RP-HPLC法测定怀槐中染料木素和芒柄花黄素含量

怀槐(Maackia amurensis Rupr.et Maxim.)广泛分布于中国东北三省及内蒙古等地,尤以黑龙江省为多.怀槐作为民间药,其茎、枝可治疗风湿性关节炎; 叶、枝皮可治疗肿瘤; 内皮可治疗深度伤口及久不愈合的溃疡.另外,怀槐树皮在民间还作为祛风湿、消炎、镇痛、健胃、止血等药广泛应用,疗效甚佳.从怀槐的根、枝、叶、果实和心材中分别分得异黄酮、生物碱、NC442类化合物,据报道,其中的异黄酮类化合物表现出对心血管系统的复合作用和明显的抗肿瘤活性[1].作者在对怀槐进行系统的化学成分研究过程中,分离鉴定了9个异黄酮类化合物[2,3],并进行了抑制肿瘤细胞增殖作用研究[4],其中,染料木素(genistein)对胃腺癌细胞(BGC)增殖有一定程度的抑制作用,芒柄花黄素(formononetin)则对白血病细胞(HL-60)增殖有一定的抑制作用.染料木素和芒柄花黄素作为马鞍树属(Maackia Rupr.)植物中普遍存在的2个异黄酮类化合物,其活性研究日益受到重视.采用HPLC法测定染料木素等异黄酮类化合物含量在大豆等其他植物中曾有文献报道[5].为了尽早开发怀槐资源,同时为其资源的综合利用提供科学依据,本文采用HPLC法对怀槐不同部位的染料木素和芒柄花黄素2个有效成分进行了含量测定.     

Isolation, characterization and molecular cloning of the bark lectins from Maackia amurensis

A detailed study was made of the bark lectins of the legume tree Maackia amurensis using a combination of protein purification and cDNA cloning. The lectins, which are the most abundant bark proteins, are a complex mixture of isoforms composed of two types of subunits of 32 and 37 kDa, respectively. Isolation and characterization of the homotetrameric isoforms indicated that the 32 kDa subunit exhibits a 100-fold stronger haemagglutinating activity than the 37 kDa subunit. Molecular cloning confirmed that the two lectin subunits are encoded by different genes. The 32 kDa subunit is apparently encoded by a single gene, whereas two highly homologous genes encode the 37 kDa subunit. A comparison of the deduced amino acid sequences of the bark lectin cDNAs and the previously described cDNA encoding the seed haemagglutinin demonstrated that they are encoded by different genes. Abbreviations: LECMAHb, cDNA clone encoding Maackia amurensis bark haemagglutinin; LECMALb, cDNA clone encoding Maackia amurensis bark leucoagglutinin; MALb, Maackia amurensis bark leucoagglutinin; MAHb, Maackia amurensis bark haemagglutinin This revised version was published online in November 2006 with corrections to the Cover Date.     

Enhanced 3-O-sulfation of galactose in Asn-linked glycans and Maackia amurensis lectin binding in a new Chinese hamster ovary cell line

We report the characterization of two Chinese hamster ovary cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient cell mutant pgsA-745 with a cDNA library prepared from wild-type cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.     

Markers of cell lineage, differentiation and activation

The most widespread use of CD markers is in the determination of cell lineage and sublineage. For example, T cells are identified by the expression of CD3 (reviewed in this issue of CD corner). A mature T cell may belong to the T4 subset, in which case it will express CD4. Similarly, there are markers for other cell populations and sub-populations. Within the lineages, it is helpful to distinguish cells at different stages of differentiation and activation. Differentiation status is particularly useful in the diagnostic analysis of the lymphoid and myeloid malignancies, and in research on the haemopoietic system. Examples include markers for na?ve or antigen-experienced cells (especially the CD45 isoforms) and molecules such as CALLA (CD9) found on B-lineage precursors, including B lineage acute lymphoblastic leukaemia. Activation status is especially interesting in studies of cell function. Activation markers include growth factor receptors such as CD25 (a component of the receptor for IL-2), and molecules who's cellular function is not fully understood, such as CD69 and CD98. These markers have revolutionised aspects of pathology and research, and the ease with which some cell populations can be identified has lead to some unrealised, and perhaps unrealistic, expectations. We expect to be able to identify T helper type 1 (TH1) and T helper type 2 (TH2) cells on the basis of a simple surface marker; we are frustrated by the lack of a single marker for all dendritic cells or for all NK cells; we are confused by the un-coordinated expression of different activation markers; we tend to over-interpret phenotype in some situations. To find solutions to these problems it is helpful to examine why the successful lineage markers work so well, and to reconsider our expectations. Lineage markers are the main focus in this commentary; the question of activation markers and markers of differentiation state will be considered in a separate paper.     

