Specific Degradation of D1 Protein during Exposure of Thylakoid Membranes to High Temperature in the Dark

Exposure of thylakoid membranes to high temperature in dark leads to the degradation of D1 protein. Maximum degradation of D1 protein occurred at 45 °C. Using N-terminal specific D1 antibody, a 23 kDa fragment of D1 protein was detected. The degradation of D1 protein could be prevented both by radical scavengers and inhibitors of serine protease and metallo-protease. These results suggest that degradation of D1 protein during exposure of thylakoid membranes to high temperature in dark is catalyzed by protease. This revised version was published online in June 2006 with corrections to the Cover Date.     

交变脉冲电场凝胶电泳与植物大分子DNA的制备

介绍了交变脉冲电场凝胶电泳的原理、方法及其在植物大分子ＤＮＡ制备方面的应用     

毛细管电泳及其在蛋白质折叠研究中的应用

简要介绍了毛细管电泳的常用分离模式及其原理,并对毛细管电泳在蛋白质化学领域中的新应用——研究蛋白质折叠和发展前景作了评述。     

Evidence of Actin in the Cytoskeleton of Microsporidia

Using transmission electron microscopy, immuno-electron microscopy, and biochemical techniques such as 2-D electrophoresis and immunoblotting, actin was found in all biological stages of the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi.     

Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis

Forty-three Listeria ivanovii isolates were collected in the UK between 1991 and 1997 from: 35 animal infections; two human infections; five foods; and one environmental source. A further two type strains of L. ivanovii (subsp. ivanovii and subsp. londoniensis) were obtained from a culture collection. These bacteria were characterised by conventional phenotypic methods and by pulsed-field gel electrophoresis (PFGE) using ApaI and SmaI. Forty-two of the isolates from the UK were identified as L. ivanovii subsp. ivanovii and the remaining culture as L. ivanovii subsp. londoniensis. Six and four PFGE profiles were obtained using ApaI and SmaI digestion respectively; six composite profiles were obtained combining the results for both enzymes. The PFGE profile of the UK L. ivanovii subsp. londoniensis (isolated from processed shrimps) was similar to the type strain of this subspecies and differed from all of the L. ivanovii subsp. ivanovii tested. The majority of isolates (38 out of 45) belonged to one profile showing that the UK population of this bacterium is much less genetically diverse than similar studies have shown for Listeria monocytogenes.     

The analysis of oligonucleotide preparations by fractal measures

In this paper we put forward improved mathematical methods for detecting synthesis parameters in connection with analyzing crude products of chemically synthesized oligonucleotides. The crude products experimentally sampled are separated by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography. The measured separation profiles of experimental syntheses can be expressed as target and nontarget yields; they are characterized by a few parameters. These parameters account for nonlinear synthesis equations that are solvable by employing iteration procedures. We provide here a theoretical as well as computational analysis based upon specific models for stepwise chain growth. Under nonconstant (nonuniform) conditions we use here an exponential form of growth, with different expressions for calculating the fractal dimension of the biochemical process under study. Step lengths of parameter variations in an interval of finite length have to be adjusted properly to find convergent solutions in a mathematical, regularly four-dimensional parameter space. It is conceivable to have most, if not all, of the calculating and plotting carefully done by a computer. This analysis represents the experimental situation up to 65-mer target oligonucleotides analyzed so far. We thus obtain the dynamics of the polymerization process limited in number by fractal models. The advantage, calculating these new methods as compared to qualitatively judged experimental methods, lies in the satisfactory evaluation of crude products, also of large amounts, of syntheses of these biopolymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 361–379, 1998     

Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method

Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources. J. Cell. Biochem. 71:363–374, 1998. © 1998 Wiley-Liss, Inc.     

Separation of enantiomers of drugs by capillary electrophoresis,part 7: Gamma-cyclodextrin as chiral solvating agent

Following an extended chiral drug screening program by capillary zone electrophoresis (CZE), the enantioseparation of 86 racemic drugs was tested with γ-cyclodextrin as a chiral solvating agent. Unified conditions were applied to all experiments. In total, 18 drug racemates were separated, 13 entries thereof that had not been separated at the lower CSA concentration applied in an earlier stage of the project. A comparison of the data with the results obtained for α- and β-cyclodextrin points to the significance of partial penetration (“side-on binding”) of aryl groups into the cyclodextrin cavity. Chirality 10:548–554, 1998. © 1998 Wiley-Liss, Inc.     

Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 μg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6–8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[³⁵S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes. Mol. Reprod. Dev. 46:401–407, 1997. © 1997 Wiley-Liss, Inc.