琼脂糖平板制备聚焦电泳法进一步提纯胸腺素组分五

用琼脂糖平板等电聚焦电泳法,由胸腺素组分五中分离出三种在聚焦电泳谱上是单一谱线的多肽成份——CP1、CP2和CP3,等电点分别为4.3、4.9和5.6。测定了这些多肽对脐带血中淋巴细胞形成羊红细胞玫瑰花的影响。与对照相比,CP1(2微克/0.6毫升),和CP3(0.2-2微克/毫升)分别在统计学上呈显著和非常显著差异。在相同测定条件下,这三种多肽成份的活性均高于化学合成的胸腺多肽——胸腺素α_1。     

Gc、Pi蛋白亚型的快速微量电泳分析

用快速微量等电聚焦技术对190名北京地区汉族健康人血清Gc蛋白亚型、Pi蛋白亚型进行分型鉴定和基因频率调查.上样量为1.5μl,电泳和染色各0.5h.Gc^1F=0.4891,Gc^1S=0.2432,Gc²=0.2678.观察值与期望值吻合良好.(∑X²=1.404,0.7<P<0.8).Pi^M₁=0.7542,Pi^M₂=0.1808,Pi^M₃=0.0650,观察值与期望值吻合也良好,(∑X²=1.1233,0.7<P<0.8).     

Observations of a set of isomeric anti-lactose antibodies directed against a group D streptococcal polysaccharide

Sets of isomeric anti-lactose antibodies with specificity for the lactose units of a cell wall polysaccharide fromStreptococcus faecalis strain N were induced in rabbits immunized with a vaccine of nonviable cells of the organism. Such sets of anti-lactose antibodies were isolated from the serum of immunized animals by affinity chromatography on lactosyl-Sepharose. Gel electrofocusing experiments showed that the preparations consisted of multiprotein components. One preparation of antibodies of 13 isomers was separated into homogeneous components by liquid isoelectrofocusing. The individual isomeric antibodies exhibit specificity for the lactose units of the antigenic polysaccharide, possess isoelectric points in the range of 5.9–8.0, and belong to the IgG class of immunoglobulins, and each member yields one light chain and one heavy chain on dissociation in sodium dodecyl sulfate (SDS) and mercaptoethanol. These results have been interpreted as evidence for the assembly of the chains of isomeric antibodies by a single-chain pairing mechanism.     

Effects of phosphinotricin treatment on glutamine synthetase isoforms in Scots pine seedlings

The occurrence of GS isoenzymes has been investigated in Scots pine (Pinus sylvestris) seedlings. A transient increase of glutamine synthetase (GS, EC 6.3.1.2) activity was observed in the cotyledon whorl of plants treated with the herbicide phosphinotricin (PPT). The increase in GS activity was accompanied by a parallel accumulation of GS1 protein, which remained at high levels throughout the PPT treatment. Two-dimensional SDS-PAGE western analysis showed that pine extracts contained two GS1 polypeptides which differ in their corresponding isoelectric points. Analysis of crude extracts by ion-exchange chromatography led to the separation of two GS isoforms. The first peak (GS1-a) eluted from the columns at a low ionic strength (0.15-0.18 M KCl), whereas the second one (GS1-b) was detected at 0.5 M KCl. A detailed molecular study of both GS holoenzymes confirmed that their subunits were similar in size (about 41 kDa) but different in charge. All these data clearly demonstrate the presence of two GS1 forms in Scots pine cotyledons. Moreover, a comparison of isolated GS isoproteins with the recombinantly expressed Scots pine cytosolic subunit suggests that GS1-a corresponds to the previously characterized cDNA (pGSP114) whereas GS1-b is a minor GS isoenzyme with increased relative abundance in phosphinotricin treated plants.     

DNA Packaging-Associated Hyper-Capsid Expansion of Bacteriophage T3

Evidence that in vivo bacteriophage T3 DNA packaging includes capsid hyper-expansion that is triggered by lengthening of incompletely packaged DNA (ipDNA) is presented here. This evidence includes observation that some of the longer ipDNAs in T3-infected cells are packaged in ipDNA-containing capsids with hyper-expanded outer shells (HE ipDNA-capsids). In addition, artificially induced hyper-expansion is observed for the outer shell of a DNA-free capsid. Detection and characterization of HE ipDNA-capsids are based on two-dimensional, non-denaturing agarose gel electrophoresis, followed by structure determination with electron microscopy and protein identification with SDS-PAGE/mass spectrometry. After expulsion from HE ipDNA-capsids, ipDNA forms sharp bands during gel electrophoresis. The following hypotheses are presented: (1) T3 has evolved feedback-initiated, ATP-driven capsid contraction/hyper-expansion cycles that accelerate DNA packaging when packaging is slowed by increase in the packaging-resisting force of the ipDNA and (2) each gel electrophoretic ipDNA band reflects a contraction/hyper-expansion cycle.     

