Comparative Histochemical and Biochemical Analysis of Endogenous Receptors for Glycoproteins in Human and Pig Peripheral Nerve

Endogenous sugar-binding proteins were localized in sections of human and pig peripheral nerves by the application of two types of labelled ligands: neoglycoproteins (chemically glycosylated carrier proteins that had proven to be histochemically inert) and desialylated, naturally occurring glycoproteins. These proteins allowed evaluation of the presence and distribution of endogenous receptors for carbohydrates, commonly present in cellular glycoconjugates. (Neo)glycoprotein binding was similar, but not identical, for the two types of mammalian peripheral nerves. The pig nerve differed from the human nerve in more pronounced staining when using different types of beta-galactoside-terminated (neo)glycoproteins and charge-carrying neoglycoproteins, such as bovine serum albumin, bearing galactose-6-phosphate residues, glucuronic acid residues, and sialic acid residues. Comparative biochemical analysis of certain classes of sugar receptors by affinity chromatography and gel electrophoresis revealed the presence of sugar receptors that can contribute to the histochemical staining in a pattern with certain significant differences among rather similar expression for the two species. The assessment of sugar receptor distribution by application of (neo)glycoprotein binding among morphologically defined regions in nerves may hold promise in detecting developmental regulation and changes during nerve degeneration and subsequent regeneration after trauma or pathological states. Correlation of these results to changes in the structure and abundance of glycoconjugates, which are the potential physiological ligands of endogenous sugar receptors commonly detected by plant lectins, may help to infer functional relationships.     

Expression of proteasomal proteins in ten different tumor cell lines

Summary. Controlled intracellular protein degradation is crucial for the maintenance of normal cell functions. An evolving concept claims that alterations in the exact timely degradation of proteins involved in growth control, apoptosis, signaling and differentiation contribute to carcinogenesis. This tightly regulated process is facilitated by the ubiquitin-26S proteasome system, a multi-enzyme complex, and inhibitors of this pathway have already been developed as potential anticancer agents.In order to generate proteasomal protein expression patterns of tumor cells and to provide an analytical tool we applied two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MALDI-TOF-TOF with LIFT technology) in ten individual tumor cell lines (Saos-2; SK-N-SH; HCT-116; Caov3; A-549; HL60; A-673; A-375; MCF-7; HeLa) widely used in tumor research. A series of 39 proteasomal/proteolytic proteins was unambiguously identified by this proteomic approach, comprising proteins of the 20S core complex, the 19S regulatory complex, the 11S regulator, components of the ubiquitin pathway and proteases.Construction of individual protein maps by 2-DE and mass spectrometry provides an analytical tool and reference base for studying the pivotal importance of the proteasome and other proteolytic enzymes in tumor cells, independent of antibody availability and specificity. This preliminary database enables for designing studies in this area of research and reveals proteins that can be used as targets for new therapeutic strategies.     

Characterization of glycosylation and adherent properties of melanoma cell lines

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins—fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases—α2,3(Galβ1,3)- and α2,3(Galβ1,4)-sialyltransferases, α1,2- and α1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing α-2,6- and α-2,3-linked sialic acid. The average number of polypeptides bearing β-1,6-branched tri- and tetra antennary glycans(characteristic of the metastatic phenotype)were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module

For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl.     

Cellular thiols in rat liver cell lines possessing different growth characteristics

Thiol levels were measured in three cell lines derived from rat hepatocytes with different growth rates and degrees of tumorigenicity: IAR20 having normal epithelial morphology and no tumour forming ability; IAR6.1 being a chemically-transformed malignant cell line; and IAR6.1RT7 derived from an epithelial tumour obtained after injection of IAR6.1 cells into a syngenic animal. The mean levels of GSH, GSSG, low molecular weight thiols (LMWT), macromolecular thiols (MT) and total reactive protein sulphur (TRPS), expressed as nmoles-SH mg-1 protein, were found to be 25.5, 7.5, 50.1, 114.5 and 143.6 respectively for IAR20; 37.6, 3.9, 65.4, 126.8 and 148.4 for IAR6.1; 17.2, 4.4, 52.3, 141.0 and 168.2 for IAR6.1RT7. Cultures were treated with D,L-buthionine-S,R-sulphoximine (BSO) to cause greater than 70 per cent depletion of GSH and the measurements of cellular thiols repeated. Although treatment with BSO caused a substantive decrease in the LMWT fraction, there were no major changes in macromolecular thiols or in total reactive protein sulphur. The respective mean values for LMWT, MT and TRPS (expressed as nmoles-SH mg-1 protein) were 19.4, 109.8, 136.3 for IAR20; 17.2, 119.3, 143.6 for IAR6.1; 21.6, 150.7 and 163.5 for IAR6.1RT7. It is concluded that significant differences in thiol levels exist between the three rat liver cell lines studied. However, severe acute depletion of GSH is not reflected by changes in the levels of macromolecular thiols which suggests that there is only a slow equilibrium between these two major thiol pools.     

与PRRSV nsp11互作的宿主细胞蛋白鉴定及生物信息学分析

【目的】研究猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)nsp11与宿主细胞蛋白之间的相互作用,对于揭示nsp11在病毒复制过程中发挥的功能至关重要。【方法】在病毒感染细胞的基础上,利用nsp11的单克隆抗体,采用免疫沉淀结合串联质谱的方法,筛选与PRRSV nsp11相互作用的宿主细胞蛋白,并对所筛选出的宿主细胞蛋白进行了GO注释、COG注释和KEGG代谢通路注释;选取筛选出的宿主细胞蛋白IRAK1,利用免疫共沉淀技术和激光共聚焦技术鉴定其与nsp11之间的相互作用。【结果】与空白对照组相比,病毒感染组中出现3条差异带;经质谱分析共筛选得到了201个与nsp11相互作用的宿主细胞蛋白,分别与蛋白质代谢、细胞信号通路转导以及病原致病性等密切相关;在生物信息学分析的基础上,实验验证了nsp11确与宿主细胞蛋白IRAK1进行相互作用。【结论】鉴定出与PRRSV nsp11相互作用的宿主细胞蛋白,生物信息学分析显示它们在病毒的复制和致病过程中发挥重要作用。研究结果为探究nsp11的生物学功能指明了方向,也为研究宿主细胞蛋白与病毒蛋白间的相互作用及其调控病毒复制和致病性的分子机制奠定了基础。     

EMT参与肿瘤侵袭转移的研究进展

在癌症类型中,上皮癌占绝大多数。从良性腺瘤过渡到恶性癌和转移期间,上皮肿瘤细胞获得去分化、迁移和入侵行为,同时上皮-间质转化(epithelial-mesenchymal transition EMT)伴随着显著的细胞形态学变化、细胞与细胞间及细胞与基质之间的粘附性丢失及重塑、并获得迁徙和侵袭能力。正如完全分化的上皮细胞转换成低分化、迁移和侵入性间质细胞,其涉及到一个高度的细胞可塑性、大量不同的基因和表观遗传学改变,因此EMT本身是一个多阶段的过程。该综述的目的是系统地总结EMT分子机制及EMT与肿瘤关系的最新进展。     

Variation of proline rich cell wall proteins in soybean lines with anthocyanin mutations

The I locus controls inhibition of anthocyanin accumulation in the epidermal cells of the soybean seed coat and affects abundance of PRP1, a proline-rich cell wall protein in the seed coat. Saline-soluble PRP1 is abundant in the developing seed coats of cultivar Richland (homozygous I, yellow), while it is significantly decreased in the pigmented isogenic mutant T157 (homozygous i, imperfect black). In this report, we examined soluble PRP1 in several cultivars containing alleles of the I locus which affect spatial distribution of pigmentation in the seed coat. We also characterized PRP1 in isolines with allelic variants of several other loci involved in seed coat pigmentation, including T and Im. The T gene is pleiotropic and affects both pubescence color and seed coat pigmentation and structure. Soluble PRP1 was abundant in the developing seed coats of lines with yellow seed (I or i ⁱ alleles) regardless of pubescence color, just as in Richland. Likewise, soluble PRP1 was decreased in pigmented seed coats (i ^k or i alleles) with grey (t) pubescence, as in T157. However, the total seed coat proteins were not extractable from pigmented seed coats with tawny pubescence (i, T genotypes) because they have proanthocyanidins that exhibit tannin properties. The dominant Im allele inhibits seed coat mottling (irregular patches of pigmentation) that occurs if plants are infected with soybean mosaic virus. PRP1 was 35 kDa in mottled (im) isolines and 34 kDa in non-mottled (Im) isolines. PRP2, which is expressed later in seed coat development and in the hypocotyl hooks of soybean seedlings, was also smaller in Im isolines. In summary, some of the anthocyanin mutations affect the quantity of soluble PRP1 polypeptides, while others correlate with structural changes in developmentally regulated proline-rich proteins.     

