Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.     

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Light-driven primary sodium ion transport in halobacterium halobium membranes

Light-induced sodium extrusion from H halobium cell envelope vesicles proceeds largely through an uncoupler-sensitive pathway involving bacteriorhodopsin and a proton/sodium antiporter. Vesicles from bacteriorhodopsin-negative strains also extrude sodium ions during illumination, but this transport is not sensitive to uncouplers and has been proposed to involve a light-energized primary sodium pump. Proton uptake in such vesicles is passive, and under steady-state illumination the large electrical potential (negative inside) is just balanced by a pH difference (acid inside), so that the protonmotive force is near zero. Action spectra indicated that this effect of illumination is attributable to a pigment absorbing near 585 nm (of 568 for bacteriorhodopsin). Bleaching of the vesicles by prolonged illumination with hydroxylamine results in inactivation of the transport; retinal addition causes partial return of the activity. Retinal addition also causes the appearance of an absorption peak at 588 nm, while the absorption of free retinal decreases. The 588 nm pigment is present in very small quantities (0.13 nmole/mg protein), and behaves differently from bacteriorhodopsin in a number of respects. Vesicles can be prepared from bacteriorhodopsin-containing H halobium strains in which primary transport for both protons and sodium can be observed. Both pumps appear to cause the outward transport of the cations. The observations indicate the existence of a second retinal protein, in addition to bacteriorhodopsin, in H halobium, which is associated with primary sodium translocation. The initial proton uptake normally observed during illumination of whole H halobium cells may therefore be a passive flux in response to the primary sodium extrusion.     

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

胰岛素不同给药方式对妊娠期糖尿病患者围手术期血糖控制的效果研究

目的:研究胰岛素不同给药方式对妊娠期糖尿病患者围手术期血糖控制的效果与安全性。方法:选择2013年11月～2016年11月于我院接受治疗的92例妊娠期糖尿病患者,所有患者按随机数字表法分为对照组和研究组,每组46例。对照组围手术期予以皮下注射胰岛素治疗,研究组围手术期予以胰岛素泵治疗。比较两组空腹血糖(FBG),餐后2 h血糖(2hPBG),血糖达标情况,抗生素使用情况,切口愈合情况、住院时间及并发症的发生情况。结果:治疗后12 h、24 h及36 h,两组FBG、2hPBG水平均较治疗前显著下降,且研究组以上指标均低于对照组(P0.05)。研究组血糖达标、抗生素使用、切口愈合及住院时间均明显短于对照组(P0.05)。两组均有低血糖、切口感染发生,研究组并发症发生率低于对照组(P0.05)。结论:胰岛素泵输注胰岛素对妊娠期糖尿病患者围手术血糖的控制效果明显优于皮下注射胰岛素,且其安全性更高。     

明胶/海藻酸钠/沙蒿胶复合水凝胶的制备及表征

为探究明胶（G）、海藻酸钠（SA）,沙蒿胶（ASKG）对复合水凝胶的力学性能、溶胀和保湿性能的影响,采用共混-离子交联法制备海藻酸钠/明胶/沙蒿胶复合水凝胶,并对制得的水凝胶进行结构表征和溶血率测试。结果表明:当G质量分数为2.5%,SA为1.5%,ASKG为0.7%时,复合水凝胶压缩强度达到427.2 kPa,拉伸强度达到563.449 kPa,断裂伸长率为117%,溶胀率为744%,且具有较好的保湿性能。红外光谱表明,由于沙蒿胶中存在大量羟基,因此加入沙蒿胶后在3 300 cm^-1～3 600 cm^-1羟基峰形变宽。G/SA/ASKG复合水凝胶溶血率低于5%,具有较好的网络孔结构和血液相容性,为复合水凝胶在医用敷料方面的应用提供一定的参考价值。