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Significance and Impact of the Study: Pullulanases have a great potential in industrial applications including the starch industry, the production of maltose syrups and high-purity glucose and fructose. In this study, a pullulanase from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 was successfully expressed in Escherichia coli and the recombinant enzyme can be purified and characterized. The high activity, broad pH range and stability implicate it as a potential enzyme for industrial applications. AbstractIn this report, a glycoside hydrolase 13 family pullulanase gene (Tk0977) was cloned from a thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1 (Pul-Tk). Pul-Tk encodes a protein of 765 amino acids including a putative 22-residue signal peptide. The protein has four consensus motives and a catalytic triad of glycoside hydrolase 13 family in the deduced amino acid sequence. The recombinant enzyme was expressed in Escherichia coli and purified to homogeneity. Pul-Tk can hydrolyse both pullulan and soluble starch. The purified enzyme was optimal at pH 5Á5-6Á0 and 100°C and exhibited good stability over a broad pH range (4-8). The V max and K m values were 118Á39 AE 1Á76 lmol mg À1 min À1 and 0Á37 AE 0Á02 mg ml À1 for pullulan and 53Á19 AE 11Á66 lmol mg À1 min À1 and 0Á36 AE 0Á05 mg ml À1 for starch.All these favourable enzymatic properties make it valuable in various industries.

Aims: To evaluate the interaction of 1-(1-naphthylmethyl)-piperazine (NMP) and ciprofloxacin (CPFX) in vitro against fluoroquinolone (FQ)-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Methods and Results: The in vitro interaction of NMP and CPFX in 12 FQresistant clinical isolates of MRSA was assessed using a checkerboard microdilution method. In the study, a synergistic antimicrobial effect between NMP and CPFX was observed in all 12 FQ-resistant strains tested, as determined by the fractional inhibitory concentration index (FICI), and in 10 strains using DE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed using the time-killing test and agar diffusion assay for the selected strain, MRSA 1862; synergistic activity was observed when NMP was combined with the first-line antimicrobial agent CPFX against Staph. aureus. Conclusions: Synergistic activity between NMP and CPFX against clinical isolates of FQ-resistant Staph. aureus was observed in vitro. Significance and Impact of the Study: This report might provide alternative methods to reduce the resistance of Staph. aureus to CPFX.

Miniature pigs have been recognized as valuable experimental animals in medical research. However, porcine models related to gene knockout of human diseases are not widely available. The objective of this study was to establish Mx1-Cre pigs using somatic cell nuclear transfer. In this study, we created transgenic pigs using somatic cell nuclear transfer (SCNT). Transfer of 210, 230, 250 and 215 zygotes to four surrogates produced 10 piglets. The Cre recombinase expression in transgenic pigs was studied using reverse transcriptase (RT)-PCR and immunohistochemistry. Mx1-Cre swine were shown to harbor the Cre gene in their genomic DNA using the PCR. In conclusion, Mx1-Cre transgenic piglets were successfully produced by SCNT. These transgenic swine, in conjunction with inducible systems for controlling Cre expression and function, are likely to have a profound impact on the study of human diseases.

We demonstrate the stimulated Raman scattering (SRS) of a binary solution of toluene and m-xylene at different volume concentrations in liquid-core optical fiber (LCOF). The results show that SRS of three vibration modes of 1002 cm −1 , 2920 cm −1 and 3058 cm −1 bands are simultaneously generated at some volume concentrations. The 2920 cm −1 band and the 3058 cm −1 band are generated at one time, the SRS thresholds of the first-order Stokes of the 2920 cm −1 and 3058 cm −1 bands are lower compared with the second-order Stokes threshold of the 1002 cm −1 band and the main peak of the 2920 cm −1 and 3058 cm −1 bands changes from the 2920 cm −1 band to the 3058 cm −1 band as the volume concentrations are changed. We assume that these phenomena are attributed to the intermolecular Fermi resonance. Raman scattering cross section (RSCS) theory is used to explain this assumption.

The golden hamster is an excellent experimental animal in many research fields. In an effort to establish experimental protocols necessary for cloning golden hamsters, optimized conditions for induced cytoplasmic protrusion and assisted enucleation of golden hamster oocytes with different concentrations of colchicine and demecolcine were examined in this study. The Golden hamsters (female, six weeks of age) were superovulated with eCG (30 IU, ip) followed by hCG (30 IU, ip) at intervals of 72 h. Hamsters were sacrificed at 13.5 h, 15 h and 18 h after hCG injection. The different ages of cumulus–oocyte complexes (COCs) were collected from oviducts and cumulus cells were removed with 0.1% hyaluronidase. (1) denuded oocytes of different ages were treated with 2.5 μg mL–1, 5 μg mL–1, and 10 μg mL–1 of colchicine for 4 h in M199TE, and they were examined each hour; (2) denuded oocytes of different ages were treated with 0.02 μg mL–1, 0.04 μg mL–1, 0.06 μg mL–1, 0.1 μg mL–1, 0.2 μg mL, 0.4 μg mL–1, and 0.6 μg mL–1 of demecolcine for 1 h in M199TE, and they were examined every 15 min; 3) according to the results of processes (1) and (2), cytoplasmic protrusions of oocytes treated with 10 μg mL–1 of colchicine (60 oocytes) or 0.4 μg mL–1 demecolcine (88 oocytes) for 1 h were removed with a micromanipulation pipette. Then the oocytes were examined by being stained with Hoechst 33342, and the percentage of assisted enucleation was compared with that of blind enucleation in which position of the chromosomes was indirectly determined by the location of the first polar body. The results showed that: (1) about 90% of oocytes at 13.5 h and 15 h post hCG injection were induced to form protrusions under the treatment of 10 μg mL–1 of colchicine for 1 h; (2) oocytes of 13.5 h post hCG were very sensive to demecolcine, even the treatment of 0.02 μg mL–1 for 1 h could also induce cytoplasmic protrusions (45.38% of 31 oocytes); (3) when treated with 0.4 μg mL–1 of demecolcine for 1 h, the cytoplasmic protrusion rate of oocytes of 13.5 h–18 h post-hCG could reach to 99–100%; (4) when the cytoplasmic protrusions induced by 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were removed, assisted enucleation rates were over 80%, significantly higher (P < 0.05) than blind enucleation (31.53% of 60 oocytes). In conclusion, the results of this study demonstrate that both colchicine and demecolcine can induce the hamster oocytes to form cytoplasmic protrusions. The treatments of 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were the best conditions to induce oocytes (13.5 h–18 h post hCG) to form protrusion. Also, the assisted enucleation rate with colchicine and demecolcine is much higher than that of blind enucleation. These results define conditions for chemical assisted enucleation and should facilitate the development of cloned golden hamsters as an animal model for human diseases.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

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