Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number-dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.     

Structure, antihyperglycemic activity and cellular actions of a novel diglycated human insulin

Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (M(r) 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly(1) and Phe(1) of the insulin A- and B-chains, respectively. Intraperitoneal (i.p.) administration of diglycated insulin to mice alone or in combination with glucose (7 nmol/kg) resulted in a 43-61% and 11-34% reduction in glucose lowering activity, respectively, compared with native insulin. Consistent with these findings, diglycated insulin (10(-9) to 10(-7) mol/liter) was 22-38% less effective (P < 0.001) than native insulin in stimulating glucose uptake, glucose oxidation and glycogen production in isolated mouse abdominal muscle.     

Site-directed mutagenesis of the substrate-binding cleft of human estrogen sulfotransferase

The sulfonation of estrogens by human estrogen sulfotransferase (humSULT1E1) plays a vital role in controlling the active levels of these hormones in the body. To understand more fully the structural and functional characteristics of humSULT1E1, we have carried out site-directed mutagenesis of critical amino acids found in the substrate-binding cleft. Three single amino acid mutations of humSULT1E1 (V145E, H107A, and K85A) were created in this study. Kinetic studies were used to provide information about the importance of these residues in substrate specificity and catalysis, using a variety of substrates. Lysine at position 85 has been proposed to be within hydrogen bonding distance to the 3alpha-phenol group of beta-estradiol, thereby stabilising the substrate in the active site. However, substitution to a neutral alanine at this position improved substrate specificity of humSULT1E1 for beta-estradiol, estrone, and dehydroepiandrosterone (DHEA). The exchange of valine 145 for negatively charged glutamic acid markedly improved the ability of humSULT1E1 to sulfonate dopamine, but caused a reduction in specificity constants toward steroids tested, in particular DHEA. The presence of a histidine residue at position 107 was shown to be essential for the production of a functional protein, as substitution of this amino acid to alanine resulted in complete loss of activity of humSULT1E1 towards all substrates tested.     

Targeted insertion of a lacZ reporter gene into the mouse Cer1 locus reveals complex and dynamic expression during embryogenesis

The mouse Cer1 (mCer1, Cer-l, Cerr1) gene encodes one member of a family of cytokines structurally and functionally related to the Xenopus head-inducing factor, Cerberus (xCer). We generated a mouse line in which the Cer1 gene was inactivated by replacing the first coding exon with a lacZ reporter gene. Mice homozygous for this allele (Cer1(lacZ)) showed no apparent perturbation of embryogenesis or later development. However, the lacZ reporter revealed a number of hitherto uncharacterised sites of Cer1 expression in late fetal and adult tissues. Preliminary analysis suggests that Cer1 is not essential for their morphogenesis, differentiation, or homeostasis.     

A Three-Dimensional Finite Element Analysis of Heat Transfer in the Forearm

The finite element method was used to analyze heat transfer within a section of the forearm while exposed to different ambient conditions and with different metabolic states. The three-dimensional model accounts for the different material properties of bone, muscle and blood and incorporates a single artery-vein pair for counter-current heat exchange. The geometry of the model was developed from anatomical cross-sectional images of the forearm. The model was used to determine the effects or rest vs. exercise, free vs. forced surface convection and 0 degrees C vs. -20 degrees C external temperatures. The results of the model were compared to experimental data and the model exhibits qualitatively correct behaviour. This model can be used to study hyperthermia, burns and cryogenic freezing of tissue.     

Effect of hydroquinone on meiotic segregation in Drosophila melanogaster females

The induction of sex chromosomes meiotic nondisjunction (ND) by hydroquinone (HQ) given orally was investigated in Drosophila melanogaster 2-7, 8-22, 24, 48, 72 and 96 h-old females. ND was assessed by a system where exceptional females (XXY) and only 1/4 of the expected regular progeny are viable. Oocytes were treated at different stages of development. 4% HQ tested only in 72 h-old females induced ND in oocytes sampled in brood I (mostly mature oocytes at metaphase I). 6% HQ increased ND in brood I of 8-22 h-old females, while other broods, (including cells treated at early prophase) were also affected in older flies, the highest significance being attained in the 48 h-old series. Newly hatched females (2-7 h-old) were refractory to the treatment, though oocytes sampled in the first three subcultures are comparable to cells showing enhancement of ND in series run with older females. Toxicity of 2, 4 and 6% HQ increased with concentration and females' age: (a) 2% was not toxic; (b) 4% was toxic only to 72 h-old females; (c) 6% was increasingly toxic to females 24, 48 and 72 h-old. The results indicate that age plays a significant role on both chromosomal segregation and toxicity and suggest that in Drosophila HQ is metabolized to its reactive species. The lack of toxic and aneugenic effect in very young females could reflect a more efficient detoxification due to the known high specific activity of glutathione-S-transferase (GST) after eclosion. The decline in GST activity around day 2 of adult life coincides with the high effect of HQ in 48 h-old females.     

Validation of the human T-lymphocyte cloning assay--ring test report from the EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.     

Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.     

非人类物种的城市化:动物也是地球上的一员

提出在日益城市化进程中人类与非人类物种之间关系的4种推测。由于非人类物种也在快速城市化,它们的空间条件大部分都是由人类为自己所构建,这可以集中并强化拉图尔的生物共生理念。风景园林师应该理清自己工作的政治影响,而其中的一种方法是处理物种之间微妙而脆弱的相互关系。当然,采用未经验证的新自由主义设计在大多数情况下对非人类物种都会带来特殊的破坏性后果。本文使用“基础设施”模型探讨不同的生物群体之间如何相互影响彼此的命运。