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Abstract: A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, maximum activity at 43°C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of K m and V m were found to be 0.37mM and 303μmolmg−1 min−1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7mM and inhibited at concentrations over 10mM. [Copyright &y& Elsevier]

Abstract: Ordered MCM-41-type mesoporous silica nanoparticles (MSNs) with pore size of 2.6nm were synthesized and were further modified with various amounts of 3-aminopropyltriethoxysilane (APTES), respectively, by a direct co-condensation method. These amine functionalized mesoporous silica nanoparticles (Am-MSNs) were employed to complex with plasmid DNA (pDNA) to study their adsorption and protection capacities. The results demonstrate the MSNs functionalized with aminopropyl groups present advanced adsorption capacities for pDNA immobilization. And Am-MSNs with high APTES amount lead to high amount of pDNA adsorption. Further investigation of pDNA protection shows that Am-MSNs with moderate APTES amount could completely protect pDNA from enzymatic degradation, while those with smaller and/or higher amount of APTES could partially provide protection of pDNA. [Copyright &y& Elsevier]

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290±8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 °C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 °C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and l-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. Km values of the recombinant enzyme are comparable for both substrates: 0.2 mM for l-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 °C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent. [Copyright &y& Elsevier]

The N-methyl-N-nitrosourea (MNU)-treated rat is typically used as an animal model of chemically-induced retinitis pigmentosa (RP). Reactive oxygen species (ROS) have been recognized as the crucial contributor to the retinal photoreceptor apoptosis seen in MNU-treated rats. In the present study, we explored the therapeutic effects of hydrogen-rich saline (HRS), a selective ROS scavenger, on MNU-induced photoreceptor degeneration. Intraperitoneal (IP) administration of HRS ameliorated MNU-induced photoreceptor degeneration in terms of morphology and function: Sharply decreased thickness of the retinal outer nuclear layer (ONL) and flattened photopic and scotopic electroretinogram (ERG) waveforms, typically seen in response to MNU treatment, were substantially rescued in rats cotreated with MNU and HRS (MNU + HRS). Moreover, the terminal deoxyuridine triphosphate nick-end labeling (TUNEL) assay revealed a smaller number of apoptotic photoreceptors in the MNU + HRS group compared that in the MNU group. Compared to MNU-treated rats, retinal malondialdehyde (MDA) content in MNU + HRS rats significantly decreased while superoxide dismutase (SOD) activity significantly increased. Morphological and multi-electrode array (MEA) analyses revealed more efficient preservation of the architecture and field potential waveforms in particularly the peripheral regions of the retinas within the MNU + HRS group, compared to that in the MNU group. However, this enhanced protection of structure and function in the peripheral retina is unlikely the result of site-dependent variation in the efficacy of HRS; rather, it is most likely due to reduced susceptibility of peripheral photoreceptors to MNU-induced degeneration. Inner retinal neuron function in the MNU + HRS rats was better preserved, with fewer apoptotic photoreceptors in the ONL. Collectively, these results support the rationale for future clinical evaluation of HRS as a therapeutic agent for human RP. [ABSTRACT FROM AUTHOR]

Abstract: The disease-causing gene which underlies a naturally occurring X-linked mutant cone dysfunction Sprague–Dawley rat model was investigated. Full-field electroretinogram (ERG) and simple sequence length polymorphism analyses were applied to 441-second filial generation rats that were derived from crossing a mutant rat and a Brown–Norway rat. After identifying a mutation mapping within the telomeric region of chromosome X, a candidate gene related to retinal cone function in this region was further screened using real-time PCR, immunohistochemistry and histological methods. The results showed that a G-to-T substitution at the splice acceptor site of intron 4 was present in the opsin 1, medium-wave sensitive (Opn1mw) gene, thereby causing down-regulated transcription and translation. These changes were consistent with abnormities seen in the ERG response. However, there was no significant histological change in the mutant rat retina. Therefore, we infer from this that the causative gene for the mutation is Opn1mw and consequently term this a middle-wavelength opsin cone dysfunction (MCD) rat model. The deficiency in vision of the MCD rat is similar to the color vision defects that occur in humans with a color vision defect but without recessive retinal degeneration. This rat model may be useful for understanding the mechanism that is responsible for color vision and for developing clinical therapies for several retinal dystrophies caused by cone opsin deficiencies. [Copyright &y& Elsevier]