Speciation of tissue and cellular iron with on-line detection by inductively coupled plasma-mass spectrometry.

Iron accumulating to excess in tissues of humans and animal models occurs mainly as complexes with transferrin, ferritin, other hemoproteins, and insoluble hemosiderin particles. To determine the distribution of Fe amongst these molecular species, we have used inductively coupled plasma-mass spectrometry as a means of on-line, isotope-specific detection for their liquid chromatographic separation. The stable isotope 57Fe is a suitable isotope for monitoring the Fe content of each fraction, and its availability at high isotopic enrichment makes it an attractive choice for tracer studies when the use of a radioisotope is undesirable, e.g., in human subjects. The detection system offers the advantages of high sensitivity (detection limits in the parts per billion range), a wide dynamic range (linearity of the calibration curve over several orders of magnitude), and on-line analysis facilitating real-time evaluation of the chromatographic separation, in addition to isotope-specific information. The Fe distributions in healthy rat livers, liver and heart tissue from Fe-loaded human subjects, and human hepatocyte cultures are reported. The ferritin:hemosiderin ratio in these samples is shown to be an indicator of the degree of Fe loading and correlates well with that determined by Zeeman-corrected electrothermal atomic absorption as an alternative means of detection.     

Immunodetection after complete destaining of coomassie blue-stained proteins on immobilon-PVDF.

A technique that simplifies the localization of an immunodetectable protein in relation to the other electrophoresed proteins is described. Proteins are transblotted onto a polyvinylidene difluoride (PVDF) membrane and visualized by staining with Coomassie brilliant blue R-250, and a photograph of the protein pattern is taken. The Coomassie blue-stained PVDF membrane is then completely destained using a 25% acetic acid/50% methanol solution that allows subsequent immunostaining on the same membrane. The technique uses common laboratory reagents, is rapid, and has been shown to be applicable for a variety of proteins using both monoclonal and polyclonal antibodies and a variety of transblots.     

Crystal structure of recombinant murine adipocyte lipid-binding protein.

Adipocyte lipid-binding protein (ALBP) is the adipocyte member of an intracellular hydrophobic ligand-binding protein family. ALBP is phosphorylated by the insulin receptor kinase upon insulin stimulation. The crystal structure of recombinant murine ALBP has been determined and refined to 2.5 A. The final R factor for the model is 0.18 with good canonical properties. Crystalline ALBP has a conformation which is essentially identical to that of intestinal fatty acid binding protein and myelin P2 protein. Although the crystal structure is of the apo- form, a cavity resembling that in other family members is present. It contains a number of bound and implied unbound water molecules and shows no large obvious portal to the external milieu. The cavity of ALBP, which by homology is the ligand-binding site, is formed by both polar and hydrophobic residues among which is tyrosine 19. Y19 is phosphorylated by the insulin receptor kinase as described in the accompanying paper [Buelt, M. K., Xu, Z., Banaszak, L. J., & Bernlohr, D. A. (1992) Biochemistry (following paper in this issue)]. By comparing ALBP with the earlier structural results on intestinal fatty acid binding protein, it is now possible to delineate conserved amino acids which help form the binding site in this family.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

兴安落叶松结实规律与长短枝习性的关系

1987年5月,大兴安岭林区发生的特大森林火灾,实属世界罕见,火灾面积达1.0×10~6ha 多。大量的火烧迹地亟待更新、无论是人工更新还是人工促进天然更新,其中关键的问题之一是种子的来源,在大兴安岭地区,兴     

西双版纳热带季节雨林植物种类多样性的一种研究方法

一、方法1.样地的选择样地分别选取热带干性季节雨林的典型代表——以箭毒木(Antiaris toxicaria)、龙果(Pouteria grandifolia)为标志的群落,以千果榄仁(Terminalia myriocarpa)、番龙眼     

多聚酶链反应技术鉴别肾综合征出血热病毒血清型的初步研究

肾综合征出血热(HFRS)为一组抗原性密切相关的布尼亚科汉坦病毒引起的急性传染病。在我国存在至少两种临床表现、动物宿主及流行特征截然不同的血清型别,即血清Ⅰ型(汉坦型)和血清Ⅱ型(汉城型)。这两型病毒间的血清学定型已有报道。近年来,除啮齿类动物外,从临床病人以及非啮齿类动物体内也分离到了HFRS病毒。同时出现两类型别毒株共存,以及从家鼠体内分离到野鼠型毒株或从野鼠体内分离到家鼠型毒株的复杂情形。为此,准确检定并鉴别不同来源毒株型别,将为深入了解其病原学、流行病学以及制定疫苗生产策略提供重要信息。     

用高碘酸盐活化葡聚糖凝胶制备亲和吸附剂的研究

 <正> 用高碘酸盐活化的Sephadex与鸡卵类粘蛋白偶合制备分离纯化胰蛋白酶的亲和吸附剂。该方法比CNBr活化Sepharose 4B制备亲和吸附剂的方法具有操作安全,价格低廉等优点。活化载体在4℃保存较长时间不失去键合能力。该亲和吸附剂可制备得比活力为11228.8u/mgBAEE单位电泳单带纯胰蛋白酶,并能反复使用十余次,仍具有较强亲和吸附能力。酶     

Expression of mammalian liver glycolytic/ gluconeogenic enzymes in Escherichia coli: Recovery of active enzyme is strain and temperature dependent

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs

The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5′-CACACCCA and (b) 5′-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA genes of the donkey and the horse, Equus caballus, were compared in detail. Total nucleotide difference outside the control region was 6.9%. Nucleotide difference between peptide-coding genes ranged from 6.4% to 9.4% with a mean of 8.0%. In the inferred protein sequences of the 13 peptide-coding genes the amino acid difference was 0.2–8.8%, and the mean for the 13 concatenated amino acid sequences was 1.9%. In the 22 tRNA genes, the mean difference was 3.5%, and that in the two rRNA genes was 4.1%. The mtDNA differences between the donkey and the horse suggest that the evolutionary separation of the two species occurred ≈9 million years ago. Analyses of differences among the mtDNAs of three other species-pairs, harbor seal/grey seal, fin whale/blue whale, and Homo/common chimpanzee, showed that the relative evolutionary rate of individual peptide-coding genes varies among different species-pairs and modes of comparison. The findings show that the superimposition of sequence data of one lineage for resolving and dating evolutionary divergences of other lineages should be performed with caution unless based on comprehensive data. Received: 15 October 1995 / Accepted: 15 April 1996