Yeast differentiation using histone promoter sequences

AIMS: To evaluate a new platform for yeast differentiation based on histone promoter regions. METHODS AND RESULTS: The histone gene amino acid sequences of a wide phylogenetic range of organisms were aligned, and primers designed that were capable of amplifying the divergent promoters of the H3-H4 and H2a-H2b loci from yeast. Analysis indicated that the promoter regions were variable in length between species and represented rapidly changing sequences flanked by highly conserved sequences. CONCLUSIONS: The histone promoter regions in yeast provide an excellent locus for the rapid and accurate identification of yeast species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes an alternative platform to the ribosomal internal transcribed spacer (ITS) sequences for the identification of yeast species.     

Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology

Recombinant soluble human complement receptor type 1 (sCR1) is a highly glycosylated glycoprotein intended for use as a drug to treat ischemia-reperfusion injury and other complement-mediated diseases and injuries. sCR1-sLe(x) produced in the FT-VI-expressing mutant CHO cell line LEC11 exists as a heterogeneous mixture of glycoforms, a fraction of which include structures with one or more antennae terminated by the sialyl Lewis X (sLe(x)) [Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc]) epitope. Such multivalent presentation of sLe(x) was shown previously to effectively target sCR1 to activated endothelial cells expressing E-selectin. Here, we describe the use of the soluble, recombinant alpha2-3 sialyltransferase ST3Gal-III and the alpha1-3 fucosyltransferase FT-VI in vitro to introduce sLe(x) moieties onto the N-glycan chains of sCR1 overexpressed in standard CHO cell lines. The product (sCR1-S/F) of these in vitro enzymatic glycan remodeling reactions performed at the 10-g scale has approximately 14 N-glycan chains per sCR1 molecule, comprised of biantennary (90%), triantennary (8.5%), and tetraantennary (1.5%) structures, nearly all of whose antennae terminate with sLe(x) moieties. sCR1-S/F retained complement inhibitory activity and, in comparison with sCR1-sLe(x) produced in the LEC11 cell line, contained twice the number of sLe(x) moieties per mole glycoprotein, exhibited a twofold increase in area under the intravenous clearance curve in a rat pharmacokinetic model, and exhibited a 10-fold increase in affinity for E-selectin in an in vitro binding assay. These results demonstrate that in vitro glycosylation of the sCR1 drug product reduces heterogeneity of the glycan profile, improves pharmacokinetics, and enhances carbohydrate-mediated binding to E-selectin.     

Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

PICKLE acts during germination to repress expression of embryonic traits

PICKLE (PKL) codes for a CHD3 chromatin remodeling factor that plays multiple roles in Arabidopsis growth and development. Previous analysis of the expression of genes that exhibit PKL-dependent regulation suggested that PKL acts during germination to repress expression of embryonic traits. In this study, we examined the expression of PKL protein to investigate when and where PKL acts to regulate development. A PKL:eGFP translational fusion is preferentially localized in the nucleus of cells, consistent with the proposed role for PKL as a chromatin remodeling factor. A steroid-inducible version of PKL [a fusion of PKL to the glucocorticoid receptor (PKL:GR)] was used to examine when PKL acts to repress expression of embryonic traits. We found that activation of PKL:GR during germination was sufficient to repress expression of embryonic traits in the primary roots of pkl seedlings, whereas activation of PKL:GR after germination had little effect. In contrast, we observed that PKL is required continuously after germination to repress expression of PHERES1, a type I MADS box gene that is normally expressed during early embryogenesis in wild-type plants. Thus, PKL acts at multiple points during development to regulate patterns of gene expression in Arabidopsis.     

Remodeling protein complexes: insights from the AAA+ unfoldase ClpX and Mu transposase

Multiprotein complexes in the cell are dynamic entities that are constantly undergoing changes in subunit composition and conformation to carry out their functions. The protein-DNA complex that promotes recombination of the bacteriophage Mu is a prime example of a complex that must undergo specific changes to carry out its function. The Clp/Hsp100 family of AAA+ ATPases plays a critical role in mediating such changes. The Clp/Hsp100 unfolding enzymes have been extensively studied for the roles they play in protein degradation. However, degradation is not the only fate for proteins that come in contact with the ATP-dependent unfolding enzymes. The Clp/Hsp100 enzymes induce structural changes in their substrates. These structural changes, which we refer to as "remodeling", ultimately change the biological activity of the substrate. These biological changes include activation, inactivation (not associated with degradation), and relocation within the cell. Analysis of the interaction between Escherichia coli ClpX unfoldase and the Mu recombination complex, has provided molecular insight into the mechanisms of protein remodeling. We discuss the key mechanistic features of the remodeling reactions promoted by ClpX and possible implications of these findings for other biological reactions.     

