Fine structural features of oyster glycogen: mode of multiple branching

The fine structural features of oyster glycogen, especially its mode of multiple branching, was investigated by repeated enzymic treatment with β-amylase and pullulanase, followed by the precise analysis of the -1,4-linked glucosyl unit-chains by high performance anion exchange chromatography (HPAEC). The purified glycogen (average mol. wt 8.5 × 10⁵, 11) obtained by DMSO-extraction from fresh oysters (Crassostrea gigas) collected in February (a time when the oysters are edible) showed a distribution of -1,4- -glucosyl unit-chains, with degrees of polymerization (dp) in the range 2–35 (dp 6, dominant), as measured by HPAEC after complete enzymic debranching. The oyster glycogen was subjected to stepwise degradations with β-amylase and pullulanase, and this procedure was repeated until complete hydrolysis was achieved (extent and degradation of 98% after five treatments). The yield of the limit dextrin formed at each trimming step and quantitative analysis of the unit-chain distributions indicated that the oyster glycogen has a highly branched structure (A:B-chain, 0.7:1), involving five or six times interlinkings of the chains (B-chains). Assuming that B1 chain carrying only A-chains, attaches by -1,6-bonds to another B-chain (B2 chain), which in turn attaches to a B3-chain, and so on, the molar ratios of the unit-chains (A, B1, B2-) of the dextrins during successive enzymic trimming showed that the ratio of A:B1:B2:B3:B4:B5-chain was 34:25:11:5:5:1, confirming the multiple ramified molecule. In connection with the digestion of oyster glycogen in the mammalian digestive tract, the glycogen was hydrolyzed by salivary and pancreatic -amylase, and several branched maltosaccharides in the digestion product were fractionated, and their structures determined using HPAEC.     

Stimulation of macrophage by enzymatically synthesized glycogen: the relationship between structure and biological activity

Glycogen is exclusively known as an energy and carbon reserve in animal cells and micro-organisms. We synthesized glycogens of varying molecular weight by using three enzymes, and investigated the relationship between the structure and immunostimulating activity of glycogen. These results indicated that glycogens with a molecular weight of more than 1.0×10⁷ hardly activated RAW264.7, a murine macrophage cell line, whereas glycogens of 5.0–6.5×10⁶ strongly stimulated RAW264.7 in the presence of interferon-γ, leading to augmented production of nitric oxide, tumour necrosis factor-α and interleukin-6. Additionally, the number-average unit chain length and the exterior and interior chain lengths of the glycogens showed a minor correlation between active and less-active glycogen derivatives. On the other hand, the binding activity of glycogen toward RAW264.7 did not depend on the molecular weight of glycogen. The available evidence suggests that the macrophage-stimulating activity of glycogen is strictly related to its molecular weight rather than to fine structural properties.     

Multiple branching in glycogen and amylopectin

The β-amylase limit dextrins of glycogen and amylopectin are completely debranched by joint action of isoamylase and pullulanase. Action of isoamylase alone results in incomplete debranching as a consequence of the inability of this enzyme to hydrolyze those A-chains that are two glucose units in length (half the total number of A-chains). From the reducing powers released by isoamylase acting (a) alone and (b) in conjunction with pullulanase, the relative numbers of A- (unsubstituted) and B- (substituted) chains in the β-dextrins, and therefore in the native polysaccharides themselves, can be calculated. Examination of a series of glycogens and amylopectins in this way showed that the ratio of A-chains: B-chains is markedly higher in amylopectins (1.5–2.6:1) than in glycogens (0.6–1.2:1). Glycogen typically contains A-chains and B-chains in approximately equal numbers; amylopectin typically contains approximately twice as many A-chains as B-chains. These polysaccharides therefore differ in degree of multiple branching as well as in average chain length. A consequence of these findings is that amylopectin cannot be formed in vivo by debranching of a glycogen precursor, as proposed by Erlander, since it is impossible to increase the A:B chain ratio by action of a debranching enzyme.     

Variation in the degree of branching of the glycogens and an approximate relationship between average exterior chain length and iodine staining

Precise evaluation of the branching characteristics of glycogen complex from a variety of sources was made by replicate determinations of end groups by a reproducible periodate oxidation method and by replicate determinations of the percentage degradation by exhaustive β-amylase activity. Also, the iodine staining characteristics of these samples were studied. The following observations were made. During the synthesis of glycogen by baker's yeast, there was a significant increase in the average chain length of the glycogen, and the maximal optical absorbance A_M of the glycogen–iodine complex doubled. The A_M decreased linearly with increasing temperature, and increases in iodine concentration caused increases in both the A_M and the wavelength of maximal absorbance. There was a general increase in the A_M of the glycogen with increasing exterior chain length, but there were minor deviations due to some dependence on the source of the glycogen. Unlike the linear relationship between the A_M per chain and the average chain length of the amyloses, the A_M per chain was exponentially related to the average exterior chain length of the glycogens.     

