Membrane protein analysis of human breast cancer cell line MCF-7 by different membrane washing methods

Membrane and membrane-associated proteins are rich in known or potential pharmaceutical drug targets for carcinogenesis. In order to systemically analyze membrane proteins of human breast cancer, we isolated membrane from MCF-7 cells by sequential extraction by washing with three different buffers, namely, phosphate buffer (5 mM, pH 8.0), Tris (40 mM, pH 9.5), and sodium carbonate (100 mM pH 11). The extracted proteins were separated by two-dimensional gel electrophoresis (2-DE) using cup-loading and were then analyzed by peptide mass fingerprinting (PMF). A total of 137 spots from the gels of the three procedures were successfully identified. They corresponded to 79 distinct proteins. Among them, 22 exclusive proteins belonging to each washing procedure were also found, including P-glycoprotein, endoplasmin, Stress-70 protein, ADAM 10, protein disulfide isomerase, and glutamate receptor. These results indicate phosphate buffer to be the most beneficial for enrichment of peripheral membrane proteins, and sodium carbonate is beneficial for the presentation of integral membrane proteins but usually with poor resolution. The reference maps and identified proteins will serve as a basis for the further investigation of breast cancer, especially the proteomic comparison among different cell types of breast cancer, or among the different stages in the drug interfering process of the MCF-7 cell line.     

羊栖菜的药用功能研究现状

对经济海藻羊栖菜的药用功能进行综述。认为其功能主要是抗癌、提高机体免疫能力和降血脂、降血压、防治心血管疾病等。     

N-Bromosuccinimide modification of tryptophan 241 at the C-terminus of the manganese stabilizing protein of plant photosystem II influences its structure and function

Life on earth depends upon the ability of oxygenic photosynthesis to oxidize water to molecular oxygen. This process is catalyzed by water–plastoquinone oxido-reductase complex. In addition to the photosystem II (PSII) reaction core, it includes a manganese stabilizing protein (MSP) that plays an important regulatory role in the process in plants and algae. Tryptophan 241, located at the carboxyl-terminus of the MSP, is its sole tryptophan. Modification of MSP by N-bromosuccinimide (NBS) was carried out to explore the role of Trp241 in maintaining its structure and function. Data and arguments are presented to show that it is Trp241, not other tyrosines in MSP, that is involved in the modification and changes observed in this study. Further, the pH-dependence of the modification and the comparison of features of fluorescence spectra of MSP suggested that Trp241 is buried in the hydrophobic interior of the protein. Hydropathy analysis revealed that Trp241 is located in the middle of the hydrophobic region at the C-terminus of MSP. Circular dichroism spectroscopy showed that NBS modification of Trp241 dramatically modified the protein structure. The affinity of MSP to PSII decreased greatly after the modification of Trp241, and no oxygen-evolving activity was recovered after its reconstitution. This study provides a novel demonstration that Trp241 at the C-terminus hydrophobic region of the MSP is critical for maintaining appropriate structure and function of MSP.     

Stabilizing mutations and calcium-dependent stability of subtilisin

Stability is a property of subtilisin which has proven particularly amenable to enhancement via random mutagenesis and screening, yet the effects of most stabilizing mutations are not understood in structural and energetic detail. This paper seeks to explain the longstanding observation that stabilizing mutations are usually calcium-dependent in their stabilizing effect, irrespective of their proximity to the calcium binding sites. Stabilizing mutations in subtilisin fall into one of three classes. The largest class of mutations stabilize only in the presence of excess calcium. A smaller number of mutations stabilize independently of [calcium], and a few mutations stabilize only in the presence of chelating agents, such as EDTA. This study compares the effects of mutations from each class when introduced into subtilisin BPN' and two calcium-free versions of subtilisin. The calcium-dependent effects of mutations can be explained by considering subtilisin to be in conformational equilibrium between two structurally similar but energetically distinct states: N and N*. The equilibrium from the N* to the N state can be altered either by calcium binding to site A or by mutation. Mutations which stabilize only in the presence of calcium stabilize the N state relative to N*. Mutations which stabilize only in the presence of chelants stabilize the N* state relative to N. As a byproduct of this analysis, we have developed a hyperstable variant of subtilisin whose inactivation at high temperature in the presence of EDTA is 10(5) times slower than wild-type subtilisin.     

Regulation of insulin sensitivity by adipose tissue-derived hormones and inflammatory cytokines

PURPOSE OF REVIEW: The aim of this review is to assess the role of adipose tissue-derived hormones and inflammatory cytokines in the pathogenesis of obesity-linked type II diabetes, with a special focus on articles published between December 2002 and December 2003. RECENT FINDINGS: Insulin resistance is widely recognized as a fundamental defect seen in obesity and type II diabetes. Although the molecular mechanisms triggering the development of insulin resistance remain elusive, recent studies have suggested that adipose tissue and adipose tissue-derived hormones and inflammatory cytokines play essential roles in the overall insulin sensitivity in vivo. Dysfunctions of adipose tissue can lead to systemic insulin resistance. SUMMARY: Understanding the regulation of the metabolic and secretory functions of adipose tissue, as well as its subsequent impact on overall insulin sensitivity, is becoming increasingly important given the therapeutic potential of targeting the root causes of insulin resistance in the treatment of type 2 diabetes and its associated complications, such as cardiovascular and cerebrovascular diseases.     

A one pot synthesis of mono- and di-lactosyl sphingosines

The importance of analogues of lactosyl ceramides as basic structures of many natural glycosphingolipids provided a rationale for developing an efficient synthetic route to these compounds. We report herein a novel approach to synthesize several members of this family. Glycosylation of N-diphenylmethylene-spingosine, which exists in an imine–oxazolidine tautomeric mixture, with acetobromolactose under a modified Koenigs-Knorr condition yielded lactosyl -(1 1) sphingosine, lactosyl -(1 3) sphingosine and dilactosyl sphingosine in good yields. A similar glycosylation could be applicable to the synthesis of other glycosphingolipids.     

Self assembly of the transmembrane domain promotes signal transduction through the erythropoietin receptor

Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor-like conformation precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR), the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240-Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR(-/-) mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.