Analysis of cyclic nucleotide phosphodiesterase by isoelectric focusing coupled to a specific activity stain

The procedure described allowed a convenient analysis of cyclic nucleotide phosphodiesterase. The different phosphodiesterase forms present in a crude cytosolic preparation from rat heart were separated by isoelectric focusing on a polyacrylamide gel plate. Phosphodiesterase activity bands were rendered evident by a specific staining method. They were then characterized by means of their substrate specificity and their sensitivity to selective phosphodiesterase inhibitors. The correspondence between the stain bands and the previously described activity peaks, obtained by a preparative technique and detected by radioisotopic enzyme assay, was also investigated.     

Consensus Brain-derived Protein,Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.     

Flow cytometry analysis of DNA ploidy levels and protein profiles distinguish between populations of Lumbriculus (Annelida: Clitellata)

Variations in DNA ploidy have been observed in Lumbriculus, a freshwater annelid, as well as in other clitellates. Interpretation and application of experimental results using these animals may be impacted as ploidy levels affect the protein expression, reproductive behavior, and response to stressors. Ploidy is typically determined by chromosome spreads, a time‐consuming and inefficient method. We adapted flow cytometry protocols used on vertebrates and plants to determine the ploidy levels in different populations of Lumbriculus, including a laboratory strain (Environmental Protection Agency), a commercial strain (Aquatic Foods), and worms collected from natural habitats. To isolate nuclei, worms were homogenized, filtered to remove cell debris, and centrifuged through Optiprep? density gradients. Nuclei were recovered, treated with RNAse, and stained with propidium iodide. Flow cytometry of the labeled nuclei showed that Lumbriculus from natural habitats in Minnesota and Iowa were diploid, with an estimated genome size of 2.7 pg. Populations from natural habitats in California and Oregon were highly polyploid, as were the laboratory and commercial strains. Chromosome spreads verified the high ploidy levels indicated by flow cytometry results, but also suggested that flow cytometry may be underestimating the DNA content levels. Staining of nuclei with diamidino‐2‐phenylindole indicated that this may be due to high levels of heterochromatin in nuclei from polyploid forms of Lumbriculus. To further compare the populations, proteins in worm homogenates were subjected to isoelectrofocusing gel electrophoresis. Distinct protein profiles were seen; one was shared in common by the diploid worms, the other was characteristic of polyploid populations. Diploid worms could also be distinguished from polyploid worms based on differences in hemoglobin linker proteins. The results further support taxonomic classification of the diploid and polyploid forms of Lumbriculus as distinct species.     

Vibrational spectra and structure of myelin membranes

Raman and infrared spectroscopy have been simultaneously applied, for the first time, to the study of myelin membranes and their proteolipid protein (PLP) so as to obtain information on the secondary structure of proteins and the ordering of lipid chains. The vibrational spectra were recorded at physiological pH using a non-denaturing detergent (n-octyl--d-glucopyranoside) in phosphate buffer. Neither the buffer nor the detergent interfere spectroscopically with the amide bands from proteins. The spectra reveal that the predominant secondary structure in the polypeptide backbone in myelin is the helix. The proteolipid protein was found to be more disordered than the polypeptide arrangement of the myelin membrane, as deduced from the relative intensities and halfwidths of characteristic infrared amide I bands. -form and turns are also present, the amount of these structures being higher in PLP. The study of the Raman spectra of vC-C and vC-H regions made it possible to obtain information on the lipid chain order.     

Conformation of brain proteolipid apoprotein

The conformation of brain proteolipid apoprotein (PLA) has been investigated using infrared spectroscopy and freeze-fracture electron microscopy. For this purpose, spectroscopic samples consisting of a mixture of liquid paraffin and wet protein have been prepared. These systems have allowed us to record the infrared spectra of PLA at neutral pH. The amide I and III regions reveal the existence of a predominantly -helical structure, as well as the presence of minor -strands and random coil forms. The effect of sonication and a non-denaturing detergent, (n-octyl--d-glucopyranoside), on the structure of the protein have also been investigated. Sonication produces an increase of the and unordered structures at the expense of the -helical conformation. These structural changes are enhanced in the presence of the non-ionic detergent n-octyl--d-glucopyranoside. Lipids protect the native protein structure from the effects of sonication. The aforementioned detergent changes the PLA conformation by increasing the -helical content at the expense of -sheet and random coil forms. Therefore the PLA structure seems to be similar to the structures of other proteins intrinsic to non-neural membranes. The effects investigated also suggest that PLA behaves in a conformationally flexible manner.