The Structure of Neurospora crassa Glycogen

The mycelia of a wild type strain of Neurospora crassa (6068, IFO) contain a polysaccharide which is stained reddish brown by iodine. The polysaccharide purified by repeated precipitation with ethanol is made up of d-glucose and has a molecular weight of about (more than) 2 × 10⁷, 101 S on ultracentrifugation analysis, an average chain length of 10, β-amylolysis limit of 33.6%, and α-amylolysis limit of 58.3%. The highly branched structure, therefore, resembles to that of a typical glycogen. The properties of the glycogen from N. crassa are discussed in comparison with the commercial glycogens from shellfish and rabbit liver.     

Trehalose determination in linseed subjected to osmotic stress. HPAEC‐PAD analysis: an inappropriate method

Trehalose is a non‐reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co‐elution of trehalose in these two species by a trehalase assay and liquid chromatography‐high resolution mass spectrometry analysis, gas chromatography–mass spectrometry (GC‐MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC‐PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC‐MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (?0.30 MPa), the quantity (31.49 nmol g^–1 dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.     

Last Step in the Conversion of Trehalose to Glycogen: A MYCOBACTERIAL ENZYME THAT TRANSFERS MALTOSE FROM MALTOSE 1-PHOSPHATE TO GLYCOGEN

We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [¹⁴C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [¹⁴C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the ¹⁴C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 μmol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named α1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [¹⁴C]maltose-1-P, but not [¹⁴C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.     

Mitochondrial ROS-K+ channel signaling pathway regulated secretion of human pulmonary artery endothelial cells

The objective was to investigate the molecular mechanism of mitochondrial reactive oxygen species (ROS) signaling regulation of pulmonary artery endothelial cell (HPAEC) secretion in the condition of oxidative stress. Acrolein (40 μM) induced HPAEC mitochondrial generation of ROS, rotenone (2 μmol/L) blocked mitochondrial respiratory chain complex I, cesium chloride (CsCl, 40 mmol/L)blocked K⁺channels, and saline (0.9 g/dl) were used as control. The generations of NOS, ET-1 and VEGF were determined with ELISA in the condition of different treatment reagents namely acrolein, acrolein plus rotenone, acrolein plus CsCl and saline. In the different reagent treatment of HPAECs, acrolein increased mitochondrial ROS, membrane potential, Kv1.5 mRNA and protein expression, intracellular calcium and the generation of NOS (determining NO production), ET-1 and VEGF, and those were reduced by rotenone. CsCl decreased the increment of membrane potential, the elevation of intracellular calcium and the upregulation of NOS, E-1 and VEGF expressions, which were induced by acrolein. The present study demonstrated that mitochondrial ROS-K⁺channel regulated HPAEC secretion of NO, ET-1 and VEGF in the condition of oxidative stress. Kv1.5 channel may be an important component of ROS-K+ channel signaling pathway, and intracellular calcium contributed to mitochondrial ROS-K⁺ channel signaling modulation of HPAEC secretion.     

The fine structure of the branched α-D-glucan from the blue-green alga Anacystis nidulans: comparison with other bacterial glycogens and phytoglycogen

The fine structure of the glycogen from the blue-green alga Anacystis nidulans has been examined. After selective hydrolysis of all (1→6)-α-D linkages by a bacterial isoamylase, the resulting mixture of linear chains was subjected to gel-permeation chromatography. For purposes of comparison, the glycogens from Escherichia coli and Arthrobacter sp., amylopectin, phytoglycogen from sweet corn, and shell-fish glycogen were treated similarly. The profiles of the unit chains of A. nidulans glycogen and phytoglycogen were closely similar. There was no close resemblance in the size distribution of unit chains for A. nidulans glycogen, other bacterial glycogens, and amylopectin.     