A role for caveolin-1 in post-injury reactive neuronal plasticity

Remodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin-1 is a cholesterol-binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up-regulation of caveolin-1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin-1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin-1-overexpressing cells revealed increases in synaptophysin and GAP-43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin-1-overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin-1-overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin-1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin-1 plays an active role in the regulation of injury-induced synaptic and terminal remodeling in the adult CNS.     

Identification of novel electroconvulsive shock-induced and activity-dependent genes in the rat brain

Electroconvulsive shock (ECS) has been used as an effective treatment for patients suffering from major depression disorders and schizophrenia. However, the exact mechanisms underlying the action of ECS are poorly understood. Using high-density oligonucleotide microarrays, we identified 60 ECS-induced genes whose gene products are involved in the neuronal signaling, neuritogenesis and tissue remodeling. In situ hybridization and depolarization-dependent expression assay were performed to characterize 4 genes (lysyl oxidase, Ab1-046, SOX11, and T-type calcium channel 1G subunit) which have not yet been reported to be induced by ECS. Interestingly, the induction of these genes was observed mainly in the dentate gyrus of hippocampal formation and piriform cortex, where ECS-induced neural activation is highlighted, and depolarization of cultured cortical neurons also induced the expression of these genes. Taken together, our results suggest that therapeutic actions of ECS may be manifested by the activity-dependent induction of genes related to the plastic changes of the brain such as neuronal signaling neuritogenesis, and tissue remodeling.     

骨桥蛋白增强自发性高血压大鼠血管外膜成纤维细胞的迁移活性

血管外膜成纤维细胞迁移参与形成新生内膜是一些血管疾病的共同发病过程。研究高血压动物模型的外膜成纤维细胞是否与对照组不同将有利于阐述高血压血管重塑的机制。本实验比较自发性高血压大鼠(spontaneously hy-pertensive rats,SHR)与正常对照大鼠(Wistar-Kyoto rats,WKY)的血管外膜成纤维细胞在体外培养条件下迁移能力的差别,并对其机制进行了探讨。采用大鼠胸主动脉的培养血管外膜成纤维细胞,用Transwell技术测定培养细胞的迁移能力。用实时定量PCR技术检测mRNA表达。结果表明,在血清和bFGF趋化作用下,SHR培养血管外膜成纤维细胞的迁移活性显著强于WKY(每个视野平均迁移细胞数目,血清:35.20±5.26 vs 22.2±3.27,P<0.05;bFGF:30.23±4.54vs 19.20±4.47,P<0.05)。进一步研究发现,SHR培养血管外膜成纤维细胞中的骨桥蛋白(osteopontin,OPN)mRNA水平显著高于WKY(1863.23±43.91 vs 326.24±68.29,P<0.01)。反义OPN(100 μmol/L)对血清诱导的SHR血管外膜成纤维细胞迁移有抑制作用(每个视野平均迁移细胞数目 38.60±5.98 vs 26.61±3.84,P<0.05)。而正义及错配义OPN组均无此效应。反义OPN对SHR细胞迁移的抑制作用呈浓度依赖性。上述结果证实SHR培养血管外膜成纤维细胞的迁移能力强于WKY,OPN在细胞迁移中     

NAP-2 is part of multi-protein complexes in HeLa cells

We previously reported that a complex of nuclear proteins from HeLa cells, among them histone H1 and casein kinase 2 co-eluted from immobilized nucleosome assembly protein 2 (NAP-2)-Sepharose. Here, using HeLa cell nuclear extracts, we found NAP-2 migrates in a blue-native polyacrylamide gel with an apparent molecular weight of 300 kDa. HeLa cell NAP-2, labeled in vivo with radioactive orthophosphate, co-precipitated with at least two phosphoproteins, with an apparent mass of 100 and 175 kDa, respectively, as determined by SDS-PAGE. NAP-2 from total HeLa cell extract co-purified with other proteins through two sequential chromatographic steps: first, a positively charged resin, Q-Sepharose, was used, which purified NAP-2 more easily with other proteins that eluted as a single peak at 0.5 M NaCl. This fraction possessed both relaxing and supercoiling activities, and it was able to assemble regularly spaced nucleosomes onto naked DNA in an ATP-dependent manner. Second, a negatively charged resin (heparin) was used, which retained small amounts of NAP-2 (a very acidic polypeptide) and topoisomerase I. This fraction, although able to supercoil relaxed DNA, did so to a lesser extent than the Q-Sepharose fraction. The data suggest that NAP-2 is in complex(es) with other proteins, which are distinct from histones.