Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Effect of oocyte mitochondrial DNA haplotype on bovine somatic cell nuclear transfer efficiency

The development capability of reconstructed bovine embryos via ovum pick-up (OPU)-somatic cell nuclear transfer (SCNT) technique has been influenced by the maternal lineage of oocyte cytoplasm, but the underlying mechanism remains unclear. Since mitochondria are the richest maternal-inherited organelle, in this study, we intended to clarify the effect of mtDNA haplotypes on cloning efficiency. By PCR-RFLP method, we identified mtDNA haplotypes A and B, differing in six restriction sites. Reconstructed embryos with haplotype A cytoplast achieved better fusion and blastocyst formation rate (64.6% and 39.4%), as compared with haplotype B (53.6% and 26.3%; P < 0.05). To further evaluate the role of mitochondria, the quantity of mtDNA, ATP content, and mRNA level of mtDNA-encoded COXI, COXIII in both oocytes were measured. Our data indicated that mtDNA copy number in haplotype A oocyte was significantly higher than that in haplotype B oocyte, both at the GV (10(5.03 +/- 0.69) vs. 10(4.81 +/- 0.86) copies/oocyte) and MII stages (10(5.31 +/- 0.71) vs. 10(5.13 +/- 0.63) copies/oocyte; logarithmically transformed values; P < 0.05). ATP content in type A oocyte was also greater at the GV (1.67 +/- 0.09 vs. 1.27 +/- 0.1 pmol) and MII stages (5.18 +/- 0.07 vs. 2.68 +/- 0.03 pmol; P < 0.05). Similarly, the mRNA expression level of mtDNA-encoded COXI and COXIII in haplotype A oocyte was significantly higher comparing to haplotype B oocyte (3.3 +/- 2.0 x 10(3) vs. 0.68 +/- 0.45 x 10(3); 24.9 +/- 10.5 x 10(3) vs. 9.4 +/- 3.3 x 10(3), respectively; P < 0.05). The data suggest that mitochondrial structure, quantity, and function may significantly affect the developmental competence of reconstructed embryos.     

Acidic hydrolysis of N-Ethoxybenzylimidazoles (NEBIs): potential applications as pH-sensitive linkers for drug delivery

This paper describes the development of a new class of N-linked imidazoles as potential pH-sensitive, cleavable linkers for use in cancer drug delivery systems. Kinetic analysis of eight derivatives of N-ethoxybenzylimidazoles (NEBIs) showed that their rates of hydrolysis are accelerated in mild aqueous acidic solutions compared to in solutions at normal, physiological pH. Incorporation of electron donating or electron withdrawing substituents on the phenyl ring of the NEBI resulted in the ability to tune the rates of hydrolysis under mild acidic conditions with half-lives ranging from minutes to months. A derivative of NEBI carrying doxorubicin, a widely used anticancer agent, also showed an increased rate of hydrolysis under mild acid compared to that at normal physiological pH. The doxorubicin analogue resulting from hydrolysis from the NEBI exhibited good cytotoxic activity when exposed to human ovarian cancer cells. These results demonstrate a potentially useful, general strategy for conjugating a wide range of drugs to imidazole-containing delivery vessels via NEBI functionalities for controlled release of therapeutics for drug delivery applications.     

Bioimaging in the mid-infrared using an organometallic carbonyl tag

Two stable and water-soluble organometallic carbonyl cluster derivatives have been prepared and shown to enter the cell with ease. The CO stretching vibrations afford strong mid-infrared signals which have been demonstrated, for the first time, to be of utility in cell imaging via an IR microscope.     

Construct synthetic gene encoding artificial spider dragline silk protein and its expression in milk of transgenic mice

Based on the known partial cDNA sequence of dragline silk protein an artificial gene monomer, a 360 bp sequence, was designed and polymerized to encode an analog of dragline silk protein. Six tandem copies of monomer were cloned into pBC1 vector and microinjected into the pronuclei of fertilized Kunming White eggs. Transgenic mice were screened by Polymerase Chain Reaction (PCR) and Southern blot which revealed that 10 mice (5 male, 5 female) among 58 mice were transgenic positive. Milk of five F0 mice and eight F1 mice was analyzed by Western blot, and two F0 mice and seven F1 mice expressed recombinant dragline silk protein. In transgenic mice milk a maximum of concentration of recombinant dragline silk protein was 11.7 mg/L by radioimmunoassay.     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.     

Temporal and spatial variations of cell-free layer width in arterioles

Separation of red blood cells and plasma in microcirculatory vessels produces a cell-free layer at the wall. This layer may be an important determinant of blood viscosity and wall shear stress in arterioles, where most of the hydraulic pressure loss in the circulatory system occurs and flow regulatory mechanisms are prominent. With the use of a newly developed method, the width of the cell-free layer was rapidly and repeatedly determined in arterioles (10- to 50-microm inner diameter) in the rat cremaster muscle at normal arterial pressure. The temporal variation of the cell-free layer width was non-Gaussian, but calculated mean and median values differed by <0.2 microm. The correlation length of the temporal variations downstream (an indication of mixing) was approximately 30 microm and was independent of pseudoshear rate (ratio of mean velocity to vessel diameter) and of vessel diameter. The cell-free layer width was significantly different on opposite sides of the vessel and inversely related. Increasing red blood cell aggregability reduced this inverse relation but had no effect on correlation length. In the diameter range studied, the mean width of the cell-free layer increased from 0.8 to 3.1 microm and temporal variations increased from 30% to 70% of the mean width. Increased aggregability did not alter either relationship. In summary, the cell-free layer width in arterioles is diameter dependent and shows substantial non-Gaussian temporal variations. The temporal variations increase as diameter increases and are inversely related on opposite sides of the vessel.     

Two new species of the sisorid Genus <Emphasis Type="Italic">Oreoglanis</Emphasis> Smith from Yunnan,China (Teleostei: Sisoridae)

Synopsis Two new species of the genus Oreoglanis, O. jingdongensis and O. immaculatus are described from the Mekong and Salween River basins, respectively, of Yunnan Province, China. Oreoglanis jingdongensis can be distinguished from other Oreoglanis species by the following characters: lower lip with a distinct median notch, posterior edge smooth or with indentations; caudal fin lunate and lower lobe longer than upper lobe. Oreoglanis immaculatus can be distinguished from its congeners by the following features: lower lip with a strait median notch, posterior edge with indentations; caudal fin emarginate and lower lobe about the same length as upper lobe, having no yellow patches on sides of body below adipose fin.