Use of a cDNA library for the study of mRNA changes during muscle differentiation

A cDNA library was used to measure changes in many individual mRNAs during muscle differentiation in culture. A library of 1000 clones was constructed from total myofiber poly(A) RNA. About 23% of these clones gave a detectable colony hybridization signal using end-labeled myofiber mRNA, the remainder containing muscle sequences too rare to be detected with this assay. The 230 positive clones were grouped into four classes based on relative visual intensity. Reconstruction experiments using pure globin mRNA enable us to determine the approximate percentage of total RNA made up by each mRNA hybridizing to a cDNA clone. Those clones containing sequences complementary to developmentally regulated mRNAs were identified by a differential hybridization procedure. The cDNA library was screened with end-labeled mRNA from both undifferentiated myoblasts and differentiated myofibers. Although the bulk of the clones hybridized essentially the same with both RNA populations, several dozen were found which hybridized differentially. Some clones contained sequences which were not present at all in myoblasts and present in very high quantities in myofibers. Others contained sequences found in both myoblasts and myofibers but in increased quantities in the differentiated cells. Still others contained sequences which decreased in quantity during muscle differentiation. The clones in the first group were chosen for immediate analysis since they likely contain contractile protein mRNA sequences. However, all the characterized cDNA clones can now be used as probes to study the chromosomal organization and developmental expression of genes active during muscle differentiation.     

The influence of the rolC gene on isoflavonoid production in callus cultures of Maackia amurensis

The polyphenolic complex of Maackia amurensis, as well as a complex of isoflavonoids from M. amurensis callus cultures, display strong hepatoprotective effects in experimental animal and human studies. To increase the yield of polyphenols in cultures of M. amurensis, calli were transformed with the rolC gene as well as with an empty vector that was used as a control. HPLC analysis revealed that the transgenic cultures produced the same complex of isoflavonoids. The complex consisted of 20 compounds, including isoflavones and their glucosides as well as pterocarpans and their glucosides. The cultures transformed with either the empty vector or the rolC gene construct produced on average 1.22 % dry weight (DW) and 1.39 % DW of isoflavonoids, respectively. Isoflavonoid production in the transformed callus lines carrying the empty vector and the rolC gene construct reached 106 and 146 mg/L, respectively. Moreover, the rolC gene construct promoted cell growth and overall cell productivity. The transgenic callus lines expressing the rolC gene exhibited higher levels of the following six isoflavonoids: daidzein, calycosin, formononetin, 4′-Ο-β-glucopyranosyldaidzin, maackiain and 6′-O-malonyl-3-O-β-D-glucopyranosylmaackiain. However, lower levels of genistin were observed in rolC calli than in those carrying the empty vector.     

Polymeric fibrous matrices for substrate-mediated human embryonic stem cell lineage differentiation

Chitosan-based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate-mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography-mediated hESC differentiation. Matrices composed of 400 nm and 1.1 μm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous-mediated hESC differentiation highlights the significant implication of tailored ECM-like substrates for hESC-based therapies.     

Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (T(H)1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particular clone to its recombinant, expressed protein. We have generated a mouse T(H)1 expression cDNA library and used protein arrays of this library to characterize the specificity and cross-reactivity of antibodies. Additionally, we have profiled the autoantibody repertoire in serum of a mouse model for systemic lupus erythematosus on these protein arrays and validated the putative autoantigens on highly sensitive protein microarrays.     

Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation

We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.     

In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.     

The gastric epithelial progenitor cell niche and differentiation of the zymogenic (chief) cell lineage

In the mammalian gastrointestinal tract, the cell fate decisions that specify the development of multiple, diverse lineages are governed in large part by interactions of stem and early lineage progenitor cells with their microenvironment, or niche. Here, we show that the gastric parietal cell (PC) is a key cellular component of the previously undescribed niche for the gastric epithelial neck cell, the progenitor of the digestive enzyme secreting zymogenic (chief) cell (ZC). Genetic ablation of PCs led to failed patterning of the entire zymogenic lineage: progenitors showed premature expression of differentiated cell markers, and fully differentiated ZCs failed to develop. We developed a separate mouse model in which PCs localized not only to the progenitor niche, but also ectopically to the gastric unit base, which is normally occupied by terminally differentiated ZCs. Surprisingly, these mislocalized PCs did not maintain adjacent zymogenic lineage cells in the progenitor state, demonstrating that PCs, though necessary, are not sufficient to define the progenitor niche. We induced this PC mislocalization by knocking out the cytoskeleton-regulating gene Cd2ap in Mist1^−/− mice, which led to aberrant E-cadherin localization in ZCs, irregular ZC-ZC junctions, and disruption of the ZC monolayer by PCs. Thus, the characteristic histology of the gastric unit, with PCs in the middle and ZCs in the base, may depend on establishment of an ordered adherens junction network in ZCs as they migrate into the base.     

Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway

Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation.