationic polystyrene latexes in the absence of emulsifiers

Preparation of cationic polystyrene latexes has been investigated in the absence of emulsifiers. It is clarified that a stable latex can be obtained by using 2,2′-azobis(2-amidinopropane) hydrochloride as initiator (initiator process). The latex seems to be stabilized with the fragments of the initiator chemically bound to the surface of particles. More stable latexes can be obtained by copolymerizing cationic monomers such as dialkyl aminoethyl methacrylates (copolymerization process). The stability of these latexes may predominantly be attributed to cationic monomers chemically bound to the surface of particles. Stable latexes with high solids content can be obtained by using the two-step polymerization technique in the copolymerization process. The particle formation process in the initiator process and the copolymerization process is discussed.     

Formation of particles in the preparation of carboxylated polystyrene latexes

Carboxylated polystyrene latexes have been prepared by copolymerizing acrylic acid at the appropriate degree of neutralization in the absence or presence of sodium dodecyl sulfate (SDS), and the particle formation process was investigated from the number and uniformity of particles. In the absence of SDS or in a concentration range of SDS lower than 6.41 mmole/l., the formation of particles can predominantly be attributed to the precipitation of growing radicals formed in the aqueous phase; whereas in a concentration range of SDS higher than 6.41 mmole/l., the formation of particles can predominantly be attributed to the initiation of polymerization in the interior of SDS micelles containing solubilized styrene by the collision of growing radicals formed in the aqueous phase. In the former range, the number of particles increases linearly with increasing concentration of SDS below the critical concentration of 1.60 mmole/l., which is sufficient to stabilize particles formed by the precipitation of growing radicals, and remains constant above the critical concentration. The effect of initiator concentration as well as amount of styrene on the formation of particles also supports the particle formation process described above.     

Resonance enhancement effect in non-linear optical susceptibility of polysilanes

We have investigated the ↑χ⁽³⁾(−3ω; ω, ω, ω)↑ spectra of polysilane films by means of third-harmonic generation. The fundamental photon energy is scanned from 0.56 to 2.2 eV. The ↑χ⁽³⁾↑ spectra show three structures, which are assigned to three- and two-photon excitonic resonances. These features indicate that the predominant excited states involved in the non-linear optical response of polysilanes are composed of three levels: the lowest one-photon allowed exciton, a one-photon forbidden exciton, and another higher-lying one-photon allowed state.     

The role of polyvinyl alcohol in emulsion polymerization

Emulsion polymerization of styrene (St) and vinyl acetate (VAc) in the presence of conventional polyvinyl alcohol (PVA), PVA modified with a terminal alkyl group or PVA modified with a terminal thiol group (HS-PVA) was compared. Whereas stable PVAc latexes were obtained, a stable PSt latex was obtained only in the case of HS-PVA. From the adsorption isotherms of these PVAs on the surface of PVAc and PSt latex particles, as well as the grafting efficiencies of VAc and St onto HS-PVA in relation to the stability of the polymerization process, the role of PVA in the emulsion polymerization was discussed.     

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on cultured porcine granulosa cells by employing the aminopeptidase assay using alanine-p-nitroanilide and histochemical staining using L-leucyl-beta-naphthylamide. Porcine granulosa cells obtained from follicles 4-5 mm in diameter were cultured for 7 days. The aminopeptidase assay showed that the porcine granulosa cell culture had aminopeptidase activity and that this activity was inhibited in a dose-dependent manner by bestatin which binds to cell surfaces and inhibits cell surface aminopeptidases. Histochemical staining also indicated that cultured granulosa cells had aminopeptidase activity. Porcine granulosa cells were cultured in the presence or absence of porcine follicle stimulating hormone (FSH, 3.125 nmol/l) and/or bestatin (0.4, 4.0 and 40.0 micrograms/ml) for 7 days, and the production of progesterone and oestradiol was measured. In the presence of porcine FSH, the production of progesterone and oestradiol by granulosa cells was increased significantly by approximately 5- and 2-fold respectively. These increases were enhanced further by bestatin (40.0 micrograms/ml). In the absence of porcine FSH, progesterone production was enhanced by bestatin (40.0 micrograms/ml), whereas no significant effect of bestatin on oestradiol secretion was observed. These findings indicate that the inhibition of membrane-bound aminopeptidase(s) on the cell surfaces affects the steroidogenesis of granulosa cells, and that these aminopeptidase(s) are important regulators of granulosa cell differentiation.     

The use of free rectus abdominis myocutaneous flap to close the empyema space with alveolar fistula--a case report

A 59-year-old man, who had had right middle and lower lobectomy for pulmonary tuberculosis, admitted for the treatment of empyema with fistula. Closure of empyema space with free rectus abdominis myocutaneous flap was performed following open window thoracotomy and thoracoplasty. As he previously underwent two major operation, lobectomy by posterolateral approach and gastrectomy for gastric ulcer, free rectus abdominis flap was chosen instead of omental flap or latissimus dorsi myocutaneous flap. Postoperative CT film showed that this flap was filled up in all interstices of the empyema cavity. The pedicle vessels to this flap are large enough to provide long stalks, so microsurgical anastomosis can be accomplished safely. The use of free rectus abdominis myocutaneous flap is one of a useful maneuver for chronic empyema with fistula.     

Gas transport in poly(vinyl-p-isopropylbenzoate): Comparison and correlation with some other poly(vinyl esters)

Gas permeability in poly(vinyl-p-isopropylbenzoate) (PVp-i-PrB) was determined by a timelag method. The transport properties were discussed from comparison with the permeability data of other poly(vinyl esters), which were studied previously. All these polymers are structurally related, and the size of a side group or the position of its substituent was changed systematically. The isopropyl group of PVp-i-PrB is attached at the para position of a phenyl ring and is the largest in size. As a result gas diffusivity and therefore permeability were increased. The effect of the substituent on gas diffusivity was explained as it increases the interchain and intrachain distances. The discussion was supported from the comparison of the density data between PVp-i-PrB and other poly(vinyl esters). The diffusion coefficients of six glassy poly(vinyl esters) were correlated at their T_g and good correlations were shown to the free volume and its fraction. On the other hand, gas solubility was little affected by the change of an alkyl group on a phenyl ring. The solubility data of PVp-i-PrB and poly(vinyl benzoate) were shown to be clearly correlated with the critical properties of the penetrants. © 1995 John Wiley & Sons, Inc.