Linker-based GnRH-PE chimeric proteins inhibit cancer growth in nude mice

Since the number of cancer-related deaths has not decreased in recent years, major efforts are being made to find new drugs for cancer treatment. In this report we introduce the gonadotropin releasing hormone-Pseudomonas exotoxin (GnRH-PE) based chimeric proteins L-GnRH-PE66 and L-GnRH-PE40. These proteins are composed of a GnRH moiety attached to modified forms ofPseudomonas exotoxin via a polylinker (gly₄ser)₂. The chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 have the ability to target and kill adenocarcinoma cell linesin vitro, whereas non-adenocarcinoma cell lines are not affected. We demonstrate that L-GnRH-PE66 and L-GnRH-PE40 efficiently inhibit cancer growth. Nude mice were injected subcutaneously with the SW-48 adenocarcinoma cell line to produce xenograft tumours. When the tumours were established and visible, the animals were injected with chimeric proteins for 10 days. At the end of this period, a reduction of up to 3-fold in tumor size was obtained in the treated mice, as compared with the control group, which received equivalent amounts of GnRH; the difference was even greater 13 days after termination of treatment. Thus, the chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 are promising candidates for treatment of a variety of adenocarcinomas and their use in humans should be considered.     

TP53 mutations in circulating tumor DNA in advanced epidermal growth factor receptor-mutant lung adenocarcinoma patients treated with gefitinib

Tumor protein p53 (TP53) is a tumor suppressor gene and TP53 mutations are associated with poor prognosis in non-small cell lung cancer. However, the in-depth classification of TP53 and its relationship with treatment response and prognosis in epidermal growth factor receptor (EGFR)-mutant tumors treated with EGFR tyrosine kinase inhibitors are unclear. Circulating tumor DNA was prospectively collected at baseline in advanced treatment-naïve EGFR-mutant lung adenocarcinoma patients treated with gefitinib in an open-label, single-arm, prospective, multicenter, phase 2 clinical trial (BENEFIT trial) and analyzed using next-generation sequencing. Survival was estimated using the Kaplan–Meier method. Of the 180 enrolled patients, 115 (63.9%) harbored TP53 mutations. The median progression-free survival (PFS) and overall survival (OS) of patients with TP53-wild type tumors were significantly longer than those of patients with TP53-mutant tumors. Mutations in exons 5–8 accounted for 80.9% of TP53 mutations. Mutations in TP53 exons 6 and 7 were significantly associated with inferior PFS and OS compared to wild-type TP53. TP53 mutation also influenced the prognosis of patients with different EGFR mutations. Patients with TP53 and EGFR exon 19 mutations had significantly longer PFS and OS than patients with TP53 and EGFR L858R mutations, and both groups had worse survival than patients with only EGFR mutations. Patients with TP53 mutations, especially in exons 6 and 7, had a lower response rate and shorter PFS and OS when treated with gefitinib. Moreover, TP53 exon 5 mutation divided TP53 mutations in disruptive and non-disruptive types.     

Interaction between viral proteins with the transmission of Potyvirus

Potyviridae is the largest family in plant viruses, in which a group of potyviruses constitutes a very important role in causing diseases in plants. The organisation of the viral genome is positive-sense RNA, ranging in size from 9000 to 12000?bp. The viral genome encodes a large polyprotein that is processed by three virus-encoded proteinases (two proteinases and helper component proteinase) to yield the mature products. This review concentrates on the interaction between viral proteins with the transmission of Potyvirus. Transmission and long-distance movement of Potyvirus is only possible through vector and that time interaction between two viral proteins takes place, named as helper component-proteinase and coat protein. Interaction between NIb, NIa, 6K2 as well as with CI (helicase activity) also involved in the replication of potyviruses. Some researchers developed a yeast two-hybrid system and biomolecular fluorescence complementation system technology which proved the interaction among the viral protein. At last all proteins are correlated with each other and play a very significant role in the transmission of Potyvirus.     

Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties

Here we report the identification of a new insulin-like growth factor binding protein homologue, provisionally designated insulin-like growth factor binding related protein-4 (IGFBP-rP4). IGFBP-rP4 was found to be most closely related to IGFBP-7 with 52% amino acid homology and 43% amino acid identity, and shares a similar domain structure. Semi-quantitative RT-PCR expression analysis demonstrated a pattern of downregulation of this gene in multiple tumor samples including lung and colon cancer, compared to matched adjacent normal tissue. Western blotting revealed a protein of approximately 38kDa expressed in both the cell pellet and secreted into the supernatant of transiently transfected Cos-7 cells. Cos-7 supernatants containing IGFBP-RP4 protein were observed to suppress the growth of HeLa cells in culture compared to vector controls. IGFBP-RP4 directly transiently transfected into HeLa cells also further confirmed the growth suppressive properties of this protein. Together these data suggest that IGFBP-RP4 may be a novel putative tumor suppressor protein.     

The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.     

Visualization and elution of unstained proteins from polyacrylamide gels

Proteins fractionated by electrophoresis on 18% polyacrylamide gels with low crosslinking can be directly visualized by ultraviolet light-induced fluorescence and can be recovered by electroelution.     

High-throughput characterization of lipopolysaccharide-binding proteins using mass spectrometry

Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the "untargeted" mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.     

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology

Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor–matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor–ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.