Antibacterial activity of naringin derivatives against pathogenic strains

Aims: To study the antimicrobial activity of naringin (NAR), a flavonoid extracted from citrus industry waste, and NAR derivatives [naringenin (NGE), prunin and alkyl prunin esters] against pathogenic bacteria such as L. monocytogenes, E. coli O157:H7 and S. aureus. The relationship between the structure of the chemical compounds and their antagonistic effect was also analysed. Methods and Results: The agar dilution technique and direct contact assaying were applied. NGE, prunin and NAR showed no antimicrobial activity at a concentration of 0·25 mmol l^?1. Similarly, fatty acids with a chain length between C2 and C18 showed no antimicrobial activity at the same concentration. However, prunin‐6″‐O‐acyl esters presented high antibacterial activity, mainly against Gram‐positive strains. This activity increased with increasing chain length (up to 10–12 carbon atoms). Alkyl prunin esters with 10–12 carbon atoms diminished viability of L. monocytogenes by about 3 log orders and S. aureus by 6 log orders after 2 h of contact at 37°C and at a concentration of 0·25 mmol l^?1. The compounds examined were not effective against any of the Gram‐negative strains assayed, even at the highest concentration. Conclusions: Addition of sugars to the aglycone did not enhance its antimicrobial activity. Attachment of a saturated aliphatic chain with 10–12 carbon atoms to the A ring of the flavonoid (or to sugars attached to this ring), seems to be the most promising modification. In conclusion, alkyl prunin esters with a chain length of C10–C12 have promising features as antimicrobial agents because of their high antilisterial and antistaphylococcal activity. Significance and Impact of the Study: This study shows that it is possible to obtain NAR derivatives with important antimicrobial activity, especially against Gram‐positive pathogenic bacteria. It also provides guidelines on the structural modifications in similar molecules to enhance the antimicrobial activity.     

A Cupheaβ‐ketoacyl‐ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases

Acyl–acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8–C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a β-ketoacyl-ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II-type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0- and 8:0-ACP at a rate four- to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0- and 16:0-ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain-specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one-third and one-half, respectively. F₂ progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4-fold more 12:0 than the FatB2 parent, and the double-transgenic progeny produced one-quarter less 14:0 and one-half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl-ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.     

Comparison of theN-glycoloylneuraminic andN-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography

N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - RPM rat promegakaryoblast - MEG-01 human megakaryoblastic leukaemia cell line - PAD pulsed amperometric detection - WGA wheat germ agglutinin - FCS foetal calf serum - PPEADF phosphatidylethanolamine dipalmitoyl - LSIMS liquid secondary ion mass spectrometry - HPAEC high performance anion exchange chromatography - TBA thiobarbituric acid     

Glycogen from Klebsiella pneumoniae M 5 al and Escherichia coli K 12

Summary 1. A continuous two stage cultivation method for two strains of Klebsiella pneumoniae and Escherichia coli yielding high cell mass and relatively high glycogen contents is described. The stage 1 cells (carbon-limited) were fed with the nitrogen source ammonia (which also neutralized simultaneously) soly via the pH-stat. In stage 2, the cells grew nitrogen-limited, a small excess of the carbon source was maintained by continuous addition of a glucose solution.2. Through the action of lysozyme, the glycogen could be quantitatively solubilized from the alkali-insoluble cell material obtained through alkaline hydrolysis of the cells in dimethylsulfoxide-3M aqueous potassium hydroxide. The possibility that the glycogen is in part covalently linked to the peptidoglycan and localized in the periplasm is discussed. 3. Analytical data for the glycogens isolated from the two bacterial strains is given.     

VCD Robustness of the Amide‐I and Amide‐II Vibrational Modes of Small Peptide Models

The rotational strengths and the robustness values of amide‐I and amide‐II vibrational modes of For(AA)_nNHMe (where AA is Val, Asn, Asp, or Cys, n = 1–5 for Val and Asn; n = 1 for Asp and Cys) model peptides with α‐helix and β‐sheet backbone conformations were computed by density functional methods. The robustness results verify empirical rules drawn from experiments and from computed rotational strengths linking amide‐I and amide‐II patterns in the vibrational circular dichroism (VCD) spectra of peptides with their backbone structures. For peptides with at least three residues (n ≥ 3) these characteristic patterns from coupled amide vibrational modes have robust signatures. For shorter peptide models many vibrational modes are nonrobust, and the robust modes can be dependent on the residues or on their side chain conformations in addition to backbone conformations. These robust VCD bands, however, provide information for the detailed structural analysis of these smaller systems. Chirality 27:625–634, 2015 © 2015 Wiley Periodicals, Inc.     

A novel tetrasaccharide, with a structure similar to the terminal sequence of an arabinogalactan-protein, accumulates in rice anthers in a stage-specific manner

Analysis of free sugars in developing rice anthers by high-performance anion-exchange chromatography (HPAEC) showed that a very high concentration of a novel oligosaccharide accumulated specifically during microsporogenesis. Structural analysis of the purified oligosaccharide by methylation analysis, mass spectrometry/mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy revealed its structure to be β-l Araf.(1→3)-α-l -Ara_f-(1→3)-β-d -Gal_p-(1→6)-d -Gal, which is closely related to a tetrasaccharide unit found in the glycan chain of a plant cell surface proteoglycan, the arabinogalactan-protein (AGP). Chilling treatment (12°C, 4 days), which injures rice anthers during microsporogenesis, decreased the concentration of the tetrasaccharide, but the sucrose level increased. This effect was especially evident in a chilling-sensitive mutant line, YM56-1. These results suggest that this unique tetrasaccharide may play an important role in both the development of the rice anther and its response to chilling.     

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.     

Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: An insight on the sulfide binding-site

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b–e) and four linker chains (L1–L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a–f) and five for C1 (a–e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a Mr of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vetimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the “strain A” family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs. Proteins 29:562–574, 1997. © 1997 Wiley-Liss, Inc.     

An enzymic method for determination of the average chain lengths of glycogens and amylopectins

A new method for the determination of the average chain lengths of glycogens and amylopectins is described. The method utilizes yeast glucosidase-transferase and sweet-potato β-amylase, and was tested successfully against a number of samples of polysaccharides of known chain lengths. The method has several advantages over previous enzymic methods in that (1) it is relatively insensitive to the presence of α-amylase, (2) the enzymes required are readily available—sweet-potato β-amylase is commercially available and we describe a relatively simple method for the isolation of the yeast glucosidase-transferase, (3) the time required for the assay is short, relative to the method of Lee and Whelan (8).     

The alpha-amylases as glycosylases, with wider catalytic capacities than envisioned or explained by their representation as hydrolases

In a study undertaken to illustrate the inadequacy of the familiar concept of carbohydrases as hydrolases, crystalline α-amylases from six different sources, as well as crude salivary amylase, were examined and found to catalyze the synthesis of maltose and maltosaccharides from α-d-glucopyranosyl fluoride, a stereoanalog of α-d-glucopyranose. These syntheses apparently involve initial formation of maltosyl fluoride and higher maltosaccharide 1-fluorides, traces of which were found in digests with certain α-amylases. That the reactions are due to the α-amylases themselves and not to some accompanying enzyme(s) appears certain from the purity and diversity of the preparations; their failure (with one exception) to attack α- or β-maltose; the correspondence of the synthesized products with the known specificity of α-amylases for α-1,4-d-glucosidic linkages (and capacity of different α-amylases to hydrolyze saccharides of different sizes). The “saccharifying” α-amylase of B. sublilis var amylosacchariticus was unique in producing maltosaccharides from both α- and β-maltose (i.e., by α-d-glucosyl transfer). However, the entire group of α-amylases had the capacity to promote α-d-glucosyl transfer from α-d-glucosyl fluoride to C₄-carbinol sites, demonstrating for the first time that the catalytic range of α-amylase extends beyond hydrolysis and its reversal. Indeed, all transferred the glucosyl group of α-d-glycosyl fluoride preferentially to C₄-carbinols rather than water—a finding neither anticipated nor explained by the representation of α-amylases as hydrolases.     

低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响*

目的:探讨低氧时人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC)的高迁移率族蛋白1(HMGB1)及相关受体和炎症因子表达,并检测HMGB1对两种细胞增殖、迁移活性的影响。方法:低氧(1%氧浓度,Hypoxia组)及常氧(Control组)条件下培养HPASMC和HPAEC,RealTime-PCR检测两种细胞HMGB1、TLR2、TLR4、TLR9、RAGE、CD24、IL-6 、TNF-a和CXCL8 mRNA等受体和炎性因子的表达。MTS法观察不同浓度HMGB1对HPASMC和HPAEC增殖的影响;划痕法观察HMGB1对HPASMC和HPAEC迁移的影响。结果:Hypoxia组HPASMC、HPAEC中HMGB1及RAGE mRNA表达量较Control 组明显升高(P＜0.05及0.01);Hypoxia组HPAEC中CD24及HPASMC中IL-6 mRNA表达明显增高(P均＜0.05)。MTS结果显示在345 pmol/L 剂量下 HMGB1明显抑制HPAEC的增殖(P＜0.01),而对HPASMC增殖无影响。划痕实验示HMGB1对HPASMC和HPAEC迁移无明显影响。结论:低氧诱导HPAEC、HPASMC 产生HMGB1;HMGB1通过抑制HPAEC增殖引起内皮屏障功能障碍;而低氧进一步刺激HPASMC产生炎症因子。